WO2018225873A1 - Nucleic-acid-containing nanoparticles - Google Patents

Abstract

The present invention pertains to nanoparticles containing: a lipid (lipid I) that includes a lipid- or water-soluble unit and has a ligand capable of binding to Siglec (sialic acid-binding immunoglobulin-like lectin), and a nucleic acid; or a polymer (polymer I) that includes a water-soluble unit and a cationic unit and has a ligand capable of binding to Siglec (sialic acid-binding immunoglobulin-like lectin), and a nucleic acid. These nanoparticles are nucleic acid-containing nanoparticles that can be delivered to immune system cells.

Description

Nucleic acid-containing nanoparticles

The present invention relates to a nucleic acid-containing nanoparticle, a method for introducing a nucleic acid into a cell, a medicine using the nucleic acid-containing nanoparticle, and a method for treating or preventing a disease.

Nucleic acid drugs such as plasmid DNA (pDNA), antisense, decoy nucleic acid, ribozyme, siRNA, miRNA, antimiRNA, and mRNA can control genes by promoting or suppressing the expression of all genes in cells. Due to its high versatility, clinical application to various diseases that have been considered difficult to treat is expected.
Nucleic acid drugs are expected as next-generation drugs after antibodies and low-molecular-weight drugs because of their high target selectivity and activity in cells.
However, it is a problem that nucleic acid drugs are difficult to deliver to target tissues.

As one effective delivery method for nucleic acid drugs in vivo, a method using lipid / nucleic acid or polymer / nucleic acid nanoparticles having a targeting compound has been reported. Targeting compounds include ligands that can bind to receptors expressed extracellularly.

In Non-Patent Document 1 and Patent Document 1, a lipid comprising an N-acetyl-D-galactosamine (GalNAc) ligand capable of binding to an asialoglycoprotein receptor (ASGPR) highly expressed in hepatocytes and a double-chain lipid / Targeting the liver with nucleic acid nanoparticles has been reported.

In Non-Patent Document 2 and Patent Document 2, cancer by a lipid / nucleic acid nanoparticle comprising a peptide ligand capable of binding to a prostate membrane specific antigen (PSMA) that is specifically highly expressed in prostate cancer patients and a double-stranded lipid. Targeting to has been reported.

Also, Non-Patent Document 3 reports targeting of cancer cells with polymer / nucleic acid nanoparticles having transferrin.

As reported in Non-Patent Documents 1 to 3 and Patent Documents 1 and 2, application of a targeting compound to a nucleic acid-containing nanoparticle can be expected to improve migration to a target tissue and pharmacological effect. However, the majority of these target organs and cells are the liver or cancer, and their application range is limited. Accordingly, there is a strong demand for the development of nucleic acid drugs that are effective for target organs and cells other than the liver.

Siglec (sialic acid-binding-immunoglobulin-likelectin) is a receptor that recognizes sialic acid-containing sugar chains belonging to the immunoglobulin superfamily and is expressed on the surface of immune system cells (Non-patent Document 4).

As a delivery technique using Siglec, Non-Patent Documents 5 and 6 report the use of a complex using an antibody capable of binding to Siglec-2, which is one of Siglec receptors.

Patent Document 3 discloses a sugar chain ligand capable of binding to Siglec. Furthermore, Patent Document 4 reports a technique for delivering a low-molecular-weight drug or an antigen by nanoparticles using a sugar chain ligand capable of binding to Siglec.

However, there are no reports of nucleic acid-containing nanoparticles using sugar chain ligands that can bind to Siglec.

International Publication No. 2010/054405 International Publication No. 2012/016188 International Publication No. 2007/056525 International Publication No. 2012/018377

Molecular Therapy, 2010, Vol. 18, p 1357-1364 Molecular Therapy Cracic Acid (Molecular Therapy Nucleic Acid), 2016, Vol. 5, pe 348 Nature, 2010, 464, p1067-1070 Nature Reviews Immunology (Nature Reviews Immunology), 2007, Vol. 7, p255-266 British Journal of Hematology (British Journal of Haematology), 2014, Vol. 167, p.487-499. Leukemia, 2010, Vol. 24, p1566-1573

An object of the present invention is to provide nucleic acid-containing nanoparticles and the like that can be delivered to immune system cells.

（N1は、１以上の整数である。）
で表され、
　高分子Iが、シグレックに結合可能なリガンドと、水溶性ユニットとが、あるいはシグレックに結合可能なリガンドと、水溶性ユニットと、カチオン性ユニットとがリンカーを介して連結した構造：

（N2は、１以上の整数である。）
で表される、［１］または［２］に記載のナノ粒子。
［４］
　前記リガンドが、糖鎖リガンドである、［１］～［３］のいずれかに記載のナノ粒子。
［５］
　糖鎖リガンドが、以下の式（１）で表される基である、［４］に記載のナノ粒子。

（式（１）中、Acはアセチル基を表し、R¹は、C6-C12アリール基、C4-C12ヘテロアリール基、または、C6-C12アリール基もしくはC4-C12ヘテロアリール基で置換されたC1-C6アルキル基を表す。）
［６］
　式（１）におけるR¹が、以下の構造で表される基である、［５］に記載のナノ粒子。

［７］
　水溶性ユニットが、ポリエチレングリコール、ポリグリセリン、ポリエチレンイミン、ポリビニルアルコール、ポリアクリル酸、ポリアクリルアミド、オリゴ糖、デキストリン、水溶性セルロース、デキストラン、コンドロイチン硫酸、ポリグリセリン、キトサン、ポリビニルピロリドン、ポリアスパラギン酸アミド、ポリ-L-リジン、マンナン、プルラン、オリゴグリセロール、およびそれらの誘導体からなる群より選択される1種以上を有するユニットである、［１］～［６］のいずれかに記載のナノ粒子。
［８］
　カチオン性ユニットが、リジン、アルギニン、ヒスチジンからなる群より選択される一種以上を含むアミノ酸ポリマーユニット、ポリエチレンイミンユニット、又は、ポリアミノアクリレートユニットである、［１］～［７］のいずれかに記載のナノ粒子。
［９］
　ナノ粒子が、さらに中性脂質を含む、［１］～［８］のいずれかに記載のナノ粒子。
［１０］
　核酸がsiRNAまたはmRNAである、［１］～［９］のいずれかに記載のナノ粒子。
［１１］
　siRNAが、コレステロール、トコフェロールまたは脂肪酸とリンカーを介して共有結合している、［１０］に記載のナノ粒子。
［１２］
　Siglec-1(CD169)陽性細胞へ送達するために使用される、［１］～［１１］のいずれかに記載のナノ粒子。
［１３］
　Siglec-1（CD169）陽性細胞が、マクロファージ、樹状細胞または単球である、［１２］に記載のナノ粒子。
［１４］
　［１］～［１１］のいずれかに記載のナノ粒子を用いる、核酸を細胞内に導入する方法。
［１５］
　細胞が、Siglec-1(CD169)陽性細胞である、［１４］に記載の方法。
［１６］
　Siglec-1（CD169）陽性細胞が、マクロファージ、樹状細胞または単球である、［１５］に記載の方法。
［１７］
　［１］～［１３］のいずれかに記載のナノ粒子を含む、医薬。
［１８］
　静脈内投与用また皮下投与用である、［１７］に記載の医薬。
［１９］
　［１７］または［１８］に記載の医薬を、それを必要とする患者に投与することを含む、疾患の治療または予防方法。
［２０］
　シグレック（Sialic acid-binding immunoglobulin-like lectin）に結合可能なリガンドを有する、脂質または水溶性ユニットを含む脂質(脂質I)、および核酸を含む、［１］に記載のナノ粒子。
［２１］
　シグレック（Sialic acid-binding immunoglobulin-like lectin）に結合可能なリガンドを有する、脂質または水溶性ユニットを含む脂質(脂質I)、核酸、およびカチオン性脂質(脂質II)を含む、[２]に記載のナノ粒子 The present invention relates to the following.
[1]
A lipid containing lipid or a water-soluble unit (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a nucleic acid, or
A polymer containing a water-soluble unit and a cationic unit (polymer I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a nucleic acid,
Nanoparticles.
[2]
Contains a lipid or lipid-containing unit with a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) (lipid I), nucleic acid, and cationic lipid (lipid II), or
Polymers (Polymer I) containing water-soluble units and cationic units, having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), nucleic acids, and high molecules containing water-soluble units and cationic units Including molecules (polymer II),
Nanoparticles.
[3]
Lipid I is a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit are linked via a linker:

(N1 is an integer of 1 or more.)
Represented by
Polymer I has a structure in which a ligand capable of binding to Siglec and a water-soluble unit, or a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are linked via a linker:

(N2 is an integer of 1 or more.)
The nanoparticle according to [1] or [2], represented by:
[4]
The nanoparticle according to any one of [1] to [3], wherein the ligand is a sugar chain ligand.
[5]
The nanoparticle according to [4], wherein the sugar chain ligand is a group represented by the following formula (1).

(In the formula (1), Ac represents an acetyl group, and R ¹ represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1 substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Represents a -C6 alkyl group.)
[6]
The nanoparticle according to [5], wherein R ¹ in formula (1) is a group represented by the following structure.

[7]
Water-soluble unit is polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, chitosan, polyvinylpyrrolidone, polyaspartic acid amide The nanoparticle according to any one of [1] to [6], which is a unit having one or more selected from the group consisting of: poly-L-lysine, mannan, pullulan, oligoglycerol, and derivatives thereof.
[8]
The cationic unit is an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit, according to any one of [1] to [7] Nanoparticles.
[9]
The nanoparticle according to any one of [1] to [8], wherein the nanoparticle further contains a neutral lipid.
[10]
The nanoparticle according to any one of [1] to [9], wherein the nucleic acid is siRNA or mRNA.
[11]
The nanoparticle according to [10], wherein the siRNA is covalently bonded to cholesterol, tocopherol or a fatty acid via a linker.
[12]
The nanoparticle according to any one of [1] to [11], which is used for delivery to Siglec-1 (CD169) positive cells.
[13]
The nanoparticle according to [12], wherein the Siglec-1 (CD169) positive cell is a macrophage, a dendritic cell or a monocyte.
[14]
[1] A method for introducing a nucleic acid into a cell, using the nanoparticles according to any one of [11].
[15]
The method according to [14], wherein the cell is a Siglec-1 (CD169) positive cell.
[16]
The method according to [15], wherein the Siglec-1 (CD169) positive cells are macrophages, dendritic cells or monocytes.
[17]
[1] A pharmaceutical comprising the nanoparticle according to any one of [13].
[18]
The medicament according to [17], which is for intravenous administration or subcutaneous administration.
[19]
[17] A method for treating or preventing a disease, comprising administering the medicine according to [18] to a patient in need thereof.
[20]
The nanoparticle according to [1], comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) and containing a lipid or a water-soluble unit (lipid I), and a nucleic acid.
[21]
The lipid according to [2], comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), including a lipid or a water-soluble unit (lipid I), a nucleic acid, and a cationic lipid (lipid II). Nanoparticles

The present invention can provide nucleic acid-containing nanoparticles that are useful as pharmaceuticals and can be delivered to immune system cells.

Hereinafter, embodiments of the present invention will be described in detail. Note that the embodiments described below do not limit the present invention.

[Nanoparticles]
The nanoparticle of the present invention includes a lipid having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), a lipid or a water-soluble unit (lipid I), and a nucleic acid, or
A polymer (polymer I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) and containing a water-soluble unit and a cationic unit (polymer I), and a nucleic acid. The nanoparticles of the present invention are nucleic acid-containing nanoparticles.
The average particle size of the nanoparticles of the present invention is preferably 1.00 to 2000 nm, more preferably 10.0 to 500 nm, still more preferably 20.0 to 300 nm, and most preferably 20.0 to 200 nm.
Siglec is a receptor belonging to the immunoglobulin superfamily and is present on the surface of immune system cells. The nanoparticles of the present invention can be targeted to monocytes, macrophages, or dendritic cells.

The nanoparticle of the present invention has, as a model, a structure in which a lipid-containing outer layer is encapsulated in a central part including a nucleic acid (that is, a structure in which a nucleic acid exists inside a particle and a lipid constitutes the outer part of the particle (outer layer)). Can be considered. At this time, lipid I and polymer I are present in the outer layer of the nanoparticle, and the ligand capable of binding to Siglec is present in the outward direction of the particle, and the surface of the particle is modified by the ligand. it is conceivable that. This allows the ligand to interact with Siglec.
At this time, the modification rate (%) by the ligand on the surface of the particle is defined as the molar concentration of lipid I or polymer I relative to the total molar concentration of lipid or polymer constituting the particle (that is, the number of moles of lipid I / particle). The total number of moles of lipid constituting or the number of moles of polymer I / the total number of moles of polymer constituting particles) can be used as an index. In the case of a nucleic acid-containing nanoparticle containing lipid I, the modification rate is preferably 0.2% or more, more preferably 0.4% or more in order for the nanoparticle to reach the target cell and transfer the nucleic acid into the cell. More preferably 1.0% or more, still more preferably 1.4% or more. The maximum value of the modification rate is not particularly limited, but is preferably 50% or less, more preferably 30% or less, still more preferably 10% or less, and even more preferably 5% or less.
In the case of the nucleic acid-containing nanoparticles containing the polymer I, the modification rate is preferably 0.2% or more, more preferably 0.4% in order for the nanoparticles to reach the target cell and transfer the nucleic acid into the cell. Or more, more preferably 1.0% or more, and still more preferably 1.4% or more. The maximum value of the modification rate may be 100% or less. The modification rate in the nucleic acid-containing nanoparticles containing the polymer I is preferably 30 to 100%, more preferably 50 to 100% or less, and even more preferably 70 to 100%.

The nanoparticle of the present invention may be a nanoparticle comprising a lipid (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), a lipid containing lipid or a water-soluble unit (lipid I), and a nucleic acid. Preferably, in nanoparticles comprising a lipid or lipid containing a water soluble unit (lipid I), a nucleic acid, and a cationic lipid (lipid II) with a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) More preferably.

(Lipid I and polymer I)
Lipid I is a lipid having a ligand capable of binding to Siglec, or a lipid comprising a water-soluble unit having a ligand capable of binding to Siglec. The lipid containing a lipid and a water-soluble unit in the present invention may be a natural type lipid or a non-natural type lipid in which a natural type structure is partially modified, that is, a lipid analog.
The polymer I is a polymer including a water-soluble unit and a cationic unit having a ligand capable of binding to Siglec.
The lipid having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec and a lipid unit are bound. The lipid containing a water-soluble unit having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec, a water-soluble unit, and a lipid unit are bound. The polymer containing a water-soluble unit and a cationic unit having a ligand capable of binding to Siglec can also be referred to as a complex in which a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are bound.
Lipid I preferably has a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit are linked via a linker. The polymer I preferably has a structure in which a ligand capable of binding to Siglec and a water-soluble unit are linked via a linker. A ligand capable of binding to Siglec and a lipid or cationic unit can be linked by a linker.

The structure having a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit is linked via a linker can be specifically represented by the following structural formula. Note that one or more ligands that can bind to Siglec in the structural formula may be bound to a linker.

（構造式中のN1、N2は、１以上の整数である。）

(N1 and N2 in the structural formula are integers of 1 or more.)

Lipid I may be a lipid containing a water-soluble unit having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), that is, a ligand capable of binding to siglec and a water-soluble unit It can also be represented by the following structural formula linked via a linker.

（構造式中のN1は、１以上の整数である。）

(N1 in the structural formula is an integer of 1 or more.)

In the polymer I, a water-soluble unit and a cationic unit may be bonded via a linker, that is, the polymer I can be represented by the following structural formula.

（構造式中のN2は、１以上の整数である。）

(N2 in the structural formula is an integer of 1 or more.)

Lipid I and polymer I may have one or more ligands capable of binding to Siglec as described above, may have two, may have three, or have four It may be. Lipid I and polymer I having two or more ligands capable of binding to Siglec can be obtained by using a linker containing a branched structure, and can be represented by the following structural formula.
Lipid I in the following structure may or may not contain a water-soluble unit between the lipid and the linker. In the polymer I having the following structure, a water-soluble unit and a cationic unit may be bonded via a linker.

The branched structure contained in the linker is not particularly limited, and examples thereof include the structures (A) to (D) shown below.

（式中、m1～m3はそれぞれ独立して、0～3の整数を表す）

(In the formula, m1 to m3 each independently represents an integer of 0 to 3)

The branched structures (A) to (D) can be provided by the following compounds. Specifically, the branched structure (A) can be provided by utilizing, for example, the branched structure possessed by the compounds (A-1) to (A-5). The branched structure (B) can be provided, for example, by utilizing the branched structure possessed by the compounds (B-1) and (B-2). The branched structure (C) can be provided by using, for example, a branched structure possessed by the compounds (C-1) to (C-3). The branched structure (D) can be provided by utilizing a branched structure possessed by the compound (D-1), for example.

It is also possible to provide a branched structure branched into five or more by combining two or more of the branched structures (A) to (D).

Water-soluble units include, for example, polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, chitosan, polyvinylpyrrolidone, polyasparagine It is composed of acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol or the like or derivatives thereof. Among these, polyethylene glycol is preferable. The number average molecular weight of the water-soluble unit is not particularly limited, but is preferably 100 to 40000, more preferably 500 to 20000, and further preferably 500 to 5000.

The lipid in the lipid I is not particularly limited as long as it is a lipid capable of forming lipid nanoparticles, and a neutral lipid is preferable.
Suitable examples of neutral lipids include phospholipids, glycerol lipids, sterols, glyceroglycolipids, glycosphingolipids and sphingoids. These neutral lipids may be used alone or in combination of two or more.

Examples of the phospholipid in the neutral lipid constituting the lipid I include phosphatidylcholine (PC) (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3 -Phosphocholine (DSPC), dipalmitoylphosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), dimyristoylphosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC) ), Phosphatidylethanolamine (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoylphosphoethanolamine). (DMPE), 16-0-monomethyl PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine ( SOPE), etc.), glycerophospholipids (specifically phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.), sphingophospholipids (specifically sphingomyelin, ceramide) Phosphoethanolamine, ceramide phosphoglycerol, ceramide phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.) or hydrogenated phospholipid (specifically hydrogen Additive soybean Scan choline etc.) phospholipids natural or synthetic, such as including but not limited to.

Examples of the glycerol lipid in the neutral lipid constituting the lipid I include, but are not limited to, diacylglycerol.

Examples of the glyceroglycolipid in the neutral lipid constituting the lipid I include, but are not limited to, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.

Examples of the glycosphingolipid in the neutral lipid constituting the lipid I include, but are not limited to, galactosyl cerebroside, lactosyl cerebroside, ganglioside, and the like.

Examples of the sphingoid in the neutral lipid constituting the lipid I include, but are not limited to, sphingan, icosasphingan, sphingosine or derivatives thereof. Derivatives include, for example, —NH ₂ such as sphingan, icosasphingan, and sphingosine —NHCO (CH ₂ ) xCH ₃ (wherein x is an integer of 0 to 18, among which 6, 12 or 18 is preferred. However, it is not limited to these.

Examples of the sterol in the neutral lipid constituting lipid I include, for example, cholesterol (Chol), dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucosterol, or 3β- [N -(N ', N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like are exemplified, but not limited thereto.

Lipid I preferably includes a structure composed of a lipid and a water-soluble unit (hereinafter also referred to as a structure derived from a lipid derivative of a water-soluble polymer). The structure composed of the lipid and the water-soluble unit is preferably derived from a lipid derivative of a water-soluble polymer.
The lipid derivative of the water-soluble polymer in the present invention may be the above-described neutral lipid polyethylene glycol lipid derivative. The polyethylene glycol lipid derivative of the neutral lipid includes, for example, a structure in which a polyethylene glycol group is bonded to a hydroxy group contained in the neutral lipid, with or without an amide group or an ester group, or the neutral lipid. In which a hydroxy group contained in is substituted with an amide group or a carboxyl group, and a polyethylene glycol group is bonded via the substituted amide group or carboxyl group.
Specific examples of structures composed of lipids and water-soluble units include structures represented by the following formulas (Z1) to (Z3).

In the formula (Z1), Ra and Rb are each independently linear or branched alkyl, alkenyl or alkynyl having 7 to 23 carbon atoms, preferably heptadecanyl, pentadecanyl, tridecanyl, (Z) -heptadecane- 8-enyl, (Z) -tridec-8-enyl, (Z) -pentadeca-8-enyl, (Z) -heptadeca-5-enyl, (Z) -heptadeca-8-enyl, (E) -heptadeca- 8-enyl, (Z) -heptadeca-10-enyl, (8Z, 11Z) -heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -heptadeca-8,11,14-trienyl, (Z)- Nonadeca-10-enyl, (10Z, 13Z) -nonadeca-10,13-dienyl, (Z) -heneicosa-12-enyl, nonane-10-ynyl, tridec-5-inyl, pentadeca-6-ynyl, pentadeca- 4,6-diynyl or pentadec-8-ynyl, and n is an integer of 1 to 300, preferably 5 to 200, more preferably 10 to 150.
Of Ra and Rb, heptadecanyl, pentadecanyl, tridecanyl, and (Z) -heptadec-8-enyl are more preferable.

In the formula (Z2), Ra ′ and Rb ′ are independently straight-chain or branched alkyl, alkenyl or alkynyl having 7 to 23 carbon atoms, preferably heptadecanyl, pentadecanyl, tridecanyl, (Z) — Heptadeca-8-enyl, (Z) -Tridec-8-enyl, (Z) -Pentadeca-8-enyl, (Z) -Heptadeca-5-enyl, (Z) -Heptadeca-8-enyl, (E)- Heptadeca-8-enyl, (Z) -Heptadeca-10-enyl, (8Z, 11Z) -Heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -Heptadeca-8,11,14-trienyl, (Z ) -Nonadeca-10-enyl, (10Z, 13Z) -nonadeca-10,13-dienyl, (Z) -heneicosa-12-enyl, nonane-10-ynyl, tridec-5-ynyl, pentadeca-6-ynyl, Pentadeca-4,6-diynyl or pentadeca-8-ynyl, and n is an integer of 1 to 300, preferably 5 to 200, more preferably 10 to 150.
Among Ra ′ and Rb ′, more preferred are heptadecanyl, pentadecanyl, tridecanyl, and (Z) -heptadec-8-enyl.

In the formula (Z3), Ra ″ and Rb ″ are each independently a linear or branched alkyl, alkenyl or alkynyl having 8 to 24 carbon atoms, preferably octadecanyl, hexadecanyl, tetradecanyl, (Z ) -Octadeca-9-enyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E ) -Octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl, (Z) -docosa-13-enyl, deca-11-ynyl, tetradec-6-ynyl, hexadec-7- Inyl, hexadeca-5,7-diynyl or octadec-9-ynyl, n is an integer from 1 to 300, preferably from 5 to 200, more preferably from 10 to 150.
Among Ra ″ and Rb ″, octadecanyl, hexadecanyl, tetradecanyl, and (Z) -octadec-9-enyl are more preferable.

The lipid derivative of the water-soluble polymer is preferably a lipid derivative of polyethylene glycol, more preferably polyethylene glycol-phosphatidylethanolamine (more specifically 1,2-distearoyl-sn-glycero-3-phospho Ethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG -DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3 -[Methoxy (polyethylene glycol) -2000]), and polyethylene glycol-diacylglycerol (more specifically, 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DPG), 1,2-dimyristoyl-sn-glycerol, methoxypolyethyleneglycol-2000 (PEG-DMG), 1,2-dioleoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DOG)), more preferably 1,2-distearoyl-sn-glycero- 3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) and 1,2-distearoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DSG).

The cationic unit in the polymer I is preferably an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit.

The amino acid polymer unit containing one or more selected from the group consisting of lysine, arginine and histidine can be obtained by polymerization of an amino acid containing one or more selected from the group consisting of lysine, arginine and histidine. When the amino acid polymer unit is composed of two or more amino acids, it may be a random copolymer or a block copolymer.
The amino acid polymer unit may contain amino acids other than lysine, arginine, and histidine. The amino acid other than lysine, arginine, and histidine is not particularly limited, but is preferably glycine.
In the amino acids constituting the amino acid polymer unit, one or more amino acid units selected from the group consisting of lysine, arginine and histidine are preferably 70% or more, more preferably 80% or more, and 90% or more. More preferably it is.
Examples of the amino acid polymer unit include the following structures.

（上記構造中、ｎ1は2以上の整数である。）

(In the above structure, n1 is an integer of 2 or more.)

The cationic unit in the polymer I is oriented so as to wrap the nucleic acid to form particles. The amount of charge of the nucleic acid contained in the nanoparticle varies depending on the size (molecular weight) of the nucleic acid, and the size of the cationic unit necessary for particle formation varies accordingly. Therefore, the number of repeating amino acid monomers represented by n1 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably. Is from 10 to 1000, more preferably from 10 to 100, even more preferably from 10 to 50, and particularly preferably from 10 to 40.

The polyethyleneimine unit is a unit made of a polymer obtained by polymerizing ethyleneimine. The polyethyleneimine unit can be represented, for example, as shown in the main chain structure below, but the polyethyleneimine main chain may be cross-linked with other polyethyleneimine structures.

（上記主鎖構造中、ｎ2は１以上の整数である。）

(In the main chain structure, n2 is an integer of 1 or more.)

The number of repeating amino acid monomers represented by n2 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably 50. ~ 1000.

The polyaminoacrylate unit is a unit made of a polymer obtained by polymerizing methacrylic acid having an amino group such as 2- (dimethylamino) ethyl methacrylate. As the polyaminoacrylate unit, for example, the following structures are preferably exemplified.

（上記構造中、ｎ3は１以上の整数である。）

(In the above structure, n3 is an integer of 1 or more.)

The number of repeating amino acid monomers represented by n3 may be adjusted based on the molecular weight of the nucleic acid and is not particularly limited, but may be in the range of 1 to 3000, preferably 10 to 2000, and more preferably 50 ~ 1000.

The ligand is preferably a sugar chain ligand. The sugar chain ligand is preferably a group represented by the following formula (1).

（式（１）中、Acはアセチル基を表し、R¹は、C6-C12アリール基、C4-C12ヘテロアリール基、または、C6-C12アリール基もしくはC4-C12ヘテロアリール基で置換されたC1-C6アルキル基を表す。）

(In the formula (1), Ac represents an acetyl group, and R ¹ represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1 substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Represents a -C6 alkyl group.)

Examples of the C6-C12 aryl group in R ¹ include a phenyl group, a biphenyl group, and a naphthyl group.

The C4-C12 heteroaryl group in R ¹ is a furanyl group, thienyl group, thiopyranyl group, isothiochromenyl group, pyrrolyl group, imidazolyl group, pyrazolyl group, pyridyl group, pyralidinyl group, pyrimidinyl group, pyridazinyl group, thiazolyl group , An isothiazolyl group, and a pyranyl group.

The C6-C12 aryl group and the C4-C12 heteroaryl group may be substituted with a substituent. Examples of the substituent include an alkyl group such as methyl, ethyl, propyl, and isopropyl; an alkoxy group such as methoxy, ethoxy, and propoxy A halogen atom such as chloro and bromo; a hydroxy group; an amino group; a C6-C12 aryl group such as a phenyl group, a biphenyl group and a naphthyl group; a C4-C12 heteroaryl group such as a furanyl group and a thienyl group; a piperidine and a chroman A heterocyclic group; and the like.
Among the above substituents, C6-C12 aryl groups such as phenyl group, biphenyl group and naphthyl group; C4-C12 heteroaryl groups such as furanyl group and thienyl group; heterocyclic groups such as piperidine and chroman; The C6-C12 aryl group and the C4-C12 heteroaryl group may be condensed to form a condensed ring composed of at least two rings.

In R ^1, the "C6-C12 aryl group" and "C4-C12 heteroaryl group" in the C1-C6 alkyl group substituted by C6-C12 aryl or C4-C12 heteroaryl group, the R ¹ above , C6-C12 aryl group and C4-C12 heteroaryl group can be exemplified.
The “C1-C6 alkyl group” in the C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group in R ¹ is methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl. , Sec-butyl, tert-butyl, cyclobutyl, cyclopropylmethyl, pentyl, isopentyl, sec-pentyl, neopentyl, tert-pentyl, cyclopentyl, hexyl, cyclohexyl and the like, preferably methyl, ethyl and propyl.

R ¹ in Formula (1) is preferably a group represented by the following structure.

The linker links the ligand and lipid that can bind to Siglec, or the ligand that can bind to Siglec and the water-soluble unit, or the water-soluble unit and the cationic unit. If it does not reduce, it will not restrict | limit in particular.
Further, from the viewpoint of ease of production of lipid I and polymer I, it is preferable that each of a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are bonded via a linker. Therefore, in order to form a linker-mediated bond, the terminal structure of the water-soluble unit, the ligand capable of binding to Siglec, and the cationic unit each have a functional group such as a hydroxy group or an amino group. It is preferable.

The linker structure includes amide group (-NHCO-), amino group (-NH-), ether group (-O-), thioether group (-S-), carbonyl group (-CO-) ester group (-CO An alkylene group that may contain at least one functional group selected from the group consisting of -O-), a heterocycle, a heteroaromatic ring, and the like is preferable. The number of carbon atoms of the alkylene group is not particularly limited, but is preferably 1 to 10.

One example of the linker structure is represented by the following linker structure I.

（リンカー構造I中、Q1及びQ2は、それぞれ独立して、アミド基（-NHCO-または-CONH-）、アミノ基（-NH-）、エーテル基（-O-）、チオエーテル基（-S-）、カルボニル基（-CO-）、エステル基（-CO-O-または-O-CO-）、ヘテロ環、及びヘテロ芳香環のいずれかであり、aおよびbは、それぞれ独立して、０～５、好ましくは１～５、より好ましくは２～３のいずれかの整数であり、cは、０または１であり、Zは１以上、好ましくは１～５、より好ましくは１～２の整数である。）

(In the linker structure I, Q1 and Q2 are each independently an amide group (-NHCO- or -CONH-), an amino group (-NH-), an ether group (-O-), a thioether group (-S- ), A carbonyl group (—CO—), an ester group (—CO—O— or —O—CO—), a heterocycle, and a heteroaromatic ring, and a and b are each independently 0 Is an integer of 1 to 5, preferably 1 to 5, more preferably 2 to 3, c is 0 or 1, and Z is 1 or more, preferably 1 to 5, more preferably 1 to 2. (It is an integer.)

The heterocycle of Q1 and Q2 is not particularly limited, and examples thereof include a 4- to 7-membered heterocycle containing one or more heteroatoms, succinimide and the like.
The heteroaromatic ring of Q1 and Q2 is not particularly limited, and examples thereof include triazole, imidazole, tetrazole, pyridine, pyrimidine and the like.
When Z is 2 or more, a plurality of Q1 and b may be the same or different.

As described above, in order to have two or more ligands in one molecule of lipid I or polymer I, the linker existing between the ligand capable of binding to Siglec and the water-soluble unit has a branch point. You may have. Further, in the polymer I, the linker existing between the water-soluble unit and the cationic unit may have a branch point. Therefore, a combination of the above-mentioned branched structures (A) to (D) and linker structure I is also a suitable linker structure.
Examples of the linker containing a branched structure include the following linker structures IA to ID.

（構造中、Q1及びQ2は、それぞれ独立して、アミド基（-NHCO-または-CONH-）、アミノ基（-NH-）、エーテル基（-O-）、チオエーテル基（-S-）、カルボニル基（-CO-）、エステル基（-CO-O-または-O-CO-）、ヘテロ環、及びヘテロ芳香環のいずれかであり、a1～a4およびb1～b4は、それぞれ独立して、０～５のいずれかの整数であり、c1～c4は、それぞれ独立して、０または１であり、Z1～Z4は、それぞれ独立して１以上の整数である。）

(In the structure, Q1 and Q2 are each independently an amide group (—NHCO— or —CONH—), an amino group (—NH—), an ether group (—O—), a thioether group (—S—), Any one of a carbonyl group (—CO—), an ester group (—CO—O— or —O—CO—), a heterocycle, and a heteroaromatic ring, wherein a1 to a4 and b1 to b4 are each independently And any one of 0 to 5, c1 to c4 are each independently 0 or 1, and Z1 to Z4 are each independently an integer of 1 or more.)

The heterocycle of Q1 and Q2 is not particularly limited, and examples thereof include a 4- to 7-membered heterocycle containing one or more heteroatoms, succinimide and the like.
The heteroaromatic ring of Q1 and Q2 is not particularly limited, and examples thereof include triazole, imidazole, tetrazole, pyridine, pyrimidine and the like.
When Z1 to Z4 are 2 or more, a plurality of Q1 and b1 to b4 may be the same or different.

As the linker structure in lipid I, linker structure I, linker structure IA, and linker structure IB described above are preferable.
At this time, Q1 and Q2 in the linker structure I are each independently any of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring. A and b are each independently an integer of 0 to 5, c is preferably 0 or 1, and Z is preferably 1 or 2.
Q1 and Q2 in the linker structure IA are each independently any one of an amide group (-NHCO- or -CONH-), an amino group (-NH-), a heterocycle, and a heteroaromatic ring, and a1 To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
Q1 and Q2 in the linker structure IB are each independently any one of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring; To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.

Specific examples of the linker structure in lipid I include the following structures.
In addition, when the ligand capable of binding to Siglec is, for example, a group represented by the formula (1), the sugar chain, A linker may be bonded.

Of the above linker structures in lipid I, the following structures are preferred.

In addition, when lipid I has a ligand capable of binding to two siglecs, examples of the linker structure include the following structures.

（構造中、L1およびL2は、同一でも異なっていてもよく、シグレックに結合可能なリガンドを表す。）

(In the structure, L1 and L2 may be the same or different and represent ligands capable of binding to Siglec.)

As the structure of the linker that links the ligand capable of binding to Siglec and the water-soluble unit in the polymer I, the above-described linker structure I, linker structure IA, and linker structure IB are preferable.
At this time, Q1 and Q2 in the linker structure I are each independently any one of an amide group (—NHCO—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring, and a and Each b is independently an integer of 0 to 5, c is preferably 0 or 1, and Z is preferably 1 or 2.
Q1 and Q2 in the linker structure IA are each independently any one of an amide group (-NHCO- or -CONH-), an amino group (-NH-), a heterocycle, and a heteroaromatic ring, and a1 To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.
Q1 and Q2 in the linker structure IB are each independently any one of an amide group (—NHCO— or —CONH—), an amino group (—NH—), a heterocycle, and a heteroaromatic ring; To a3 and b1 to b3 are each independently an integer of 0 to 5, c1 to c3 are independently 0 or 1, and Z1 to Z3 are independently 1 or 2. It is preferable.

Specific examples of the structure of the linker that links the ligand capable of binding to Siglec and the water-soluble unit in the polymer I include the following structures.
In addition, when the ligand capable of binding to Siglec is, for example, a group represented by the formula (1), the sugar chain, A linker may be bonded.

Among the above linker structures in polymer I, the following structures are preferred.

Further, when the polymer I has a ligand capable of binding to two siglecs, examples of the linker structure include the following structures.

（構造中、L1およびL2は、同一でも異なっていてもよく、シグレックに結合可能なリガンドを表す。）

(In the structure, L1 and L2 may be the same or different and represent ligands capable of binding to Siglec.)

The linker present in the polymer I for linking the water-soluble unit and the cationic unit is preferably the linker structure I described above, and more preferably represented by the following structure.

（aおよびbは、それぞれ独立して、０～５、好ましくは１～５、より好ましくは２～３のいずれかの整数である。）

(A and b are each independently an integer of 0 to 5, preferably 1 to 5, more preferably 2 to 3.)

In the polymer I, the linker structure I existing for linking the water-soluble unit and the cationic unit is more preferably the following structure.

When the polymer I has a ligand capable of binding to two or more siglecs, a branch point may be provided in a linker that is present for linking a water-soluble unit and a cationic unit.
At this time, examples of the structure of the linker include the following structures.

Specific examples of lipid I include the following structures.

In the formula, R ¹ represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Linker represents a linker, PEG is a unit containing polyethylene glycol, Lipid is a lipid, and N1 is an integer of 1 or more. Ac represents acetyl.
The structure of -PEG-Lipid in the formula is preferably any of the structures represented by the formulas (Z1) to (Z3) described above, and is preferably a structure represented by the formula (Z2). More preferred.
The Linker is not particularly limited, but is preferably any of the linker structure I, linker structure IA, and linker structure IB described above, more preferably the linker structure I, and still more preferably the following structure: It is.

Specifically, lipid I preferably has the following structure.

（構造中、R¹、Q1、Q2、a、b、c、Z、PEG、Lipid、およびAcは上記と同様である。）

(In the structure, R ¹ , Q ¹ , Q 2, a, b, c, Z, PEG, Lipid, and Ac are the same as above.)

Specifically, the lipid I is more preferably the following structure.

（構造中、R¹、PEG、Lipid、およびAcは、上記と同様である。）

(In the structure, R ¹ , PEG, Lipid, and Ac are the same as above.)

As lipid I, the following structure is more preferable.

（Acは、アセチルを表す。nは、1～300の整数である。）

(Ac represents acetyl. N is an integer of 1 to 300.)

Specific examples of the polymer I include the following structures.

（式中、R¹は、C6-C12アリール基、C4-C12ヘテロアリール基、または、C6-C12アリール基もしくはC4-C12ヘテロアリール基で置換されたC1-C6アルキル基を表す。PEGは、ポリエチレングリコールを含むユニットであり、Polyは、リジン、アルギニン、ヒスチジンからなる群より選択される一種以上を含むアミノ酸ポリマーユニット、ポリエチレンイミンユニット、又は、ポリアミノアクリレートユニットである。Linker 1及びLinker 2は、それぞれ独立してPolyとPEGを連結するリンカーである。N2は１以上の整数である。Acはアセチルを表す。）

(Wherein R ¹ represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1-C6 alkyl group substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. PEG represents A unit containing polyethylene glycol, Poly is an amino acid polymer unit, a polyethyleneimine unit, or a polyaminoacrylate unit containing at least one selected from the group consisting of lysine, arginine, and histidine Linker 1 and Linker 2 are (Linker that independently links Poly and PEG. N2 is an integer greater than or equal to 1. Ac represents acetyl.)

Linker IV 1 is preferably the above-described linker structure I, linker structure IA, and linker structure IB.

Preferred examples of the polymer I include the following structures.

（構造中、R¹、PEG、Poly、Q1、Q2、a、b、c、およびAcは、上記と同様である。複数の、Q1、Q2、a、b、cは、同一であっても、異なっていてもよい。）

(In the structure, R ¹ , PEG, Poly, Q 1, Q 2, a, b, c, and Ac are the same as described above. Plural Q 1, Q 2, a, b, c may be the same , May be different.)

（構造中、R¹、PEG、Poly、Q1、Q2、a、b、c、a1～a3、b1～b3、c1～c3、およびAcは、上記と同様である。複数の、R¹、Q1、Q2は、同一であっても、異なっていてもよい。）

(In the structure, R ¹ , PEG, Poly, Q 1, Q 2, a, b, c, a 1 to a 3, b 1 to b 3, c 1 to c 3, and Ac are the same as described above. A plurality of R ¹ , Q 1 Q2 may be the same or different.)

（構造中、R¹、PEG、Poly、Q1、Q2、b、c、a1～a3、b1～b3、c1～c3、およびAcは、上記と同様である。複数の、R¹、Q1、Q2、a1～a3、b1～b3、c1～c3は、同一であっても、異なっていてもよい。a’及びa”は、それぞれ独立して、０～５のいずれかの整数である。）

(In the structure, R ¹ , PEG, Poly, Q 1, Q 2, b, c, a 1 to a 3, b 1 to b 3, c 1 to c 3, and Ac are the same as described above. Multiple R ¹ , Q ¹ , Q 2 , A1 to a3, b1 to b3, and c1 to c3 may be the same or different. A ′ and a ″ are each independently an integer of 0 to 5.)

More preferable polymer I includes the following structures.

（R¹、PEG、Poly、およびAcは、上記と同様である。）

(R ¹ , PEG, Poly, and Ac are the same as above.)

（R¹、PEG、Poly、およびAcは、上記と同様である。）

(R ¹ , PEG, Poly, and Ac are the same as above.)

More preferable polymer I includes the following structures.

（構造中、Acはアセチルを表し、nは、1～300の整数であり、Polyは、以下の構造：

（n1は、1～3000、好ましくは10～2000であり、より好ましくは10～1000であり、よりさらに好ましくは10～100であり、さらにより好ましくは10～50であり、特に好ましくは10～40である。）のいずれかで表される。）

(In the structure, Ac represents acetyl, n is an integer of 1 to 300, and Poly has the following structure:

(N1 is 1 to 3000, preferably 10 to 2000, more preferably 10 to 1000, even more preferably 10 to 100, still more preferably 10 to 50, and particularly preferably 10 to 1000. 40)). )

（構造中、Acはアセチルを表し、nは、1～300の整数であり、Polyは、以下の構造：

（n1は、1～3000、好ましくは10～2000であり、より好ましくは10～1000であり、よりさらに好ましくは10～100であり、さらにより好ましくは10～50であり、特に好ましくは10～40である。）のいずれかで表される。）

(In the structure, Ac represents acetyl, n is an integer of 1 to 300, and Poly has the following structure:

(N1 is 1 to 3000, preferably 10 to 2000, more preferably 10 to 1000, even more preferably 10 to 100, still more preferably 10 to 50, and particularly preferably 10 to 1000. 40)). )

In addition, several types of isomers may exist as lipid I and polymer I, respectively, but lipid I and polymer I may be any one of isomers, and these are optional. The mixture contained in the ratio may be sufficient. Specific examples of isomers include optical isomers.
The formula (1) is preferably an isomer represented by the following formula (1 ′).

（式（１’）中、R¹は、式（１）におけるR¹と同義である。Acはアセチル基である。）

(In formula (1 ′), R ¹ has the same meaning as R ¹ in formula (1). Ac is an acetyl group.)

Lipid I can be synthesized using organic synthesis techniques with reference to J. Am. Chem. Soc., 2012, 134, 15696.
Specifically, as shown in the scheme below, a reaction introduced by performing general derivatization on a reactive group contained in a lipid containing a lipid or a water-soluble unit, or a lipid containing a lipid or a water-soluble unit. The lipid I is obtained by reacting the reactive group with a reactive group in an organic group containing a reactive group bonded to a ligand capable of binding to Siglec (the organic group is a linker in lipid I). However, the synthesis method is not particularly limited.
Examples of the combination of reactive groups include a combination of an amino group and a carboxyl group, a combination of a hydroxyl group and a carboxyl group, etc., preferably a combination of an amino group and a carboxyl group, and more preferably a lipid or water-soluble group. This is a combination in which a reactive group of a lipid containing a sex unit is an amino group and a reactive group bonded to a ligand capable of binding to Siglec is a carboxyl group.
The reactive carboxyl group may be activated by acid chloride, N-hydroxysuccinimide, N, N'-dicyclohexylcarbodiimide, etc., and applies the general conditions for amide bond formation and dehydration condensation reactions. can do.
For ligands that can bind to lipids or water-soluble units containing reactive groups and Siglecs containing organic groups containing reactive groups, see J. Am. Chem. Soc., 2012, 134, 15696. A commercially available lipid or ligand may be derivatized using an organic synthesis technique.

Polymer I can also be synthesized using organic synthesis techniques.
Specifically, as shown in the scheme below, the reactive group contained in the polymer containing a water-soluble unit and a cationic unit, or the reactivity introduced by general derivatization of the water-soluble unit. By reacting the group with a reactive group in an organic group containing a reactive group bonded to a ligand capable of binding to Siglec (the organic group is a linker in the polymer I), the polymer I Although it can be obtained, the synthesis method is not particularly limited.
The combination of the reactive groups is not particularly limited, and examples thereof include a combination of an amino group and a carboxyl group, and a combination of a hydroxyl group and a carboxyl group.
The reactive carboxyl group may be activated by acid chloride, N-hydroxysuccinimide, N, N'-dicyclohexylcarbodiimide, etc., and applies the general conditions for amide bond formation and dehydration condensation reactions. can do.
Ligand capable of binding to a water-soluble unit containing a reactive group and a polymer containing a cationic unit and a Siglec containing an organic group containing a reactive group are described in J. Am. Chem. Soc., 2012, 134, 15696. And commercially available lipids and ligands may be derivatized using organic synthesis techniques.

In addition, as shown below, after reacting an organic group containing a reactive group bound to a ligand capable of binding to Siglec with a water-soluble unit containing a reactive group, a cationic unit containing a reactive group The method of making it react can also be mentioned.

For example, J. 結合 Am. 含 む Chem. Soc., 2008, 130, 6680-6681, International Publication No. 2007/056525 and the like are synthesized as ligands that can bind to Siglec containing an organic group containing a reactive group. You can also.

By introducing an organic group containing a reactive group into a ligand that can bind to Siglec, a ligand that can bind to Siglec can be converted into a lipid containing a lipid or a water-soluble unit, or a high water containing a water-soluble unit and a cationic unit. Linkage with molecules becomes possible.
The method for introducing an organic group containing a reactive group into a ligand that can bind to Siglec is not particularly limited, and the ligand capable of binding to Siglec and a compound that becomes an organic group containing a reactive group are etherified. (4th edition Experimental Chemistry Course 20 Synthesis of Organic Compounds II ”, 4th edition, p.187, Maruzen (1992)), Amination (4th edition Experimental Chemistry Course 20 Synthesis of Organic Compounds II”, 4th edition Edition, p.279, Maruzen (1992), etc.), esterification (4th edition, Experimental Chemistry Lecture 22, Synthesis of Organic Compounds IV ”, 4th edition, p.43, Maruzen (1992), etc.), amidation ( 4th edition, Experimental Chemistry Lecture 22, Synthesis of Organic Compounds IV ”, 4th edition, p.137, Maruzen (1992), etc.) may be performed.

When the ligand capable of binding to Siglec is a sugar chain ligand, for example, by reacting a compound that becomes an organic group containing a reactive group at the anomeric position at the end of the sugar chain (eg, acetalization), An organic group containing a reactive group can be introduced into a ligand capable of binding.

(Lipid II)
When the nanoparticles of the present invention are lipid nanoparticles containing lipid, in addition to lipid I, a cationic lipid (lipid II) may be contained.
The cationic lipid includes a lipophilic region containing one or more optionally substituted hydrocarbon groups, and at least one primary amino group, secondary amino group, tertiary amino group and / or quaternary ammonium group. It is not particularly limited as long as it is an amphiphilic molecule having a cationic hydrophilic region containing, but may be substituted with a hydrophilic part having one amino group or one quaternary ammonium group which may be substituted, and may be substituted. Mention may be made of lipids having a hydrophobic part having two independent hydrocarbon groups.

Examples of the cationic lipid used in the present invention include International Publication No. 2013/089151, International Publication No. 2011/136368, International Publication No. 2014/007398, International Publication No. 2010/042877 or International Publication No. 2010 / And cationic lipids described in No. 054401.

Specific examples of the cationic lipid used in the present invention include the following formulas (CL-I) to (CL-XVI).

Formula (CL-I)

(式中、
　R¹⁰¹およびR¹⁰²は同一または異なって直鎖状もしくは分岐状のC10-C24アルキル、C10-C24アルケニルまたはC10-C24アルキニルであり、
　L¹⁰¹およびL¹⁰²は水素原子であるか、または一緒になって単結合もしくはC2-C8アルキレンを形成し、
　L¹⁰³は単結合、-CO-または-CO-O-であり、
　L¹⁰³が単結合である場合には、
　　X¹⁰¹は水素原子、C1-C6アルキル、C3-C6アルケニル、ピロリジン-3-イル、ピペリジン-3-イル、ピペリジン-4-イル、または同一もしくは異なって1～3つのアミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイル、ピロリジニル、ピペリジルもしくはモルホリニルで置換されたC1-C6アルキルもしくはC3-C6アルケニルであり、
　L¹⁰³が-CO-または-CO-O-である場合には、
　　X¹⁰¹はピロリジン-2-イル、ピロリジン-3-イル、ピペリジン-2-イル、ピペリジン-3-イル、ピペリジン-4-イル、モルホリン-2-イル、モルホリン-3-イル、または同一もしくは異なって1～3つのアミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイル、ピロリジニル、ピペリジルもしくはモルホリニルで置換されたC1-C6アルキルもしくはC3-C6アルケニルであり、該置換基の少なくとも1つは、アミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ピロリジニル、ピペリジルまたはモルホリニルである)

(Where
R ¹⁰¹ and R ¹⁰² are the same or different and are linear or branched C10-C24 alkyl, C10-C24 alkenyl or C10-C24 alkynyl,
L ¹⁰¹ and L ¹⁰² are hydrogen atoms or together form a single bond or C2-C8 alkylene;
L ¹⁰³ is a single bond, -CO- or -CO-O-,
When L ¹⁰³ is a single bond,
X ¹⁰¹ is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino, dialkyl C1-C6 alkyl or C3-C6 alkenyl substituted with amino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl,
When L ¹⁰³ is -CO- or -CO-O-
X ¹⁰¹ is pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or the same or different With 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl And at least one of the substituents is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl)

Formula (CL-II)

(式中、
　R¹⁰³およびR¹⁰⁴は同一または異なって直鎖状もしくは分岐状のC12-C24アルキル、C12-C24アルケニルまたはC12-C24アルキニルであり、
　p¹⁰¹およびp¹⁰²は同一または異なって0～3の整数であり、
　L¹⁰⁶およびL¹⁰⁷は水素原子であるか、または一緒になって単結合もしくはC2-C8アルキレンを形成し、
　L¹⁰⁴およびL¹⁰⁵は同一または異なって-O-、-CO-O-または-O-CO-であり、
　L¹⁰⁸は単結合、-CO-または-CO-O-であり、
　L¹⁰⁸が単結合である場合には、
　　X¹⁰²は水素原子、C1-C6アルキル、C3-C6アルケニル、ピロリジン-2-イル、ピロリジン-3-イル、ピペリジン-3-イル、ピペリジン-4-イル、または同一もしくは異なって1～3つのアミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイル、ピロリジニル、ピペリジルもしくはモルホリニルで置換されたC1-C6アルキルもしくはC3-C6アルケニルであり、
　L¹⁰⁸が-CO-または-CO-O-である場合には、
　　X¹⁰²はピロリジン-2-イル、ピロリジン-3-イル、ピペリジン-2-イル、ピペリジン-3-イル、ピペリジン-4-イル、モルホリン-2-イル、モルホリン-3-イル、または同一もしくは異なって1～3つのアミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイル、ピロリジニル、ピペリジルもしくはモルホリニルで置換されたC1-C6アルキルもしくはC3-C6アルケニルであり、該置換基の少なくとも1つは、アミノ、モノアルキルアミノ、ジアルキルアミノ、トリアルキルアンモニオ、ピロリジニル、ピペリジルまたはモルホリニルである)

(Where
R ¹⁰³ and R ¹⁰⁴ are the same or different and are linear or branched C12-C24 alkyl, C12-C24 alkenyl or C12-C24 alkynyl,
p ¹⁰¹ and p ¹⁰² are the same or different and are integers of 0 to 3,
L ¹⁰⁶ and L ¹⁰⁷ are hydrogen atoms or together form a single bond or C2-C8 alkylene;
L ¹⁰⁴ and L ¹⁰⁵ are the same or different and are -O-, -CO-O- or -O-CO-,
L ¹⁰⁸ is a single bond, -CO- or -CO-O-
When L ¹⁰⁸ is a single bond,
X ¹⁰² is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino acids C1-C6 alkyl or C3-C6 alkenyl substituted with monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl,
When L ¹⁰⁸ is -CO- or -CO-O-
X ¹⁰² is pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or the same or different With 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl And at least one of the substituents is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl)

Formula (CL-III)

(式中、
　R¹⁰⁵は直鎖状もしくは分岐状のC8-C24アルキル、C8-C24アルケニルまたはC8-C24アルキニルであり、
　R¹⁰⁶は直鎖状または分岐状のC8-C24アルキル、C8-C24アルケニル、C8-C24アルキニル、C8-C24アルキルオキシエチル、C8-C24アルキルオキシプロピル、C8-C24アルケニルオキシエチル、C8-C24アルケニルオキシプロピル、C8-C24アルキニルオキシエチルまたはC8-C24アルキニルオキシプロピルであり、
　X¹⁰³およびX¹⁰⁴は同一または異なってC1-C3アルキルであるか、または一緒になってC2-C8アルキレンを形成するか、またはX¹⁰³はL¹¹¹と一緒になってC2-C8アルキレンを形成し、
　L¹¹¹は水素原子、C1-C6アルキル、C3-C6アルケニル、アミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイル、ジアルキルカルバモイル、または同一もしくは異なって1～3つのアミノ、モノアルキルアミノ、ヒドロキシ、アルコキシ、カルバモイル、モノアルキルカルバモイルもしくはジアルキルカルバモイルで置換されたC1-C6アルキルもしくはC3-C6アルケニルであるか、またはX¹⁰³と一緒になってC2-C8アルキレンを形成し、
　L¹⁰⁹はC1-C6アルキレンであり、
　L¹¹⁰は単結合であるか、またはC1-C6アルキレンであり、ただし、L¹⁰⁹とL¹¹⁰の炭素数の和は7以下であり、L¹¹¹が、水素原子の場合、L¹¹⁰は単結合であり、L¹¹¹がX¹⁰³と一緒になってC2-C6アルキレンを形成する場合、L¹¹⁰は単結合であるか、またはメチレンもしくはエチレンである)

(Where
R ¹⁰⁵ is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl,
R ¹⁰⁶ is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl,
X ¹⁰³ and X ¹⁰⁴ are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene, or X ¹⁰³ is taken together with L ¹¹¹ to form C2-C8 alkylene. ,
L ¹¹¹ is a hydrogen atom, C1-C6 alkyl, C3-C6 alkenyl, amino, monoalkylamino, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, or the same or different 1 to 3 amino, monoalkylamino, C1-C6 alkyl or C3-C6 alkenyl substituted with hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl or dialkylcarbamoyl, or together with X ¹⁰³ to form C2-C8 alkylene;
L ¹⁰⁹ is C1-C6 alkylene,
L ¹¹⁰ is a single bond or C1-C6 alkylene, provided that the sum of the carbon numbers of L ¹⁰⁹ and L ¹¹⁰ is 7 or less, and when L ¹¹¹ is a hydrogen atom, L ¹¹⁰ is a single bond. And when L ¹¹¹ together with X ¹⁰³ forms C2-C6 alkylene, L ¹¹⁰ is a single bond or is methylene or ethylene)

Formula (CL-IV)

(式中、
　R¹⁰⁷は直鎖状もしくは分岐状のC8-C24アルキル、C8-C24アルケニルまたはC8-C24アルキニルであり、
　R¹⁰⁸は直鎖状もしくは分岐状のC8-C24アルキル、C8-C24アルケニル、C8-C24アルキニル、C8-C24アルキルオキシエチル、C8-C24アルキルオキシプロピル、C8-C24アルケニルオキシエチル、C8-C24アルケニルオキシプロピル、C8-C24アルキニルオキシエチル、C8-C24アルキニルオキシプロピル、C8-C24アルキルオキシエトキシエチル、C8-C24アルケニルオキシエトキシエチルまたはC8-C24アルキニルオキシエトキシエチルであり、
　X¹⁰⁵は水素原子または置換されていても良いC1-C4アルキルである)

(Where
R ¹⁰⁷ is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl;
R ¹⁰⁸ is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl, C8-C24 alkynyloxypropyl, C8-C24 alkyloxyethoxyethyl, C8-C24 alkenyloxyethoxyethyl or C8-C24 alkynyloxyethoxyethyl,
X ¹⁰⁵ is a hydrogen atom or an optionally substituted C1-C4 alkyl)

Formula (CL-V)

(式中、
　R¹⁰⁹は直鎖状もしくは分岐状のC8-C24アルキル、C8-C24アルケニルまたはC8-C24アルキニルであり、
　R¹¹⁰は直鎖状もしくは分岐状のC8-C24アルキル、C8-C24アルケニル、C8-C24アルキニル、C8-C24アルキルオキシエチル、C8-C24アルキルオキシプロピル、C8-C24アルケニルオキシエチル、C8-C24アルケニルオキシプロピル、C8-C24アルキニルオキシエチルまたはC8-C24アルキニルオキシプロピルであり、
　L¹¹²はC1-C3アルキレンであり、
　X^105’は水素原子またはC1-C3アルキルである)

(Where
R ¹⁰⁹ is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl,
R ¹¹⁰ is linear or branched C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyl Oxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl,
L ¹¹² is C1-C3 alkylene;
X ^{105 ′} is a hydrogen atom or C1-C3 alkyl)

Formula (CL-VI)

(式中、
　R¹¹¹およびR¹¹²は同一または異なって直鎖状もしくは分岐状の置換されていても良いC8-C24アルキル、C8-C24アルケニルまたはC8-C24アルキニルであり、
　X¹⁰⁶およびX¹⁰⁷は同一または異なってC1-C3アルキルであるか、または一緒になってC2-C8アルキレンを形成し、
　p¹⁰³、p¹⁰⁴およびp¹⁰⁵は同一または異なって0または1であり、ただしp¹⁰³, p¹⁰⁴およびp¹⁰⁵は同時には0ではなく、
　L¹¹³およびL¹¹⁴は同一または異なってO、SまたはNHである)

(Where
R ¹¹¹ and R ¹¹² are the same or different and may be linear or branched, optionally substituted C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl,
X ¹⁰⁶ and X ¹⁰⁷ are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene;
p ¹⁰³ , p ¹⁰⁴ and p ¹⁰⁵ are the same or different and are 0 or 1, provided that p ¹⁰³ , p ¹⁰⁴ and p ¹⁰⁵ are not 0 at the same time,
L ¹¹³ and L ¹¹⁴ are the same or different and are O, S or NH)

Formula (CL-VII)

(式中、
　R¹¹³およびR¹¹⁴は同一または異なって直鎖もしくは分岐状の置換されていても良いC8-C24アルキル、C8-C24アルケニルまたはC8-C24アルキニルであり、
　R¹¹⁵は水素原子、ヒドロキシ、置換されていても良いC1-C4アルキル、C1-C4アルコキシまたはC1-C4アシルオキシであり、
　X¹⁰⁹およびX¹¹⁰は同一または異なってC1-C3アルキルであるか、または一緒になってC2-C8アルキレンを形成し、
　L¹¹⁵は-CO-O-、-O-CO-、-NHCO-または-CONH-であり、
　p¹⁰⁶は0～3の整数であり、
　p¹⁰⁷は1～4の整数である）

(Where
R ¹¹³ and R ¹¹⁴ are the same or different, linear or branched optionally substituted C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl,
R ¹¹⁵ is a hydrogen atom, hydroxy, optionally substituted C1-C4 alkyl, C1-C4 alkoxy or C1-C4 acyloxy,
X ¹⁰⁹ and X ¹¹⁰ are the same or different and are C1-C3 alkyl or taken together to form C2-C8 alkylene;
L ¹¹⁵ is -CO-O-, -O-CO-, -NHCO- or -CONH-
p ¹⁰⁶ is an integer from 0 to 3,
p ¹⁰⁷ is an integer from 1 to 4)

Formula (CL-VIII)

（式中、
　R¹¹⁶およびR¹¹⁷は同一または異なって直鎖状もしくは分岐状の置換されていても良いC8-C24アルキル、C8-C24アルケニル、C8-C24アルキニル、C7-C20アルキルオキシC1-C3アルキル、C7-C20アルケニルオキシC1-C3アルキルまたはC7-C20アルキニルオキシC1-C3アルキルであり、
　B¹⁰⁰は、水素原子、C1-C3アルキル、ヒドロキシC2-C4アルキル、C1-C3ジアルキルアミノC2-C4アルキル、式(A)

(式中、X¹¹¹およびX¹¹²は同一または異なって水素原子もしくはC1-C3アルキルであるか、またはX¹¹¹およびX¹¹²が結合する窒素原子と一緒になってC2-C6含窒素ヘテロ環を形成してもよく、p¹¹⁰は2～6の整数である)、または式(B)

(式中、X¹¹³およびX¹¹⁴は同一または異なって水素原子もしくはC1～C3アルキルであるか、またはX¹¹³およびX¹¹⁴が結合する窒素原子と一緒になってC2-C6含窒素ヘテロ環を形成してもよく、p¹¹¹は1～6の整数である)であり、
　P¹⁰⁸は0～4の整数であり、P¹⁰⁹は1～4の整数であり(但し、P¹⁰⁸が0であり、P¹⁰⁹が1である場合を除く)、
　L¹¹⁶は結合する炭素ごとに同一または異なって水素原子またはC1-C3アルキルであり、
　L¹¹⁷は結合する炭素ごとに同一または異なって水素原子またはC1-C3アルキルである）

(Where
R ¹¹⁶ and R ¹¹⁷ are the same or different and may be linear or branched substituted C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C7-C20 alkyloxy C1-C3 alkyl, C7- C20 alkenyloxy C1-C3 alkyl or C7-C20 alkynyloxy C1-C3 alkyl,
B ¹⁰⁰ is a hydrogen atom, C1-C3 alkyl, hydroxy C2-C4 alkyl, C1-C3 dialkylamino C2-C4 alkyl, formula (A)

(In the formula, X ¹¹¹ and X ¹¹² are the same or different and are a hydrogen atom or C1-C3 alkyl, or together with the nitrogen atom to which X ¹¹¹ and X ¹¹² are bonded, a C2-C6 nitrogen-containing heterocycle is formed. P ¹¹⁰ is an integer from 2 to 6), or formula (B)

(Wherein X ¹¹³ and X ¹¹⁴ are the same or different and are a hydrogen atom or C1-C3 alkyl, or together with the nitrogen atom to which X ¹¹³ and X ¹¹⁴ are bonded form a C2-C6 nitrogen-containing heterocycle And p ¹¹¹ is an integer from 1 to 6),
P ¹⁰⁸ is an integer from 0 to 4, P ¹⁰⁹ is an integer from 1 to 4 (except when P ¹⁰⁸ is 0 and P ¹⁰⁹ is 1),
L ¹¹⁶ is the same or different for each carbon to be bonded and is a hydrogen atom or C1-C3 alkyl;
L ¹¹⁷ is the same or different for each carbon to be bonded and is a hydrogen atom or C1-C3 alkyl)

Formula (CL-IX)

（式中、
　X¹¹⁵およびX¹¹⁶は同一または異なって水素原子またはC1-C3アルキルであり、
　L¹¹⁸およびＬ¹¹⁹は同一または異なって直鎖状もしくは分岐状の置換されていても良いC8-C24アルキレンもしくはC8-C24アルケニレンであり、
　M¹⁰¹およびM¹⁰²は同一または異なって-C=C-、-OC(O)-、-C(O)O-、-SC(O)-、-C(O)S-、-OC(S)-、-C(S)O-、-SS-、-C(R^’’)=N-、-N=C(R^’’)-、-C(R^’’)=N-O-、-O-N=C(R^’’)-、-N(R^’’)C(O)-、-C(O)N(R^’’)-、-N(R^’’)C(S)-、-C(S)N(R^’’)-、-N(R^’’)C(O)N(R^’’’)-、-N(R^’’)C(O)O-、-OC(O)N(R^’’)-および-OC(O)O-からなる群から選ばれ、
　R^’’およびR^’’’は、同一または異なって、水素原子またはC1-C3アルキルであり、
　R¹¹⁸およびR¹¹⁹は同一または異なって直鎖状もしくは分岐状の置換されていても良いC1-C16アルキルまたはC2-C16アルケニルである）

(Where
X ¹¹⁵ and X ¹¹⁶ are the same or different and are a hydrogen atom or C1-C3 alkyl,
L ¹¹⁸ and L ¹¹⁹ are the same or different and may be linear or branched C8-C24 alkylene or C8-C24 alkenylene,
M ¹⁰¹ and M ¹⁰² are the same or different, and -C = C-, -OC (O)-, -C (O) O-, -SC (O)-, -C (O) S-, -OC (S )-, -C (S) O-, -SS-, -C (R ^'' ) = N-, -N = C (R ^'' )-, -C (R ^'' ) = NO-, -ON = C (R ^'' )-, -N (R ^'' ) C (O)-, -C (O) N (R ^'' )-, -N (R ^'' ) C (S)-, -C (S) N (R ^'' )-, -N (R ^'' ) C (O) N (R ^''' )-, -N (R ^'' ) C (O) O-, -OC (O) Selected from the group consisting of N (R ^'' )-and -OC (O) O-
R ^'' and R ^''' are the same or different and are a hydrogen atom or C1-C3 alkyl;
R ¹¹⁸ and R ¹¹⁹ are the same or different and may be linear or branched C1-C16 alkyl or C2-C16 alkenyl.

Formula (CL-X)

（式中
　X¹¹⁷およびX¹¹⁸は同一または異なって水素原子、置換されていても良いC1-C6アルキル、ヘテロシクリルまたはポリアミンであるか、またはX¹¹⁷およびX¹¹⁸はそれらが結合している窒素と一緒に、該窒素に加えて、N、OおよびSから選択される1または2個のさらなるヘテロ原子を含有していてもよい4～7員の単環式ヘテロ環を形成してもよく、
　R¹²⁰およびR¹²¹は同一または異なって直鎖状もしくは分岐状の置換されていても良いC4-C24アルキルまたはC4-C24アルケニルである）

^Wherein X ¹¹⁷ and X ¹¹⁸ are the same or different hydrogen atoms, optionally substituted C1-C6 alkyl, heterocyclyl or polyamine, or X ¹¹⁷ and X ¹¹⁸ together with the nitrogen to which they are attached. In addition to the nitrogen, it may form a 4-7 membered monocyclic heterocycle which may contain 1 or 2 additional heteroatoms selected from N, O and S;
R ¹²⁰ and R ¹²¹ are the same or different and may be linear or branched, optionally substituted C4-C24 alkyl or C4-C24 alkenyl)

Formula (CL-XI)

(式中、
　X¹¹⁹およびX¹²⁰は同一または異なって水素原子、直鎖状もしくは分岐状の置換されていても良いC1-C20アルキル、C1-C20アルケニル、C1-C20アルキニルまたはC6-C20アシルであり、
　R¹²²およびR¹²³は同一または異なって直鎖状もしくは分岐状の置換されていても良いC1-C30アルキル、C2-C30アルケニルまたはC2-C30アルキニルであり、
　p¹¹²、p¹¹³およびp¹¹⁴は同一または異なって0であるか、任意の正の整数である）

(Where
X ¹¹⁹ and X ¹²⁰ are the same or different and each represents a hydrogen atom, a linear or branched C1-C20 alkyl, C1-C20 alkenyl, C1-C20 alkynyl or C6-C20 acyl,
R ¹²² and R ¹²³ are the same or different, linear or branched optionally substituted C1-C30 alkyl, C2-C30 alkenyl or C2-C30 alkynyl,
p ¹¹² , p ¹¹³ and p ¹¹⁴ are the same or different and are 0 or any positive integer)

Formula (CL-XII)

（式中、
　X¹²¹およびX¹²²は同一または異なって水素原子、シクロアルキル、シクロアルケニルであるか、またはX¹²¹およびX¹²²はそれらが結合する窒素原子と一緒になってC2-C6含窒素ヘテロ環を形成してもよく
　L¹²⁰およびＬ¹²¹は同一または異なって-O-、-OC(O)-または-(O)CO-であり、
　R¹²⁴およびR¹²⁵は同一または異なって直鎖状もしくは分岐状の置換されていても良いC8-C24アルキルまたはC8-C24アルケニルである）、

(Where
X ¹²¹ and X ¹²² are the same or different and are a hydrogen atom, cycloalkyl, cycloalkenyl, or X ¹²¹ and X ¹²² together with the nitrogen atom to which they are bonded form a C2-C6 nitrogen-containing heterocycle. L ¹²⁰ and L ¹²¹ may be the same or different and are —O—, —OC (O) — or — (O) CO—,
R ¹²⁴ and R ¹²⁵ are the same or different and are linear or branched optionally substituted C8-C24 alkyl or C8-C24 alkenyl)),

Formula (CL-XIII)

（式中、
　R¹²⁶およびR¹²⁷は同一または異なって直鎖状もしくは分岐状の置換されていても良いC8-C24アルキル、C8-C24アルケニル、C8-C24アルキニル、C8-C24ヘテロアルキル、C8-C24ヘテロアルケニルまたはC8-C24ヘテロアルキニルであり、
　X¹²³は水素原子または置換されていても良いC1-C6アルキルであり、
　X¹²⁴はC1-C6アルキル、-NR^4aR^4bで置換される置換C1-C6アルキルまたは置換されていても良いC3-C7ヘテロシクリルであり、
　R^4aおよびR^4bは同一または異なって水素原子、C(=NH)NH₂または置換されていても良いC1-C6アルキルであるか、またはR^4aおよびR^4bは、置換されていても良いC3-C7ヘテロシクリルを形成してもよく、
　X¹²³およびX¹²⁴はそれらが結合する窒素原子と一緒になって置換されていても良いC3-C7ヘテロシクリルを形成してもよく、
　ただし、X¹²³およびX¹²⁴はイミダゾリル、ベンズイミダゾリル、またはたスクシンイミジルを形成せず、および１つだけの１級アミンがX¹²³およびX¹²⁴のいずれか一方の上に存在することができ、またはいかなる１級アミンもX¹²³およびX¹²⁴のいずれか一方の上に存在せず、X¹²³およびX¹²⁴は置換されたアミドではなく、
　R¹²⁶およびR¹²⁷が飽和C11アルキルまたは飽和C15アルキルであるとき、X¹²³は水素原子ではなく、
　R¹²⁶およびR¹²⁷が飽和C16アルキルまたは飽和C17アルキルであるとき、R¹²⁶およびR¹²⁷はOHと置換されず、
　R¹²⁶およびR¹²⁷が飽和C17アルキルであるとき、X¹²³およびX¹²⁴はOHと置換されず、
　R¹²⁶およびR¹²⁷が飽和C18アルキルであるとき、X¹²⁴は置換されていても良いイ
ミダゾリルと置換されない）

(Where
R ¹²⁶ and R ¹²⁷ are the same or different and may be linear or branched substituted C8-C24 alkyl, C8-C24 alkenyl, C8-C24 alkynyl, C8-C24 heteroalkyl, C8-C24 heteroalkenyl or C8-C24 heteroalkynyl,
X ¹²³ is a hydrogen atom or an optionally substituted C1-C6 alkyl,
X ¹²⁴ is C1-C6 alkyl, substituted C1-C6 alkyl substituted with -NR ^4a R ^4b or optionally substituted C3-C7 heterocyclyl;
R ^4a and R ^4b are the same or different and are a hydrogen atom, C (= NH) NH ₂ or an optionally substituted C1-C6 alkyl, or R ^4a and R ^4b are optionally substituted C3 -C7 heterocyclyl may be formed,
X ¹²³ and X ¹²⁴ together with the nitrogen atom to which they are attached may form an optionally substituted C3-C7 heterocyclyl;
However, X ¹²³ and X ¹²⁴ do not form imidazolyl, benzimidazolyl, or succinimidyl, and only one primary amine can be present on either of X ¹²³ and X ¹²⁴ , or any primary amines also not present on either one of X ¹²³ and X ^124, X ¹²³ and X ¹²⁴ is not substituted amides,
When R ¹²⁶ and R ¹²⁷ are saturated C11 alkyl or saturated C15 alkyl, X ¹²³ is not a hydrogen atom,
When R ¹²⁶ and R ¹²⁷ are saturated C16 alkyl or saturated C17 alkyl, R ¹²⁶ and R ¹²⁷ are not substituted with OH;
When R ¹²⁶ and R ¹²⁷ are saturated C17 alkyl, X ¹²³ and X ¹²⁴ are not substituted with OH;
When R ¹²⁶ and R ¹²⁷ are saturated C18 alkyl, X ¹²⁴ is not substituted with an optionally substituted imidazolyl)

Formula (CL-XIV)

（式中、
　X¹²⁵およびX¹²⁶は同一または異なって水素原子、置換されていても良いC1-C6アルキル、ヘテロシクリルまたはポリアミンであるか、またはX¹²⁵およびX¹²⁶はそれらが結合している窒素と一緒に、該窒素に加えて、N、OおよびSから選択される1または2個のさらなるヘテロ原子を含有していてもよい4～7員の単環式ヘテロ環を形成してもよく、
　R¹³⁰は水素原子またはC1-C6アルキルであり、
　R¹²⁸およびR¹²⁹は同一または異なって直鎖状もしくは分岐状の置換されていても良いC4-C24アルキルまたはC4-C24アルケニルである）

(Where
X ¹²⁵ and X ¹²⁶ are the same or different hydrogen atoms, optionally substituted C1-C6 alkyl, heterocyclyl or polyamine, or X ¹²⁵ and X ¹²⁶ together with the nitrogen to which they are attached, In addition to nitrogen, it may form a 4-7 membered monocyclic heterocycle which may contain 1 or 2 additional heteroatoms selected from N, O and S;
R ¹³⁰ is a hydrogen atom or C1-C6 alkyl;
R ¹²⁸ and R ¹²⁹ are the same or different and may be linear or branched, optionally substituted C4-C24 alkyl or C4-C24 alkenyl)

Formula (CL-XV)

（式中、
　X¹²⁷およびX¹²⁸はそれぞれ独立して、C1-C6アルキル、C2-C6アルケニル、C2-C6アルキニルであるか、
　X¹²⁷およびX¹²⁸は、それらが結合している窒素原子と一体になって、1から2個の窒素原子を有する複素環を形成し、
　L¹²²は－C(O)O－、－OC(O）－、－C(O)N(X¹³⁰)－、－N（X¹³⁰）C(O)－、－OC(O)O－、－OC(O)N(X¹³⁰)－、－N(X¹³⁰)C(O)N(X¹³⁰)－、または－N(X¹³⁰)C(O)O－であり、
　X¹³⁰の各存在は独立して、水素原子またはC1-C3アルキルであり、
　aは1、2、3、4、5、または6であり、
　bは0、1、2、または3であり、
　X¹²⁹は存在しないか、水素またはC1-C3アルキルであり、
　R¹³¹およびR¹³²のそれぞれは独立して、１つ以上の生分解性基を有する炭素数12～24のアルキル、炭素数12～24のアルケニル、または炭素数12～24のアルコキシであり、各生分解性基は独立して、上記の炭素数12～24のアルキル基、アルケニル基、またはアルコキシ基に割り込んでいるか、炭素数12～24のアルキル基、アルケニル基、またはアルコキシ基の末端で置換されており（上記生分解性基は、割り込んでいるものは、‐C(O)O-、‐OC（O）－、－C(O)N(X¹³⁰)－、または‐N(X¹³⁰)C(O)－であり、末端のものは、‐C(O)O‐C1-C4アルキル、－OC(O)－C1-C4アルキル、－C(O)N(X¹³⁰)－C1-C4アルキル、または－N(X¹³⁰)C(O)－C1-C4アルキルである）、
　R¹³¹およびR¹³²は、上記生分解性基とアスタリスク（＊）の付された第３級炭素原子との間に少なくとも4つの炭素原子を有する鎖である）

(Where
X ¹²⁷ and X ¹²⁸ are each independently C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl,
X ¹²⁷ and X ¹²⁸ together with the nitrogen atom to which they are attached form a heterocyclic ring having 1 to 2 nitrogen atoms;
L ¹²² is -C (O) O-, -OC (O)-, -C (O) N (X ¹³⁰ )-, -N (X ¹³⁰ ) C (O)-, -OC (O) O-, -OC (O) N (X ¹³⁰ )-, -N (X ¹³⁰ ) C (O) N (X ¹³⁰ )-, or -N (X ¹³⁰ ) C (O) O-
Each ^occurrence of X ¹³⁰ is independently a hydrogen atom or C1-C3 alkyl;
a is 1, 2, 3, 4, 5, or 6;
b is 0, 1, 2, or 3;
X ¹²⁹ is absent or is hydrogen or C1-C3 alkyl;
Each of R ¹³¹ and R ¹³² is independently alkyl having 12 to 24 carbons, alkenyl having 12 to 24 carbons, or alkoxy having 12 to 24 carbons having one or more biodegradable groups, The biodegradable group is independently inserted into the alkyl group, alkenyl group or alkoxy group having 12 to 24 carbon atoms, or substituted at the terminal of the alkyl group, alkenyl group or alkoxy group having 12 to 24 carbon atoms. (The above-mentioned biodegradable group is interrupted by -C (O) O-, -OC (O)-, -C (O) N (X ¹³⁰ )-, or -N (X ¹³⁰ ) C (O)-, and the terminal ones are -C (O) O-C1-C4 alkyl, -OC (O) -C1-C4 alkyl, -C (O) N (X ¹³⁰ ) -C1- C4 alkyl, or —N (X ¹³⁰ ) C (O) —C1-C4 alkyl),
R ¹³¹ and R ¹³² are chains having at least 4 carbon atoms between the biodegradable group and a tertiary carbon atom marked with an asterisk (*))

Formula (CL-XVI)

（式中、
　R¹³³およびR¹³⁴は同一または異なり、それぞれ直鎖もしくは分岐のC1-C9アルキル、C2-C11アルケニルもしくはC2-C11アルキニルであり、
　L¹²³およびL¹²⁴は同一または異なり、それぞれ直鎖のC5-C18アルキレンもしくは直鎖のC5-C18アルケニレンであるか、またはNと複素環を形成しており、
　L¹²⁵は、単結合であるか、または-CO-O-であり、それによって-L¹²⁴-CO-OR¹³⁴が形成されており、
　L¹²⁷はSまたはOであり、
　Ｌ¹²⁶は単結合であるか、または直鎖状もしくは分岐状のC1-C6アルキレンであるか、またはNと複素環を形成しており、
　L¹²⁸は直鎖状もしくは分岐状のC1-C6アルキレンであり、そして
　X¹³¹およびX¹³²は同一または異なり、それぞれ水素または直鎖状もしくは分岐状のC1-C6アルキルである）

(Where
R ¹³³ and R ¹³⁴ are the same or different and are each linear or branched C1-C9 alkyl, C2-C11 alkenyl or C2-C11 alkynyl,
L ¹²³ and L ¹²⁴ are the same or different and are each linear C5-C18 alkylene or linear C5-C18 alkenylene, or form a heterocyclic ring with N,
L ¹²⁵ is a single bond or -CO-O-, thereby forming -L ¹²⁴ -CO-OR ¹³⁴ ;
L ¹²⁷ is S or O,
L ¹²⁶ is a single bond, or a linear or branched C1-C6 alkylene, or forms a heterocyclic ring with N.
L ¹²⁸ is linear or branched C1-C6 alkylene, and X ¹³¹ and X ¹³² are the same or different and are each hydrogen or linear or branched C1-C6 alkyl)

In the definition of each group of the formula (CL-I), examples of the linear or branched C10-C24 alkyl include decyl, undecyl, dodecyl, tridecyl, 6,10-dimethylundec-2-yl, tetradecyl, Pentadecyl, hexadecyl, heptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl and the like, preferably decyl, undecyl, dodecyl, tridecyl, tetradecyl, Examples include pentadecyl, hexadecyl, heptadecyl, and octadecyl, and more preferable examples include tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, and octadecyl.

The linear or branched C10-C24 alkenyl may be a linear or branched C10-C24 alkenyl containing 1 to 3 double bonds, such as (Z) -dodec-7-enyl. , (Z) -tetradec-7-enyl, (Z) -tetradec-9-enyl, (Z) -hexadec-4-enyl, (Z) -hexadeca-7-enyl, (E) -hexadeca-7-enyl , (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadec-7,10,13-trienyl, (Z) -octadeca-6 -Enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (Z) -octadeca-11-enyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl, etc., preferably (Z) -dodeca -7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl, (Z) -hexadec-7-enyl, (E) -hexa Deca-7-enyl, (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadec-7,10,13-trienyl, (Z ) -Octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (11Z, 14Z) -icosa-11,14-dienyl, and more preferably (7Z, 10Z) -hexadeca -7,10-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl.

The linear or branched C10-C24 alkynyl may be a linear or branched C10-C24 alkynyl containing 1 to 3 triple bonds, and examples thereof include deca-9-ynyl and dodeca-4. -Inyl, dodec-11-ynyl, tetradec-5-ynyl, tetradec-6-ynyl, hexadec-7-ynyl, hexadec-3,5-diynyl, hexadec-5,7-diynyl or octadec-9-ynyl Preferably hexadeca-7-ynyl or octadec-9-ynyl, and more preferably octadec-9-ynyl.

In the formula (CL-I), R ¹⁰¹ and R ¹⁰² are preferably the same linear or branched C10-C24 alkyl, C10-C24 alkenyl or C10-C24 alkynyl, and the same linear It is more preferably a straight or branched C10-C24 alkyl or C10-C24 alkenyl, and even more preferably the same linear C10-C24 alkenyl.

Examples of C1-C3 alkylene include methylene, ethylene, or propylene, preferably methylene or ethylene, and more preferably methylene.

Examples of the C1-C6 alkyl include methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert-butyl, cyclobutyl, cyclopropylmethyl, pentyl, isopentyl, sec-pentyl, neopentyl, tert- Examples include pentyl, cyclopentyl, hexyl, cyclohexyl, etc., preferably methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, sec-pentyl, tert-pentyl, neopentyl, hexyl, etc. More preferably, methyl, ethyl, propyl, etc. are mentioned.

Examples of C3-C6 alkenyl include allyl, 1-propenyl, butenyl, pentenyl, hexenyl and the like, preferably allyl and the like.

Monoalkylamino and dialkylamino are each one or two identical or different C1-C6 alkyls (as defined above) or amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl Any amino substituted with C1-C6 alkyl (as defined above) substituted with, for example, methylamino, ethylamino, propylamino, butylamino, pentylamino, hexylamino, dimethylamino, diethylamino, ethylmethylamino Methylpropylamino, butylmethylamino, methylpentylamino, hexylmethylamino, aminoethylamino, aminopropylamino, (aminoethyl) methylamino, bis (aminoethyl) amino, etc., preferably methylamino Bruno, ethylamino, dimethylamino, diethylamino, aminopropyl amino or bis (aminoethyl) amino and the like, more preferably methyl or dimethylamino and the like.

Trialkylammonio includes the same or different C1-C6 alkyl (as defined above), or C1-C6 substituted with amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl. Any ammonio substituted with alkyl (as defined above) may be used, for example, trimethylammonio, ethyldimethylammonio, diethylmethylammonio, triethylammonio, tripropylammonio, tributylammonio, tripentylammonio, tripentylammonio, Hexylammonio, tris (aminoethyl) ammonio, (aminoethyl) dimethylammonio, bis (aminoethyl) methylammonio and the like can be mentioned, preferably trimethylammonio, triethylammonio, tris (aminoethyl) ammonio (Aminoethyl) dimethyl ammonio or bis (aminoethyl) methyl ammonio, and the like, or more preferably trimethylammonio like.

In the compound (CL-I), the trialkylammonio may form a salt with a pharmaceutically acceptable anion (as defined above).

Alkoxy is substituted with C1-C6 alkyl (as defined above) or C1-C6 alkyl (as defined above) substituted with amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl. It may be hydroxy, for example, methoxy, ethoxy, propyloxy, butyloxy, pentyloxy, hexyloxy, aminoethoxy or methylaminoethoxy, preferably methoxy, ethoxy, aminoethoxy or methylaminoethoxy, More preferably, methoxy etc. are mentioned.

Monoalkylcarbamoyl and dialkylcarbamoyl are each one or two identical or different C1-C6 alkyls (as defined above) or amino, methylamino, ethylamino, dimethylamino, diethylamino, pyrrolidinyl, piperidyl or morpholinyl Any carbamoyl substituted with C1-C6 alkyl substituted with (as defined above), for example, methylcarbamoyl, ethylcarbamoyl, propylcarbamoyl, butylcarbamoyl, pentylcarbamoyl, hexylcarbamoyl, dimethylcarbamoyl, diethylcarbamoyl, ethyl Methylcarbamoyl, methylpropylcarbamoyl, butylmethylcarbamoyl, methylpentylcarbamoyl, hexylmethylcarbamoyl, aminoethylcarbamoyl, amino Examples include propylcarbamoyl, (aminoethyl) methylcarbamoyl, or bis (aminoethyl) carbamoyl, preferably methylcarbamoyl, ethylcarbamoyl, dimethylcarbamoyl, and the like, more preferably methylcarbamoyl, dimethylcarbamoyl, and the like. .

L ¹⁰¹ and L ¹⁰² are more preferably hydrogen atoms. In this case, R ¹⁰¹ and R ¹⁰² are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl. (Z) -hexadec-7-enyl, (E) -hexadeca-7-enyl, (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadeca-7,10,13-trienyl, preferably (Z) -octadeca-9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl, (Z) -tetradec-7- More preferably, they are enyl, (Z) -hexadec-7-enyl, (7Z, 10Z) -hexadeca-7,10-dienyl, or (9Z, 12Z) -octadec-9,12-dienyl. Z) -tetradec-7-enyl, (Z) -hexadeca-7-enyl or (7Z, 10Z) -hexadeca-7,10-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl Further preferred.
In the case where L ¹⁰¹ and L ¹⁰² are hydrogen atoms, X ¹⁰¹ is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different from 1 to Must be C1-C6 alkyl or C3-C6 alkenyl substituted with 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl Is more preferably a hydrogen atom, methyl, or C1-C6 alkyl or C3-C6 alkenyl substituted with 1 to 3 amino, hydroxy or carbamoyl, which are the same or different, and more preferably a hydrogen atom, methyl, etc. More preferably it is.

When L ¹⁰¹ and L ¹⁰² together form a single bond or C1-C3 alkylene, R ¹⁰¹ and R ¹⁰² are the same or different and are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadec-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, or (11Z, 14Z) -icosa -11,14-dienyl, more preferably (Z) -octadec-9-enyl, or (9Z, 12Z) -octadec-9,12-dienyl, and identically (Z) -octadeca More preferred is -9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl.

When L ¹⁰¹ and L ¹⁰² together form a single bond or C1-C3 alkylene, X ¹⁰¹ is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl Or C1-C6 alkyl substituted with 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, the same or different Or a C3-C6 alkenyl, more preferably a hydrogen atom, methyl, or a C1-C6 alkyl or C3-C6 alkenyl substituted with 1 to 3 amino, hydroxy or carbamoyl, which are the same or different. And most preferably a hydrogen atom or methyl.
When L ¹⁰¹ and L ¹⁰² together form a single bond, L ¹⁰³ is —CO— or —CO—O—, preferably —CO—. One. In this case, X ¹⁰¹ is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-aminopropyl, 1,4-diaminobutyric, 1,5 Jiaminopenchiru, 3-aminopropyl, 4 -Aminobutyl or 5-aminopentyl is preferable, and 1,2-diaminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl or 1,5-diaminopentyl is more preferable. R ¹⁰¹ and R ¹⁰² are the same or different and are tetradecyl, hexadecyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadec-6-enyl, (Z)- Octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca- Preferably it is 9,12,15-trienyl or (Z) -icosa-11-enyl or (11Z, 14Z) -icosa-11,14-dienyl, (Z) -octadeca-9-enyl or (9Z, More preferably, they are 12Z) -octadeca-9,12-dienyl, and more preferably (Z) -octadeca-9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl.

L ¹⁰³ is more preferably a single bond.

When L ¹⁰³ is a single bond, X ¹⁰¹ is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino , Dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl, etc., more preferably a hydrogen atom , Methyl, hydroxymethyl, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-3-methoxypropyl, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2- (N, N-dimethylamino) ethyl 3- (N, N-dimethylamino) propyl, 2-carbamoylethyl, 2-dimethylcarbamoylethyl, 1-methylpiperidin-4-yl, and the like, more preferably a hydrogen atom or methyl. preferable.

When L ¹⁰³ is —CO— or —CO—O—, X ¹⁰¹ is pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different one to three amino, mono Alkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl substituted C1-C6 alkyl or C3-C6 alkenyl, at least of the substituents More preferably, one is amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl, morpholinyl or the like, and R ³ is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1 , 3-Diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl, 4-aminobutyl 1,5-diaminopentyl, 5-aminopentyl, (N, N-dimethylamino) methyl, 2- (N, N-dimethylamino) ethyl, 3- (N, N-dimethylamino) propyl, 1-hydroxy- 2-aminoethyl or 1-amino-2-hydroxyethyl is more preferable, such as 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl, 3-aminopropyl, 1,4-diamino. Most preferred is butyl, 4-aminobutyl, 1,5-diaminopentyl or 5-aminopentyl.

One of the more preferred embodiments of the present invention is that L ¹⁰³ is a single bond and X ¹⁰¹ is a hydrogen atom. In this case, R ¹⁰¹ and R ¹⁰² are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl. (Z) -hexadec-7-enyl, (E) -hexadeca-7-enyl, (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadeca-7,10,13-trienyl, (Z) -octadeca-9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl, preferably the same or different, (Z) More preferred are -tetradec-7-enyl or (7Z, 10Z) -hexadec-7,10-dienyl, and (Z) -tetradec-7-enyl, (Z) -hexadeca-7-enyl or ( 7Z, 10Z) -hexadec-7,10-dienyl is more preferable.

L ¹⁰³ is a single bond and X ¹⁰¹ is methyl is one of the more preferable embodiments of the present invention. In this case, R ¹⁰¹ and R ¹⁰² are the same or different and dodecyl, tetradecyl, (Z) -dodec-7-enyl, (Z) -tetradec-7-enyl, (Z) -hexadec-4-enyl. (Z) -hexadec-7-enyl, (E) -hexadeca-7-enyl, (Z) -hexadec-9-enyl, (7Z, 10Z) -hexadec-7,10-dienyl, (7Z, 10Z, 13Z) -hexadeca-7,10,13-trienyl, (Z) -octadeca-9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl, preferably the same or different, (Z) More preferred are tetradeca-7-enyl, (7Z, 10Z) -hexadec-7,10-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl, and identically (Z) -tetradec-7 More preferred is -enyl, (7Z, 10Z) -hexadec-7,10-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl.

In the definition of each group of the formula (CL-II), linear or branched C12-C24 alkyl includes, for example, dodecyl, tridecyl, tetradecyl, 2,6,10-trimethylundecyl, pentadecyl, 3,7, 11-trimethyldodecyl, hexadecyl, heptadecyl, octadecyl, 6,10,14-trimethylpentadecan-2-yl, nonadecyl, 2,6,10,14-tetramethylpentadecyl, icosyl, 3,7,11,15-tetra Examples include methylhexadecyl, henicosyl, docosyl, tricosyl, tetracosyl, etc., preferably dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, etc., more preferably dodecyl, tridecyl, tetradecyl, pentadecyl Hexadecyl, heptadecyl, octadecyl, etc. It is.

The linear or branched C12-C24 alkenyl may be a linear or branched C12-C24 alkenyl containing 1 to 3 double bonds, such as (Z) -tridec-8-enyl. , (Z) -tetradec-9-enyl, (Z) -pentadeca-8-enyl, (Z) -hexadeca-9-enyl, (Z) -heptadeca-5-enyl, (Z) -octadeca-6-enyl , (Z) -heptadeca-8-enyl, (Z) -octadeca-9-enyl, (E) -heptadeca-8-enyl, (E) -octadeca-9-enyl, (Z) -heptadeca-10-enyl , (Z) -octadeca-11-enyl, (8Z, 11Z) -heptadeca-8,11-dienyl, (9Z, 12Z) -octadeca-9,12-dienyl, (8Z, 11Z, 14Z) -octadeca-8 , 11,14-trienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -nonadec-10-enyl, (Z) -icosa-11-enyl, (10Z, 13Z) -Nonadeca-10,13-dienyl, (11Z, 14Z) -icosa-11,14-dienyl, 2,6,10-trimethylundeca-1,5,9-trienyl 3,7,11-trimethyldodeca-2,6,10-trienyl, 2,6,10,14-tetramethylpentadec-1-enyl or 3,7,11,15-tetramethylhexadec-2- Preferably include (Z) -pentadeca-8-enyl, (Z) -hexadeca-9-enyl, (Z) -heptadeca-5-enyl, (Z) -octadeca-6-enyl, (Z ) -Heptadeca-8-enyl, (Z) -octadeca-9-enyl, (8Z, 11Z) -heptadeca-8,11-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl, etc. More preferably (Z) -heptadeca-8-enyl, (Z) -octadeca-9-enyl, (8Z, 11Z) -heptadeca-8,11-dienyl or (9Z, 12Z) -octadeca-9,12-dienyl Etc.

The linear or branched C12-C24 alkynyl may be a linear or branched C12-C24 alkynyl containing 1 to 3 triple bonds, such as dodeca-11-ynyl, tridec-12- Inyl, pentadec-6-ynyl, hexadec-7-ynyl, pentadec-4,6-diynyl, hexadec-5,7-diynyl, heptadec-8-ynyl, octadec-9-ynyl and the like, preferably pentadec-9 6-Inyl, hexadec-7-ynyl, pentadec-4,6-diynyl, hexadec-5,7-diynyl, heptadec-8-ynyl, octadec-9-ynyl, etc., more preferably heptadeca-8-inyl Or octadec-9-inyl etc. are mentioned.

C1-C3 alkylene, C1-C6 alkyl and C3-C6 alkenyl in the definition of each group of formula (CL-II) are respectively synonymous with those in formula (CL-I).

Monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl have the same meanings as those in formula (CL-I), respectively.

R ¹⁰³ and R ¹⁰⁴ are preferably the same linear or branched C12-C24 alkyl, C12-C24 alkenyl or C12-C24 alkynyl, the same linear or branched C12-C24 alkyl, Or C12-C24 alkenyl.

L ¹⁰⁴ and L ¹⁰⁵ are the same -O -, - more preferably CO-O- or -O-CO-.

When at least one of L ¹⁰⁴ and L ¹⁰⁵ is —O— or —O—CO—, R ¹⁰³ and R ¹⁰⁴ are the same or different and are dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11- Enyl, (11Z, 14Z) -icosa-11,14-dienyl, 3,7,11-trimethyldodeca-2,6,10-trienyl or 3,7,11,15-tetramethylhexadec-2-enyl More preferably, tetradecyl, hexadecyl, octadecyl, (Z) -hexadec-9-enyl, (Z) -octadec-6-enyl, (Z) -octadec-9-enyl or (9Z, 12Z) -octadec- 9, More preferred is 12-dienyl.

When at least one of L ¹⁰⁴ and L ¹⁰⁵ is -CO-O-, R ¹⁰³ and R ¹⁰⁴ are each tridecyl, pentadecyl, heptadecyl, nonadecyl, henicosyl, tricosyl, (Z) -tridec-8-enyl, ( (Z) -pentadeca-8-enyl, (Z) -heptadeca-5-enyl, (Z) -heptadeca-8-enyl, (E) -heptadeca-8-enyl, (Z) -heptadeca-10-enyl, ( 8Z, 11Z) -heptadeca-8,11-dienyl, (8Z, 11Z, 14Z) -octadeca-8,11,14-trienyl, (Z) -nonadec-10-enyl, (10Z, 13Z) -nonadec-10 , 13-dienyl, (11Z, 14Z) -icosa-11,14-dienyl, 2,6,10-trimethylundeca-1,5,9-trienyl or 2,6,10,14-tetramethylpentadeca- More preferred is 1-enyl, tridecyl, pentadecyl, heptadecyl, (Z) -pentadec-8-enyl, (Z) -heptadeca-5-enyl, (Z) -heptadeca-8-enyl or (8Z, 11Z ) -Heptadeca-8,11- Further preferably enyl.

More preferably, p ¹⁰¹ and p ¹⁰² are 0 or 1 at the same time.

More preferably, L ¹⁰⁶ and L ¹⁰⁷ together form a single bond or C1-C3 alkylene. When L ¹⁰⁶ and L ¹⁰⁷ together form a single bond or C1-C3 alkylene, X ¹⁰² is a hydrogen atom, methyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl , Piperidin-3-yl, piperidin-4-yl, morpholin-2-yl, morpholin-3-yl, or the same or different 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, More preferably it is C1-C6 alkyl or C3-C6 alkenyl substituted with alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl, hydrogen atom, methyl, or the same or different from 1 to 3 C1-C6 alkyl substituted with amino, trialkylammonio, hydroxy or carbamoyl Or more preferably a C3-C6 alkenyl, a hydrogen atom, methyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diamino. Most preferred is propyl, 1,4-diaminobutyl, 1,5-diaminopentyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl or 2-carbamoylethyl. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl. Two or three alkyls in dialkylamino, trialkylammonio and dialkylcarbamoyl may be the same or different.

When L ¹⁰⁶ and L ¹⁰⁷ together form a single bond, L ¹⁰⁸ is preferably —CO— or —CO—O—, preferably —CO—.

When L ¹⁰⁶ and L ¹⁰⁷ together form a single bond, p ¹⁰¹ and p ¹⁰² are preferably the same or different and are 1 to 3.

When L ¹⁰⁶ and L ¹⁰⁷ are hydrogen atoms, X ¹⁰² is a hydrogen atom, methyl, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, piperidine-4 -Yl, morpholin-2-yl, morpholin-3-yl or the same or different 1 to 3 amino, monoalkylamino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, C1-C6 alkyl or C3-C6 alkenyl substituted with pyrrolidinyl, piperidyl or morpholinyl, preferably hydrogen atom, methyl or the same or different from 1 to 3 amino, trialkylammonio, hydroxy or carbamoyl More preferred is a substituted C1-C6 alkyl or C3-C6 alkenyl. Hydrogen atom, methyl, 2,3-dihydroxypropyl, 3-hydroxypropyl, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl, 1,4-diaminobutyl, 1, More preferred are 5-diaminopentyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2-carbamoylethyl and the like. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl. Two or three alkyls in dialkylamino, trialkylammonio, and dialkylcarbamoyl may be the same or different.

L ¹⁰⁸ is preferably a single bond. When L ¹⁰⁸ is a single bond, L ¹⁰⁴ and L ¹⁰⁵ are preferably —O—.

When L ¹⁰⁸ is a single bond, X ¹⁰² is a hydrogen atom, methyl, pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl, or the same or different 1 to 3 amino, monoalkylamino, C1-C6 alkyl or C3-C6 alkenyl substituted with dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl is preferred. , Hydroxymethyl, 2-hydroxyethyl, 2,3-dihydroxypropyl, 2-hydroxypropyl, 3-hydroxypropyl, 2-hydroxy-3-methoxypropyl, aminomethyl, 2-aminoethyl, 3-aminopropyl, 4- Aminobutyl, 5-aminopentyl, 2- (N, N-dimethylamino) ethyl, 3- (N, N-dimethylamino) propyl, 2-carbamoylethyl, 2-dimethylcarbamoylethyl, 1-methylpiperidin-4-yl and the like are more preferable, such as a hydrogen atom, methyl, 2,3-dihydroxypropyl, 3-hydroxypropyl. 2-aminoethyl, 3-aminopropyl, 4-aminobutyl, 5-aminopentyl, 2-carbamoylethyl and the like are more preferable. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl. Two or three alkyls in dialkylamino, trialkylammonio, and dialkylcarbamoyl may be the same or different.

More preferably, L ¹⁰⁴ and L ¹⁰⁵ are —O—. However, when L ¹⁰⁸ is a single bond and X ¹⁰² is a hydrogen atom, L ¹⁰⁴ and L ¹⁰⁵ are preferably the same —CO—O— or —O—CO—, and —CO—O— More preferably.

When L ¹⁰⁸ is —CO— or —CO—O—, L ¹⁰⁴ and L ¹⁰⁵ are preferably the same —CO—O— or —O—CO—, and are —CO—O—. Is more preferable.

When L ¹⁰⁸ is —CO— or —CO—O—, X ¹⁰² is pyrrolidin-3-yl, piperidin-3-yl, piperidin-4-yl or the same or different 1 to 3 amino, monoalkyl Preferably it is C1-C6 alkyl or C3-C6 alkenyl substituted with amino, dialkylamino, trialkylammonio, hydroxy, alkoxy, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, pyrrolidinyl, piperidyl or morpholinyl. Is preferably amino, monoalkylamino, dialkylamino, trialkylammonio, pyrrolidinyl, piperidyl or morpholinyl, and X ¹⁰² is aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1 , 3-Diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl, 4-a Nobutyl, 1,5-diaminopentyl, 5-aminopentyl, (N, N-dimethylamino) methyl, 2- (N, N-dimethylamino) ethyl, 3- (N, N-dimethylamino) propyl or 1- More preferred are amino-2-hydroxyethyl and the like, aminomethyl, 1,2-diaminoethyl, 2-aminoethyl, 1,3-diaminopropyl, 3-aminopropyl, 1,4-diaminobutyl, 4- More preferred are aminobutyl, 1,5-diaminopentyl, 5-aminopentyl and the like. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, trialkylammonio, alkoxy, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C6 alkyl. Two or three alkyls in dialkylamino, trialkylammonio and dialkylcarbamoyl may be the same or different.

L ¹⁰⁴ and L ¹⁰⁵ are preferably the same —CO—O— or —O—CO—, and more preferably —CO—O—.

In the definitions of the groups of formulas (CL-III), (CL-IV) and (CL-V), linear or branched C8-C24 alkyl, C8-C24 alkenyl and C8-C24 alkynyl are the same as defined above. As defined in formulas (CL-I) to (CL-II), the same groups are preferred.

In the definition of each group of the formulas (CL-III), (CL-IV) and (CL-V), the alkyl moiety in C8-C24 alkyloxyethyl and C8-C24 alkyloxypropyl may be, for example, the linear or Examples thereof include those exemplified for branched C8-C24 alkyl.

Examples of the alkynyl moiety in alkynyloxyethyl and alkynyloxypropyl include those exemplified for the linear or branched C8-C24 alkynyl.

R ¹⁰⁵ and R ¹⁰⁶ are preferably the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, and are the same or different linear or branched C8-C24 alkenyl. More preferably, it is more preferably the same or different linear C8-C24 alkenyl. R ¹⁰⁵ and R ¹⁰⁶ are more preferably the same, and in that case, linear or branched C12-C24 alkyl, C12-C24 alkenyl, or C12-C24 alkynyl is preferable. More preferably, the chain is C12-C24 alkenyl. Linear or branched C12-C24 alkyl, C12-C24 alkenyl, and C12-C24 alkynyl have the same meanings as those in formula (CL-II), respectively.

R ¹⁰⁵ and R ¹⁰⁶ are preferably the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, and are the same or different linear or branched C8-C24 alkenyl. More preferably, it is more preferably the same or different linear C8-C24 alkenyl. R ¹⁰⁵ and R ¹⁰⁶ are more preferably the same, and in that case, linear or branched C15-C20 alkyl, C15-C20 alkenyl, or C15-C20 alkynyl is preferable. More preferably, the chain is C15-C20 alkenyl. Linear or branched C15-C20 alkyl, C15-C20 alkenyl, and C15-C20 alkynyl have the same meanings as those in formulas (I) to (IV), respectively, and the same groups are preferable.

When R ¹⁰⁵ and R ¹⁰⁶ are different, R ¹⁰⁵ is a linear or branched C15-C20 alkyl, C15-C20 alkenyl or C15-C20 alkynyl, and R ¹⁰⁶ is a linear or branched C8. -C12 alkyl is preferred. Here, examples of the linear or branched C8-C12 alkyl include octyl, nonyl, decyl, undecyl, and dodecyl, and preferably octyl, decyl, and dodecyl.

More preferably, R ¹⁰⁵ is linear C15-C20 alkenyl, R ¹⁰⁶ is linear C8-C12 alkyl, and R ¹⁰⁵ is (Z) -octadec-9-enyl or (9Z, 12Z) -octadeca More preferably, it is -9,12-dienyl and R ¹⁰⁶ is octyl, decyl or dodecyl.

When R ¹⁰⁵ and R ¹⁰⁶ are different, R ¹⁰⁵ is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, R ¹⁰⁶ is C8-C24 alkyloxyethyl, C8-C24 alkyloxypropyl, C8-C24 alkenyloxyethyl, C8-C24 alkenyloxypropyl, C8-C24 alkynyloxyethyl or C8-C24 alkynyloxypropyl is also preferred. In this case, R ¹⁰⁵ is a C8-C24 linear alkenyl, R ¹⁰⁶ is more preferably a C8-C24 alkenyloxy ethyl, R ¹⁰⁵ is, (Z) - octadec-9-enyl, (9Z , 12Z) -octadeca-9,12-dienyl or (11Z, 14Z) -icosa-11,14-dienyl, R ¹⁰⁶ is (Z) -octadec-9-enyloxyethyl, (9Z, 12Z)- More preferably, it is octadeca-9,12-dienyloxyethyl or (11Z, 14Z) -icosa-11,14-dienyloxyethyl, and R ¹⁰⁵ is (9Z, 12Z) -octadeca-9,12- Most preferably, it is dienyl and R ¹⁰⁶ is (9Z, 12Z) -octadeca-9,12-dienyloxyethyl.

When R ¹⁰⁵ and / or R ¹⁰⁶ are the same or different linear or branched C8-C24 alkyl or C8-C24 alkenyl, the same or different tetradecyl, hexadecyl, (Z) -tetradec-9 -Enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11 -Enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -Icosa-11,14-dienyl or (Z) -docosa-13-enyl, the same or different, hexadecyl, (Z) -hexadec-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (Z) -icosa-11-enyl or (11Z, 14Z) -icosa-11,14-dienyl Are more preferred, identical or More preferably (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl or (11Z, 14Z) -icosa-11,14-dienyl, more preferably identical (9Z , 12Z) -octadeca-9,12-dienyl is most preferred.

R ¹⁰⁷ and R ¹⁰⁸ have the same meanings as R ¹⁰⁵ and R ¹⁰⁶ , respectively, and the same groups as R ¹⁰⁵ and R ¹⁰⁶ are preferred. Provided that when R ¹⁰⁷ is linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, R ¹⁰⁷ and R ¹⁰⁸ are identically (9Z, 12Z) -octadeca-9, 12-dienyl is preferred.

R ¹⁰⁹ and R ¹¹⁰ are synonymous with R ¹⁰⁵ and R ¹⁰⁶ , respectively, and the same groups as R ¹⁰⁵ and R ¹⁰⁶ are preferred. However, R ¹⁰⁹ and R ¹¹⁰ are preferably the same linear or branched C15-C20 alkyl, C15-C20 alkenyl or C15-C20 alkynyl, and identically (9Z, 12Z) -octadeca-9, More preferred is 12-dienyl.

The C1-C3 alkyl in X ¹⁰³ and X ¹⁰⁴ includes, for example, methyl, ethyl, propyl, isopropyl or cyclopropyl, preferably methyl or ethyl, and more preferably methyl.

Examples of the C2-C8 alkylene formed by combining X ¹⁰³ and X ¹⁰⁴ include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, preferably butylene, pentylene, hexylene, and the like. More preferred is hexylene.

Examples of the C2-C8 alkylene formed by X ¹⁰³ together with L ¹¹¹ include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, and preferably propylene, butylene, pentylene, and the like. More preferably, propylene or butylene is used, and propylene is more preferably used.

X ¹⁰³ and X ¹⁰⁴ are the same or different and are methyl or ethyl, together form butylene, pentylene or hexylene, or X ¹⁰³ together with L ¹¹¹ form ethylene, propylene or butylene It is preferable to do. X ¹⁰³ and X ¹⁰⁴ are the same or different and are preferably methyl or ethyl, or together, form butylene, pentylene or hexylene, and X ¹⁰³ together with L ¹¹¹ is ethylene, propylene or It is also preferred that it forms butylene and X ¹⁰⁴ is methyl or ethyl. And it is more preferred that X ¹⁰³ and X ¹⁰⁴ are identically methyl or together form hexylene, X ¹⁰³ together with L ¹¹¹ forms propylene or butylene, and X ¹⁰⁴ is methyl. It is further more preferable.

In L ^111, C1-C6 alkyl, C3-C6 alkenyl, monoalkylamino, alkoxy, mono- alkylcarbamoyl and dialkylcarbamoyl have the same meanings as those in each of the formulas (CL-I).

L ¹¹¹ is a hydrogen atom, C1-C6 alkyl, amino, monoalkylamino, hydroxy, alkoxy or C1-C6 alkyl substituted with 1 to 3 amino, monoalkylamino, or hydroxy or alkoxy, the same or different. Or preferably together with X ¹⁰³ to form C2-C6 alkylene, substituted with 1 to 3 amino or hydroxy atoms, hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy, or the same or different More preferably, together with X ¹⁰³ to form ethylene, propylene or butylene, which is a hydrogen atom, C1-C3 alkyl, or hydroxy, or together with X ¹⁰³ More preferably it forms propylene or butylene, together with a hydrogen atom or X ¹⁰³ Most preferably, propylene is formed.

In L ¹⁰⁹ and L ^110, as the C1-C6 alkylene, such as methylene, ethylene, propylene, butylene, etc. pentylene or hexylene and the like, preferably methylene or ethylene and the like.

L ¹⁰⁹ is preferably methylene, ethylene, propylene, or the like, more preferably methylene, ethylene, or the like. L ¹¹⁰ is preferably a single bond, methylene, ethylene, or the like, and is a single bond, methylene, or the like. More preferably. The sum of the carbon numbers of L ¹⁰⁹ and L ¹¹⁰ is preferably 1 to 3, and more preferably 2. In any of these cases, X ¹⁰³ and X ¹⁰⁴ are the same or different, such as methyl or ethyl, and L ¹¹¹ is a hydrogen atom, methyl, amino, methylamino, hydroxy, methoxy, or the same or different 1 ˜3 amino or hydroxy substituted methyls or the like, or X ¹⁰³ and X ¹⁰⁴ together form pentylene, hexylene or heptylene, etc., and L ¹¹¹ is a hydrogen atom, methyl, amino, methylamino, X ¹⁰³ and L ¹¹¹ together form propylene, butylene, pentylene or the like, such as hydroxy, methoxy or methyl substituted with 1 to 3 amino or hydroxy identically or differently, or X ¹⁰⁴ preferably is methyl or ethyl and the like, X ¹⁰³ and X ¹⁰⁴ is methyl, or L ¹¹¹ is a hydrogen atom, X ¹⁰³ and X ¹⁰⁴ are together It forms a pentylene or hexylene, L ¹¹¹ is hydrogen atom or X ¹⁰³ and L ^111, form a propylene together, X ¹⁰⁴ is more preferably methyl and the like.

In the definition of each group of the formula (CL-V), the C1-C4 alkyl in X ¹⁰⁵ ′ includes, for example, methyl, ethyl, propyl, isopropyl, cyclopropyl and the like, preferably methyl, ethyl, isopropyl and the like More preferably, methyl or ethyl is exemplified. X ¹⁰⁵ ′ is more preferably a hydrogen atom or methyl, and most preferably a hydrogen atom.

In L ^112, as the C1-C3 alkylene, e.g., methylene, ethylene or propylene and the like, preferably methylene or ethylene and the like.

In the definition of each group of formula (CL-VI) and formula (CL-VII), linear or branched optionally substituted C8-C24 alkyl, C8-C24 alkenyl and C8-C24 alkynyl are respectively The meanings are the same as those in the formulas (CL-I) to (CL-V).

Examples of the C1-C4 alkyl in the optionally substituted C1-C4 alkyl in R ¹¹⁵ of the formula (CL-VII) include, for example, methyl, ethyl, propyl, isopropyl, cyclopropyl, butyl, isobutyl, sec-butyl, tert -Butyl, cyclobutyl, cyclopropylmethyl, etc. are mentioned, Preferably methyl, ethyl etc. are mentioned, More preferably, methyl is mentioned.
The alkyl part of C1-C4 alkoxy which may be substituted has the same meaning as the C1-C4 alkyl.
Examples of the substituent in the optionally substituted C1-C4 alkyl include amino, monoalkylamino, dialkylamino, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl, and piperidine 4-yl, morpholin-2-yl, morpholin-3-yl, hydroxy, alkoxy, alkoxycarbonyl, hydroxycarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, nitro, cyano, fluoro, chloro, bromo and the like. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, alkoxy, alkoxycarbonyl, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl. Two alkyls in dialkylamino and dialkylcarbamoyl may be the same or different.

Examples of the acyl in the optionally substituted C1-C4 acyloxy include formyl, acetyl, propanoyl, 2-methylpropanoyl, cyclopropanoyl, butanoyl, and preferably acetyl and the like.
Examples of the substituent in C1-C4 acyloxy which may be substituted include amino, monoalkylamino, dialkylamino, pyrrolidin-2-yl, pyrrolidin-3-yl, piperidin-2-yl, piperidin-3-yl and piperidine 4-yl, morpholin-2-yl, morpholin-3-yl, hydroxy, alkoxy, alkoxycarbonyl, hydroxycarbonyl, carbamoyl, monoalkylcarbamoyl, dialkylcarbamoyl, nitro, cyano, fluoro, chloro, bromo and the like. Of these substituents, the alkyl moiety in monoalkylamino, dialkylamino, alkoxy, alkoxycarbonyl, monoalkylcarbamoyl and dialkylcarbamoyl has the same meaning as the C1-C4 alkyl. Two alkyls in dialkylamino and dialkylcarbamoyl may be the same or different.

In the formula (CL-VI), R ¹¹¹ and R ¹¹² are preferably the same linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, and the same linear or branched More preferably, it is C8-C24 alkyl or C8-C24 alkenyl.

R ¹¹¹ and R ¹¹² are the same or different and are octyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl, icosyl, docosyl, tetracosyl, (Z) -tetradec-9-enyl, (Z) -hexadec-9-enyl, ( (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12 -Dienyl, (9Z, 12Z, 15Z) -octadeca-9,12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl, 3,7,11 -Trimethyldodeca-2,6,10-trienyl or 3,7,11,15-tetramethylhexadec-2-enyl or the like, and the same or different, dodecyl, tetradecyl, (Z) -hexadec-9 -Enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl or (9Z, 12Z) -octadeca-9,12-dienyl are more preferred and identical. More preferred are (Z) -hexadec-9-enyl, (Z) -octadeca-6-enyl, (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, and the like. .

X ¹⁰⁶ and X ¹⁰⁷ are preferably the same or different and are preferably methyl or ethyl, more preferably methyl.

Examples of the C2-C8 alkylene formed by combining X ¹⁰⁶ and X ¹⁰⁷ include ethylene, propylene, butylene, pentylene, hexylene, heptylene, octylene, and the like, preferably butylene, pentylene, hexylene, and the like. More preferred is butylene or pentylene.

X ¹⁰⁶ and X ¹⁰⁷ are preferably the same methyl or taken together to form butylene, pentylene or hexylene.

p ¹⁰³ and ¹⁰⁴ are preferably 0 at the same time, and p ¹⁰⁵ is preferably 1.

L ¹¹³ and L ¹¹⁴ are preferably O at the same time.

R ¹¹³ and R ¹¹⁴ are preferably the same linear or branched C8-C24 alkyl, C8-C24 alkenyl or C8-C24 alkynyl, the same linear or branched C8-C24 alkyl or More preferably, it is C8-C24 alkenyl.

C1-C3 alkyl and C2-C8 alkylene in X ¹⁰⁹ and X ¹¹⁰ are respectively synonymous with those in the formula (CL-VI).

R ¹¹⁵ is preferably a hydrogen atom, hydroxy, methyl, methoxy or the like, more preferably a hydrogen atom or hydroxy, and even more preferably a hydrogen atom.

L ¹¹⁵ is preferably —O—CO—. In this case, p ¹⁰⁶ is 0 or 1, preferably p ¹⁰⁷ is an integer of 2 ~ 4, p ¹⁰⁶ is 0 or 1, and more preferably p ¹⁰⁷ is 3.

L ¹¹⁵ is the case of -CO-O-, p ¹⁰⁶ is 0, it is preferable that p ¹⁰⁷ is an integer of 2 ~ 4, p ¹⁰⁶ is 0, more that p ¹⁰⁷ is 3 preferable.

The definition of each group of formula (CL-VIII) to formula (CL-XVI) may be synonymous with that in formula (CL-I) to formula (CL-VII).

In addition, each group in the formula (CL-VIII) is in WO 2016/002753, each group in the formula (CL-X) is in WO 2009/129385, each group in the formula (CL-XI) is In International Publication No.2013 / 149140, each group in formula (CL-XII) is in International Publication No.2009 / 129395, each group in formula (CL-XIII) is in International Publication No.2013 / 059496, -XIV) in International Publication No. 2011/149733, each group in Formula (CL-XV) in International Publication No.2011 / 153493, each group in Formula (CL-XVI) in International Publication No. In 074085, a preferred embodiment of each group described correspondingly may be used.

L ¹¹⁸ and L ¹¹⁹ in formula (CL-IX) are the same or different and are preferably linear or branched C8-C24 alkylene or C8-C24 alkenylene, more preferably linear or branched C8. -C20 alkylene or C8-C20 alkenylene.

Wherein C1-C6 alkyl (CL-X) in X ¹¹⁷ and X ^118, heterocyclyl or polyamines, halogen atom, R ', OR', SR 1 is selected ', CN, CO ₂ R' or from CONR _'2 It may be substituted with up to 3 substituents.

Together with the nitrogen to which X ¹¹⁷ and X ¹¹⁸ in formula (CL-X) are bonded them, in addition to the nitrogen, containing one or two further heteroatoms selected from N, O and S In the case of forming a 4- to 7-membered monocyclic heterocycle, the monocyclic heterocycle is a halogen atom, R ′, OR ′, SR ′, CN, CO ₂ R ′ or CONR ′. It may be substituted with 1 to 3 substituents selected from ₂ .
Here, R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.

R ¹²⁰ and R ¹²¹ in formula (CL-X) are the same or different and are preferably linear or branched C4-C24 alkyl or C4-C24 alkenyl, more preferably linear or branched C4- C20 alkyl or C4-C20 alkenyl.
C4-C24 alkyl or C4-C24 alkenyl may be substituted with one or more substituents selected from a halogen atom, R ′, OR ′, SR ′, CN, CO ₂ R ′ or CONR ′ _2. .
Here, R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.

When an expression (CL-XI) in X ¹¹⁹ and X ¹²⁰ linear or branched optionally substituted by C6-C20 acyl, a structure other than the carbonyl group in the C6-C20 acyl, C5-C19 alkyl, It can be C5-C19 alkenyl or C5-C19 alkynyl.

R ¹²⁴ and R ¹²⁵ in formula (CL-XII) are the same or different and are preferably linear or branched C8-C24 alkyl or C8-C24 alkenyl, more preferably linear or branched C14. -C20 alkyl or C14-C20 alkenyl.

X ¹²⁵ and X ¹²⁶ C1-C6 alkyl, heterocyclyl or polyamine in the formula (CL-XIV) are selected from halogen atoms, R ′, OR ′, SR ′, CN, CO ₂ R ′ or CONR ′ ₂ It may be substituted with up to 3 substituents.
X ¹²⁵ and X ¹²⁶ in formula (CL-XIV) contain, in addition to the nitrogen to which they are attached, one or two additional heteroatoms selected from N, O and S. In the case of forming a 4- to 7-membered monocyclic heterocycle, the monocyclic heterocycle is a halogen atom, R ′, OR ′, SR ′, CN, CO ₂ R ′ or CONR ′. It may be substituted with 1 to 3 substituents selected from ₂ .
Here, R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.

R ¹²⁸ and R ¹²⁹ in formula (CL-XIV) are the same or different and are preferably linear or branched C4-C24 alkyl or C4-C24 alkenyl, more preferably linear or branched C4. -C20 alkyl or C4-C20 alkenyl.
C4-C24 alkyl or C4-C24 alkenyl may be substituted with one or more substituents selected from a halogen atom, R ′, OR ′, SR ′, CN, CO ₂ R ′ or CONR ′ _2. .
Here, R ′ is a hydrogen atom or C1-C6 alkyl, and the C1-C6 alkyl as R ′ may be substituted with a halogen atom or OH.
R ¹³⁰ in formula (CL-XIV) is a hydrogen atom or C1-C6 alkyl.

The cationic lipid in the present invention is preferably the formula (CL-I), (CL-II), (CL-III), (CL-IV), (CL-V), (CL-VIII), (CL- IX) is a cationic lipid.

Specific examples of the cationic lipid used in the present invention are shown in Tables 1 to 7 below, but the cationic lipid of the present invention is not limited thereto.

Next, a method for producing a cationic lipid in the present invention will be described. In addition, in the production method shown below, when a defined group changes under the conditions of the production method or is inappropriate for carrying out the production method, introduction and removal of a protective group commonly used in organic synthetic chemistry Methods [e.g., Methods described in Protective Groups in Organic Synthesis, third edition, by TWGreene, John Wiley & Sons Inc. (1999)], etc. Can be used to obtain the target compound. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.

In addition, etherification (4th edition, Experimental Chemistry Course 20, Synthesis of Organic Compounds II, 4th edition, p.187, Maruzen (1992), etc.), amination (4th edition) Edition Experimental Chemistry Lecture 20 “Synthesis of Organic Compounds II”, 4th Edition, p.279, Maruzen (1992), etc., Esterification (4th Edition Experimental Chemistry Course 22 “Synthesis of Organic Compounds IV”, 4th Edition, p. .43, Maruzen (1992), etc.), amidation (4th edition, Experimental Chemistry Lecture 22, Synthesis of Organic Compounds IV, 4th edition, p.137, Maruzen (1992), etc.) Can also be carried out using general reaction conditions described in the existing literature.

The lipid represented by the formula (CL-I) can be obtained by the method described in International Publication No. 2013/089151, or a method analogous thereto.

The lipid represented by the formula (CL-II) can be obtained by the method described in International Publication No. 2011/136368 or a method analogous thereto.

The lipids represented by formula (CL-III), formula (CL-IV) and formula (CL-V) can be obtained by the method described in International Publication No. 2014/007398 or a method analogous thereto.

The lipid represented by the formula (CL-VI) can be obtained by the method described in International Publication No. 2010/042877 or a method analogous thereto.

The lipid represented by the formula (CL-VII) can be obtained by the method described in International Publication No. 2010/054401, or a method analogous thereto.

The lipid represented by the formula (CL-VIII) can be obtained by the method described in International Publication No. 2016/002753 or a method analogous thereto.

The lipid represented by the formula (CL-IX) can be obtained by the method described below or a method analogous thereto.

The production method of the compound of the present invention will be described. In addition, in the production method shown below, when a defined group changes under the conditions of the production method or is inappropriate for carrying out the production method, introduction and removal of a protective group commonly used in organic synthetic chemistry Methods [e.g., Methods described in Protective Groups in Organic Synthesis, third edition, by TWGreene, John Wiley & Sons Inc. (1999)], etc. Can be used to produce the target compound. Further, the order of reaction steps such as introduction of substituents can be changed as necessary.

Production method 1
Of the lipids represented by the formula (CL-IX), the compound (CL-IXa) in which both X ¹¹⁵ and X ¹¹⁶ are hydrogen atoms, and the compound (CL-IXb) in which X ¹¹⁵ and X ¹¹⁶ are the same are as follows: It can manufacture by the method of.

(式中、R¹¹⁸、R¹¹⁹、M¹⁰¹、M¹⁰²、L¹¹⁸およびL¹¹⁹はそれぞれ前記と同義であり、IX-IIIaおよびIX-IIIbにおけるXは同一または異なって、塩素原子、臭素原子、ヨウ素原子、トリフルオロメタンスルホニルオキシ、メタンスルホニルオキシ、ベンゼンスルホニルオキシ、p-トルエンスルホニルオキシ等の脱離基を表し、R¹³⁵は水素原子、メチルまたはエチルであり、R¹³⁶は水素原子またはメチルであるか、またはR¹³⁵とR¹³⁶は隣接する炭素と一緒になってシクロプロピル環を形成する（ただし、R¹³⁵が水素原子またはエチルであるとき、R¹³⁶はメチルでない)）

(Wherein R ¹¹⁸ , R ¹¹⁹ , M ¹⁰¹ , M ¹⁰² , L ¹¹⁸ and L ¹¹⁹ have the same meanings as defined above, and X in IX-IIIa and IX-IIIb is the same or different, Represents a leaving group such as iodine atom, trifluoromethanesulfonyloxy, methanesulfonyloxy, benzenesulfonyloxy, p-toluenesulfonyloxy, R ¹³⁵ is a hydrogen atom, methyl or ethyl, and R ¹³⁶ is a hydrogen atom or methyl Or R ¹³⁵ and R ¹³⁶ together with adjacent carbons form a cyclopropyl ring (provided that when R ¹³⁵ is a hydrogen atom or ethyl, R ¹³⁶ is not methyl))

Step 26 and Step 27
Compound (IX-IIa) is obtained by reacting 2-amino-2-methyl-1,3-propanediol and compound (IX-IIIa) at room temperature in the presence of 1 to 10 equivalents of a base without solvent or in a solvent. It can be produced by reacting at a temperature between 200 ° C. for 5 minutes to 100 hours. Further, compound (CL-IXa) is obtained by reacting compound (IX-IIa) and compound (IX-IIIb) in the presence of 1 to 10 equivalents of base without solvent or in a solvent at a temperature between room temperature and 200 ° C. Thus, it can be produced by reacting for 5 minutes to 100 hours.

Examples of the solvent include dichloromethane, 1,2-dichloroethane, toluene, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, pyridine and the like, and these may be used alone or in combination. it can.

Examples of the base include sodium methoxide, potassium tert-butoxide, sodium hydride, lithium diisopropylamide, hexamethyldisilazane lithium, hexamethyldisilazane sodium, n-butyllithium and the like.

Compound (IX-IIIa) and Compound (IX-IIIb) are commercially available products or known methods (for example, “5th edition Experimental Chemistry Course 13 Synthesis of Organic Compounds I”, 5th edition, p.374, Maruzen ( 2005)) or a method based thereon.

Compound (CL-IXa) when R ¹¹⁸ -M ¹⁰¹ -L ¹¹⁸ and R ¹¹⁹ -M ¹⁰² -L ¹¹⁹ are the same can be obtained by using 2 equivalents or more of compound (IX-IIIa) in Step 26 Can do.

2-Amino-2-methyl-1,3-propanediol can be obtained as a commercial product.

Step 28
Compound (CL-IXb) is obtained by mixing compound (CL-IXa) with 2 to 20 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of reducing agent and preferably 1 to 10 equivalents in a solvent. It can be produced by reacting at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of an equivalent amount of acid.

Examples of the solvent include methanol, ethanol, tert-butyl alcohol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N , N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, water and the like, and these may be used alone or in combination.

Examples of the reducing agent include sodium triacetoxyborohydride and sodium cyanoborohydride.

Examples of the acid include hydrochloric acid and acetic acid.

Compound (IX-IV) can be obtained as a commercial product.

Production method 2
Among the lipids of Formula (CL-IX), compound X ¹¹⁵ and X ¹¹⁶ are different (CL-IXc) and (CL-IXd) can be prepared by the following method.

(式中、R¹¹⁸、R¹¹⁹、M¹⁰¹、M¹⁰²、L¹¹⁸、L¹¹⁹、R¹³⁵、R¹³⁶およびXはそれぞれ前記と同義であり、R¹³⁷はX¹¹⁵と同義であり、PGは保護基を表わす。)

(Wherein R ¹¹⁸ , R ¹¹⁹ , M ¹⁰¹ , M ¹⁰² , L ¹¹⁸ , L ¹¹⁹ , R ¹³⁵ , R ¹³⁶ and X are as defined above, R ¹³⁷ is as defined in X ¹¹⁵ , and PG is protected) Represents a group.)

Step 29
Compound (IX-IIb) is compound (CL-IXa) that is commonly used in synthetic organic chemistry (e.g., Protective Groups in Organic Synthesis, third edition, Green (TWGreene ) By John Wiley & Sons Inc. (1999), etc.].

Process 30
Compound (IX-IIc) is obtained by reacting Compound (IX-IIb) and Compound (IX-IIIc) in the presence of 1 to 10 equivalents of a base without solvent or in a solvent at a temperature between −20 ° C. and 150 ° C. Thus, it can be produced by reacting for 5 minutes to 72 hours.

Examples of the solvent include dichloromethane, 1,2-dichloroethane, toluene, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, pyridine, N, N-dimethylformamide, N, N-dimethylacetamide and the like. These may be used alone or in admixture.

Examples of the base include sodium methoxide, potassium tert-butoxide, sodium hydride, lithium diisopropylamide, hexamethyldisilazane lithium, hexamethyldisilazane sodium, n-butyllithium, potassium carbonate, cesium carbonate, triethylamine and the like. .

Compound (IX-IIIc) can be obtained as a commercial product.

Step 31
Compound (CL-IXc) can be obtained by removing protecting group PG of compound (IX-IIc) by an appropriate method. Methods for removing protecting groups include those commonly used in organic synthetic chemistry (for example, Protective Groups in Organic Synthesis, third edition, TW Greene, John The removal method described in Wiley & Sons Inc. (1999), etc.] can be used, whereby the target compound can be produced.

Process 32
Compound (CL-IXd) is obtained by combining compound (CL-IXc) with 1 to 10 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of a reducing agent and preferably 1 to 10 equivalents in a solvent. It can be produced by reacting at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of an equivalent amount of acid.

Examples of the solvent, reducing agent, and acid include those exemplified in Step 28.

Production method 3
Among the lipids represented by the formula (CL-IX), the compounds (CL-IXc ′) and (CL-IXd ′) in which M ¹⁰¹ and M ¹⁰² are each —OC (O) — are also produced by the following method can do.

(式中、R¹¹⁸、R¹¹⁹、M¹⁰¹、M¹⁰²、L¹¹⁸、L¹¹⁹、R¹³⁵、R¹³⁶、R¹³⁷およびPGはそれぞれ前記と同義であり、BおよびB^’は直鎖状または分岐状のC1-C16アルキルまたはC2-C16アルケニルである。)

(Wherein R ¹¹⁸ , R ¹¹⁹ , M ¹⁰¹ , M ¹⁰² , L ¹¹⁸ , L ¹¹⁹ , R ¹³⁵ , R ¹³⁶ , R ¹³⁷ and PG are as defined above, and B and B ^′ are linear or branched. C1-C16 alkyl or C2-C16 alkenyl in the form of

Step 33
Compound (IX-IId) can be produced by reacting compound (IX-IIc ′) and an oxidizing agent in a solvent at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.

Examples of the oxidizing agent include ozone, osmium tetroxide / sodium periodate, osmium tetroxide / lead tetraacetate, and the like.

Examples of the solvent include those exemplified in Step 28.

Compound (IX-IIc ′) can be produced by the method described in Production Method 2.

Step 34
Compound (IX-IIe) can be produced by reacting compound (IX-IId) and an oxidizing agent in a solvent at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours.

Examples of the oxidizing agent include Jones reagent, pyridinium dichromate, ruthenium tetroxide, sodium chlorite and the like.

Solvents include tert-butyl alcohol, dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetone, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, 1,4-dioxane, N, N- Examples thereof include dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, water and the like, and these can be used alone or in combination.

Step 35 and Step 36
Compound (IX-IIf) is obtained by reacting Compound (IX-IIe) and Compound (IX-Va) at room temperature and in the presence of 1 to 10 equivalents of a condensing agent and 1 to 10 equivalents of a base without solvent or in a solvent. It can be produced by reacting at a temperature between 0 ° C. and 5 minutes to 100 hours. Further, Compound (IX-IIc ″) is obtained by combining Compound (IX-IIf) and Compound (IX-Vb) in the absence of solvent or in a solvent in the presence of 1 to 10 equivalents of condensing agent and 1 to 10 equivalents of base. The reaction can be carried out at a temperature between room temperature and 200 ° C. for 5 minutes to 100 hours.

Examples of the solvent include dichloromethane, chloroform, 1,2-dichloroethane, toluene, ethyl acetate, acetonitrile, diethyl ether, tetrahydrofuran, 1,2-dimethoxyethane, dioxane, N, N-dimethylformamide, N, N-dimethylacetamide, N-methylpyrrolidone, pyridine and the like can be mentioned, and these can be used alone or in combination.

Examples of the condensing agent include 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride, N, N′-dicyclohexylcarbodiimide, 4- (4,6-dimethoxy-1,3,5-triazine-2- ジ ン yl). ) -4-Methylmorpholinium chloride n hydrate, 1H-benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate, O- (7-azabenzotriazol-1-yl) -N , N, N ′, N ′,-tetramethyluronium hexafluorophosphate and the like.

Examples of the base include potassium carbonate, cesium carbonate, triethylamine, N, N-diisopropylethylamine, N-methylmorpholine, pyridine and the like.

Compound (IX-Va) and compound (IX-Vb) can be obtained as commercial products.

The compound (IX-IIc ″) in the case where R ¹¹⁸ and R ¹¹⁹ are the same can be obtained by using 2 equivalents or more of the compound (IX-Va) in Step 35.

Step 37
Compound (CL-IXc ′) is obtained by removing the protecting group PG of compound (IX-IIc ″) by an appropriate method. Methods for removing protecting groups include those commonly used in organic synthetic chemistry (for example, Protective Groups in Organic Synthesis, third edition, TW Greene, John The removal method described in Wiley & Sons Inc. (1999), etc.] can be used, whereby the target compound can be produced.

Step 38
Compound (CL-IXd ′) is obtained by combining compound (CL-IXc ′) with 1 to 10 equivalents of compound (IX-IV), preferably 1 equivalent to a large excess of reducing agent in a solvent and preferably 1 It can be produced by reacting at a temperature between −20 ° C. and 150 ° C. for 5 minutes to 72 hours in the presence of ˜10 equivalents of acid.

Examples of the solvent and the acid include those exemplified in Step 28.

Of the compounds (CL-IX), compounds other than the compounds (CL-IXa) to (CL-IXd) can be obtained by adopting materials and reagents suitable for the structure of the target compound. It can be produced according to the production method or by applying a general production method commonly used in organic synthetic chemistry.

The intermediates and target compounds in each of the above production methods should be isolated and purified by subjecting them to separation and purification methods commonly used in organic synthetic chemistry, such as filtration, extraction, washing, drying, concentration, and various recrystallization chromatography. Can do. The intermediate can be subjected to the next reaction without any particular purification.

X ¹¹⁵ and X ¹¹⁶ are the same or different and each represents a hydrogen atom or C1-C3 alkyl.
X ¹¹⁵ and X ¹¹⁶ are the same or different and are preferably a hydrogen atom, methyl, ethyl, or propyl, more preferably a hydrogen atom or methyl.
The combination of (X ¹¹⁵ , X ¹¹⁶ ) is preferably (hydrogen atom, hydrogen atom), (hydrogen atom, methyl), (methyl, methyl), more preferably (hydrogen atom, methyl), (methyl, Methyl).

L ¹¹⁸ and L ¹¹⁹ are the same or different and are linear or branched C8-C24 alkylene or C8-C24 alkenylene.
When L ¹¹⁸ and L ¹¹⁹ are the same or different and are alkylene, they are preferably linear C8-C24 alkylene, more preferably linear C8-C20 alkylene, and even more preferably linear C8-C12 alkylene.
L ¹¹⁸ and L ¹¹⁹ are the same or different and are preferably octylene, nonylene, undecylene, tridecylene, pentadecylene, and more preferably octylene, nonylene, undecylene.
When L ¹¹⁸ and L ¹¹⁹ are the same or different and are alkenylene, they are preferably linear C8-C24 alkenylene, more preferably linear C10-C20 alkenylene, and even more preferably linear C10-C12 alkenylene.
L ¹¹⁸ and L ¹¹⁹ are the same or different, preferably (Z) -undec-9-enylene, (Z) -tridec-11-enylene, (Z) -tetradec-9-enylene, (Z) -hexadeca-9 -Enylene, (Z) -octadeca-9-enylene, (Z) -octadeca-11-enylene, (9Z, 12Z) -octadeca-9,12-dienylene.
L ¹¹⁸ and L ¹¹⁹ are preferably the same.

M ¹⁰¹ and M ¹⁰² are the same or different, and -C = C-, -OC (O)-, -C (O) O-, -SC (O)-, -C (O) S-, -OC ( S)-, -C (S) O-, -SS-, -C (R ^'' ) = N-, -N = C (R ^'' )-, -C (R ^'' ) = NO-,- ON = C (R ^'' )-, -N (R ^'' ) C (O)-, -C (O) N (R ^'' )-, -N (R ^'' ) C (S)-,- ^{C (S) N (R '} ') -, - N (R '') C (O) N (R ''') -, - N (R 3) C (O) O -, - OC (O) N (R ^'' )-and -OC (O) O-.
M ¹⁰¹ and M ¹⁰² are the same or different and are preferably -C = C-, -OC (O)-, -C (O) O-, -C (O) (NR ^'' )-, -N (R ^'' ) C (O)-, -N (R ^'' ) C (O)-, -N (R ^'' ) C (O) N (R ^''' )-, -N (R ^'' ) C (O) O-, -OC (O) N (R ^'' )-, -OC (O) O-, more preferably -C = C-, -OC (O)-, -C (O) O-.
The bond of each structure of M ¹⁰¹ and M ¹⁰² will be described by taking —OC (O) — as an example, which means that the structure is R ¹¹⁸ —OC (O) —L ¹¹⁸ .
M ¹⁰¹ and M ¹⁰² are preferably the same.

R ^'in M ¹⁰¹ and M ^102' and R ^'''the same or different, is a hydrogen atom or a C1-C3 alkyl.
R ^″ and R ^{′ ″} are preferably a hydrogen atom, methyl, ethyl or propyl, more preferably a hydrogen atom or methyl, and even more preferably a hydrogen atom.

R ¹¹⁸ and R ¹¹⁹ are the same or different and are linear or branched C1-C16 alkyl or C2-C16 alkenyl.
When R ¹¹⁸ and R ¹¹⁹ are the same or different and are alkyl, they are preferably linear C1-C16 alkyl, more preferably linear C2-C9 alkyl.
R ¹¹⁸ and R ¹¹⁹ are the same or different and are preferably pentyl, octyl, nonyl, decyl, dodecyl.
When R ¹¹⁸ and R ¹¹⁹ are the same or different and are alkenyl, they are preferably linear C 2 -C 16 alkenyl, more preferably linear C 3 -C 9 alkenyl.
R ¹¹⁸ and R ¹¹⁹ are the same or different, preferably (Z) -hept-2-ene, (Z) -oct-2-ene, (Z) -non-2-ene, (Z) -nona-3 -Ene, nona-8-ene, (Z) -dodec-2-ene, (Z) -tridec-2-ene.
R ¹¹⁸ and R ¹¹⁹ are preferably the same.

R ¹¹⁸ -M ¹⁰¹ -L ¹¹⁸ and R ¹¹⁹ -M ¹⁰² -L ¹¹⁹ are the same or different, and R ¹¹⁸ and R ¹¹⁹ , M ¹⁰¹ and M ¹⁰² , and L ¹¹⁸ and L ¹¹⁹ are from the structure described for each group. It may be a combination of
R ¹¹⁸ -M ¹⁰¹ -L ¹¹⁸ and R ¹¹⁹ -M ¹⁰² -L ¹¹⁹ are preferably the same.
R ¹¹⁸ -M ¹⁰¹ -L ¹¹⁸ and R ¹¹⁹ -M ¹⁰² -L ¹¹⁹ are the same or different, preferably (Z) -tetradec-9-enyl, (Z) -hexadeca-9-enyl, (Z) -octadeca -9-enyl, (E) -octadeca-9-enyl, (Z) -octadeca-11-enyl, (9Z, 12Z) -octadeca-9,12-dienyl, (9Z, 12Z, 15Z) -octadeca-9 , 12,15-trienyl, (Z) -icosa-11-enyl, (11Z, 14Z) -icosa-11,14-dienyl and (Z) -docosa-13-enyl, more preferably The group consisting of (Z) -hexadec-9-enyl, (Z) -octadeca-9-enyl, (9Z, 12Z) -octadeca-9,12-dienyl and (11Z, 14Z) -icosa-11,14-dienyl Chosen from.
R ¹¹⁸ -M ¹⁰¹ -L ¹¹⁸ and R ¹¹⁹ -M ¹⁰² -L ¹¹⁹ are the same or different and are preferably the following structures (1) to (5), more preferably the following structures (1) to (5): (5).

[式中、nは1-4の整数である]

[Where n is an integer of 1-4]

The lipid represented by the formula (CL-X) can be obtained by the method described in International Publication No. 2009/129385 or a method analogous thereto.

The lipid represented by the formula (CL-XI) can be obtained by the method described in International Publication No. 2013/1491401, or a method analogous thereto.

The lipid represented by the formula (CL-XII) can be obtained by the method described in International Publication No. 2009/129395 or a method analogous thereto.

The lipid represented by the formula (CL-XIII) can be obtained by the method described in International Publication No. 2013/059496 or a method analogous thereto.

The lipid represented by the formula (CL-XIV) can be obtained by the method described in International Publication No. 2011/149733 or a method analogous thereto.

The lipid represented by the formula (CL-XV) can be obtained by the method described in International Publication No. 2011/153493 or a method analogous thereto.

The lipid represented by the formula (CL-XVI) can be obtained by the method described in International Publication No. 2015/074085 or a method analogous thereto.

As described above, even when the lipid II is contained in the nanoparticles in the present invention, it is preferable that a neutral lipid is further contained.
Suitable examples of neutral lipids include phospholipids, glycerol lipids, sterols, glyceroglycolipids, glycosphingolipids, lipids containing water-soluble units, and sphingoids. These neutral lipids may be used alone or in combination of two or more.

When the nucleic acid-containing lipid nanoparticles of the present invention contain neutral lipids, the total number of molecules of neutral lipids is not particularly limited, but is preferably 0.05 times the molar amount or more relative to the total number of moles of lipids. Preferably it is 0.10 times mole amount or more, More preferably, it is 0.20 times mole amount or more, More preferably, it is 0.30 times mole amount or more. The total number of neutral lipid molecules is not particularly limited, but is preferably 0.75 times or less, more preferably 0.70 times or less, more preferably 0.65 times or less, and even more preferably 0.60 times or less. It is.

Examples of the phospholipid in the neutral lipid include phosphatidylcholine (PC) (specifically soybean phosphatidylcholine, egg yolk phosphatidylcholine (EPC), distearoylphosphatidylcholine, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC)). , Dipalmitoyl phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyl oleoyl phosphatidylcholine (POPC), dimyristoyl phosphatidylcholine (DMPC), dioleoylphosphatidylcholine (DOPC), etc.), phosphatidylethanol Amines (specifically distearoylphosphatidylethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dioleoylphosphatidylethanolamine (DOPE), dimyristoyl phosphoethanolamine (DMPE), 16-0-mono Chill PE, 16-0-dimethyl PE, 18-1-trans PE, palmitoyl oleoyl-phosphatidylethanolamine (POPE), 1 -stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), etc.), glycerophospholipid (specific) Phosphatidylserine, phosphatidic acid, phosphatidylglycerol, phosphatidylinositol, palmitoyloleoylphosphatidylglycerol (POPG), lysophosphatidylcholine, etc.), sphingophospholipids (specifically sphingomyelin, ceramide phosphoethanolamine, ceramide phosphoglycerol, ceramide) Phosphoglycerophosphate, etc.), glycerophosphonolipid, sphingophosphonolipid, natural lecithin (specifically egg yolk lecithin, soybean lecithin, etc.) or hydrogenated phospholipid (specifically hydrogenated soybean phosphatidyl). While natural phospholipids or synthetic phosphorus and the like), and the like without limitation.

Examples of the glyceroglycolipid in the neutral lipid include, but are not limited to, sulfoxyribosyl glyceride, diglycosyl diglyceride, digalactosyl diglyceride, galactosyl diglyceride, glycosyl diglyceride and the like.

Examples of the glycosphingolipid in the neutral lipid include, but are not limited to, galactosyl cerebroside, lactosyl cerebroside, ganglioside, and the like.

Examples of the sphingoid in the neutral lipid include, but are not limited to, sphingan, icosasphingan, sphingosine, and derivatives thereof. As the derivative, for example, —NH ₂ such as sphingan, icosasphingan or sphingosine —NHCO (CH ₂ ) xCH ₃ (wherein x is an integer of 0 to 18, among which 6, 12 or 18 is preferable. However, it is not limited to these.

Examples of sterols in neutral lipids include cholesterol (Chol), dihydrocholesterol, lanosterol, β-sitosterol, campesterol, stigmasterol, brassicasterol, ergocasterol, fucostosterol, or 3β- [N- (N ′, N'-dimethylaminoethyl) carbamoyl] cholesterol (DC-Chol) and the like, but are not limited thereto.

The lipid containing a water-soluble unit is a lipid derivative or a fatty acid derivative of a water-soluble polymer.
Examples of the water-soluble polymer lipid derivative or fatty acid derivative include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, and polyglycerin. , Chitosan, polyvinyl pyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol etc. Examples thereof include salts formed by binding with fatty acids such as acid, myristic acid or lauric acid, salts thereof, and the like. More preferably, lipid derivatives or fatty acid derivatives such as polyethylene glycol or polyglycerin and the like. Cited et salts, more preferable example is a lipid derivative or fatty acid derivatives and salts thereof polyethylene glycol.

Examples of lipid derivatives or fatty acid derivatives of polyethylene glycol include polyethylene glycolated lipids [specifically, polyethylene glycol-phosphatidylethanolamine and polyethylene glycol-diacylglycerol (more specifically, 1,2-distearoyl-sn-glycero -3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol)- 2000] (PEG-DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn -Glycerol, methoxypolyethylene glycol-2000 (PEG-DSG), 1,2-dipalmitoyl-sn-glycerol, methoxypolyethyleneglycol-2000 (PEG-DPG) , 1,2-dimyristoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DMG), 1,2-oleoyl-sn-glycerol, methoxypolyethylene glycol-2000 (PEG-DOG))), polyoxy Ethylene hardened castor oil 60, Cremophor EL, etc.], polyethylene glycol sorbitan fatty acid esters (specifically, polyoxyethylene sorbitan monooleate) or polyethylene glycol fatty acid esters, and the like, more preferably And polyethylene glycolated lipids.

Examples of the lipid derivative or fatty acid derivative of polyglycerin include polyglycerinized lipids (specifically polyglycerin-phosphatidylethanolamine) or polyglycerin fatty acid esters, and more preferably, polyglycerinized lipids. Can be mentioned.

In the nucleic acid-containing nanoparticles of the present invention, the total number of water-soluble polymer lipid derivatives and fatty acid derivative molecules in the nucleic acid-containing nanoparticles is not particularly limited, but is 0.005 times the molar amount of the total lipid or more. The molar amount is preferably 0.01 to 0.30 times the molar amount, more preferably 0.02 to 0.25 times the molar amount, still more preferably 0.03 to 0.20 times the molar amount, and more preferably 0.04 to 0.15 times the molar amount. It is even more preferred that it is 0.04 to 0.12 times the molar amount.

The nanoparticles of the present invention may contain a polymer, for example, protein, albumin, dextran, polyfect, chitosan, dextran sulfate, such as poly-L-lysine, polyethyleneimine, polyaspartic acid. A micelle comprising at least one polymer such as styrene maleic acid copolymer, isopropylacrylamide-acryl pyrrolidone copolymer, polyethylene glycol modified dendrimer, polylactic acid, polylactic acid polyglycolic acid or polyethylene glycolated polylactic acid, or a salt thereof. However, it is not limited to these.

Here, the polymer salt includes, for example, metal salts, ammonium group salts, acid addition salts, organic amine addition salts, amino acid addition salts, and the like. Examples of the metal salt include, but are not limited to, alkali metal salts such as lithium salt, sodium salt and potassium salt, alkaline earth metal salts such as magnesium salt and calcium salt, aluminum salt and zinc salt. Examples of ammonium group salts include, but are not limited to, salts such as ammonium groups or tetramethylammonium groups. Examples of the acid addition salt include inorganic acid salts such as hydrochloride, sulfate, nitrate or phosphate, and organic acid salts such as acetate, maleate, fumarate or citrate. It is not limited to. Examples of organic amine addition salts include, but are not limited to, addition salts such as morpholine or piperidine. Examples of amino acid addition salts include, but are not limited to, addition salts such as glycine, phenylalanine, aspartic acid, glutamic acid, or lysine.

Any of the nucleic acid-containing lipid nanoparticles of the present invention may contain, for example, a lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant. . The neutral lipid in the present invention includes the above lipid derivatives and fatty acid derivatives.

The lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or the surfactant, preferably a lipid or fatty acid derivative of a glycolipid or a water-soluble polymer. More preferred are lipid derivatives or fatty acid derivatives of water-soluble polymers. A lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant is a part of the molecule and other components of the composition, such as hydrophobic affinity, static It is a substance with a two-sided property that has the property of binding by electrical interaction, etc., and the other part has the property of binding to the solvent at the time of production of the composition, for example, hydrophilic affinity, electrostatic interaction, etc. Preferably there is.

Examples of lipid derivatives or fatty acid derivatives of sugars, peptides or nucleic acids include sugars such as sucrose, sorbitol, and lactose, such as casein-derived peptides, egg white-derived peptides, soybean-derived peptides, peptides such as glutathione, or, for example, DNA, Nucleic acids such as RNA, plasmid, siRNA, ODN and the like and neutral lipids listed in the definition of the composition or fatty acids such as stearic acid, palmitic acid, myristic acid, lauric acid, etc. Is mentioned.

Examples of the sugar lipid derivative or fatty acid derivative include glyceroglycolipid and glycosphingolipid mentioned in the definition of the composition.

Examples of the surfactant include polyoxyethylene sorbitan monooleate (specifically polysorbate 80 etc.), polyoxyethylene polyoxypropylene glycol (specifically Pluronic F68 etc.), sorbitan fatty acid ester (specifically Sorbitan monolaurate, sorbitan monooleate, etc.), polyoxyethylene derivatives (specifically polyoxyethylene hydrogenated castor oil 60, polyoxyethylene lauryl alcohol, etc.), glycerin fatty acid ester or polyethylene glycol alkyl ether, etc. Preferably, polyoxyethylene polyoxypropylene glycol, glycerin fatty acid ester, polyethylene glycol alkyl ether or the like is used.

As the cationic lipid, in addition to the above-described cationic lipid (lipid II), another cationic lipid may be used. (Hereinafter, lipid II and another cationic lipid are combined and simply referred to as a cationic lipid.)
Examples of cationic lipids other than lipid II include N- [1- (2,3-dioleyloxy) propyl] disclosed in JP-A-61-161246 (US Pat. No. 5,049,386). -N, N, N-trimethylammonium chloride (DOTMA), N- (2,3-di- (9- (Z) -octadecenoyloxy))-prop-1-yl-N, N, N- N- [1- (2,3-dioleyloxypropyl)]-N, N-, such as trimethylammonium chloride (DOTAP), disclosed in WO 91/16024 and WO 97/019675 Dimethyl-N-hydroxyethylammonium bromide (DORIE), 2,3-dioleyloxy-N- [2- (sperminecarboxamido) ethyl] -N, N-dimethyl-1-propanaminium trifluoroacetic acid (DOSPA ), Etc., disclosed in International Publication No. 2005/121348, DLinDMA, etc., disclosed in International Publication No. 2009/086558, DLin-K-DMA, disclosed in International Publication No. 2010/042877 , DLin-KC2-DMA, International Publication No. 2010/054401 DLin-MC3-DMA, disclosed in WO 2011/136368, (3R2R) -3,4-bis ((Z) -hexadec-9-enyloxy) -1-methyl Pyrrolidine, N-methyl-N, N-bis (2-((Z) -octadeca-6-enyloxy) ethyl) amine and the like, preferably DOTMA, DOTAP, DORIE, DOSPA, 1,2-dilinoleyl Oxy-N, N-dimethylaminopropane (DLinDMA), 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane (DLin-K-DMA), 2,2-dilinoleyl-4-dimethylamino Ethyl- [1,3] -dioxolane (DLin-KC2-DMA), (6Z, 9Z, 28Z, 31Z) -heptatriconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino) Cationic lipids having a tertiary amine moiety having two unsubstituted alkyl groups or a quaternary ammonium moiety having three unsubstituted alkyl groups, such as butanoate (DLin-MC3-DMA), and more preferably Has a secondary amine moiety And cationic lipids. The unsubstituted alkyl group of the tertiary amine moiety and the quaternary ammonium moiety is more preferably a methyl group.

One embodiment of the present invention is a lipid nanoparticle containing a lipid (lipid I) having a ligand capable of binding to Siglec and containing a nucleic acid, for example, a lipid composed of lipid I, a cationic lipid, and a nucleic acid A nanoparticle; a lipid nanoparticle containing a complex of a lipid I and a cationic lipid and a neutral lipid and a nucleic acid; a lipid nanoparticle containing the complex and a lipid membrane containing lipid I; And the like, and the like. Lipid nanoparticles containing the complex in lipid nanoparticles containing a lipid membrane.
Examples of the complex include a complex of a membrane composed of a nucleic acid and a lipid bilayer, a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, and preferably a complex of a nucleic acid and a micelle or Examples include a complex of a nucleic acid and a liposome. The lipid membrane may be a lipid single membrane (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane). The lipid membrane may contain a cationic lipid or a neutral lipid.

Examples of the form of the complex include a complex of a nucleic acid and a membrane composed of a single lipid (single molecule) layer (reverse micelle), a complex of a nucleic acid and a liposome, a complex of a nucleic acid and a micelle, and the like. Preferably, a complex of a nucleic acid and a membrane composed of a lipid monolayer or a complex of a nucleic acid and a liposome can be mentioned. Examples of the lipid nanoparticle containing the complex and the lipid membrane that encapsulates the complex include liposomes that encapsulate the complex and the complex with a lipid bilayer.

In the lipid nanoparticles of the present invention, one or more kinds of cationic lipids may be mixed and used.

The lipid nanoparticles of the present invention can contain nucleic acids, but can also contain compounds that are chemically similar to nucleic acids (for example, peptide nucleic acids).

In the present invention, the total lipid is the total of lipid I and lipids other than lipid I.

In addition, the nucleic acid-containing nanoparticles of the present invention can be optionally subjected to surface modification with a water-soluble polymer, for example [Radasic, edited by F. Martin, “Stealth Liposomes”. (Stealth Liposomes) ”(USA), CRC Press Inc, 1995, p.93-102]. Examples of water-soluble polymers that can be used for surface modification include polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, poly Examples include glycerin, chitosan, polyvinylpyrrolidone, polyaspartic acid amide, poly-L-lysine, mannan, pullulan, oligoglycerol, etc., preferably polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, etc. More preferably, polyethylene glycol, polyglycerin and the like can be mentioned, but not limited thereto. For the surface modification, a lipid derivative or fatty acid derivative (as defined above) of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant or the like can be used. The surface modification is one of methods in which the lipid derivative or fatty acid derivative of one or more substances selected from sugars, peptides, nucleic acids, and water-soluble polymers, or a surfactant is contained in the nucleic acid-containing lipid nanoparticles of the present invention. One.

(Polymer II)
The polymer II is a polymer including a water-soluble unit and a cationic unit.
In the polymer II, the water-soluble unit and the cationic unit may be bonded via a linker, that is, can be represented by the following structural formula.

Examples of water-soluble units include polyethylene glycol units, polyglycerin units, polyethyleneimine units, polyvinyl alcohol units, polyacrylic acid units, polyacrylamide units, oligosaccharide units, dextrin units, water-soluble cellulose units, dextran units, and chondroitin sulfate. Examples thereof include units, polyglycerin units, chitosan units, polyvinylpyrrolidone units, polyaspartic acid amide units, poly-L-lysine units, mannan units, pullulan units, oligoglycerol units and the like. Among these, a polyethylene glycol unit is preferable. The number average molecular weight of the water-soluble unit is not particularly limited, but is preferably 100 to 5000, and more preferably 500 to 3000.

The cationic unit in polymer II is preferably an amino acid polymer unit containing at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit.

The amino acid polymer unit containing one or more selected from the group consisting of lysine, arginine and histidine can be obtained by polymerization of an amino acid containing one or more selected from the group consisting of lysine, arginine and histidine. When the amino acid polymer unit is composed of two or more amino acids, it may be a random copolymer or a block copolymer.
Examples of the amino acid polymer unit include the following structures.

（上記構造中、ｎ1は2以上の整数である。ｎ1は、好ましくは２～１００であり、より好ましくは５～５０である。）

(In the above structure, n1 is an integer of 2 or more. N1 is preferably 2 to 100, more preferably 5 to 50.)

The polyethyleneimine unit is a unit made of a polymer obtained by polymerizing ethyleneimine. The polyethyleneimine unit can be represented, for example, as shown in the main chain structure below, but the polyethyleneimine main chain may be cross-linked with other polyethyleneimine structures.

（上記主鎖構造中、ｎ2は１以上の整数である。ｎ2は、好ましくは２～１００であり、より好ましくは５～５０である。）

(In the main chain structure, n2 is an integer of 1 or more. N2 is preferably 2 to 100, more preferably 5 to 50.)

The polyaminoacrylate unit is a unit made of a polymer obtained by polymerizing methacrylic acid having an amino group such as 2- (dimethylamino) ethyl methacrylate. As the polyaminoacrylate unit, for example, the following structures are preferably exemplified.

（上記構造中、ｎ3は１以上の整数である。）

(In the above structure, n3 is an integer of 1 or more.)

The linker in polymer II is not particularly limited as long as it can link a water-soluble unit and a cationic unit, but a linker suitably used for lipid I and polymer I is also suitable for a linker in polymer II. is there.

(Nucleic acid)
The nucleic acid used in the present invention may be any molecule as long as it is a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides, for example, ribonucleic acid that is a polymer of ribonucleotides. (RNA), deoxyribonucleic acid (DNA) which is a polymer of deoxyribonucleotides, chimeric nucleic acids composed of RNA and DNA, and nucleotides in which at least one nucleotide of these nucleic acids is replaced with a molecule having a function equivalent to that nucleotide A polymer etc. are mentioned. In addition, the nucleic acid of the present invention also includes a derivative containing at least a part of the structure of a molecule obtained by polymerizing nucleotides and / or molecules having functions equivalent to nucleotides. In the present invention, uracil U and thymine T can be replaced with each other.

Examples of molecules having a function equivalent to nucleotides include nucleotide derivatives.

The nucleotide derivative may be any molecule as long as it is a modified nucleotide, for example, it improves nuclease resistance or stabilizes from other degradation factors compared to RNA or DNA. In order to increase the affinity with the complementary strand nucleic acid, to increase cell permeability, or to visualize the molecule, a molecule in which ribonucleotides or deoxyribonucleotides are modified is preferably used.

Examples of nucleotide derivatives include sugar moiety-modified nucleotides, phosphodiester bond-modified nucleotides, base-modified nucleotides, and the like.

The sugar moiety-modified nucleotide may be any nucleotide as long as it is modified or substituted with an arbitrary substituent on a part or all of the sugar sugar chemical structure, or substituted with an arbitrary atom. 2'-modified nucleotides are preferably used.

Examples of the modifying group in the sugar moiety-modified nucleotide include 2′-cyano, 2′-alkyl, 2′-substituted alkyl, 2′-alkenyl, 2′-substituted alkenyl, 2′-halogen and 2′-O-cyano. 2'-O-alkyl, 2'-O-substituted alkyl, 2'-O-alkenyl, 2'-O-substituted alkenyl, 2'-S-alkyl, 2'-S-substituted alkyl, 2'-S -Alkenyl, 2'-S-substituted alkenyl, 2'-amino, 2'-NH-alkyl, 2'-NH-substituted alkyl, 2'-NH-alkenyl, 2'-NH-substituted alkenyl, 2'-SO -Alkyl, 2'-SO-substituted alkyl, 2'-carboxy, 2'-CO-alkyl, 2'-CO-substituted alkyl, 2'-Se-alkyl, 2'-Se-substituted alkyl, 2'-SiH ₂ -alkyl, 2'-SiH ₂ -substituted alkyl, 2'-ONO ₂ , 2'-NO ₂ , 2'-N ₃ , 2'-amino acid residues (hydroxy was removed from the carboxyl group constituting the amino acid) And 2'-O-amino acid residues (synonymous with the above amino acid residues) and the like.

Examples of sugar-modified nucleotides include, for example, a crosslinked structure-type artificial nucleic acid (BNA) having a structure in which a modification group at the 2 ′ position is crosslinked to a carbon atom at the 4 ′ position, more specifically, the 2 ′ position. Locked Nucleic Acid (LNA) in which the oxygen atom and 4'-position carbon atom are bridged via methylene, and Ethylene bridged nucleic acid (ENA) [Nucleic Acid Research, 32, e175 (2004)] and the like, and these are included in 2′-modified nucleotides.

Peptide nucleic acid (PNA) [Acc. Chem. Res., 32, 624 (1999)], oxypeptide nucleic acid (OPNA) [J. Am. Chem. Soc., 123, 4653 (2001)] And peptide ribonucleic acid (PRNA) [J. Am. Chem. Soc., 122, 6900 (2000)].

2'-cyano, 2'-halogen, 2'-O-cyano, 2'-alkyl, 2'-substituted alkyl, 2'-O-alkyl, 2'-O-substituted as a modifying group in sugar modified nucleotide Preferred are alkyl, 2′-O-alkenyl, 2′-O-substituted alkenyl, 2′-Se-alkyl or 2′-Se-substituted alkyl, 2′-cyano, 2′-fluoro, 2′-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O-methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (Methoxy) ethyl], 2'-O- (3-aminopropyl), 2'-O- [2- (N, N-dimethylaminooxy) ethyl], 2'-O- [3- (N, N -Dimethylamino) propyl], 2'-O- {2- [2- (N, N-dimethylamino) ethoxy] ethyl}, 2'-O- [2- (methylamino) -2-oxoethyl] or 2 '-Se-methyl and the like are more preferable, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro and the like are more preferable, and 2'-O-methyl Or 2'-O-ethyl is most preferred.

The preferred range of the modifying group in the sugar moiety-modified nucleotide can also be defined from its size, preferably from the size of fluoro to the size of -O-butyl, and from the size of -O-methyl- Those corresponding to the size of O-ethyl are more preferred.

Examples of the alkyl in the modifying group in the sugar-modified nucleotide include C1-C6 alkyl, and more specifically, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl. And C1-C6 alkyl such as neopentyl or hexyl.

Examples of the alkenyl in the modifying group in the sugar moiety-modified nucleotide include C3-C6 alkenyl, and more specifically, C3-C6 alkenyl such as allyl, 1-propenyl, butenyl, pentenyl, hexenyl and the like.

Examples of the halogen in the modifying group in the sugar moiety-modified nucleotide include a fluorine atom, a chlorine atom, a bromine atom or an iodine atom.

Examples of amino acids in amino acid residues include aliphatic amino acids (specifically, glycine, alanine, valine, leucine, isoleucine, etc.), hydroxy amino acids (specifically, serine, threonine, etc.), acidic amino acids (specifically, Aspartic acid, glutamic acid, etc.), acidic amino acid amides (specifically, asparagine, glutamine, etc.), basic amino acids (specifically, lysine, hydroxylysine, arginine, ornithine, etc.), sulfur-containing amino acids (specifically, Specifically, cysteine, cystine, methionine and the like) or imino acid (specifically, proline, 4-hydroxyproline and the like) and the like.

Examples of the substituted alkyl in the modified group in the sugar-modified nucleotide and the substituent in the substituted alkenyl include halogen (as defined above), hydroxy, sulfanyl, amino, oxo, -O-alkyl (the alkyl portion of the -O-alkyl is The same as C1-C6 alkyl in the modifying group), -S-alkyl (the alkyl part of the -S-alkyl is synonymous with C1-C6 alkyl in the modifying group), -NH-alkyl (the alkyl of -NH-alkyl) Part is synonymous with C1-C6 alkyl in the modifying group), dialkylaminooxy (the two alkyl parts of the dialkylaminooxy are the same or different and are synonymous with C1-C6 alkyl in the modifying group), dialkylamino (the dialkylamino The two alkyl moieties are the same or different and have the same meaning as C1-C6 alkyl in the above-mentioned modifying group) or dialkylaminoalkyl Xi (the two alkyl moieties of the dialkylaminoalkyloxy are the same or different and have the same meaning as the C1-C6 alkyl in the modifying group, and the alkylene moiety has one hydrogen atom removed from the C1-C6 alkyl in the modifying group. The number of substitutions is preferably 1 to 3.

The phosphodiester bond-modified nucleotide is any nucleotide that has been modified or substituted with an arbitrary substituent for a part or all of the chemical structure of the phosphodiester bond of the nucleotide, or with any atom. For example, a nucleotide in which a phosphodiester bond is replaced with a phosphorothioate bond, a nucleotide in which a phosphodiester bond is replaced with a phosphorodithioate bond, a nucleotide in which a phosphodiester bond is replaced with an alkylphosphonate bond, or a phosphate Examples include nucleotides in which a diester bond is substituted with a phosphoramidate bond.

As the base-modified nucleotide, any or all of the nucleotide base chemical structure modified or substituted with an arbitrary substituent or substituted with an arbitrary atom may be used. In which oxygen atom is substituted with sulfur atom, hydrogen atom is substituted with C1-C6 alkyl group, methyl group is substituted with hydrogen atom or C2-C6 alkyl group, amino group is C1-C6 Examples include those protected with a protecting group such as an alkyl group and a C1-C6 alkanoyl group.

Further, as nucleotide derivatives, nucleotides or sugar moieties, nucleotide derivatives modified with at least one of phosphodiester bonds or bases, lipids, phospholipids, phenazine, folate, phenanthridine, anthraquinone, acridine, fluorescein, rhodamine, coumarin and Examples include dyes and other chemical substances added. Specifically, 5'-polyamine-added nucleotide derivatives, cholesterol-added nucleotide derivatives, steroid-added nucleotide derivatives, bile acid-added nucleotide derivatives, vitamin-added nucleotide derivatives, green Examples include fluorescent dye (Cy3) addition nucleotide derivatives, red fluorescent dye (Cy5) addition nucleotide derivatives, fluorescein (6-FAM) addition nucleotide derivatives, and biotin addition nucleotide derivatives. The

In the nucleic acid used in the present invention, a nucleotide or a nucleotide derivative is an alkylene structure, a peptide structure, a nucleotide structure, an ether structure, an ester structure, or a structure combining at least two of these with other nucleotides or nucleotide derivatives in the nucleic acid. A cross-linked structure such as

The nucleic acid used in the present invention preferably has a molecular weight of 1,000 kDa or less, more preferably 100 kDa or less, and even more preferably 30 kDa or less. The nucleic acid used in the present invention preferably includes a nucleic acid that supplements a target gene expressed in normal cells and a nucleic acid that suppresses the expression of the target gene, and more preferably RNA interference ( Examples include nucleic acids having an inhibitory effect on target gene expression using RNAi).

The nucleic acid used in the present invention may be mRNA. mRNA refers to mRNA transcribed from template DNA, and the encoded protein (including peptide) is not particularly limited. The mRNA may encode a glycoprotein or a fusion protein. The number of bases of mRNA is not particularly limited. The mRNA may be a homologous sequence capable of synthesizing the target protein, and may have a plurality of bases deleted, substituted, inserted or added.

The nucleic acid in the nanoparticles of the present invention is introduced into the cell. Such cells are preferably mammalian Siglec-1 (CD169) positive cells. By introducing the nucleic acid into the cell, the expression of the target gene can be promoted or suppressed in the cell. Examples of Siglec-1 (CD169) positive cells include macrophages, dendritic cells or monocytes.
The target gene in the present invention may be appropriately selected according to the type of disease intended for treatment, for example, a gene capable of repairing or correcting a defect causing the disease that is the purpose of treatment, and the cause of the disease. And genes that suppress the expression of factors and receptors. Examples of the disease include diseases in which Siglec-1 (CD169) -positive cells are seen, and diseases involving macrophages, dendritic cells or monocytes.

As the nucleic acid, for example, a nucleic acid containing a base sequence complementary to a partial base sequence of mRNA of a gene encoding a protein (target gene) and suppressing the expression of the target gene, for example, Any nucleic acid may be used, such as double-stranded nucleic acid such as siRNA (short interference RNA) and miRNA (micro RNA), single-stranded nucleic acid such as shRNA (short hairpin RNA), antisense nucleic acid, and ribozyme. Double-stranded nucleic acids are preferred.

A nucleic acid containing a base sequence complementary to a part of the base sequence of the target gene mRNA is called an antisense strand nucleic acid, and a nucleic acid containing a base sequence complementary to the base sequence of the antisense strand nucleic acid is a sense strand. Also called nucleic acid. A sense strand nucleic acid refers to a nucleic acid capable of forming a double strand forming part by pairing with an antisense strand nucleic acid, such as a nucleic acid itself consisting of a partial base sequence of a target gene.

A double-stranded nucleic acid refers to a nucleic acid in which two strands are paired and have a duplex forming part. The double-stranded forming part refers to a part where nucleotides constituting the double-stranded nucleic acid or a derivative thereof constitute a base pair to form a double strand. The base pair constituting the duplex forming part is usually 15 to 27 base pairs, preferably 15 to 25 base pairs, more preferably 15 to 23 base pairs, further preferably 15 to 21 base pairs, and 15 to 19 base pairs. Base pairs are particularly preferred.

As the antisense strand nucleic acid of the duplex forming part, for example, a nucleic acid comprising a partial sequence of the mRNA of the target gene, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base in the nucleic acid is substituted. A nucleic acid that is deleted or added and has the activity of suppressing the expression of the target protein is preferably used. The single-stranded nucleic acid constituting the double-stranded nucleic acid usually consists of a series of 15 to 30 bases (nucleosides), preferably 15 to 29 bases, more preferably 15 to 27 bases, and further preferably 15 to 25 bases. 17 to 23 bases are particularly preferred, and 19 to 21 bases are most preferred.

The antisense strand, the sense strand, or both of the nucleic acids constituting the double-stranded nucleic acid have an additional nucleic acid that does not form a duplex on the 3 ′ side or 5 ′ side following the duplex forming part. May be. The part that does not form a double chain is also referred to as a protrusion (overhang).

As the double-stranded nucleic acid having an overhang, for example, one having an overhang of 1 to 4 bases, usually 1 to 3 bases at the 3 ′ end or 5 ′ end of at least one strand is used. Those having a protruding portion made of a base are preferably used, and those having a protruding portion made of dTdT or UU are more preferably used. The overhang can have only the antisense strand, only the sense strand, and both the antisense strand and the sense strand, but a double-stranded nucleic acid having an overhang on both the antisense strand and the sense strand is preferably used. .

Sequence that matches part or all of the base sequence of the target gene mRNA following the duplex formation part, or part or all of the base sequence of the complementary strand of the target gene mRNA following the duplex formation part Sequences that match may be used. Furthermore, as a nucleic acid that suppresses the expression of a target gene, for example, a nucleic acid molecule that generates the above double-stranded nucleic acid by the action of a ribonuclease such as Dicer (International Publication No. 2005/089287), a 3 ′ end or a 5 ′ A double-stranded nucleic acid or the like that does not have a terminal protruding portion can also be used.

When the double-stranded nucleic acid is siRNA, preferably, the antisense strand has a sequence of at least the 1st to 17th bases (nucleosides) from the 5 ′ end to the 3 ′ end, and the mRNA of the target gene More preferably, the antisense strand has a sequence of bases 1 to 19 from the 5 ′ end to the 3 ′ end, The base sequence is complementary to the 19-base sequence of the gene mRNA, or the base sequence 1 to 21 is the base sequence complementary to the 21-base sequence of the target gene mRNA. Alternatively, the sequence of the 1st to 25th bases is a sequence of bases complementary to the sequence of 25 consecutive bases of the mRNA of the target gene.

Furthermore, when the nucleic acid used in the present invention is siRNA, ribose substituted with a modifying group at the 2'-position is preferably included. In the present invention, ribose substituted with a modifying group at the 2′-position means that the hydroxyl at the 2′-position of ribose is substituted with the modifying group, and the configuration is the same as the hydroxy at the 2′-position of ribose. Although it may be present or different, the configuration is preferably the same as that of the 2′-hydroxy of ribose. Examples of the modifying group in ribose substituted with a modifying group at the 2′-position include those exemplified in the definition of the modifying group in the 2′-modified nucleotide in the sugar moiety-modified nucleotide and the hydrogen atom, such as 2′-cyano, 2 ′ -Halogen, 2'-O-cyano, 2'-alkyl, 2'-substituted alkyl, 2'-O-alkyl, 2'-O-substituted alkyl, 2'-O-alkenyl, 2'-O-substituted alkenyl , 2'-Se-alkyl, 2'-Se-substituted alkyl, etc. are preferred, 2'-cyano, 2'-fluoro, 2'-chloro, 2'-bromo, 2'-trifluoromethyl, 2'-O -Methyl, 2'-O-ethyl, 2'-O-isopropyl, 2'-O-trifluoromethyl, 2'-O- [2- (methoxy) ethyl], 2'-O- (3-aminopropyl ), 2'-O- [2- (N, N-dimethyl) aminooxy] ethyl, 2'-O- [3- (N, N-dimethylamino) propyl], 2'-O- {2- [ 2- (N, N-dimethylamino) ethoxy] ethyl}, 2'-O- [2- (methyl Amino) -2-oxoethyl], 2'-Se-methyl, hydrogen atom and the like are more preferable, 2'-O-methyl, 2'-O-ethyl, 2'-fluoro, hydrogen atom and the like are more preferable, and 2'- -O-methyl, 2'-O-fluoro is most preferred.

The nucleic acid used in the present invention includes a derivative in which an oxygen atom or the like contained in a phosphoric acid part, an ester part or the like in the structure of the nucleic acid is substituted with another atom such as a sulfur atom.

The sugar that binds to the 5 'terminal base of the antisense strand and the sense strand has a 5'-positioned hydroxy group, either a phosphate group or the above-mentioned modifying group, or an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted to a modifying group.

The sugar that binds to the base at the 3 ′ end of the antisense strand and the sense strand is such that the 3′-position hydroxy is a phosphate group or the above-mentioned modifying group, or an in vivo nucleolytic enzyme, etc. It may be modified by a group that is converted to a modifying group.

As the single-stranded nucleic acid, for example, 15 to 27 bases (nucleoside) of the target gene, preferably 15 to 25 bases, more preferably 15 to 23 bases, still more preferably 15 to 21 bases, particularly preferably 15 A nucleic acid comprising a sequence complementary to a sequence consisting of ˜19 bases, or 1 to 3 bases, preferably 1 to 2 bases, more preferably 1 base is substituted, deleted or added in the nucleic acid, and the target protein expression inhibitory activity Any nucleic acid may be used as long as it has a nucleic acid. The single-stranded nucleic acid is preferably composed of a series of 10 to 30 bases (nucleosides), more preferably 10 to 27 bases, further preferably 10 to 25 bases, particularly preferably 10 to 23 bases. Nucleic acids are preferably used.

As the single-stranded nucleic acid, one obtained by linking the antisense strand and the sense strand constituting the double-stranded nucleic acid via a spacer sequence (spacer oligonucleotide) may be used. The spacer oligonucleotide is preferably a 6- to 12-base single-stranded nucleic acid molecule, and the sequence at the 5 'end is preferably 2 Us. An example of a spacer oligonucleotide is a nucleic acid having the sequence UUCAAGAGA. Either the antisense strand or the sense strand connected by the spacer oligonucleotide may be on the 5 'side. The single-stranded nucleic acid is preferably, for example, a single-stranded nucleic acid such as shRNA having a duplex forming part by a stem-loop structure. Single-stranded nucleic acids such as shRNA are usually 50 to 70 bases in length.

A nucleic acid having a length of 70 bases or less, preferably 50 bases or less, more preferably 30 bases or less, designed to produce the above single-stranded nucleic acid or double-stranded nucleic acid by the action of ribonuclease or the like may be used. Good.

The nucleic acid used in the present invention can be obtained using a known RNA or DNA synthesis method and RNA or DNA modification method.

The nucleic acid-containing lipid nanoparticles of the present invention can contain not only nucleic acids but also compounds that are chemically similar to nucleic acids (anionic polymers such as anionic peptides).

[Method for producing nucleic acid-containing lipid nanoparticles]
The lipid nanoparticles of the present invention can be produced by a known production method or a method similar thereto, and may be produced by any production method. For example, a known method for preparing liposomes can be applied to the production of a composition containing liposome, which is one of the compositions. Known liposome preparation methods include, for example, Bangham et al.'S liposome preparation method [“J. Mol. Biol.”, 1965, Vol. 13, p.238- 252], ethanol injection method [“J. Cell Biol.”, 1975, Vol. 66, pp. 621-634], French press method [“FBS Letters (FEBS Lett.), 1979, Vol. 99, p. 210-214], freeze-thaw method [“Arch. Biochem. Biophys.”, 1981 Year 212, p.186-194], reverse phase evaporation [“Proceedings of the National Academy of Sciences United States of America” (Proc. Natl. Acad. Sci. USA) ”, 1978, Vol. 75, p. 4194-4198] or pH gradient method (for example, Japanese Patent No. 2572554, Japanese Patent No. 2659136) Broadcast, etc. reference), and the like. As a solution in which the liposome is dispersed in the production of the liposome, for example, water, acid, alkali, various buffers, physiological saline, amino acid infusion, or the like can be used. In the production of liposomes, for example, an antioxidant such as citric acid, ascorbic acid, cysteine or ethylenediaminetetraacetic acid (EDTA), for example, an isotonic agent such as glycerin, glucose or sodium chloride can be added. is there. Liposomes can also be produced by dissolving lipids or the like in an organic solvent such as ethanol and distilling off the solvent, and then adding physiological saline or the like and stirring to form liposomes.

The lipid nanoparticles of the present invention may be prepared, for example, by dissolving a cationic lipid in chloroform in advance, and then adding an aqueous solution of nucleic acid and methanol and mixing to form a complex of cationic lipid / nucleic acid. A layer is taken out, and lipid I, polyethylene glycolated phospholipid, neutral lipid, and water are added to form a water-in-oil (W / O) emulsion and processed by the reverse phase evaporation method (special method). (See Table 2002-508765) and nucleic acid is dissolved in an acidic aqueous electrolyte solution.For example, a mixture of lipid I and cationic lipid or lipid I, cationic lipid and neutral lipid (in ethanol) is added. The concentration of the encapsulated lipid nanoparticles such as the nucleic acid is reduced to 20 v / v%, sizing filtration, and excess ethanol is removed by dialysis. grain A method for removing nucleic acid attached to the surface of a child (Japanese Patent Publication No. 2002-501511 and “Biochimica et Biophysica Acta”, 2001, 1510, p.152- 166) and the like.

Among the lipid nanoparticles of the present invention, a lipid nanoparticle containing a complex of lipid I and a combination of cationic lipid and / or neutral lipid and a nucleic acid or the like or a complex of cationic lipid and nucleic acid is encapsulated Lipid nanoparticles composed of lipid membranes containing lipid I can be produced, for example, according to the production methods described in WO02 / 28367 and WO2006 / 080118.

Among the lipid nanoparticles of the present invention, for example, a complex of lipid I, a cationic lipid, and a nucleic acid, or a combination of a lipid I, a cationic lipid, a neutral lipid, and a nucleic acid, or a cationic Lipid nanoparticles encapsulating lipid / nucleic acid complexes with lipid membranes containing lipid I and / or cationic lipids and neutral lipids are described in WO 02/28367 and WO 2006/080118. Each composite can be manufactured according to the manufacturing method. For example, a cationic lipid and a nucleic acid are mixed in water or a 0 to 40% ethanol aqueous solution, and the complex containing the cationic lipid and the nucleic acid is dispersed without dissolving (the first lipid solution). A lipid membrane component containing a water-soluble unit having a ligand capable of binding to Siglec (lipid I), a cationic lipid, and a neutral lipid, for example, dissolved in an aqueous ethanol solution (second lipid solution) It can be obtained by mixing the first lipid solution and the second lipid solution in an equal amount or volume ratio of 1: 1 to 10: 1 and adding water appropriately. One or more kinds of cationic lipids may be used as the cationic lipid in the first lipid solution and the second lipid solution.

In the present invention, a complex of lipid I, a cationic lipid and a nucleic acid, a complex of lipid I and a cationic lipid in combination with a neutral lipid and a nucleic acid, or a complex of a cationic lipid and a nucleic acid During and after production of lipid nanoparticles encapsulated with lipid membranes containing lipid I and / or cationic lipids and neutral lipids, and electrostatic interaction between the nucleic acid in the complex and the cationic lipid in the lipid membrane The lipid nanoparticles of the present invention also include those in which the structure of the complex and the membrane is mutated due to the action or fusion of the cationic lipid in the complex and the cationic lipid in the lipid membrane.

When the nucleic acid-containing lipid nanoparticle of the present invention contains lipid II, it can be produced, for example, by a production method including the following steps (a) to (c).
(a) a first lipid containing a cationic lipid (lipid II) and a nucleic acid by mixing the cationic lipid (lipid II) and the nucleic acid in a mixed solvent of water and an organic solvent miscible with water Preparing a solution;
(b) a lipid containing lipid or a water-soluble unit (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a second lipid solution containing a cationic lipid (lipid II) A step of preparing
(c) a step of mixing the first lipid solution and the second lipid solution and further adding water or an aqueous buffer solution

Specific examples of the method for producing the nucleic acid-containing lipid nanoparticles of the present invention include the following methods.
Production of a complex of a nucleic acid (as defined above), preferably a double-stranded nucleic acid and a liposome containing a cationic lipid, according to the production method described in WO 02/28367 and WO 2006/080118 Then, the complex is dispersed in water or an aqueous solution of 0 to 40% ethanol without dissolving it (first lipid solution), and a lipid containing a water-soluble unit having a ligand capable of binding to Siglec separately (lipid I) Cationic lipid and neutral lipid are dissolved in an aqueous ethanol solution (second lipid solution), and the first lipid solution and the second lipid in an equal amount or volume ratio of 1: 1 to 10: 1 Lipid nanoparticles containing the nucleic acid and lipid I can also be obtained by mixing the solution or adding water as appropriate. The lipid nanoparticle is preferably a composition containing a lipid membrane containing lipid I encapsulating a complex of lipid I, a cationic lipid and a nucleic acid and a complex of a cationic lipid and a nucleic acid, or the nucleic acid And a lipid membrane containing lipid I encapsulating the complex (reverse micelle) composed of a lipid monolayer containing the cationic lipid. The lipid membrane in these cases may be a lipid monolayer (lipid monomolecular membrane) or a lipid bilayer membrane (lipid bimolecular membrane).

In mixing the lipid solution of the first lipid solution or the second lipid solution, the former may be added to the latter, or the latter may be added to the former. Moreover, you may add the former and the latter simultaneously to a container, stirring. Furthermore, the former and the latter can be mixed in-line. In this case, for example, a T-connector or the like can be used as the in-line mixing device.

The liposome in the complex of the nucleic acid of the present disclosure and the liposome is preferably a liposome whose size is adjusted in advance to an average particle size of 10 nm to 400 nm, more preferably 20 nm to 110 nm, and further preferably 30 nm to 80 nm. . Further, the complex and / or lipid membrane may contain a neutral lipid and / or a polymer. The first lipid solution may have an ethanol concentration of 0 to 70% as long as a complex of liposomes and the nucleic acid can be formed.

Also, instead of mixing equal amounts of the first lipid solution and the second lipid solution, the complex does not dissolve after mixing the first lipid solution and the second lipid solution, and the second lipid solution You may mix by the ratio used as the ethanol density | concentration which does not melt | dissolve the lipid. Preferably, the first lipid solution and the second lipid solution are in such a ratio that the complex does not dissolve, the lipid in the second lipid solution does not dissolve, and the ethanol concentration becomes an ethanol aqueous solution of 10 to 60%. Alternatively, the first lipid solution and the second lipid may be mixed in a ratio that results in an ethanol concentration that does not dissolve the complex after mixing the first lipid solution and the second lipid solution. The solution may be mixed and water may be added to obtain an ethanol concentration at which the lipid in the second lipid solution is not dissolved.

The complex of the nucleic acid and the liposome in the first lipid solution of the present disclosure is prepared by mixing the first lipid solution and the second lipid solution, and further adding water appropriately, and then adding the cationic lipid. The form may be changed to a complex of a membrane (reverse micelle) composed of a lipid monolayer and a nucleic acid. The composition containing lipid I, the nucleic acid and the cationic lipid obtained by the production method of the present disclosure is preferably a composition containing a complex of a cationic lipid and a nucleic acid and a lipid membrane encapsulating the complex Or a complex of a lipid monolayer containing a cationic lipid (reverse micelle) and a nucleic acid, and a lipid membrane encapsulating the complex, and the lipid membrane contains lipid I and a cationic lipid. Is a composition containing the above, and its manufacturability (yield and / or uniformity) is excellent.

The average particle size of the complex in the lipid nanoparticle of the present invention or the lipid membrane encapsulating the complex can be freely selected as desired, but the average particle size described below is preferable. As a method for adjusting the average particle size, for example, an extrusion method, a method of mechanically crushing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin, a microfluidizer, etc.) [Müller (RH Muller, S. Benita, B. Bohm, “Emulsion and Nanosuspensions for the Formulation of Poorly Soluble Drugs) ”, Scientific Publishers Stuttgart, 1998, p.267-294].

The nucleic acid in step (a) of the production method of the present invention may be the nucleic acid itself, or may be dissolved in distilled water in advance to form a nucleic acid aqueous solution. When an aqueous nucleic acid solution is used in step (a), the nucleic acid concentration in the aqueous solution is preferably 0.1 to 1000 mg / mL, more preferably 1 to 500 mg / mL, and further preferably 10 to 100 mg / mL. .
The nucleic acid concentration in the first solution may be appropriately adjusted depending on the type and molecular weight of the nucleic acid, but is usually 0.01 to 150 mg / mL, preferably 0.05 to 30 mg / mL, more preferably 0.3 to 10 mg / mL. It is.

The concentration of the cationic lipid (lipid II) in the first solution may be appropriately adjusted according to the type of nucleic acid, molecular weight, and amount used, but is usually 0.01 to 400 mmol / L, preferably 0.2 to 50 mmol. / L, more preferably 1 to 20 mmol / L.

The concentration of lipid I in the second solution may be appropriately adjusted according to the target modification rate by lipid I in the nanoparticles, but is usually 0.00001 to 6 mmol / L, preferably 0.02 to 4 mmol / L, and more Preferably it is 0.05-3 mmol / L.
The concentration of the cationic lipid (lipid II) in the second solution may be appropriately adjusted according to the type of nucleic acid, molecular weight, and amount used, but is usually 0.01 to 100 mmol / L, preferably 0.1 to 80 mmol. / L, more preferably 0.8 to 50 mmol / L.
Further, when the second solution further contains lipids other than lipid I and lipid II, the lipid may be one or more, preferably a neutral lipid, usually 0.01 to 100 mmol / L, preferably 0.1 to 80 mmol / L, more preferably 0.8 to 50 mmol / L.

The temperature at which the organic solvent solution containing the nucleic acid and lipid is prepared is not particularly limited as long as the nucleic acid and lipid are dissolved, but is preferably 10 to 60 ° C, more preferably 20 to 50 ° C, and further preferably 20 to 30 ° C. preferable. In addition, when heating at 30 degreeC or more, the solubility of a nucleic acid and a lipid increases and a lipid nanoparticle can be manufactured with a smaller solvent amount.

The water-miscible organic solvent is preferably alcohol, dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile or a mixture thereof, and more preferably an alcohol. The alcohol is preferably methanol, ethanol, propanol, butanol or a mixture thereof.
As a mixed solvent of water and an organic solvent miscible with water, C1-C6 alcohols such as methanol, ethanol, propanol, butanol and the like containing 0 to 50% (v / v) water or a mixture thereof are preferable. Ethanol or propanol containing 0 to 50% (v / v) water is more preferred, and ethanol containing 0 to 50% (v / v) water is more preferred. Here, “% (v / v)” indicates the volume percentage of the solute in the total volume of the solution, and so on.

An inorganic acid such as hydrochloric acid, acetic acid, phosphoric acid, or a salt of these acids can be added to the solvent in the organic solvent solution containing nucleic acid and lipid. In this case, the pH of the solvent is preferably 1 to 7, more preferably 1 to 5, and further preferably 2 to 4.

In the operation of adding water or an aqueous buffer solution to an organic solvent solution containing nucleic acid and lipid, the volume of water or aqueous buffer solution used is not particularly limited, but is 0.5% relative to the volume of the organic solvent solution of nucleic acid and lipid. ~ 100 times is preferable, 1.5 to 20 times is more preferable, and 2.0 to 10 times is more preferable.

In this case, the concentration of the organic solvent after adding water or an aqueous buffer solution is not particularly limited, but is preferably 50% (v / v) or less, more preferably 40% (v / v) or less with respect to the obtained solution. Preferably, it is more preferably 30% (v / v) or less, and most preferably 20% / (v / v) or less. The aqueous buffer solution is not particularly limited as long as it has a buffering action, and examples thereof include a phosphate buffer aqueous solution, a citrate buffer aqueous solution, and an acetate buffer aqueous solution.

The temperature at the time of the above addition operation is not particularly limited, but is preferably 10 to 60 ° C, more preferably 20 to 50 ° C, and further preferably 20 to 30 ° C.

In the above addition operation, it is important to quickly lower the organic solvent solution. Specifically, it is preferable to change the organic solvent concentration from 70% 70 (v / v) to 50% (v / v) within 1 minute, more preferably within 0.5 minutes, 0.1% More preferably, it is changed within minutes, and most preferably within 0.05 minutes.

When the nucleic acid-containing lipid nanoparticle of the present invention does not contain a cationic lipid (lipid II), it can be produced, for example, by a production method including the following steps (a ′) to (c ′).
(a ') Aqueous solution A is prepared by mixing water and a lipid having a ligand capable of binding to nucleic acid and siglec (Sialic acid-binding immunoglobulin-like lectin) or a lipid containing a water-soluble unit (lipid I). Process,
(b ′) preparing a lipid solution B of an organic solvent miscible with water of other lipids; and
(c ′) contacting the aqueous solution A with the lipid solution B

In the nucleic acid-containing lipid nanoparticles of the present invention, the ratio of the number of moles of total lipid to the number of moles of nucleic acid (total number of moles of lipid / number of moles of nucleic acid) is preferably 50 or more, and preferably 100 to 1,000. More preferably, it is more preferably from 120 to 800, even more preferably from 140 to 600, and most preferably from 200 to 500.

The average particle size of the nucleic acid-containing nanoparticles of the present invention can be further adjusted after the preparation of lipid nanoparticles. As a method for adjusting the average particle size, for example, an extrusion method, a method of mechanically pulverizing large multilamellar liposomes (MLV) or the like (specifically using a manton gourin, a microfluidizer, etc.) RHMuller, S. Benita, B. Bohm, “Emulsion and サ Nanosuspensions for the Emulsion and Nanosuspensions for the Formulation of Poorly Soluble Drugs) ”, Scientific Publishers Stuttgart, 1998, p.267-294].

The size of the nucleic acid-containing nanoparticles of the present invention can be measured by, for example, a dynamic light scattering method.

[Method for producing nucleic acid-containing polymer nanoparticles]
The nanoparticles of the present invention can be produced by mixing nucleic acid, polymer I and polymer II. That is, one of the present invention is a method for producing nucleic acid-containing polymer nanoparticles. The method for producing nucleic acid-containing polymer nanoparticles includes a step of mixing nucleic acid, polymer I and polymer II. In the above mixing, the nucleic acid, the polymer I, and the polymer II may be mixed in any order, and examples thereof include the following methods.
(Method 1)
Method of adding polymer I and polymer II to nucleic acid solution or adding nucleic acid to solution containing polymer I and polymer II (Method 2)
Method of simultaneously mixing nucleic acid solution, solution containing polymer I, and solution containing polymer II

In the method 1, when the polymer I and the polymer II are added to the nucleic acid solution, the polymer I and the polymer II may be a solution of a mixture of the polymer I and the polymer II.
In the method 1, when the nucleic acid is added to the solution containing the polymer I and the polymer II, the nucleic acid is preferably a nucleic acid solution.
Examples of the solvent of each solution in Method 1 and Method 2 include water or a mixed solvent of water and an organic solvent miscible with water. The concentration of nucleic acid, polymer I and polymer II in each solution in the above method 1 and method 2 depends on the type of nucleic acid, molecular weight, amount used, or target modification rate by polymer I in nanoparticles. What is necessary is just to adjust suitably.

The water-miscible organic solvent is preferably alcohol, dimethyl sulfoxide, tetrahydrofuran, acetone, acetonitrile or a mixture thereof, and more preferably an alcohol. The alcohol is preferably methanol, ethanol, propanol, butanol or a mixture thereof.
As a mixed solvent of water and an organic solvent miscible with water, C1-C6 alcohols such as methanol, ethanol, propanol, butanol and the like containing 0 to 50% (v / v) water or a mixture thereof are preferable. Ethanol or propanol containing 0 to 50% (v / v) water is more preferred, and ethanol containing 0 to 50% (v / v) water is more preferred.

In the method 1, when a solution of a mixture of polymer I and polymer II is added to the nucleic acid solution, or when a nucleic acid solution is added to a solution containing polymer I and polymer II, the solution of the mixture, Alternatively, the speed at which the nucleic acid solution is added is not particularly limited.
In addition, in the method 2, even when the nucleic acid solution, the solution containing the polymer I, and the solution containing the polymer II are added simultaneously, the speed of adding each solution is not particularly limited.

The nucleic acid-containing nanoparticles of the present invention can be introduced into cells by introducing the nucleic acid-containing nanoparticles of the present invention into mammalian cells.

The introduction of the nucleic acid-containing nanoparticles of the present invention into mammalian cells in vivo may be performed according to known transfection procedures that can be performed in vivo. For example, the nucleic acid-containing nanoparticles of the present invention are intravenously administered to mammals including human beings, for example, delivered to organs or sites where tumors or inflammation has occurred, and the cells of the present invention are delivered into cells of the delivery organs or sites. Nucleic acids in nucleic acid-containing nanoparticles can be introduced. The organ or site where cells expressing Siglec-1 are not particularly limited, but for example, liver, lung, lymph, spleen, stomach, large intestine, intestine, liver, lung, spleen, pancreas, kidney, bladder, skin , Blood vessels, eyeballs and the like. In addition, the nucleic acid-containing nanoparticles of the present invention can be intravenously administered to mammals including humans, for example, delivered to the liver, stomach, lungs, kidneys, pancreas and / or spleen, and within the cells of the delivery organ or site. The nucleic acid in the nucleic acid-containing nanoparticle of the present invention can be introduced.
Among the above organs or sites, the liver, lung, lymph, kidney, intestine and / or spleen are preferable.

If the nucleic acid in the nucleic acid-containing nanoparticle of the present invention is a nucleic acid having a target gene expression suppressing action using RNA interference (RNAi), the nucleic acid that suppresses the expression of the target gene in a mammalian cell in vivo. Etc. can be introduced, and the expression of the target gene can be suppressed. The administration subject is preferably a human.

The target gene in the nucleic acid-containing nanoparticle of the present invention is a gene expressed in cells positive for Siglec-1, and is a gene expressed in, for example, the liver, lung, lymph, kidney, intestine and / or spleen If present, the nucleic acid-containing nanoparticles of the present invention are used as a therapeutic or prophylactic agent for diseases related to the liver, lung, lymph, kidney, intestinal tract and / or spleen, preferably as a therapeutic or prophylactic agent for diseases related to the liver. Can be used. That is, the present invention also provides a method for treating diseases related to liver, lung, lymph, kidney, intestinal tract and / or spleen, etc., wherein the nucleic acid-containing nanoparticles of the present invention described above are administered to mammals. The administration subject is preferably a person, more preferably a person suffering from a disease related to the liver, lung, lymph, kidney, intestine and / or spleen.

The nucleic acid-containing nanoparticles of the present invention involve cells positive for Siglec-1, and are evaluated in vivo for therapeutic or prophylactic agents for diseases related to liver, lung, lymph, kidney, intestinal tract and / or spleen. It can also be used as a tool to verify the effectiveness of suppressing target genes in a model.

The nucleic acid-containing nanoparticles of the present invention can be used, for example, to stabilize a nucleic acid in a biological component such as a blood component (for example, blood, digestive tract, etc.), to reduce side effects, or to a tissue or organ containing a target gene expression site. It can also be used as a preparation for the purpose of increasing drug accumulation.

When the nucleic acid-containing nanoparticles of the present invention are used as a therapeutic or prophylactic agent for diseases related to the liver, lung, lymph, kidney, intestine and / or spleen, the administration route is the most effective administration in the treatment. It is desirable to use the route, and examples thereof include parenteral or oral administration such as buccal, intratracheal, rectal, subcutaneous, intramuscular or intravenous, preferably intravenous, subcutaneous or intramuscular. Intravenous administration can be mentioned, and intravenous administration is more preferable.

The dose varies depending on the disease state, age, administration route, etc. of the administration subject, but for example, it may be administered so that the daily dose converted to nucleic acid is about 0.1 μg to 1000 mg.

As a preparation suitable for intravenous administration or intramuscular administration, for example, an injection can be mentioned, and a dispersion of the composition prepared by the above-described method can be used as it is, for example, in the form of an injection or the like. The dispersion can be used after removing the solvent by, for example, filtration, centrifugation, etc. For example, the dispersion can be used after lyophilization, and / or mannitol, lactose, trehalose, maltose, glycine, etc. It is also possible to use a dispersion obtained by adding the above-mentioned excipient by lyophilization.

In the case of injections, for example, water, acid, alkali, various buffers, physiological saline or amino acid infusion, etc. are mixed with the dispersion of the composition or the composition after removing or lyophilizing the solvent. It is preferable to prepare an injection. Further, for example, an injection can be prepared by adding an antioxidant such as citric acid, ascorbic acid, cysteine or EDTA or an isotonic agent such as glycerin, glucose or sodium chloride. Further, for example, it can be cryopreserved by adding a cryopreservative such as glycerin.

The present invention also relates to a nanoparticle for use in the treatment of a disease; a pharmaceutical composition for use in the treatment of a disease; use of a nanoparticle for the treatment of a disease; a nanoparticle in the manufacture of a medicament for the treatment of a disease A nanoparticle for use in the manufacture of a medicament for the treatment of a disease; a method of treating or preventing a disease, comprising administering an effective amount of the nanoparticle to a subject in need thereof.

Next, the present invention will be specifically described with reference to Examples, Reference Examples, Comparative Examples, and Test Examples. However, the present invention is not limited to these examples, reference examples, comparative examples, and test examples.
The proton nuclear magnetic resonance spectra ( ¹ H NMR) shown in the Examples and Reference Examples were measured at 270 MHz, 300 MHz, or 400 MHz, and exchangeable protons were clearly observed depending on the compound and measurement conditions. It may not be done. In addition, although what is used normally is used as the notation of the multiplicity of signals, br represents an apparently wide signal.

Reference Examples 1 to 50 show the synthesis of cationic lipids.

Reference example 1
N, N-dimethyl-2,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propan-1-amine (compound CL-1)
CL-1 was synthesized by the method described in “Junal Op Controlled Release” (2005, Vol. 107, p.276-287).

Reference example 2
N-methyl-N, N-bis (2-((Z) -hexadec-9-enyloxy) ethyl) amine (compound CL-2)
To a toluene (2 mL) suspension of sodium hydride (oil, 60%, 222 mg, 5.55 mmol) in a toluene (2 mL) solution of N-methyldiethanolamine (Tokyo Chemical Industry Co., Ltd., 82.6 mg, 0.693 mmol) Was added with stirring, and a solution of (Z) -hexadeca-9-enyl methanesulfonate (530 mg, 1.66 mmol) in toluene (2 mL) was added dropwise. The resulting mixture was stirred for 2 hours under heating to reflux. After cooling to room temperature, the reaction was quenched with water. To the obtained mixture was added saturated brine, and the mixture was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 98/2) to give the title compound (199 mg, 0.353 mmol, yield 51%).
ESI-MS m / z: 565 (M + H) ⁺ ;

Reference Example 3
trans-1-Methyl-3,4-bis ((((Z) -octadec-9-en-1-yl) oxy) methyl) pyrrolidine (Compound CL-3)
CL-3 was synthesized by the method described in International Publication No. 2011/136368.

Reference example 4
trans-1-methylpyrrolidine-3,4-diyl) bis (methylene) (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12-dienoate) (compound CL-4)
CL-4 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 5
(6Z, 9Z, 28Z, 31Z) -Heptatriaconta-6,9,28,31-tetraen-19-yl 4- (dimethylamino) butanoate (Compound CL-5)
CL-5 was synthesized by a method according to the method described in International Publication No. 2010/054401.
ESI-MS m / z: 642

Reference Example 6
3- (Di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) propan-1-ol (Compound CL-6)
CL-6 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 7
(9Z, 12Z) -N- (2-(((Z) -Octadeca-9-en-1-yl) oxy) ethyl) octadeca-9,12-dien-1-amine (Compound CL-7)
CL-7 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 8
1-Methyl-3,3-di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) azetidine (Compound CL-8)
CL-8 was synthesized by the method described in International Publication No. 2016/002753.

Reference Example 9
N, 2-Dimethyl-1,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propan-2-amine (Compound CL-9)
Process 1
2-Amino-2-methylpropane-1,3-diol (Tokyo Chemical Industry Co., Ltd., 0.300 g, 4.76 mmol) was dissolved in tetrahydrofuran (3 mL) and sodium hydride (oil 60%, 0.171 g, 7.13 mmol) ) Was added at room temperature. After the foaming stopped, (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 2.458 g, 7.13 mmol) was added, and the mixture was stirred for 2 hours under heating reflux. A saturated aqueous ammonium chloride solution was added to the reaction mixture, and the mixture was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 90/10) to give 2-methyl-1,3-bis ((9Z, 12Z) -Octadeca-9,12-dien-1-yloxy) propan-2-amine (0.280 g, yield 16%) was obtained.
ESI-MS m / z: 602 (M + H) ⁺
Process 2
2-Methyl-1,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-amine (0.500 g, 0.831 mmol) obtained in Step 1 was added to dichloromethane (3 mL ), Triethylamine (Wako Pure Chemical Industries, 2.55 mL, 18.3 mmol) and 2-nitrobenzene-1-sulfonyl chloride (Sigma-Aldrich, 0.368 g, 1.66 mmol) were added under ice-cooling, and the mixture was returned to room temperature. After that, the mixture was stirred for 1 hour. Water was added to the reaction mixture, and the mixture was extracted with hexane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 99/1 to 85/15) to give N- (2-methyl-1,3-bis (( 9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-yl) -2-nitrobenzenesulfonamide (0.400 g, 61% yield) was obtained.
ESI-MS m / z: 787 (M + H) ⁺
Process 3
N- (2-Methyl-1,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-yl) -2-nitrobenzenesulfonamide obtained in Step 2 ( 0.200 g, 0.274 mmol) was dissolved in tetrahydrofuran (3 mL), and cesium carbonate (Wako Pure Chemical Industries, 0.248 g, 0.726 mmol) and methyl iodide (Tokyo Chemical Industry, 0.048 mL, 0.762 mmol) were added. In addition, the mixture was stirred at 70 ° C. for 1 hour using a microwave reactor. Water was added to the reaction mixture, and the mixture was extracted with hexane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, and filtered. The filtrate was concentrated under reduced pressure, and N-methyl-N- (2-methyl-1,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propane as a crude product -2-yl) -2-nitrobenzenesulfonamide (0.200 g, 91% yield) was obtained.
ESI-MS m / z: 801 (M + H) ⁺
Process 4
N-methyl-N- (2-methyl-1,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-yl) -2-obtained in step 3 Nitrobenzenesulfonamide (0.200 g, 0.250 mmol) is dissolved in acetonitrile (2 mL), 1-dodecanethiol (Tokyo Chemical Industry Co., Ltd., 0.149 mL, 0.624 mmol), 1,8-diazabicyclo [5.4.0] -7 -Undecene (manufactured by Nacalai Tesque, 0.0940 mL, 0.624 mmol) was added and stirred at 80 ° C. for 1 hour. Water was added to the reaction mixture, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by NH silica gel column chromatography (hexane / ethyl acetate = 90/10 to 75/25) to give compound CL-9 (0.070 g, yield 46%).
ESI-MS m / z: 616 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.8 Hz, 6H), 1.02 (s, 3H), 1.25-1.40 (m, 32H), 1.50-1.59 (m, 4H), 2.05 (q, J = 6.8 Hz, 8H), 2.32 (s, 3H), 2.77 (t, J = 6.3 Hz, 4H), 3.26 (s, 4H), 3.40 (t, J = 6.6 Hz, 4H), 5.28-5.43 (m, 8H).

Reference Example 10
Methyldi ((9Z, 12Z) -octadeca-9,12-dienyl) amine (Compound CL-10)
(9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (1.03 g, 3.00 mmol) was added to methylamine (Sigma-Aldrich, approximately 2 mol / L tetrahydrofuran solution, 10.5 mL, 21.0 mmol), The mixture was heated and stirred at 150 ° C. for 90 minutes using a microwave reactor. The reaction solution is diluted with ethyl acetate, washed with 2 mol / L aqueous sodium hydroxide solution and then with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give methyl ((9Z, 12Z) -octadeca A crude product of -9,12-dienyl) amine was obtained.
To the obtained crude product, add (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (0.93 g, 2.70 mmol) and 50% aqueous sodium hydroxide solution (0.960 g, 12.0 mmol). The mixture was heated and stirred at 135 ° C. for 60 minutes. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water and then with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 97/3) to obtain Compound CL-10 (1.07 g, 2.03 mmol, overall yield 67%).
ESI-MS m / z: 529 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.7 Hz, 6H), 1.29 (br s, 32H), 1.40-1.51 (m , 4H), 1.97-2.06 (m, 8H), 2.20 (s, 3H), 2.30 (t, J = 7.6 Hz, 4H), 2.77 (t, J = 5.8 Hz, 4H), 5.28-5.43 (m, 8H).

Reference Example 11
N-methyl-2-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) -N- (2-((((9Z, 12Z) -octadeca-9,12-diene- 1-yl) oxy) ethyl) ethan-1-amine (compound CL-11)
CL-11 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 12
(3R, 4R) -3,4-bis (((Z) -hexadec-9-en-1-yl) oxy) -1-methylpyrrolidine (Compound CL-12)
CL-12 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 13
2- (Dimethylamino) -N-((6Z, 9Z, 28Z, 31Z) -Heptatriconta-6,9,28,31-tetraen-19-yl) acetamide (Compound CL-13)
CL-13 was synthesized by the method described in International Publication No. 2013/059496.

Reference Example 14
3- (Dimethylamino) propane-1,2-diyl (9Z, 9'Z, 12Z, 12'Z) -bis (octadeca-9,12-dienoate) (Compound CL-14)
CL-14 was synthesized by the method described in “BioChemistry”, 1994, Vol. 33, p.12573-12580.

Reference Example 15
(9Z, 12Z) -di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amine (Compound CL-15)
CL-15 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 16
Bis (2-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) ethyl) amine (Compound CL-16)
CL-16 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 17
(9Z, 12Z) -N-Methyl-N- (2-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) ethyl) octadeca-9,12-dien-1-amine (Compound CL-17)
2- (Methylamino) ethanol (Tokyo Chemical Industry Co., Ltd., 0.125 g, 1.66 mmol) was dissolved in toluene (2.5 mL) and sodium hydride (oil, 60%, 0.333 g, 8.32 mmol), (9Z, 12Z ) -Octadeca-9,12-dien-1-yl methanesulfonate (manufactured by Nu-Chek Prep, Inc., 1.32 g, 3.83 mmol) in toluene (2.5 mL) was added in order, and the mixture was stirred for 2 hours under reflux with heating. The reaction mixture was cooled to room temperature, ethanol and water were added, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 97/3) to give compound CL-17 (0.211 g, yield 22%). It was.
ESI-MS m / z: 572 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.24-1.38 (m, 32H), 1.43-1.49 ( m, 2H), 1.53-1.59 (m, 2H), 2.05 (q, J = 7.2 Hz, 8H), 2.27 (s, 3H), 2.37 (t, J = 7.7 Hz, 2H), 2.57 (t, J = 6.2 Hz, 2H), 2.78 (t, J = 6.8 Hz, 4H), 3.42 (t, J = 6.8 Hz, 2H), 3.52 (t, J = 6.2 Hz, 2H), 5.30-5.41 (m, 8H ).

Reference Example 18
(9Z, 12Z) -N- (3-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propyl) octadeca-9,12-dien-1-amine (Compound CL- 18)
CL-18 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 572

Reference Example 19
(1-Methylpiperidin-3-yl) methyl di ((11Z, 14Z) -icosa-11,14-dien-1-yl) carbamate (Compound CL-19)
CL-19 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 20
(13Z, 16Z) -N, N-Dimethyl-4-((9Z, 12Z) -octadeca-9,12-dien-1-yl) docosa-3,13,16-trien-1-amine (Compound CL- 20)
Process 1
To a solution of heptatriaconta-6,9,28,31-tetraen-19-one (0.353 g, 0.186 mmol) synthesized in the method described in WO2009 / 132131 in tetrahydrofuran (0.882 mL) under an argon atmosphere, Anhydrous cerium (III) chloride (manufactured by Tokyo Chemical Industry Co., Ltd., 0.174 g, 0.706 mmol) was added. Thereafter, cyclopropylmagnesium bromide (manufactured by Sigma-Aldrich, 0.5 mmol / L. 1.06 mL, 0.529 mmol) was added under ice cooling, and the mixture was stirred for 5 minutes and then stirred at room temperature for 1 hour. A saturated aqueous ammonium chloride solution was added to the reaction mixture, and the aqueous layer was extracted with ethyl acetate. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (hexane / ethyl acetate = 97 / 3-94 / 6) to give (6Z, 9Z, 28Z, 31Z) -19-cyclopropyl. Heptatriaconta-6,9,28,31-tetraen-19-ol (0.141 g, yield 70%) was obtained.
ESI-MS m / z: 569
Process 2
Dichloromethane (2 mL) of (6Z, 9Z, 28Z, 31Z) -19-cyclopropylheptatrita-6,9,28,31-tetraen-19-ol (0.141 g, 0.248 mmol) obtained in step 1 Lithium bromide (Sigma-Aldrich, 0.108 g, 1.24 mmol) and chlorotrimethylsilane (Tokyo Kasei Kogyo, 0.135 g, 1.24 mmol) were added to the solution at room temperature, and the mixture was stirred for 1 hour. Thereafter, lithium bromide (Sigma-Aldrich, 0.108 g, 1.24 mmol) and chlorotrimethylsilane (Tokyo Kasei Kogyo, 0.135 g, 1.24 mmol) were additionally added, and the mixture was stirred for 1 hour.
A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the aqueous layer was extracted with hexane. The organic layer was dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by silica gel column chromatography (hexane / ethyl acetate = 100/0 to 90/10 to give (6Z, 9Z, 28Z, 31Z) -19- (3- Bromopropylidene) heptatriaconta-6,9,28,31-tetraene (0.074 g, 47% yield) was obtained.
ESI-MS m / z: 632
Process 3
The (6Z, 9Z, 28Z, 31Z) -19- (3-bromopropylidene) heptatriconta-6,9,28,31-tetraene (0.074 g, 0.117 mmol) obtained in Step 2 was added to dimethylamine (Sigma -Aldrich, 2.0 mmol / L tetrahydrofuran solution, 1.5 mL, 3.0 mmol) was added, and the mixture was heated and stirred at 130 ° C. for 90 minutes under microwave irradiation. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the aqueous layer was extracted with hexane. The organic layer was washed with saturated brine, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by NH silica gel column chromatography (hexane / ethyl acetate = 97 / 3-88 / 12) to obtain CL-20 (0.062 g, yield 69%). It was.
ESI-MS m / z: 596

Reference Example 21
(S) -2-Amino-3-hydroxy-N, N-bis (2-(((Z) -octadeca-9-en-1-yl) oxy) ethyl) propanamide (Compound CL-21)
CL-21 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 22
(3R, 4R) -3,4-bis (((11Z, 14Z) -icosa-11,14-dien-1-yl) oxy) pyrrolidine (Compound CL-22)
CL-22 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 23
trans-3,4-bis ((((11Z, 14Z) -icosa-11,14-dien-1-yl) oxy) methyl) -1-methylpyrrolidine (Compound CL-23)
CL-23 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 24
1-((S) -2,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propyl) pyrrolidine (Compound CL-24)
CL-24 was synthesized by the method described in International Publication No. 2009/129395.

Reference Example 25
2- (2,2-di ((9Z, 12Z) -octaeca-9,12-dien-1-yl) -1,3-dioxolan-4-yl) -N, N-dimethylethan-1-amine ( Compound CL-25)
CL-25 was synthesized by the method described in International Publication No. 2010/042877.

Reference Example 26
3- (Dimethylamino) propyl di ((9Z, 12Z) -oxadec-9,12-dien-1-yl) carbamate (Compound CL-26)
CL-26 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 27
4- (Dimethylamino) butyl di ((9Z, 12Z) -oxadec-9,12-dien-1-yl) carbamate (Compound CL-27)
CL-27 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 28
2- (Di ((9Z, 12Z) -octadeca-9,12-dien-1-yl) amino) ethane-1-ol (Compound CL-28)
CL-28 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 29
2- (Dimethylamino) -3-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) -2-(((((9Z, 12Z) -octadeca-9,12-diene -1-yl) oxy) methyl) propan-1-ol (compound CL-29)
CL-29 was synthesized by the method described in International Publication No. 2011/149733.

Reference Example 30
(6Z, 9Z, 28Z, 31Z) -N, N-Dimethylheptatriaconta-6,9,28,31-tetraene-19-amine (Compound CL-30)
CL-30 was synthesized by the method described in International Publication No. 2010/054405.

Reference Example 31
N, N, 2-trimethyl-1,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propan-2-amine (Compound CL-31)
2-Methyl-1,3-bis ((9Z, 12Z) -octadeca-9,12-dien-1-yloxy) propan-2-amine (0.240 g, 0.399 mmol) obtained in Step 1 of Reference Example 9 Is dissolved in a mixed solvent of 1,2-dichloroethane (1 mL) and methanol (1 mL), and formaldehyde (Wako Pure Chemical Industries, 37% aqueous solution, 0.144 mL, 1.99 mmol), sodium triacetoxyborohydride (Tokyo) Kasei Kogyo Co., Ltd., 0.211 g, 0.997 mmol) was added, and the mixture was stirred overnight at room temperature. Water was added to the reaction mixture, and the aqueous layer was extracted with ethyl acetate. The organic layer was washed with saturated sodium bicarbonate, dried over anhydrous magnesium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the obtained residue was purified by NH silica gel column chromatography (hexane / ethyl acetate = 99 / 1-80 / 20) to give compound 2 (0.191 g, yield 76%). It was.
ESI-MS m / z: 630 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 7.0 Hz, 6H), 0.95 (s, 3H), 1.26-1.39 (m, 32H), 1.53-1.58 (m, 4H), 2.05 (q, J = 6.9 Hz, 8H), 2.31 (s, 6H), 2.77 (t, J = 6.3 Hz, 4H), 3.33-3.42 (m, 8H ), 5.27-5.43 (m, 8H).

Reference Example 32
N-methyl-2-(((Z) -octadeca-6-en-1-yl) oxy) -N- (2-(((Z) -octadeca-6-en-1-yl) oxy) ethyl) Ethan-1-amine (Compound CL-32)
CL-32 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 33
(3R, 4R) -3- (Dimethylamino) propyl 3,4-bis ((9Z, 12Z) -octadeca-9,12-dienyloxy) pyrrolidine-1-carboxylate (Compound CL-33)
Process 1
To a suspension of sodium hydride (oil, 60%, 5.80 g, 145 mmol) in toluene (100 mL), (3R, 4R) -1-benzylpyrrolidine-3,4-diol (Diverchim SA, 3.50 g , 18.1 mmol) in toluene (70 mL) was added with stirring, and (9Z, 12Z) -octadeca-9,12-dienyl methanesulfonate (15.6 g, 45.3 mmol) in toluene (30 mL) was added. It was dripped. The resulting mixture was stirred overnight with heating under reflux. After cooling to room temperature, the reaction was quenched with a saturated aqueous ammonium chloride group solution. Saturated brine was added to the resulting mixture, and the mixture was extracted twice with ethyl acetate. The organic layers were combined, dried over anhydrous magnesium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (methanol / chloroform = 0/100 to 2/98) to give (3R, 4R) -1-benzyl-3,4-bis ((9Z, 12Z) -octadeca-9, 12-Dienyloxy) pyrrolidine (6.96 g, 10.1 mmol, yield 56%) was obtained.
ESI-MS m / z: 691 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.26-1.38 (m, 30H), 1.52-1.62 ( m, 6H), 2.05 (q, J = 6.3 Hz, 8H), 2.50 (dd, J = 9.9, 4.3 Hz, 2H), 2.77 (t, J = 5.8 Hz, 4H), 2.85 (dd, J = 9.6 , 5.9 Hz, 2H), 3.37-3.45 (m, 4H), 3.52-3.66 (m, 2H), 3.83 (t, J = 4.6 Hz, 2H), 5.28-5.43 (m, 8H), 7.23-7.33 ( m, 5H).
Process 2
The (3R, 4R) -1-benzyl-3,4-bis ((9Z, 12Z) -octadeca-9,12-dienyloxy) pyrrolidine (6.96 g, 10.1 mmol)) obtained in Step 1 was replaced with 1,2- It was dissolved in dichloroethane (100 mL), 1-chloroethyl chloroformate (3.30 ml, 30.3 mmol) was added, and the mixture was stirred at 130 ° C. for 1 hr. Methanol (100 mL) was added to the reaction solution, and the mixture was further stirred at 130 ° C. for 1 hour. After cooling to room temperature, the mixture was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 92/8). The obtained organic layer was washed with a saturated aqueous sodium hydrogen carbonate solution and then with a saturated aqueous sodium chloride solution, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to give (3R, 4R) -3,4-bis ((9Z , 12Z) -octadeca-9,12-dienyloxy) pyrrolidine (5.56 g, 9.27 mmol, 92% yield).
ESI-MS m / z: 601 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.29-1.41 (m, 30H), 1.49-1.60 ( m, 4H), 1.67 (br s, 3H), 2.05 (q, J = 6.5 Hz, 8H), 2.75-2.85 (m, 6H), 3.09 (dd, J = 12.4, 5.1 Hz, 2H), 3.37- 3.49 (m, 4H), 3.76 (dd, J = 5.0, 3.3 Hz, 2H), 5.28-5.43 (m, 8H).
Process 3
In the same manner as in Step 2 of Reference Example 4, instead of di ((Z) -octadeca-9-enyl) amine, (3R, 4R) -3,4-bis ((9Z, 12Z) obtained in Step 2 was used. ) -Octadeca-9,12-dienyloxy) pyrrolidine (0.111 g, 0.185 mmol) was used to obtain the title compound (0.101 g, 0.139 mmol, 75%).
ESI-MS m / z: 730 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.89 (t, J = 6.9 Hz, 6H), 1.24-1.40 (m, 32H), 1.50-1.57 ( m, 4H), 1.77-1.83 (m, 2H), 2.02-2.08 (m, 8H), 2.23 (s, 6H), 2.34 (t, J = 7.4 Hz, 2H), 2.77 (t, J = 6.8 Hz , 4H), 3.38-3.56 (m, 8H), 3.83-3.86 (m, 2H), 4.11 (t, J = 6.5 Hz, 2H), 5.30-5.42 (m, 8H).

Reference Example 34
(9Z, 12Z) -N- (2-(((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) ethyl) octadeca-9,12-dien-1-amine (compound CL- 34)
CL-34 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 35
(9Z, 12Z) -N- (2-((((Z) -Hexadeca-9-en-1-yl) oxy) ethyl) octadeca-9,12-dien-1-amine (Compound CL-35)
CL-35 was synthesized by the method described in International Publication No. 2014/007398.

Reference Example 36
N, N-dimethyl-1,3-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) propan-2-amine (Compound CL-36)
CL-36 was synthesized by the method described in International Publication No. 2009/129385.

Reference Example 37
3- (Dimethylmino) propyl di ((Z) -octadeca-9-enyl) carbamate (Compound CL-37)
Process 1
Add (Z) -octadeca-9-enyl methanesulfonate (1.04 g, 3.00 mmol) to ammonia (Tokyo Chemical Industry Co., Ltd., approx. 2 mol / L methanol solution, 12.0 mL, 24.0 mmol) And stirred at 130 ° C. for 3 hours. A saturated aqueous sodium hydrogen carbonate solution was added to the reaction mixture, and the mixture was extracted 5 times with chloroform. The organic layers were combined, washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product of (Z) -octadeca-9-enylamine.
(Z) -octadeca-9-enyl methanesulfonate (0.832 g, 2.40 mmol) and 50% aqueous sodium hydroxide solution (0.960 g, 12.0 mmol) were added to the resulting crude product, and the mixture was heated at 110 ° C. on an oil bath at 60 ° C. Stir for minutes. After cooling to room temperature, the reaction mixture was diluted with ethyl acetate, washed with water and then with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 95/5) to give di ((Z) -octadeca-9-enyl) amine (0.562 g, 1.085 mmol, yield) 36%) was obtained.
ESI-MS m / z: 519 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.7 Hz, 6H), 1.29 (br s, 45H), 1.41-1.52 (m , 4H), 1.97-2.05 (m, 8H), 2.58 (t, J = 7.2 Hz, 4H), 5.28-5.40 (m, 4H).
Process 2
Di ((Z) -octadeca-9-enyl) amine (0.156 g, 0.301 mmol) obtained in Step 1 was dissolved in chloroform (3 mL), and “Journal of American Chemical Society (J. Am. Chem. Soc.) ”, 1981, Vol. 103, p. 4194-4199, 3- (dimethylamino) propyl 4-nitrophenyl carbonate hydrochloride (0.138 g, 0.452 mmol) And triethylamine (0.168 mL, 1.21 mmol) were added, and the mixture was stirred at 110 ° C. for 60 minutes using a microwave reactor. 3- (Dimethylamino) propyl 4-nitrophenyl carbonate hydrochloride (22.9 mg, 0.0753 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. 3- (Dimethylamino) propyl 4-nitrophenyl carbonate hydrochloride (22.9 mg, 0.0753 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. 3- (Dimethylamino) propyl 4-nitrophenyl carbonate hydrochloride (22.9 mg, 0.0753 mmol) was added to the reaction solution, and the mixture was stirred at 110 ° C. for 20 minutes using a microwave reactor. The reaction mixture was diluted with chloroform, washed with saturated aqueous sodium hydrogen carbonate solution, then washed with saturated brine, dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The obtained residue was dissolved in a small amount of n-hexane / ethyl acetate (1/4), adsorbed on a pad of amino-modified silica gel, eluted with n-hexane / ethyl acetate (1/4), and concentrated under reduced pressure. The obtained residue was purified by silica gel column chromatography (chloroform / methanol = 100/0 to 95/5) to give the title compound (0.173 g, 0.267 mmol, yield 89%).
ESI-MS m / z: 648 (M + H) ⁺ ; ¹ H-NMR (CDCl ₃ ) δ: 0.88 (t, J = 6.6 Hz, 6H), 1.28 (br s, 44H), 1.45-1.55 (m , 4H), 1.75-1.85 (m, 2H), 1.97-2.04 (m, 8H), 2.23 (s, 6H), 2.34 (t, J = 7.6 Hz, 2H), 3.13-3.24 (m, 4H), 4.10 (t, J = 6.4 Hz, 2H), 5.28-5.40 (m, 4H).

Reference Example 38
(11Z, 14Z) -N- (2-(((Z) -Octadeca-9-en-1-yl) oxy) ethyl) icosa-11,14-dien-1-amine (Compound CL-38)
CL-38 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 588

Reference Example 39
(9Z, 12Z) -N- (2-(((Z) -Icosa-11-en-1-yl) oxy) ethyl) octadeca-9,12-dien-1-amine (Compound CL-39)
CL-39 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 588

Reference Example 40
(11Z, 14Z) -N- (2-(((Z) -Icosa-11-en-1-yl) oxy) ethyl icosa-11,14-dien-1-amine (Compound CL-40)
CL-40 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 616

Reference Example 41
(Z) -N- (2-(((Z) -octadeca-9-en-1-yl) oxy) ethyl) octadeca-9-en-1-amine (Compound CL-41)
CL-41 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 562

Reference Example 42
Bis (2-(((11Z, 14Z) -icosa-11,14-dien-1-yl) oxy) ethyl) amine (Compound CL-42)
CL-42 was synthesized by a method according to the method described in International Publication No. 2011/136368.
ESI-MS m / z: 658

Reference Example 43
(Z) -N- (2-(((Z) -octadeca-9-en-1-yl) oxy) ethyl) hexadeca-9-en-1-amine (Compound CL-43)
CL-43 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 534

Reference Example 44
(Z) -N- (2- (Octadec-9-en-1-yloxy) ethyl) octadecan-1-amine (Compound CL-44)
CL-44 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 564

Reference Example 45
(Z) -N- (2- (Octadec-9-en-1-yloxy) ethyl) tetradecan-1-amine (Compound CL-45)
CL-45 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 508

Reference Example 46
3-((3R, 4R) -3,4-bis (((9Z, 12Z) -octadeca-9,12-dien-1-yl) oxy) pyrrolidin-1-yl) propane-1,2-diol ( Compound CL-46)
CL-46 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 47
Bis (2-((((Z) -octadeca-9-en-1-yl) oxy) ethyl) amine (Compound CL-47)
CL-47 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 48
3- (Bis (2-(((Z) -octadeca-9-en-1-yl) oxy) ethyl) amino) propane-1,2-diol (Compound CL-48)
CL-48 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 49
3- (Bis (2-(((Z) -octadeca-9-en-1-yl) oxy) ethyl) amino) propanamide (Compound CL-49)
CL-49 was synthesized by the method described in International Publication No. 2011/136368.

Reference Example 50
(9Z, 12Z) -N- (2- (2-(((Z) -Octadeca-9-en-1-yl) oxy) ethoxy) ethyl) octadeca-9,12-dien-1-amine (Compound CL -50)
CL-50 was synthesized by a method according to the method described in International Publication No. 2014/007398.
ESI-MS m / z: 604

Reference Example 51 (Synthesis of lipid having ligand capable of binding to Siglec)
A lipid having a ligand capable of binding to Siglec represented by the following formula was synthesized according to the method described in J. Am. Chem. Soc., 2012, 134, 15696. Hereinafter, the lipid synthesized in Reference Example 51 is also referred to as Siglec-1L-PEG-DSPE.
Siglec-1L-PEG-DSPE obtained by synthesis agreed with the spectrum data described in J. Am. Chem. Soc., 2012, 134, 15696.

Hereinafter, Reference Examples 52 to 63 show the synthesis of a cationic polymer SPP-1 to 12 having a ligand 可能 な (Siglec-1L) capable of binding to Siglec. Each PEG part described below is not a single molecular weight but has a distribution in the molecular weight, but in the MS spectrum, it was calculated by using the average molecular weight value (5000 kg or 2000) of each PEG part.

Reference Example 52 Synthesis Step 1 of Siglec-1L-PEG5000-K ₁₅ GC (Compound SPP-1)
Siglec-1L (4.9 mg, 5.3 μmol, retention time; 0.94 min (HPLC condition A), 5.16 min (HPLC condition B) synthesized by the method described in J. Am. Chem. Soc., 2012, 134, 15696 ) In N, N-dimethylformamide (200 μL) solution, SUNBRIGHT MA-050TS (manufactured by NOF Corporation, PEG unit average molecular weight 5000, 19 mg, 3.5 μmol, retention time; 1.42 minutes (HPLC condition A), 4.46 Min (HPLC condition B)) in N, N-dimethylformamide (200 μL) was added and allowed to stand for 1 hour. After confirming the formation of compound Int-1 (retention time; 1.41 minutes (HPLC condition A), 5.16 minutes (HPLC condition B)) and disappearance of SUNBRIGHT MA-050TS by HPLC, the reaction solution was used as it was in the next step. .

HPLC condition A
Column; Waters ACQUITY UPLC BEH C18, 1.7 μm, 2.1 x 50 mm
Column temperature; 40 ° C
Solvent A; 0.1% formic acid aqueous solution, solvent B; acetonitrile, flow rate; 0.8 mL / min gradient; solvent A / solvent B = 90/10 → 10/90 (1.7 min)

HPLC condition B
Column; Agilent Porshell 120 EC-C8, 2.7 μm, 4.6 x 50 mm
Column temperature; 40 ° C
Solvent A; 0.1% aqueous formic acid, Solvent B; Isopropanol, flow rate; 0.8 mL / min gradient; Solvent A / Solvent B = 95/5 → 60/40 (8 min)

Process 2-1
After adding 10 M acetic acid-N, N-dimethylformamide solution (2.0 μL) to the compound Int-1 solution obtained in step 1, K ₁₅ GC peptide (Toray, 11 mg, 5.3 μmol, N, N-dimethylformamide (100 μL) solution having a retention time of 0.17 minutes (HPLC condition A)) was added and allowed to stand for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was diluted with water, and the solvent was replaced with water by Vivaspin6 5K (manufactured by Sartorius). Purified with Sep-Pak (registered trademark) 6cc C18 Cartridge (Waters, eluted with 30% acetonitrile in water) and lyophilized to compound SPP-1 (17 mg, 2.0 μmol, 2 steps 57%, retention time; 1.12 Minute (HPLC condition A)) was obtained as a white solid.

Reference Example 53 Synthesis step 2-2 of Siglec-1L-PEG5000-K ₃₀ GC (compound SPP-2) 2-2
A compound Int-1 solution was obtained in the same manner as in Step 1 of Reference Example 52 using Siglec-1L (5.2 mg, 5.6 μmol) and SUNBRIGHT MA-050TS (20 mg, 3.8 μmol). Using the reaction solution and K ₃₀ GC peptide (Toray, 15 mg, 3.8 μmol, retention time; 0.15 minutes (HPLC condition A)), compound SPP-2 in the same manner as in step 2-1 of Reference Example 52 (25 mg, 2.5 μmol, 66% for 2 steps, retention time; 1.01 min (HPLC condition A)) was obtained as a white solid.

Reference Example 54 Synthesis process 2-3 of Siglec-1L-PEG5000-R ₃₀ GC (compound SPP-3) 2-3
A compound Int-1 solution was obtained in the same manner as in Step 1 of Reference Example 52 using Siglec-1L (5.2 mg, 5.6 μmol) and SUNBRIGHT MA-050TS (20 mg, 3.8 μmol). Using the reaction solution and R ₃₀ GC peptide (Toray Industries, Inc., 18 mg, 3.8 μmol, retention time; 0.15 min (HPLC condition A)), compound SPP-3 in the same manner as in step 2-1 of Reference Example 52 (28 mg, 2.5 μmol, 66% for 2 steps, retention time; 1.01 min (HPLC condition A)) was obtained as a white solid.

Reference Example 55 Synthesis Step 1-1 of PEG5000-K ₁₅ GC (Compound SPP-4) 1-1
10 M for N, N-dimethylformamide (100 μL) solution of SUNBRIGHT ME-050MA (manufactured by NOF Corporation, PEG unit average molecular weight 5000, 7.0 mg, 1.3 μmol, retention time; 4.61 min (HPLC condition B)) After adding an acetic acid-N, N-dimethylformamide solution (2.0 μL), a solution of K ₁₅ GC peptide (4.2 mg, 2.0 μmol) in N, N-dimethylformamide (100 μL) was added and allowed to stand for 2 hours. After confirming the completion of the reaction by HPLC, the reaction solution was diluted with water, and the solvent was replaced with water by Amicon Ultra-4 3K (manufactured by Waters). Purified with Sep-Pak (registered trademark) 6cc C18 Cartridge (Waters, eluted with 30% acetonitrile aqueous solution), and freeze-dried compound SPP-4 (7.2 mg, 1.0 μmol, 77%, retention time; 3.30 minutes ( HPLC condition B)) was obtained as a white solid.

Reference Example 56 Synthesis step 1-2 of PEG5000-K ₃₀ GC (compound SPP-5)
Using SUNBRIGHT ME-050MA (6.0 mg, 1.1 μmol) and K ₃₀ GC peptide (6.9 mg, 1.7 μmol), Compound SPP-5 was obtained as a white solid in the same manner as in Step 1-1 of Reference Example 55 ( 6.0 mg, 0.65 μmol, 59%, retention time; 3.04 minutes (HPLC condition B)).

Reference Example 57 Synthesis step 1-3 of PEG5000-R ₃₀ GC (compound SPP-6)
Using SUNBRIGHT ME-050MA (5.0 mg, 1.0 μmol) and R ₃₀ GC peptide (6.0 mg, 1.2 μmol), Compound SPP-6 was obtained as a white solid in the same manner as in Step 1-1 of Reference Example 55 ( (5.8 mg, 0.57 μmol, 57%, Retention time; 3.09 min (HPLC condition B))

Reference Example 58 (Siglec-1L) ₂ -PEG5000-K ₁₅ GC (SPP-7) Synthesis Step 1
N- (tert-butoxycarbonyl) -L-glutamic acid (250 mg, 1.0 mmol) was suspended in dichloromethane (5.1 mL), and N, N-diisopropylethylamine (1.8 mL, 10 mmol) and pentafluorophenyl trifluoroacetate were suspended. Ester (700 μL, 4.0 mmol) was added and stirred at room temperature for 30 minutes. The reaction was quenched with saturated aqueous sodium bicarbonate and extracted with ethyl acetate. The obtained organic layer was washed 3 times with saturated aqueous sodium hydrogen carbonate, once with water and once with saturated brine, then dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give compound Int-2 (600 mg). Obtained as a white solid.
In mass analysis of Int-2, a peak of ESI-MS m / z: 480.2 (M-Boc + H) ⁺ was observed.
Process 2
Siglec-1L (71 mg, 76 μmol) was dissolved in N, N-dimethylformamide (1.4 mL), and N, N-diisopropylethylamine (30 μL, 170 μmol) and the compound Int-2 ( 20 mg, 35 μmol) was added, and the mixture was stirred at room temperature for 2 hours. After adding Siglec-1L (4.8 mg, 8.3 μmol), the mixture was further stirred at room temperature for 30 minutes. The reaction solution was diluted with water (16 mL) and then purified by Sep-Pak (registered trademark) Vac 35cc C18 Cartridge (manufactured by Waters, eluted with 40% acetonitrile aqueous solution) to give compound Int-3 (76 mg) as white Obtained as a solid.
In int-3 mass spectrometry, ESI-MS m / z: 2071.8 (M-2Na + H) - peak of was observed.
Process 3
Compound Int-3 (73 mg, 35 μmol) obtained in step 2 was dissolved in water (880 μL), 6 M aqueous hydrochloric acid solution (880 uL) was added, and the mixture was stirred at room temperature for 30 minutes. After neutralization with 2 M aqueous sodium hydroxide solution (2.6 mL), purification was performed with Sep-Pak (registered trademark) Vac 35cc C18 Cartridge (Waters, eluted with 40% acetonitrile aqueous solution) to obtain compound Int-4 (60 mg , 30 μmol, 86%) was obtained as a white solid.
In mass analysis of Int-4, a peak of ESI-MS m / z: 1971.5 (M-2Na + H) ⁻ was observed.
Process 4
Using compound Int-4 obtained in Step 3 (3.3 mg, 1.7 μmol, retention time; 1.12 minutes (HPLC condition A), 5.95 minutes (HPLC condition B)) and SUNBRIGHT MA-050TS (6.0 mg, 1.1 μmol) In the same manner as in Step 1 of Reference Example 52, a compound Int-5 solution (retention time; 5.54 minutes (HPLC condition B)) was obtained.
Process 5-1
Using the compound Int-5 solution obtained in step 4 and K ₁₅ GC peptide (3.5 mg, 1.7 μmol), compound SPP-7 (1.2 mg, 0.13 μmol, 2 steps) in the same manner as in step 2 of Reference Example 52 12%, retention time; 1.12 min (HPLC condition A)) was obtained as a white solid.

Reference Example 59 (Siglec-1L) ₂ -PEG5000-K ₃₀ GC (SPP-8) Synthesis Process 5-2
Compound Int-5 was obtained in the same manner as in Step 4 of Reference Example 58 using Compound Int-4 (2.8 mg, 1.4 μmol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT MA-050TS (5.0 mg, 0.93 μmol). A solution was obtained. Using the reaction solution and K ₃₀ GC peptide (5.6 mg, 1.4 μmol), compound SPP-8 (1.8 mg, 0.16 μmol, 2 steps 17%, retention time; 1.04, in the same manner as in Step 5-1 of Reference Example 58, 1.04 Minute (HPLC condition A)) was obtained as a white solid.

Reference Example 60 (Siglec-1L) ₂ -PEG5000-R ₃₀ GC (SPP-9) Synthesis Process 5-3
Compound Int-5 was prepared in the same manner as in Step 4 of Reference Example 58 using Compound Int-4 (2.5 mg, 1.3 μmol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT MA-050TS (4.5 mg, 0.84 μmol). A solution was obtained. Using the reaction solution and R ₃₀ GC peptide (6.1 mg, 1.3 μmol), compound SPP-9 (5.0 mg, 0.41 μmol, 2 steps 49%, retention time; retention time; 1.02 Minute (HPLC condition A)) was obtained as a white solid.

Reference Example 61 ((Siglec-1L) ₂ -PEG2000) ₂ -K ₁₅ GC (SPP-10) Synthesis Step 1
Compound Int-4 (5.9 mg, 3.0 μmol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT 2TS-GL2-020MA4 (manufactured by NOF Corporation, average molecular weight of PEG part 2000 × 2, 3.0 mg, 1.2 μmol, retained) Time: 1.32 minutes (HPLC condition A), 4.01 minutes (HPLC condition B)) and using the same method as in step 1 of Reference Example 52, Compound Int-6 solution (retention time; 1.42 minutes (HPLC condition A), 4.46 Minute (HPLC condition B)).

Process 2-1
Using the compound Int-6 solution obtained in step 1 and K ₁₅ GC peptide (3.8 mg, 1.8 μmol), in the same manner as in step 2-1 of Reference Example 52, compound SPP-10 (7.4 mg, 0.89 μmol, Two steps 74%, retention time; 1.03 min (HPLC condition A)) was obtained as a white solid.

Reference Example 62 ((Siglec-1L) ₂ -PEG2000) ₂ -K ₃₀ GC (SPP-11) Synthesis Step 2-2
Compound Int-4 (4.9 mg, 2.5 μmol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT 2TS-GL2-020MA4 (2.5 mg, 1.0 μmol) were used in the same manner as in Step 1 of Reference Example 61. -6 solution was obtained. Using the reaction solution and K ₃₀ GC peptide (4.8 mg, 1.2 μmol), compound SPP-11 (1.6 mg, 0.15 μmol, 2 steps 15%, retention time; retention time; 0.93 in the same manner as in step 2-1 of Reference Example 61 Minute (HPLC condition A)) was obtained as a white solid.

Reference Example 63 ((Siglec-1L) ₂ -PEG2000) ₂ -R ₃₀ GC (SPP-12) Synthesis Process 2-3
Compound Int-4 (4.9 mg, 2.5 μmol) obtained in Step 3 of Reference Example 58 and SUNBRIGHT 2TS-GL2-020MA4 (2.5 mg, 1.0 μmol) were used in the same manner as in Step 1 of Reference Example 61. -6 solution was obtained. Using the reaction solution and R ₃₀ GC peptide (5.8 mg, 1.2 μmol), compound SPP-12 (6.3 mg, 0.57 μmol, 2 steps 57%, retention time; retention time; 0.92 in the same manner as in step 2-1 of Reference Example 61 Minute (HPLC condition A)) was obtained as a white solid.

Hereinafter, Examples 1 to 6 and Comparative Example 1 are preparation examples in which the content of Siglec-1L-PEG-DSPE in nanoparticles, that is, the modification rate of Siglec-1L-PEG-DSPE in nanoparticles was changed. is there.

[Example 1]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows.
The nucleic acid used was a siRNA that suppresses the expression of the β2-microglobulin gene, which comprises a sense strand (5′-AGCAAGGACUGGUCUUUCUAUCUCU-3 ′) and an antisense strand (5′-AGAGAUAGAAAGACCAGUCCUUGCU-3 ′) base sequence (hereinafter, “ Also referred to as “B2M siRNA”). PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
B2M siRNA was dissolved in distilled water and prepared to 24 mg / mL.
Each lipid contains hydrochloric acid and ethanol so that the concentration ratio of CL-8 to PEG-DSPE (CL-8 / PEG-DSPE Na) is 57.26 (mmol / L) /5.521 (mmol / L) It was suspended in an aqueous solution and stirred and heated repeatedly with a vortex mixer to obtain a uniform suspension. This suspension was passed through a 0.2 μm polycarbonate membrane filter and a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of lead particles. The average particle size of the lead particles obtained with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern Instruments) was measured and confirmed to be within the range of 30 nm to 100 nm. The siRNA solution is mixed with the obtained lead particle dispersion at a volume ratio of 3: 1 (= lead particle dispersion: siRNA solution), and 3 times the amount of distilled water is added and mixed. A dispersion of nucleic acid complex particles was prepared.
On the other hand, the concentration ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol, and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG- Each lipid was weighed so that DSPE) was 14.72 (mmol / L) /1.082 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.007 (mmol / L), and 90 vol% A solution of lipid membrane constituents was prepared by dissolving in ethanol.
After heating the obtained lipid membrane constituent solution, the lipid membrane constituent solution and the nucleic acid complex particle dispersion are mixed at a volume ratio of 1: 1, and then several times the amount. Of distilled water was mixed to obtain a crude preparation.
The obtained crude preparation was concentrated using Amicon Ultra (manufactured by Millipore), further replacing the solvent with physiological saline, and filtered in a clean bench using a 0.2 μm filter (manufactured by Toyo Roshi Kaisha, Ltd.). Furthermore, the siRNA concentration of the obtained preparation was measured, and the preparation 1 was obtained by diluting with physiological saline so that the siRNA concentration was 10 μM.

[Example 2]
A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /1.067 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.022 (mmol / L), Formulation 2 was obtained in the same manner as Example 1.

[Example 3]
A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /1.024 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.065 (mmol / L), Formulation 3 was obtained in the same manner as Example 1.

[Example 4]
A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /0.893 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.196 (mmol / L), Formulation 4 was obtained in the same manner as Example 1.

[Example 5]
A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentrate ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE) / Cholesterol / Siglec-1L-PEG-DSPE) was changed to 14.72 (mmol / L) /0.500 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.589 (mmol / L), Formulation 5 was obtained in the same manner as Example 1.

[Example 6]
A nucleic acid-containing preparation obtained by changing the content of Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentration ratio of CL-8, DOPE, cholesterol, and Siglec-1L-PEG-DSPE for the lipid membrane component solution of Formulation 1 (CL-8 / DOPE / cholesterol / Siglec-1L-PEG-DSPE) A formulation 6 was obtained in the same manner as in Example 1 except that 14.72 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /1.089 (mmol / L) was used.

[Comparative Example 1]
The nucleic acid-containing preparation containing no Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentration ratio of CL-8, PEG-DSPE Na, DOPE, and cholesterol (CL-8 / PEG-DSPE Na / DOPE / cholesterol) at 14.72 (mmol / L) ) /1.089 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) In the same manner as in Example 1, Preparation 7 was obtained.

[Test Example 1]
Measurement of average particle size of nucleic acid-containing lipid nanoparticles The average particle size and zeta potential of nucleic acid-containing lipid nanoparticles in the preparation were measured with a particle size measurement device (Zetasizer Nano ZS, manufactured by Malvern) (Table 8). PDI in the table represents a polydispersity index.

[Test Example 2]
The target gene expression inhibitory effect on nucleic acid-containing lipid nanoparticles on human cell lines was evaluated.
About each nucleic acid-containing lipid nanoparticle obtained in Examples 1 to 6 and Comparative Example 1, the target gene expression inhibitory action on human-derived monocytic cell line THP-1 (derived from American Type Culture Collection) by the following method, respectively Was measured.
The THP-1 cell line is maintained and passaged in RMP1640 medium (RPMI1640 medium, manufactured by Nacalai Tesque, 30264-56) (hereinafter referred to as 10% FBS RPMI1640 medium) containing 10% fetal bovine serum (FBS). , Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 500 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 24 hours. -1 described as THP-1).
Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 30, 10, 3 or 1 nmol / L, and a 96-well suspension cell culture plate After dispensing 20 μL each into (Corning, 3474), Siglec-1-induced THP-1 suspended in Optimem was added so that the cell number would be 12500/80 μL / well, 37 ° C., 5% Each nucleic acid-containing lipid nanoparticle was introduced into cells by culturing for 4 hours under CO ₂ conditions. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner. The cultured cells were centrifuged at room temperature at 1800 rpm for 3 minutes, the culture supernatant was carefully removed, 10% FBS RPMI1640 medium was added, and the cells were further cultured at 37 ° C., 5% CO ₂ for 20 hours. The cultured cells were washed with ice-cold phosphate buffered saline (Nacalai Tesque, 14249-24), and Super Prep Cellulosis and RT Kit Forkyu PC (catalog number SCQ-201, manufactured by Toyobo Co., Ltd.) In accordance with the method described in the instructions attached to the product, total RNA was collected and cDNA was prepared by reverse transcription using the obtained total RNA as a template.
Using the obtained cDNA as a template, Tackman Gene Expression Assays probe (Applied Biosystems) as a probe, Quant Studio 12 Kaflex Real-Time PCR System (ABI), and the attached instruction manual By carrying out PCR reaction according to the described method, beta-2 microglobulin (hereinafter referred to as B2M) gene and constitutively expressed gene glyceraldehyde 3-phosphate dehydrogenase (D-glyceraldehyde- 3-phosphate dehydrogenase (hereinafter referred to as GAPDH) gene was subjected to PCR reaction to measure the amount of mRNA amplification, and the quasi-quantitative value of B2M mRNA was calculated using Gapdh mRNA amplification amount as an internal control. Similarly, the B2M mRNA expression rate was determined from the B2M mRNA semi-quantitative value, with the B2M mRNA semi-quantitative value in the negative control measured as 1. Table 9 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.

It was calculated by dividing the number of moles of Siglec-1L -PEG-DSPE by the total number of moles of lipid in the lipid nanoparticles.

From these results, Formulation 1-6, into which the targeting element (Siglec-1L-PEG-DSPE) was introduced, showed improved gene expression suppression activity as compared with Formulation 7 not containing the targeting element. Moreover, the improvement of the gene expression suppression activity depending on the introduction amount of the targeting element was shown.

Hereinafter, Examples 7 to 8 and Comparative Example 2 are preparation examples in which PEG lipid derivatives are changed.

[Example 7]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-8 (CL-8) obtained in Reference Example 8, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
PEG-DMPE was obtained from NOF.
A preparation 8 was obtained in the same manner as in Example 5 except that the lead particle preparation of the preparation 1 was changed to CL-8 / PEG-DMPE Na = 57.26 / 5.521 mmol / L.

[Example 8]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-8 (CL-8), 1,2-distearoyl-sn-glycero-3- [methoxy (polyethylene) obtained in Reference Example 8 Glycol) -2000] (PEG-DSG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
PEG-DSG was obtained from NOF.
A preparation 9 was obtained in the same manner as in Example 5 except that the PEG-DSPE used in the preparation 5 was changed to PEG-DSG.

[Comparative Example 2]
A preparation 10 was obtained in the same manner as in Comparative Example 1, except that the lead particle preparation of the preparation 7 was changed to CL-8 / PEG-DMPE Na = 57.26 / 5.521 mmol / L.

[Test Example 3]
Measurement of average particle size of nucleic acid-containing lipid nanoparticles The average particle size and zeta potential of nucleic acid-containing lipid nanoparticles in the preparation were measured with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern) (Table 10). PDI in the table represents a polydispersity index.

[Test Example 4]
The target gene expression inhibitory effect on nucleic acid-containing lipid nanoparticles on human cell lines was evaluated.
For each nucleic acid-containing lipid nanoparticle obtained in Examples 7 and 8 and Comparative Example 2, the target gene expression inhibitory action was evaluated in the same manner as in Test Example 2.
Table 11 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.

※表中、NDは、Not Detectedを表す。

* In the table, ND represents Not Detected.

Based on these results, preparation 8 with the targeting element (Siglec-1L -PEG-DSPE) improved gene expression suppression activity compared to preparation 10 without the targeting element (Siglec-1L -PEG-DSPE) showed that. In addition, Preparation 9 in which the PEG lipid derivative was converted showed the same gene expression inhibitory activity as Preparation 8.

Hereinafter, preparation examples using various cationic lipids are shown in Examples 9 to 13 and Comparative Examples 3 to 7.

[Example 9]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-10 (CL-10) obtained in Reference Example 10, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
Preparation 11 was obtained in the same manner as in Example 7, except that CL-8 of preparation 8 was changed to CL-10.

[Example 10]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and Compound CL-26 (CL-26) obtained in Reference Example 26, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
A preparation 12 was obtained in the same manner as in Example 7, except that CL-8 of the preparation 8 was changed to CL-26.

[Example 11]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-6 (CL-6) obtained in Reference Example 6, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
Formulation 13 was obtained in the same manner as in Example 7, except that CL-8 in formulation 8 was changed to CL-6.

[Example 12]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and compound CL-7 (CL-7) obtained in Reference Example 7, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine- N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
Formulation 14 was obtained in the same manner as in Example 7, except that CL-8 of formulation 8 was changed to CL-7.

[Example 13]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51 and Compound CL-9 (CL-9), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine obtained in Reference Example 9 N- [methoxy (polyethylene glycol) -2000] (PEG-DMPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol were used to produce nucleic acid-containing lipid nanoparticles as follows.
Formulation 15 was obtained in the same manner as in Example 7, except that CL-8 was replaced with CL-9.

[Comparative Example 3]
A preparation 16 was obtained in the same manner as in Comparative Example 2 except that CL-8 of the preparation 10 was changed to CL-10.

[Comparative Example 4]
Formulation 17 was obtained in the same manner as in Comparative Example 2, except that CL-8 in Formulation 10 was changed to CL-26.

[Comparative Example 5]
A preparation 18 was obtained in the same manner as in Comparative Example 2 except that CL-8 of the preparation 10 was changed to CL-6.

[Comparative Example 6]
A preparation 19 was obtained in the same manner as in Comparative Example 2, except that CL-8 of the preparation 10 was changed to CL-7.

[Comparative Example 7]
A preparation 20 was obtained in the same manner as in Comparative Example 2, except that CL-8 of the preparation 10 was changed to CL-9.

[Test Example 5]
Measurement of average particle size of nucleic acid-containing lipid nanoparticles The average particle size and zeta potential of nucleic acid-containing lipid nanoparticles in the preparation were measured with a particle size measurement device (Zetasizer Nano ZS, manufactured by Malvern) (Table 12). PDI in the table represents a polydispersity index.

[Test Example 6]
The target gene expression inhibitory effect on nucleic acid-containing lipid nanoparticles on human cell lines was evaluated.
For each of the nucleic acid-containing lipid nanoparticles obtained in Examples 9 to 13 and Comparative Examples 3 to 7, the target gene expression inhibitory action was evaluated in the same manner as in Test Example 2.
Table 13 shows the results of expressing the expression rate of the obtained B2M mRNA as a suppression rate with respect to the B2M mRNA expression rate of the negative control.

From these results, the preparations 11 to 15 in which the targeting element (Siglec-1L-PEG-DSPE) was introduced were compared with the preparations 16 to 20 that did not contain the corresponding targeting element (Siglec-1L -PEG-DSPE). The gene expression suppression activity was improved.

[Test Example 7]
The target gene expression inhibitory action of nucleic acid-containing lipid nanoparticles on human primary monocyte cells was evaluated.
About each nucleic acid-containing lipid nanoparticle obtained in Example 10 and Comparative Example 4, the target gene for healthy human-derived CD14-positive monocyte cells (Untouched Frozen NPB-CD14 + Monocytes, manufactured by Allcells, PB011F) by the following method, respectively. The expression inhibitory action was measured.
Healthy human-derived CD14 positive monocyte cells are 10% fetal bovine serum, 1% MEM Non-Essential Amino Acids Solution (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360-070), 50 μM Monocyte cells using RPMI1640 medium (hereinafter referred to as human monocyte basal medium) and DNase I solution (DNase I Solution, StemCell Technology, 07900) containing 2-mercaptoethanol (Gibco, 21985-023) Thawed according to the protocol attached to After that, inoculate a 96-well suspension cell culture plate at a density of 100,000 cells / 100 μL / well in human monocyte basal medium containing 1000 U / mL Recombinant Human IFN-alpha 2 protein, 37 ° C, 5% CO _By culturing for 24 hours under _two conditions, Siglec-1 was induced on the cell membrane (hereinafter referred to as Siglec-1-induced human primary monocyte cells). After 24 hours of culture, the cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and Optimem was added at 80 μL / well.
Subsequently, each nucleic acid-containing lipid nanoparticle is diluted with Optimem to a final concentration of 30, 10, 3 or 1 nmol / L, and 20 μL is added to each well under conditions of 37 ° C and 5% CO ₂ Each of the nucleic acid-containing lipid nanoparticles was introduced into the cells by culturing for 4 hours. Further, Siglec-1-induced primary human monocytes that were not treated as a negative control group were cultured in the same manner. Centrifuge the cultured cells at 1800 rpm for 3 minutes at room temperature, carefully remove the culture supernatant, add 100 μL / well human monocyte basal medium, and further under 37 ° C, 5% CO ₂ conditions. Cultured for 20 hours.
Wash the cultured cells with ice-cold phosphate buffered saline, add 20 μL / well of 0.02% EDTA solution (Nacalai Tesque, 14367-74), and allow to stand at 37 ° C for several minutes. To float Siglec-1-derived primary human monocytes. 1% bovine serum albumin-containing phosphate buffered saline containing 0.05% sodium azide (Nacalai Tesque, 13160-94), 0.02% EDTA (Ambion, AM9269G) Flow cytometry containing 20% FcR Blocking Reagent human (Miltenyi Biotech, 130-059-901) after washing twice with water (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer) The buffer for use was added at 100 μL / well and allowed to stand at 4 ° C. for 15 minutes.
Subsequently, FITC anti-human β2-microglobulin Antibody (Biolegend, 316304) was diluted 4-fold with flow cytometry buffer, and dispensed into a 96-well U-bottom plate (Falcon, 353077) at 20 μL / well. Thereafter, Siglec-1-derived human primary monocytes after standing for 15 minutes were added at 80 μL / well, and the mixture was allowed to stand at 4 ° C. for 60 minutes to react the B2M protein on the cell membrane with the antibody.
Subsequently, the Siglec-1-derived primary human monocytes after the antibody reaction were washed three times with a flow cytometry buffer, and then the fluorescence intensity at 530 nm with respect to the excitation light at 488 nm of each cell was measured using the BD FACS Canto II Flow Cytometer ( The expression level of B2M protein on the cell membrane was measured by measuring using BD). Similarly, the expression level of B2M protein was determined with the B2M protein expression level in the negative control measured in the same manner as 1. Table 14 shows the results of expressing the expression rate of the obtained B2M protein as a suppression rate with respect to the B2M mRNA expression rate of the negative control.

From these results, it can be seen that, even in Siglec-1 positive activated monocyte cells derived from human primary monocytes, the preparation 12 containing Siglec-1L-PEG-DSPE is the corresponding preparation 17 containing no Siglec-1L -PEG-DSPE. Compared with, the gene expression inhibitory activity was remarkably strong.

[Example 14]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows.
As the nucleic acid, CleanCap ^™ EGFP mRNA (5moU) expressing green fluorescent protein (GFP) obtained from TriLink BioTechnologies, LLC was used (hereinafter also referred to as “GFP mRNA”). PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
GFP mRNA was dissolved in distilled water and adjusted to 1 mg / mL for use.
Each lipid contains hydrochloric acid and ethanol so that the concentration ratio of CL-8 to PEG-DSPE (CL-8 / PEG-DSPE Na) is 57.26 (mmol / L) /5.521 (mmol / L) It was suspended in an aqueous solution and stirred and heated repeatedly with a vortex mixer to obtain a uniform suspension. This suspension was passed through a 0.2 μm polycarbonate membrane filter and a 0.05 μm polycarbonate membrane filter at room temperature to obtain a dispersion of lead particles. The average particle size of the lead particles obtained with a particle size measuring device (Zetasizer Nano ZS, manufactured by Malvern Instruments) was measured and confirmed to be within the range of 30 nm to 100 nm. GFP mRNA solution is mixed with the obtained lead particle dispersion at a volume ratio of 1: 8 (= lead particle dispersion: GFP mRNA solution), and 0.185 times the amount of distilled water is added and mixed. Thus, a dispersion of nucleic acid complex particles was prepared.
On the other hand, the concentration ratio of CL-8, PEG-DSPE Na, DOPE, cholesterol, and Siglec-1L-PEG-DSPE (CL-8 / PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG- Each lipid is weighed so that DSPE) is 14.72 (mmol / L) /0.500 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) /0.589 (mmol / L), and 90 vol% A solution of lipid membrane constituents was prepared by dissolving in ethanol.
After heating the obtained lipid membrane constituent solution, the lipid membrane constituent solution and the nucleic acid complex particle dispersion are mixed at a volume ratio of 1: 1, and then several times the amount. Of distilled water was mixed to obtain a crude preparation.
The obtained crude preparation was concentrated using Vivaspin (GE Healthcare), further substituted with physiological saline, and filtered in a clean bench using a 0.2 μm filter (Toyo Roshi Kaisha, Ltd.). Furthermore, the GFP mRNA concentration of the obtained preparation was measured, and the preparation 21 was obtained by diluting with physiological saline so that the GFP mRNA concentration was 60 μg / mL.

[Example 15]
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, Compound CL-8 obtained in Reference Example 8, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy ( Polyethylene glycol) -2000] (PEG-DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and cholesterol were used to produce nucleic acid-containing nanoparticles as follows.
As the nucleic acid, CleanCap ^™ FLuc mRNA (5moU) expressing firefly luciferase obtained from TriLink BioTechnologies, LLC was used (hereinafter also referred to as “FLuc mRNA”). PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
FLuc mRNA was dissolved in distilled water and adjusted to 1 mg / mL.
A preparation 22 was obtained in the same manner as in Example 14 except that the GFP mRNA in the preparation 21 was changed to FLuc mRNA.

[Comparative Example 8]
The nucleic acid-containing preparation containing no Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentration ratio of CL-8, PEG-DSPE Na, DOPE, and cholesterol (CL-8 / PEG-DSPE Na / DOPE / cholesterol) at 14.72 (mmol / L) ) /1.089 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) In the same manner as in Example 14, Preparation 23 was obtained.

[Comparative Example 9]
The nucleic acid-containing preparation containing no Siglec-1L-PEG-lipid obtained in Reference Example 51 was produced as follows.
Concentrate ratio of CL-8, PEG-DSPE Na, DOPE and cholesterol (CL-8 / PEG-DSPE Na / DOPE / cholesterol) at 14.72 (mmol / L) ) /1.089 (mmol / L) /4.008 (mmol / L) /9.619 (mmol / L) In the same manner as in Example 15, Preparation 24 was obtained.

[Test Example 8]
Measurement of average particle size of nucleic acid-containing lipid nanoparticles The average particle size and zeta potential of nucleic acid-containing lipid nanoparticles in the preparation were measured with a particle size measurement device (Zetasizer Nano ZS, manufactured by Malvern) (Table 15). PDI in the table represents a polydispersity index.

[Test Example 9]
The effect of nucleic acid-containing lipid nanoparticles on the target cell expression enhancement of human cell lines was evaluated.
For each nucleic acid-containing lipid nanoparticle obtained in Example 14 and Comparative Example 8, the target gene expression enhancing action on human-derived monocyte cell line THP-1 (derived from American Type Culture Collection) was measured by the following methods, respectively. did.
The THP-1 cell line is maintained and passaged in RMP1640 medium (RPMI1640 medium, manufactured by Nacalai Tesque, 30264-56) (hereinafter referred to as 10% FBS RPMI1640 medium) containing 10% fetal bovine serum (FBS). , Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 500 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 48 hours (hereinafter, Siglec -1 described as THP-1).
Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 1, 0.3, 0.1, or 0.03 μg / mL, and a 96-well suspension cell culture plate After dispensing 20 μL each into (Corning, 3474), Siglec-1-induced THP-1 suspended in Optimem was added so that the cell number would be 12500/80 μL / well, 37 ° C., 5% Each nucleic acid-containing lipid nanoparticle was introduced into cells by culturing for 24 hours under CO ₂ conditions. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner. The cultured cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and then the cells were treated with 0.05% sodium azide (Nacalai Tesque, 13160-94), 0.02% EDTA (Ambion, AM9269G). And 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer). By measuring the fluorescence intensity at 530 nm against the 488 nm excitation light of each obtained cell using the BD FACS Canto II Flow Cytometer (BD), the expression level of green fluorescent protein (GFP) protein in the cell can be determined. It was measured. Table 16 shows the relative fluorescence intensity of each cell when the fluorescence intensity of the negative control cells measured in the same manner is 1.

Based on these results, compared to the preparation 23 containing no targeting element, the preparation 21 containing the targeting element (Siglec-1L -PEG-DSPE) was compared to the human cell line THP-1 that induced the receptor Siglec-1 It showed strong gene expression enhancing activity.

[Test Example 10]
The target gene expression inhibitory action of nucleic acid-containing lipid nanoparticles on human primary monocyte cells was evaluated.
About each nucleic acid-containing lipid nanoparticle obtained in Example 14 and Comparative Example 8, the target gene for CD14-positive monocyte cells derived from healthy subjects (Untouched Frozen NPB-CD14 + Monocytes, manufactured by Allcells, PB011F) by the following methods, respectively. The expression enhancing action was measured.
Healthy human-derived CD14 positive monocyte cells are 10% fetal bovine serum, 1% MEM Non-Essential Amino Acids Solution (Gibco, 11140-050), 1 mM sodium pyruvate (Gibco, 11360-070), 50 μM Using RPMI1640 medium (hereinafter referred to as human monocyte basal medium) containing 2-mercaptoethanol (Gibco, 21985-023) and DNase I solution (DNase I Solution, StemCell Technology, 07900), monocytes Thawed according to the protocol attached to the cells. After that, inoculate a 96-well suspension cell culture plate at a density of 100,000 cells / 100 μL / well in human monocyte basal medium containing 1000 U / mL Recombinant Human IFN-alpha 2 protein, 37 ° C, 5% CO _By culturing for 48 hours under _two conditions, Siglec-1 was induced on the cell membrane (hereinafter referred to as Siglec-1-induced human primary monocyte cells). After 48 hours of culture, the cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and Optimem was added at 80 μL / well.
Subsequently, each nucleic acid-containing lipid nanoparticle is diluted with Optimem to a final concentration of 1, 0.3 or 0.1 μg / mL, and 20 μL is added to each well, and the mixture is added under conditions of 37 ° C. and 5% CO _2. By culturing for a period of time, each nucleic acid-containing lipid nanoparticle was introduced into a cell. Further, Siglec-1-induced primary human monocytes that were not treated as a negative control group were cultured in the same manner. The cultured cells were centrifuged at 1800 rpm for 3 minutes at room temperature, the culture supernatant was carefully removed, and then the cells were treated with 0.05% sodium azide (Nacalai Tesque, 13160-94), 0.02% EDTA (Ambion, AM9269G). And 1% bovine serum albumin-containing phosphate buffered saline (Nacalai Tesque, 09968-35) (hereinafter, flow cytometry buffer). By measuring the fluorescence intensity at 530 nm against the 488 nm excitation light of each obtained cell using the BD FACS Canto II Flow Cytometer (BD), the expression level of green fluorescent protein (GFP) protein in the cell can be determined. It was measured. Table 17 shows the relative fluorescence intensity of each cell when the fluorescence intensity of the negative control cells measured in the same manner is 1.

From this result, even in Siglec-1 positive activated monocyte cells derived from human primary monocytes, the preparation 21 using Siglec-1L-PEG-DSPE is the corresponding preparation 23 containing no Siglec-1L -PEG-DSPE. Compared with, it showed strong gene expression enhancing activity.

[Test Example 11]
The effect of nucleic acid-containing lipid nanoparticles on the target cell expression enhancement of human cell lines was evaluated.
For each nucleic acid-containing lipid nanoparticle obtained in Example 15 and Comparative Example 9, the target gene expression enhancing effect on human-derived monocyte cell line THP-1 (derived from American Type Culture Collection) was measured by the following methods, respectively. did.
The THP-1 cell line is maintained and passaged in RMP1640 medium (RPMI1640 medium, manufactured by Nacalai Tesque, 30264-56) (hereinafter referred to as 10% FBS RPMI1640 medium) containing 10% fetal bovine serum (FBS). , Siglec-1 was induced on the cell membrane by culturing in 10% FBS RPMI1640 medium containing 2000 U / mL Recombinant Human IFN-alpha 2 protein (R & D, 11105-1) for 48 hours (hereinafter Siglec -1 described as THP-1).
Each nucleic acid-containing lipid nanoparticle is diluted with Optimem (Opti-MEM, GIBCO, 31985) to a final concentration of 0.3 or 0.1 μg / mL, and a 96-well suspension cell culture plate (coning) 3474), and then add Siglec-1-induced THP-1 suspended in Optimem to a cell number of 12500/80 μL / well, and at 37 ° C and 5% CO ₂ Each of the nucleic acid-containing lipid nanoparticles was introduced into the cells by culturing for 24 hours. Moreover, Siglec-1-induced THP-1 that was not treated as a negative control group was cultured in the same manner. After culture, use Steady-Gluciferase Assay System (Promega, E2550) in the culture medium containing cells as per the product manual, and luminescence measurement using Multilabel Reader 2030 ALVOX 3 (2030 ALVO X3, Perkin Elmer) The amount of firefly luciferase (FLuc) protein expression in the culture was measured. Table 18 shows the relative luminescence intensity of each cell when the luminescence intensity of the negative control cells measured in the same manner is 1.

From this result, compared to the preparation 24 that does not contain the targeting element, the preparation 22 that introduced the targeting element (Siglec-1L -PEG-DSPE) against the human cell line THP-1 that induced the receptor Siglec-1 It showed strong gene expression enhancing activity.

[Example 16]
Formulation 25 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows.
Compound Siglec-1L-PEG-lipid obtained in Reference Example 51, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy (polyethylene glycol) -2000] (PEG-DSPE), Nucleic acid-containing nanoparticles were produced using 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and cholesterol as follows.
The nucleic acid has a base sequence of a sense strand (5'-GCCAGACUUUGUUGGAUUUGA-3 ') and an antisense strand (5'-AAmAUmCCmAAmCAmAAmGUmCUmGGmCmUmU-3') (m represents 2'-OMe-RNA), and the antisense strand 3 ' A siRNA that suppresses the expression of hypoxanthine-guanine phosphoribosyltransferase 1 gene, which is modified with cholesterol at the terminal, was used (hereinafter also referred to as “HPRT1 siRNA-cho”). PEG-DSPE, DOPE, and cholesterol were obtained from NOF.
HPRT-1 siRNA-cho was dissolved in distilled water and stored at 24 mg / mL.
Each lipid was adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) was 0.4730 (mmol / L) /9.119 (mmol / L) /2.977 (mmol / L). Dissolved in ethanol. On the other hand, the concentration ratio of Siglec-1L-PEG-lipid and HPRT-1 siRNA-cho (Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho) is 0.06058 (mmol / L) /0.001048 (mmol / L). Diluted with distilled water.
Syringe pumps (YMC) are filled with lipid solutions and aqueous solutions containing Siglec-1L-PEG-lipid and nucleic acid in syringes (manufactured by Hamilton), and the flow rates of each solution are 3.000 mL / min and 9.000 mL / min. And a crude preparation was obtained through a microreactor mixer (manufactured by YMC). The obtained crude preparation was concentrated using Vivaspin (GE Healthcare), further substituted with physiological saline, and filtered in a clean bench using a 0.2 μm filter (Toyo Roshi Kaisha, Ltd.). Further, the HPRT-1 siRNA-cho concentration of the obtained preparation was measured, and diluted with physiological saline so that the HPRT-1 siRNA-cho concentration was 10 mM, whereby a preparation 25 was obtained.

[Example 17]
Formulation 26 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows.
Lipids and nucleic acids were the same as in Example 16.
Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.2365 (mmol / L) /4.559 (mmol / L) /1.489 (mmol / L). Dissolved in ethanol. On the other hand, the concentration ratio of Siglec-1L-PEG-lipid and HPRT-1 siRNA-cho (Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho) is 0.02983 (mmol / L) /0.005159 (mmol / L). Diluted with distilled water.
Otherwise, the preparation 26 was obtained in the same manner as in Example 16.

[Example 18]
Formulation 27 containing Siglec-1L-PEG-lipid containing no cationic lipid was prepared as follows.
Lipids and nucleic acids were the same as in Example 16.
PEG-DSPE / DOPE / Cholesterol / Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho molar ratio (PEG-DSPE Na / DOPE / cholesterol / Siglec-1L-PEG-lipid / HPRT-1 siRNA-cho) Was dissolved in a mixed solution of t-BuOH / distilled water so as to be 0.5202 (μmol) /9.914 (μmol) /3.237 (μmol) /0.1946 (μmol) /0.03365 (μmol) and freeze-dried.
To the obtained lipid / nucleic acid powder, physiological saline was added so as to have a nucleic acid concentration of 1 mg / mL, followed by stirring with a vortex stirring mixer to obtain a suspension. This suspension was passed through a 0.4 μm polycarbonate membrane filter, a 0.2 μm polycarbonate membrane filter, and a 0.1 μm polycarbonate membrane filter at room temperature to prepare a dispersion of nucleic acid complex particles.
The resulting nucleic acid complex particle dispersion was filtered in a clean bench using a 0.2 μm filter (Toyo Roshi Kaisha, Ltd.). Furthermore, the HPRT1 siRNA-cho concentration of the obtained preparation was measured, and diluted with physiological saline so that the HPRT1 siRNA-cho concentration was 10 mM, whereby a preparation 27 was obtained.

[Comparative Example 10]
Formulation 28 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows.
Lipids and nucleic acids were the same as in Example 16.
Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.6676 (mmol / L) /9.260 (mmol / L) /3.023 (mmol / L). Dissolved in ethanol. On the other hand, it was diluted with distilled water so that HPRT-1 siRNA-cho was 0.001048 (mmol / L).
Otherwise, the preparation 28 was obtained in the same manner as in Example 16.

[Comparative Example 11]
Formulation 29 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows.
Lipids and nucleic acids were the same as in Example 16.
Each lipid is adjusted so that the concentration ratio of PEG-DSPE, DOPE and cholesterol (PEG-DSPE Na / DOPE / cholesterol) is 0.3338 (mmol / L) /4.630 (mmol / L) /1.512 (mmol / L). Dissolved in ethanol. On the other hand, it was diluted with distilled water so that HPRT-1 siRNA-cho was 0.005239 (mmol / L).
Otherwise, the preparation 29 was obtained in the same manner as in Example 16.

[Comparative Example 12]
Formulation 30 containing no cationic lipid and Siglec-1L-PEG-lipid was prepared as follows.
Lipids and nucleic acids were the same as in Example 18.
The molar ratio of PEG-DSPE, DOPE, cholesterol, and HPRT-1 siRNA-cho (PEG-DSPE Na / DOPE / cholesterol / HPRT-1 siRNA-cho) is 0.7148 (μmol) /9.914 (μmol) /3.237 (μmol) It was dissolved in a mixed solution of t-BuOH / distilled water so as to be /0.03365 (μmol) and lyophilized.
Otherwise, the procedure of Example 18 was repeated to obtain a preparation 30.

[Test Example 12]
Measurement of average particle size of nucleic acid-containing lipid nanoparticles The average particle size and zeta potential of nucleic acid-containing lipid nanoparticles in the preparation were measured with a particle size measurement device (Zetasizer Nano ZS, manufactured by Malvern) (Table 19). PDI in the table represents a polydispersity index.

[Test Example 12]
The target gene expression inhibitory action of nucleic acid-containing lipid nanoparticles on human primary monocyte cells was evaluated.

[Example 19]
A preparation containing a polymer (polymer I) containing a water-soluble unit and a cationic unit and having a ligand capable of binding to nucleic acid and siglec (Sialic acid-binding immunoglobulin-like lectin) was prepared as follows.
Using the Siglec-1L-PEG5000-K ₁₅ GC (compound SPP-1) obtained in Reference Example 52, a nucleic acid complex was produced as follows.
Nucleic acid is sense strand (5'-A (F) ^ G (M) ^ G (F) A (M) C (F) U (M) G (F) G (M) U (F) C (M) U (F) U (F) U (M) C (F) U (M) A (F) U (M) C (F) U (M) ^ C (F) ^ U (M) -3 ') And antisense strand (5'-A (F) ^ G (M) ^ A (F) G (M) A (F) U (M) A (F) G (M) A (F) A (M ) A (M) G (F) A (M) C (F) C (M) A (F) G (M) U (F) C (M) C (F) U (M) ^ U (F) ^ G (M) -3 ') (M is 2'-OMe-RNA, (F) is 2'-F-RNA, ^ is Phosphorothioated) SiRNA that suppresses the above was used (hereinafter also referred to as “B2M siRNA-2”).
B2M siRNA-2 was dissolved in distilled water, stored at 24 mg / mL, and diluted with 10 mM HEPES as necessary.
Siglec-1L-PEG5000-K15GC (compound SPP-1) was dissolved in 10 mM HEPES so as to be 10 mg / mL. Siglec-1L-PEG5000-K15GC (compound SPP-1) and B2M so that the ratio of the number of amino groups (N) of compound SPP-1 to the number of phosphate groups (P) of siRNA is N / P = 1 Formulation 31 was made by mixing siRNA-2 and adjusting the nucleic acid concentration to a final concentration of 10 μM.

[Example 20]
A preparation 32 was obtained in the same manner as in Example 19 except that the condition of N / P = 1 of the preparation 31 was changed to N / P = 2.5.

[Example 21]
A preparation 33 was obtained in the same manner as in Example 19 except that the condition of N / P = 1 of the preparation 31 was changed to N / P = 5.

[Example 22]
A preparation 34 was obtained in the same manner as in Example 19 except that the condition of N / P = 1 of the preparation 31 was changed to N / P = 10.

[Example 23]
Using the Siglec-1L-PEG5000-K ₃₀ GC (compound SPP-2) obtained in Reference Example 53, a nucleic acid complex was produced as follows.
Siglec-1L-PEG5000-K ₃₀ GC (compound SPP-2) was dissolved in 10 mM HEPES to a concentration of 10 mg / mL. Formulation 35 was prepared by mixing Siglec-1L-PEG5000-K ₃₀ GC (compound SPP-2) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Example 24]
A preparation 36 was obtained in the same manner as in Example 23 except that the condition of N / P = 1 of the preparation 35 was changed to N / P = 2.5.

[Example 25]
A preparation 37 was obtained in the same manner as in Example 23 except that the condition of N / P = 1 of the preparation 35 was changed to N / P = 5.

[Example 26]
A preparation 38 was obtained in the same manner as in Example 23 except that the condition of N / P = 1 of the preparation 35 was changed to N / P = 10.

[Example 27]
Using the Siglec-1L-PEG5000-R ₃₀ GC (compound SPP-3) obtained in Reference Example 54, a nucleic acid complex was produced as follows.
Siglec-1L-PEG5000-R ₃₀ GC (compound SPP-3) was dissolved in 10 mM HEPES to a concentration of 10 mg / mL. Formulation 39 was carried out by mixing Siglec-1L-PEG5000-R ₃₀ GC (compound SPP-3) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Example 28]
A preparation 40 was obtained in the same manner as in Example 27 except that the condition of N / P = 1 of the preparation 36 was changed to N / P = 2.5.

[Example 29]
A formulation 41 was obtained in the same manner as in Example 27 except that the condition of N / P = 1 of the formulation 36 was changed to N / P = 5.

[Example 30]
A preparation 42 was obtained in the same manner as in Example 27 except that the condition of N / P = 1 of the preparation 36 was changed to N / P = 10.

[Example 31]
Using (Siglec-1L) ₂ -PEG5000-K ₁₅ GC (SPP-7) obtained in Reference Example 58, a nucleic acid complex was produced as follows.
(Siglec-1L) ₂ -PEG5000-K ₁₅ GC (SPP-7) was dissolved in water for injection to 2 mg / mL. Formulation 43 was prepared by mixing Siglec-1L-PEG5000-R ₃₀ GC (compound SPP-3) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Example 32]
A preparation 44 was obtained in the same manner as in Example 31, except that the condition of N / P = 1 of the preparation 43 was changed to N / P = 5.

[Example 33]
A preparation 45 was obtained in the same manner as in Example 31 except that the condition of N / P = 1 of the preparation 36 was changed to N / P = 5.

[Example 34]
A preparation 46 was obtained in the same manner as in Example 31 except that the condition of N / P = 1 of the preparation 36 was changed to N / P = 10.

[Example 35]
Using (Siglec-1L) ₂ -PEG5000-K ₃₀ GC (SPP-8) obtained in Reference Example 59, a nucleic acid complex was produced as follows.
(Siglec-1L) ₂ -PEG5000-K ₃₀ GC (SPP-8) was dissolved in water for injection to 2 mg / mL. Formulation 47 was made by mixing (Siglec-1L) ₂ -PEG5000-K ₃₀ GC (SPP-8) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Example 36]
A preparation 48 was obtained in the same manner as in Example 35 except that the condition of N / P = 1 of the preparation 47 was changed to N / P = 2.5.

[Example 37]
A preparation 49 was obtained in the same manner as in Example 35 except that the condition of N / P = 1 in the preparation 47 was changed to N / P = 5.

[Example 38]
A preparation 50 was obtained in the same manner as in Example 35 except that the condition of N / P = 1 in the preparation 47 was changed to N / P = 10.

[Example 39]
Using (Siglec-1L) ₂ -PEG5000-R ₃₀ GC (SPP-9) obtained in Reference Example 60, a nucleic acid complex was produced as follows.
(Siglec-1L) ₂ -PEG5000-R ₃₀ GC (SPP-9) was dissolved in water for injection at 5 mg / mL. Formulation 51 was made by mixing (Siglec-1L) ₂ -PEG5000-R ₃₀ GC (SPP-9) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Example 40]
A preparation 52 was obtained in the same manner as in Example 39 except that the condition of N / P = 1 in the preparation 51 was changed to N / P = 2.5.

[Example 41]
A preparation 53 was obtained in the same manner as in Example 39 except that the condition of N / P = 1 of the preparation 51 was changed to N / P = 5.

[Example 42]
A preparation 54 was obtained in the same manner as in Example 39 except that the condition of N / P = 1 of the preparation 51 was changed to N / P = 10.

[Example 43]
Using ((Siglec-1L) ₂ -PEG2000) ₂ -K ₁₅ GC (SPP-10) obtained in Reference Example 61, a nucleic acid complex was produced as follows.
((Siglec-1L) ₂ -PEG2000) ₂ -K ₁₅ GC (SPP-10) was dissolved in water for injection to a concentration of 10 mg / mL. Formulation 55 by mixing ((Siglec-1L) ₂ -PEG2000) ₂ -K ₁₅ GC (SPP-10) and B2M siRNA-2 to a final concentration of 10 μM so that N / P = 1. did.

[Example 44]
A preparation 56 was obtained in the same manner as in Example 43 except that the condition of N / P = 1 of the preparation 55 was changed to N / P = 2.5.

[Example 45]
A preparation 57 was obtained in the same manner as in Example 43 except that the condition of N / P = 1 of the preparation 55 was changed to N / P = 5.

[Example 46]
A preparation 58 was obtained in the same manner as in Example 43 except that the condition of N / P = 1 of the preparation 55 was changed to N / P = 10.

[Example 47]
Using ((Siglec-1L) ₂ -PEG2000) ₂ -K ₃₀ GC (SPP-11) obtained in Reference Example 62, a nucleic acid complex was produced as follows.
((Siglec-1L) ₂ -PEG2000) ₂ -K ₃₀ GC (SPP-11) was dissolved in water for injection to 2 mg / mL. Formulation 59 by mixing ((Siglec-1L) ₂ -PEG2000) ₂ -K ₃₀ GC (SPP-11) and B2M siRNA-2 to a final concentration of 10 μM so that N / P = 1. did.

[Example 48]
A preparation 60 was obtained in the same manner as in Example 47 except that the condition of N / P = 1 in the preparation 59 was changed to N / P = 2.5.

[Example 49]
A preparation 61 was obtained in the same manner as in Example 47 except that the condition of N / P = 1 in the preparation 59 was changed to N / P = 5.

[Example 50]
A preparation 62 was obtained in the same manner as in Example 47 except that the condition of N / P = 1 in the preparation 59 was changed to N / P = 10.

[Example 51]
Using ((Siglec-1L) ₂ -PEG2000) ₂ -R ₃₀ GC (SPP-12) obtained in Reference Example 63, a nucleic acid complex was produced as follows.
((Siglec-1L) ₂ -PEG2000) ₂ -R ₃₀ GC (SPP-12) was dissolved in water for injection to a concentration of 10 mg / mL. Formulation 63 by mixing ((Siglec-1L) ₂ -PEG2000) ₂ -R ₃₀ GC (SPP-12) and B2M siRNA-2 to a final concentration of 10 μM so that N / P = 1. did.

[Example 52]
A preparation 64 was obtained in the same manner as in Example 51 except that the condition of N / P = 1 of the preparation 63 was changed to N / P = 2.5.

[Example 53]
A preparation 65 was obtained in the same manner as in Example 51 except that the condition of N / P = 1 in the preparation 63 was changed to N / P = 5.

[Example 54]
A preparation 66 was obtained in the same manner as in Example 51 except that the condition of N / P = 1 in the preparation 63 was changed to N / P = 10.

[Comparative Example 13]
Using the PEG5000-K ₁₅ GC (compound SPP-4) obtained in Reference Example 55, a nucleic acid complex was produced as follows.
PEG5000-K ₁₅ GC (compound SPP-4) was dissolved in 10 mM HEPES so as to be 10 mg / mL. Formulation 67 was made by mixing PEG5000-K ₁₅ GC (compound SPP-4) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Comparative Example 14]
A preparation 68 was obtained in the same manner as in Comparative Example 13, except that the condition of N / P = 1 of the preparation 67 was changed to N / P = 2.5.

[Comparative Example 15]
A preparation 69 was obtained in the same manner as in Comparative Example 13, except that the condition of N / P = 1 of the preparation 67 was changed to N / P = 5.

[Comparative Example 16]
A preparation 70 was obtained in the same manner as in Comparative Example 13, except that the N / P = 1 condition of the preparation 67 was changed to N / P = 10.

[Comparative Example 17]
Using the PEG5000-K ₃₀ GC (compound SPP-5) obtained in Reference Example 56, a nucleic acid complex was produced as follows.
PEG5000-K ₃₀ GC (compound SPP-5) was dissolved in 10 mM HEPES so as to be 10 mg / mL. Formulation 71 was made by mixing PEG5000-K ₃₀ GC (compound SPP-5) and B2M siRNA-2 so that N / P = 1, to a final nucleic acid concentration of 10 μM.

[Comparative Example 18]
A preparation 72 was obtained in the same manner as in Comparative Example 17, except that the condition of N / P = 1 of the preparation 71 was changed to N / P = 2.5.

[Comparative Example 19]
A preparation 73 was obtained in the same manner as in Comparative Example 17, except that the condition of N / P = 1 of the preparation 71 was changed to N / P = 5.

[Comparative Example 20]
A preparation 74 was obtained in the same manner as in Comparative Example 18, except that the condition of N / P = 1 of the preparation 71 was changed to N / P = 10.

[Comparative Example 21]
Using the PEG5000-R ₃₀ GC (compound SPP-6) obtained in Reference Example 57, a nucleic acid complex was produced as follows.
PEG5000-R ₃₀ GC (compound SPP-6) was dissolved in 10 mM HEPES so as to be 10 mg / mL. PEG5000-R ₃₀ GC (compound SPP-6) and B2M siRNA-2 were mixed so that N / P = 1, and a formulation 75 was prepared by setting the nucleic acid concentration to a final concentration of 10 μM.

[Comparative Example 22]
A preparation 76 was obtained in the same manner as in Comparative Example 21, except that the condition of N / P = 1 of the preparation 75 was changed to N / P = 2.5.

[Comparative Example 23]
A preparation 77 was obtained in the same manner as in Comparative Example 21, except that the condition of N / P = 1 of the preparation 75 was changed to N / P = 5.

[Comparative Example 24]
A preparation 78 was obtained in the same manner as in Comparative Example 21, except that the condition of N / P = 1 of the preparation 75 was changed to N / P = 10.

SEQ ID NO: 1 shows the base sequence of the B2M siRNA sense strand.
SEQ ID NO: 2 shows the base sequence of B2M siRNA antisense strand.
SEQ ID NO: 3 shows the base sequence of HPRT1 siRNA-cho sense strand.
SEQ ID NO: 4 shows the base sequence of HPRT1 siRNA-cho antisense strand.
SEQ ID NO: 5 shows the base sequence of B2M-2 siRNA sense strand.
SEQ ID NO: 6 shows the base sequence of the B2M-2 siRNA antisense strand.

Claims (19)

A lipid containing lipid or a water-soluble unit (lipid I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a nucleic acid, or
A polymer containing a water-soluble unit and a cationic unit (polymer I) having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), and a nucleic acid,
Nanoparticles. Contains a lipid or lipid-containing unit with a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin) (lipid I), nucleic acid, and cationic lipid (lipid II), or
Polymers (Polymer I) containing water-soluble units and cationic units, having a ligand capable of binding to siglec (Sialic acid-binding immunoglobulin-like lectin), nucleic acids, and high molecules containing water-soluble units and cationic units Including molecules (polymer II),
Nanoparticles. Lipid I is a structure in which a ligand capable of binding to Siglec and a lipid or water-soluble unit are linked via a linker:

(N1 is an integer of 1 or more.)
Represented by
Polymer I has a structure in which a ligand capable of binding to Siglec and a water-soluble unit, or a ligand capable of binding to Siglec, a water-soluble unit, and a cationic unit are linked via a linker:

(N2 is an integer of 1 or more.)
The nanoparticle of Claim 1 or 2 represented by these. The nanoparticle according to any one of claims 1 to 3, wherein the ligand is a sugar chain ligand. The nanoparticle according to claim 4, wherein the sugar chain ligand is a group represented by the following formula (1).

(In the formula (1), Ac represents an acetyl group, and R ¹ represents a C6-C12 aryl group, a C4-C12 heteroaryl group, or a C1 substituted with a C6-C12 aryl group or a C4-C12 heteroaryl group. Represents a -C6 alkyl group.) The nanoparticle according to claim 5, wherein R ¹ in the formula (1) is a group represented by the following structure.

Water-soluble unit is polyethylene glycol, polyglycerin, polyethyleneimine, polyvinyl alcohol, polyacrylic acid, polyacrylamide, oligosaccharide, dextrin, water-soluble cellulose, dextran, chondroitin sulfate, polyglycerin, chitosan, polyvinylpyrrolidone, polyaspartic acid amide The nanoparticle according to any one of claims 1 to 6, which is a unit having one or more selected from the group consisting of: poly-L-lysine, mannan, pullulan, oligoglycerol, and derivatives thereof. The cationic unit is an amino acid polymer unit comprising at least one selected from the group consisting of lysine, arginine, and histidine, a polyethyleneimine unit, or a polyaminoacrylate unit according to any one of claims 1 to 7. Nanoparticles. The nanoparticle according to any one of claims 1 to 8, wherein the nanoparticle further contains a neutral lipid. The nanoparticle according to any one of claims 1 to 9, wherein the nucleic acid is siRNA or mRNA. The nanoparticle according to claim 10, wherein the siRNA is covalently bonded to cholesterol, tocopherol or a fatty acid via a linker. The nanoparticle according to any one of claims 1 to 11, which is used for delivery to Siglec-1 (CD169) positive cells. The nanoparticle according to claim 12, wherein the Siglec-1 (CD169) positive cell is a macrophage, a dendritic cell or a monocyte. A method for introducing a nucleic acid into a cell, using the nanoparticles according to any one of claims 1 to 11. The method according to claim 14, wherein the cells are Siglec-1 (CD169) positive cells. The method according to claim 15, wherein the Siglec-1 (CD169) positive cells are macrophages, dendritic cells or monocytes. A medicament comprising the nanoparticles according to any one of claims 1 to 13. The medicament according to claim 17, which is for intravenous administration or subcutaneous administration. A method for treating or preventing a disease, comprising administering the medicine according to claim 17 or 18 to a patient in need thereof.