Peg lipid for preparing lipid nanoparticles

Abstract

The purpose of the present invention is to provide a PEG lipid that makes it possible to prepare an LNP with which the production amount of an anti-PEG antibody in vivo is suppressed. A PEG lipid for preparing lipid nanoparticles according to the present invention is represented by formula (I): R1-L1-R2. (In the formula, R1 represents a zwitterionic moiety having a positive charge and a negative charge; L1 represents a polyethylene glycol chain; and R2 represents a lipid moiety.)

Description

PEG lipids for lipid nanoparticle preparation

[0001] This invention relates to PEG lipids used in the preparation of lipid nanoparticles.

[0002] Lipids containing polyethylene glycol (sometimes abbreviated as "PEG" in this specification) chains (sometimes abbreviated as "PEG lipids" in this specification) are used in food products, pharmaceutical compositions, cosmetic compositions, and drug delivery. In particular, PEG lipids are used as components of liposome formulations and lipid nanoparticles (sometimes abbreviated as "LNPs" in this specification) for encapsulating drugs such as therapeutic agents and delivering them to the affected area.

[0003] LNPs include, for example, cationic lipids, phospholipids, sterols or their derivatives, and PEG lipids, the latter being present on the outer shell of such LNPs. PEG lipids are used to (i) prevent aggregation due to steric hindrance, (ii) prevent proteins from binding to LNPs, or (iii) mediate the indirect targeting function of LNPs. LNP formulations containing PEG lipids and drugs (e.g., nucleic acids) are called "stealth agents" because they can bypass the reticulocyte endothelial clearance system and enter systemic circulation.

[0004] Taking advantage of these properties, LNP preparations containing PEG lipids have been marketed as follows: In 2018, ONPATTRO, an siRNA-supported LNP, was approved by the U.S. Food and Drug Administration (FDA) as a treatment for transthyretin-type familial amyloid polyneuropathy. In 2020, as COVID-19 vaccine preparations, (i) Kominati, developed by BIONTECK and PFIZER, and (ii) Spikebax, developed by MODERNA, received Emergency Use Authorization (EUA) in the United States or the United Kingdom. The PEG lipids used in these preparations are 2-[(polyethylene glycol)-2000]-N,N-ditetradecylacetamide or 1,2-dimiristoyl-rac-glycero-3-methylpolyoxyethylene.

[0005] Preparations containing PEG lipids and drugs have been shown to cause an increase in anti-drug antibodies (ADAs), and among these ADAs, anti-PEG IgG and IgM are responsible for decreased efficacy due to accelerated drug clearance in the blood (ABC phenomenon) and hypersensitivity reactions (HSR) accompanied by severe allergic symptoms.

[0006] The following methods are known to mitigate the effects of anti-PEG antibodies (Non-Patent Literature 1): (a) a method to modify the terminal functional groups of PEG lipids; (b) a method to add immunomodulatory drugs such as ImmTOR particles containing rapamycin; (c) a method to change PEG to another polymer; (d) a method to extend the administration interval or reduce the dose; (e) a method to pre-administer empty PEG-modified liposomes, etc., to saturate existing anti-PEG antibodies; (f) a method to create special PEG-modified preparations such as PEG-modified dendrimers or micelles.

[0007] Nano Today 55 (2024) 102163

[0008] As mentioned above, methods for reducing anti-PEG antibody production are known, but the mechanism of anti-PEG antibody production is still under investigation. There is a need to develop methods that can avoid binding with existing anti-PEG antibodies in vivo, thereby avoiding accelerated blood clearance of PEG lipid-containing preparations and the occurrence of side effects such as injection reactions.

[0009] The present invention has been made in view of the above circumstances, and aims to provide a PEG lipid that can prepare LNPs that suppress the amount of anti-PEG antibody produced in vivo.

[0010] As a result of diligent research to achieve the above objective, the present inventors have found that by preparing LNPs using the following PEG lipids, the amount of anti-PEG antibody produced in vivo due to said LNPs can be suppressed. Based on this finding, the present invention is as follows.

[0011] [1] PEG lipid for the preparation of lipid nanoparticles, wherein formula (I): R １ -L １ -R ２ (I) (In equation (I), R １represents a zwitterionic site having a positive charge and a negative charge, and L １ represents a polyethylene glycol chain, and R ２ represents a lipid moiety. A PEG lipid represented by).

[0012] [2] Formula (Ia):

[0013]

[0014] (In formula (Ia), R １ is as defined above, n represents a number of 10 to 300, and R ２ａ represents any group represented by formulas (1) to (6):

[0015]

[0016] (In formulas (1) to (6), * represents a binding position, R ３ to R １３ each independently represent an aliphatic hydrocarbon group having 5 to 30 carbon atoms, L ２ , L ５ and L ６ each independently represent an alkylene group having 1 to 3 carbon atoms, and L ３ and L ４ each independently represent a single bond or an amide bond. The PEG lipid according to [1] above, which is represented by).

[0017] [3] R １ is any group represented by formulas (7) to (9):

[0018]

[0019] (In formulas (7) to (9), * represents a binding position, and L ７ to L ９ each independently represent an alkylene group having 1 to 3 carbon atoms. The PEG lipid according to [1] or [2] above, which is a group represented by any one of).

[0020] [4] Formulas (Ib) to (Ii):

[0021]

[0022] (In formulas (Ib) to (Ii), R １As stated above, n represents a number between 10 and 300, and R ３ ~R １３ Each of these independently represents an aliphatic hydrocarbon group having 5 to 30 carbon atoms.) A PEG lipid according to any one of the above [1] to [3], which is at least one selected from the group consisting of PEG lipids represented by any one of the above [1] to [3].

[0023] [5] Lipid nanoparticles containing the PEG lipid described in any one of [1] to [4] above. [6] A nucleic acid delivery agent containing the PEG lipid described in any one of [1] to [4] above. [7] The nucleic acid delivery agent described in [6] above, further comprising nucleic acid. [8] A method for introducing nucleic acids contained in the nucleic acid delivery agent into cells, comprising contacting the nucleic acid delivery agent described in [7] above with cells in vitro. [9] A method for introducing nucleic acids contained in the nucleic acid delivery agent into target cells in a living organism, comprising administering the nucleic acid delivery agent described in [7] above to a living organism.

[0024] LNPs prepared using the PEG lipids of the present invention can suppress the production of anti-PEG antibodies in vivo. As a result, these LNPs are expected to exert sufficient therapeutic effects. Furthermore, by reducing the production of anti-PEG antibodies, these LNPs are expected to avoid side effects such as injection reactions in the clinical drug administration process.

[0025] The present invention will be described in order below. Note that the descriptions herein can be combined with each other unless it is clearly stated that they cannot be combined.

[0026] The PEG lipid of the present invention is a PEG lipid for preparing lipid nanoparticles. Here, "PEG lipid for preparing lipid nanoparticles" means a PEG lipid used to prepare lipid nanoparticles. The PEG lipid of the present invention may be used alone or in combination of two or more types.

[0027] The PEG lipid of the present invention is of formula (I): R １ -L １ -R ２ (I) (In equation (I), R １L represents a zwitterionic moiety that has both positive and negative charges. １ represents the polyethylene glycol chain, and R ２ This represents the lipid portion. ) This is a PEG lipid indicated by .

[0028] In this specification and the claims (hereinafter referred to as "this specification"), "PEG lipid represented by formula (I)" may be abbreviated as PEG lipid (I). Other PEG lipids, compounds, or groups represented by other formulas may also be abbreviated in the same way as "PEG lipid represented by formula (I)". PEG lipid (I) may be used alone or in combination of two or more types.

[0029] Formula (I) is preferably formula (Ia):

[0030]

[0031] (In formula (Ia), R １ As stated above, n represents a number between 10 and 300, and R ２ａ Equations (1) to (6):

[0032]

[0033] (In equations (1) to (6), * indicates the bonding position, R ３ ~R １３ Each of these independently represents an aliphatic hydrocarbon group having 5 to 30 carbon atoms, L ２ , L ５ , and L ６ Each of these independently represents an alkylene group having 1 to 3 carbon atoms, and L ３ and L ４ Each of these independently represents either a single bond or an amide bond. ) represents a group represented by either ). That is, PEG lipid (I) is preferably PEG lipid (Ia). PEG lipid (Ia) may be used alone or in combination of two or more types. The definitions of the symbols in formula (Ia) will be explained in order below.

[0034] Formula (Ia), and formulas (1) to (6) are all skeletal structural formulas, and the carbon atoms (C) and hydrogen atoms (H) are omitted. However, the "*" in formulas (1) to (6) does not represent a carbon atom, but rather a bond position. Therefore, for example, "*-L ２ The "*-" in the expression indicates a single bond. The "*" in other expressions in this specification has the same meaning.

[0035] R in equation (Ia) １ R in equation (I) １ Similarly, it is a zwitterionic moiety that has both positive and negative charges.

[0036] In formula (Ia), n is the number of ethyleneoxy groups (i.e., the average polymerization of polyethylene glycol chains) and is a number from 10 to 300. Since n is the average degree of polymerization, it may be a decimal. To adjust the particle size of the LNP to a preferred range, n is preferably a number from 10 to 200, more preferably from 10 to 150, even more preferably from 10 to 150, and particularly preferably from 30 to 60.

[0037] In formula (Ia), R ２ａ is one of the groups (1) to (6), and preferably group (3).

[0038] R in equations (1) to (6) ３ ~R １３ Each of these is independently an aliphatic hydrocarbon group having 5 to 30 carbon atoms, preferably an alkyl group having 5 to 30 carbon atoms, an alkenyl group having 5 to 30 carbon atoms, or an alkynyl group having 5 to 30 carbon atoms, more preferably an alkyl group having 5 to 30 carbon atoms, and particularly preferably an alkyl group having 6 to 26 carbon atoms.

[0039] In this specification, alkyl groups may be linear or branched. Examples of alkyl groups include pentyl, isopentyl, neopentyl, tert-pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, henicosyl, docosyl, tricosyl, tetracosyl, pentacosyl, hexacosyl, heptacosyl, octacosyl, nonacosyl, and triacontyl groups.

[0040] In this specification, the alkenyl group may be linear or branched. Also, in this specification, the number of olefinic carbon-carbon double bonds in the alkenyl group may be only one or two or more. Examples of alkenyl groups include pentenyl, hexenyl, heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icocenyl, henicocenyl, dococenyl, tricocenyl, tetracocenyl, pentacocenyl, hexacocenyl, heptacocenyl, octacocenyl, nonacocenyl, and triacontenyl groups.

[0041] In this specification, the alkynyl group may be linear or branched. Also, in this specification, the number of carbon-carbon triple bonds in the alkynyl group may be only one or two or more. Examples of alkynyl groups include pentynyl, hexynyl, heptynyl, octinyl, noninyl, desinyl, undecynyl, dodecynyl, tridecynyl, tetradecynyl, pentadecynyl, hexadecynyl, heptadecynyl, octadecynyl, nonadesynyl, icosinyl, henicosinyl, docosinyl, tricosinyl, tetracosinyl, pentacosinyl, hexacosinyl, heptacosinyl, octacosinyl, nonacosinyl, and triacontinyl groups.

[0042] L in equation (1) ２, L in equation (4) ５ , and L in formula (6) ６ Each of these is an alkylene group having 1 to 3 carbon atoms. In this specification, the alkylene group may be linear or branched. Examples of alkylene groups include a methylene group, an ethylene group, and a trimethylene group (-(CH4). ２ ) ３ -), propylene group (-CH(CH ３ )CH ２ -ien-CH ２ CH (CH ３ )-) are examples (the "-" in the above formula represents a single bond). L ２ , L ５ , and L ６ Preferably, each of these is independently a methylene group, an ethylene group, or a trimethylene group.

[0043] L in equation (2) ３ and L in equation (3) ４ Each of these is independently a single bond or an amide bond (*-CO-NH-*). Here, "L ３ "It is a single bond" means that in equation (2) "*-L ３ -CH ２ " is "*-CH ２ This means "L ４ "It is a single bond" means that in equation (3) "*-L ４ -CH ２ " is "*-CH ２ This means "L". ３ If the bond is an amide bond, the imino group (NH) is preferably the methylene group (CH) in formula (2). ２ ) combines with L. ４ If the bond is an amide bond, the imino group (NH) is preferably the methylene group (CH) in formula (3). ２ It combines with ).

[0044] In equations (I) and (Ia), R １ Preferably, the group is represented by any of the following formulas (7) to (9):

[0045]

[0046] (In equations (7) to (9), * indicates the bonding position, and L ７ ~L ９ Each of these independently represents an alkylene group with 1 to 3 carbon atoms.

[0047] L ７ ~L ９ Preferably, each is independently a methylene group, an ethylene group, or a trimethylene group, and more preferably, each is independently an ethylene group or a trimethylene group.

[0048] In one embodiment of the present invention, R １ L is preferably group (7) or group (8), and more preferably group (7). In this embodiment, L is in formulas (7) and (8). ７ and L ８ The explanation is as stated above.

[0049] In one embodiment of the present invention, the PEG lipid of the present invention is preferably at least one selected from the group consisting of PEG lipids represented by any of the following formulas (Ib) to (Ii), more preferably at least one selected from the group consisting of PEG lipid (Ib), PEG lipid (Ic), PEG lipid (Ie), and PEG lipid (Ii), and even more preferably PEG lipid (Ie):

[0050]

[0051] R in equations (Ib) to (Ii) １ , R ３ ~R １３ The explanations for , and n are as described above. Note that although "n" is used to represent the number of ethyleneoxy groups in formulas (Ib) to (Ii), the "n" in formula (Ib) to the "n" in formula (Ii) may be the same or different.

[0052] In one embodiment of the present invention, the PEG lipid of the present invention is preferably at least one selected from the group consisting of PEG lipids represented by any of the following formulas (I-1) to (I-24), more preferably at least one selected from the group consisting of PEG lipid (I-4), PEG lipid (I-5), PEG lipid (I-6), PEG lipid (I-7), and PEG lipid (I-8), even more preferably at least one selected from the group consisting of PEG lipid (I-4), PEG lipid (I-5), PEG lipid (I-7), and PEG lipid (I-8), particularly preferably at least one selected from the group consisting of PEG lipid (I-4), PEG lipid (I-5), and PEG lipid (I-8), and most preferably PEG lipid (I-4).

[0053]

[0054]

[0055]

[0056]

[0057]

[0058]

[0059]

[0060]

[0061]

[0062]

[0063]

[0064]

[0065]

[0066]

[0067]

[0068]

[0069]

[0070]

[0071]

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] The explanation for n in formulas (I-1) to (I-24) is as described above. Note that while "n" is used to represent the number of ethyleneoxy groups in all of formulas (I-1) to (I-24), the "n" in formulas (I-1) to (I-24) may be the same or different.

[0078] Next, the method for producing PEG lipids according to the present invention will be described. As an example of the method for producing PEG lipid (I), (α)HO-L １ -R ２ One method involves producing an alcohol (i.e., an alcohol having a PEG chain and a lipid moiety) and then adding a zwitterionic moiety to it, or synthesizing a compound having a (β) zwitterionic moiety and a PEG chain, and a compound having a lipid moiety, and then coupling them together. Furthermore, PEG lipid (I) can be produced by the synthesis method described in the examples below or a similar synthesis method.

[0079] For example, as shown in the following synthesis route, the compound represented by the following formula (R1) (i.e., R ２ａ A PEG lipid having a (lipid moiety) and a compound represented by the following formula (R2) (i.e., R １ａ By reacting a carboxylic acid (having a zwitterionic moiety with positive and negative charges) with the following formula (II-1), a PEG lipid (i.e., R １ａ(In the formula below, n and R are the same.) ２ａ As stated above, and also R １ａ This represents a zwitterionic moiety that possesses both positive and negative charges.

[0080]

[0081] The ratio of compound (R2) is preferably 1 to 50 mol, and more preferably 1 to 10 mol, per 1 mol of compound (R1).

[0082] The reaction between compound (R1) and compound (R2) may be carried out using a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, or 4-dimethylaminopyridine (hereinafter sometimes abbreviated as "DMAP"), or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid. The reaction may also be carried out without a catalyst.

[0083] When a catalyst is used, its amount ratio is preferably 0.05 to 100 mol, more preferably 0.1 to 20 mol, and even more preferably 0.1 to 5 mol, per 1 mol of compound (R1).

[0084] Alternatively, compound (R1) and compound (R2) may be reacted directly using a condensing agent such as dicyclohexylcarbodiimide (hereinafter sometimes abbreviated as "DCC"), diisopropylcarbodiimide (hereinafter sometimes abbreviated as "DIC"), or 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (hereinafter sometimes abbreviated as "EDC hydrochloride"), or compound (R2) may be converted to an acid anhydride or the like using a condensing agent and then reacted with compound (R1).

[0085] The solvent used in the reaction between compound (R1) and compound (R2) can be any solvent that does not inhibit the reaction, and is not particularly limited. Examples of solvents include water, dichloromethane, chloroform, acetonitrile, and toluene. Among these, chloroform and toluene are preferred.

[0086] The temperature of the above reaction is preferably 0 to 150°C, more preferably 0 to 80°C, and still more preferably 10 to 50°C. The reaction time is preferably 1 to 48 hours, more preferably 1 to 24 hours.

[0087] The PEG lipid (II-1) obtained by the above reaction can be appropriately purified by common purification methods such as extraction purification, recrystallization, adsorption purification, reprecipitation, column chromatography, and ion exchange chromatography.

[0088] For example, R １ is *-CO-O － and *-N ＋ (CH ３ ) ２ -*, which is a zwitterionic moiety, the PEG lipid represented by the following formula (II-3) can be produced by the following synthetic route (in the following formula, n and R ２ａ are as defined above, R １ｂ and R １ｃ each represent a divalent organic group that does not inhibit the following reaction, and R １４ represents a leaving group (e.g., chlorine atom, bromine atom, iodine atom, mesyloxy group, tosyloxy group)).

[0089]

[0090] As shown in the above synthetic route, first, compound (R1) is reacted with compound (R3) to obtain a PEG lipid (II-2) having a dimethylamino group and R ２ａ (lipid moiety), and then the PEG lipid (II-2) is reacted with compound (R4) having a carboxy group and a leaving group R １４ to obtain the PEG lipid (II-3).

[0091] The quantitative ratio of compound (R3) is preferably 1 to 50 mol, more preferably 1 to 10 mol, per 1 mol of compound (R1).

[0092] The reaction between compound (R1) and compound (R3) may be carried out using a base catalyst such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, or DMAP, or an acid catalyst such as p-toluenesulfonic acid or methanesulfonic acid. The reaction may also be carried out without a catalyst.

[0093] When a catalyst is used, its amount ratio is preferably 0.05 to 100 mol, more preferably 0.1 to 20 mol, and even more preferably 0.1 to 5 mol, per 1 mol of compound (R1).

[0094] Alternatively, compound (R1) and compound (R3) may be reacted directly using a condensing agent such as DCC, DIC, or EDC hydrochloride, or compound (R3) may be converted to an anhydride or the like using a condensing agent and then reacted with compound (R1).

[0095] The solvent used in the reaction between compound (R1) and compound (R3) can be any solvent that does not inhibit the reaction, and is not particularly limited. Examples of solvents include ethyl acetate, dichloromethane, chloroform, acetonitrile, and toluene. Among these, chloroform and toluene are preferred.

[0096] The reaction temperature between compound (R1) and compound (R2) is preferably 0 to 150°C, more preferably 0 to 80°C, and even more preferably 10 to 50°C. The reaction time between compound (R1) and compound (R2) is preferably 1 to 48 hours, more preferably 1 to 24 hours.

[0097] The obtained PEG lipid (II-2) can be purified as appropriate by common purification methods such as extraction, recrystallization, adsorption, reprecipitation, column chromatography, and ion exchange chromatography.

[0098] The amount ratio of compound (R4) is preferably 1 to 50 mol, and more preferably 1 to 10 mol, per 1 mol of PEG lipid (II-2).

[0099] Bases such as potassium carbonate, sodium carbonate, potassium hydroxide, triethylamine, DMAP, and diisopropylamine may be used in the reaction between PEG lipid (II-2) and compound (R4).

[0100] The solvent used in the reaction between PEG lipid (II-2) and compound (R4) can be any solvent that does not inhibit the reaction, and is not particularly limited. Examples of solvents include water, ethyl acetate, dichloromethane, chloroform, acetonitrile, toluene, and tetrahydrofuran. Among these, acetonitrile, tetrahydrofuran, and toluene are preferred.

[0101] The reaction temperature between PEG lipid (II-2) and compound (R4) is preferably 0 to 150°C, more preferably 10 to 100°C. The reaction time between PEG lipid (II-2) and compound (R4) is preferably 1 to 48 hours, more preferably 1 to 24 hours.

[0102] The present invention also provides lipid nanoparticles containing the PEG lipid of the present invention. The lipid nanoparticles of the present invention may further contain nucleic acids.

[0103] The content of the PEG lipid of the present invention in the lipid nanoparticles of the present invention is not particularly limited, but preferably, it contains a sufficient amount of the PEG lipid of the present invention to stabilize the lipid nanoparticles when the lipid nanoparticles are used as a nucleic acid delivery agent as described later. The content of the PEG lipid of the present invention in the lipid nanoparticles of the present invention is preferably 0.1 to 10 mol%, more preferably 0.1 to 8 mol%, and even more preferably 0.1 to 5 mol%, relative to the total lipids.

[0104] The lipid nanoparticles of the present invention may further contain other components in addition to the PEG lipids of the present invention. Examples of such other components include other lipids different from the PEG lipids of the present invention (phospholipids, glycolipids, peptide lipids, sterols or their derivatives, cationic lipids, other PEG lipids different from the PEG lipids of the present invention, etc.), surfactants (e.g., 3-[(3-colamidopropyl)dimethylammonio]propanesulfonate, sodium cholate, octyl glycoside, N-D-gluco-N-methylalkaneamides, etc.), proteins, and the like. The content of other lipids in the lipid nanoparticles of the present invention is preferably 90 to 99.9 mol%, more preferably 92 to 99.9 mol%, and even more preferably 95 to 99.9 mol%, relative to the total lipids.

[0105] The lipid nanoparticles of the present invention can be prepared by dispersing the PEG lipids and other constituent components (lipids, etc.) of the present invention in a suitable solvent or dispersion medium, such as an aqueous solvent or an alcoholic solvent, and performing an operation to induce organization as necessary.

[0106] "Operations that induce organization" include, for example, ethanol dilution using microfluidics or vortexing, simple hydration, sonication, heating, vortexing, ether injection, French press method, cholic acid method, Ca ２＋ Examples of known methods include, but are not limited to, the fusion method, the freeze-thaw method, and the reverse-phase evaporation method.

[0107] The present invention provides a nucleic acid delivery agent containing the PEG lipid of the present invention. The nucleic acid delivery agent of the present invention is preferably a lipid nanoparticle of the present invention. The nucleic acid delivery agent of the present invention preferably contains nucleic acid. By bringing the nucleic acid delivery agent of the present invention containing nucleic acid into contact with cells, the nucleic acid can be introduced into the cells in vivo and / or in vivo.

[0108] The nucleic acid delivery agent of the present invention can introduce any nucleic acid into cells. Examples of nucleic acid types include, but are not limited to, DNA, RNA, RNA chimeric nucleic acids, and DNA / RNA hybrids. Furthermore, any nucleic acid with one to three strands can be used, but single-stranded or double-stranded nucleic acids are preferred. The nucleic acid may be other types of nucleotides that are N-glycosides of purine or pyrimidine bases, or other oligomers having a non-nucleotide backbone (e.g., commercially available peptide nucleic acids (PNA), etc.), or other oligomers having special bonds (provided that the oligomer contains nucleotides having a configuration that allows for base pairing and base attachment, as found in DNA and RNA). Furthermore, the nucleic acid may be, for example, a nucleic acid with known modifications, a nucleic acid with a label known in the art, a capped nucleic acid, a methylated nucleic acid, a nucleic acid in which one or more natural nucleotides are replaced with analogs, a nucleic acid with intramolecular nucleotide modifications, a nucleic acid having an uncharged bond (e.g., methyl sulfonate, phosphotriester, phosphoramidate, carbamate, etc.), a nucleic acid having a charged bond or a sulfur-containing bond (e.g., phosphorothioate, phosphorodithioate, etc.), a nucleic acid having a side chain group such as a protein (e.g., nuclease, nuclease inhibitor, toxin, antibody, signal peptide, poly-L-lysine, etc.) or a sugar (e.g., monosaccharide, etc.), a nucleic acid having an intercurrent compound (e.g., acridine, psoralen, etc.), a nucleic acid containing a chelating compound (e.g., metal, radioactive metal, boron, oxidizing metal, etc.), a nucleic acid containing an alkylating agent, a nucleic acid having a modified bond (e.g., α-anomeric nucleic acid, etc.), etc.

[0109] The type of DNA that can be used in the present invention is not particularly limited and can be appropriately selected depending on the purpose of use. Examples include plasmid DNA, cDNA, antisense DNA, chromosomal DNA, PAC, BAC, CpG oligo, etc. Plasmid DNA, cDNA, and antisense DNA are preferred, and plasmid DNA is more preferred. Circular DNA such as plasmid DNA can be digested with restriction enzymes as appropriate and used as linear DNA.

[0110] The types of RNA that can be used in the present invention are not particularly limited and can be appropriately selected depending on the purpose of use. Examples include siRNA, miRNA, shRNA, antisense RNA, messenger RNA (mRNA), single-stranded RNA genome, double-stranded RNA genome, RNA replicon, transfer RNA, ribosomal RNA, etc. Preferably, siRNA, miRNA, shRNA, mRNA, antisense RNA, and RNA replicon.

[0111] In the present invention, the nucleic acids used are preferably purified by methods commonly used by those skilled in the art.

[0112] The nucleic acid-containing nucleic acid-transfer agents of the present invention can be administered in vivo, for example, for the purpose of preventing and / or treating diseases. Therefore, the nucleic acids used in the present invention are preferably those that have preventive and / or therapeutic activity against a certain disease (preventive and therapeutic nucleic acids). Examples of such nucleic acids include those used in so-called gene therapy.

[0113] To introduce nucleic acids into cells using the nucleic acid delivery agent of the present invention, for example, the target nucleic acid is made to coexist with the lipid nanoparticles of the present invention when forming the lipid nanoparticles of the present invention, thereby forming lipid nanoparticles of the present invention containing the nucleic acid (i.e., the nucleic acid delivery agent of the present invention containing the nucleic acid). For example, when forming liposomes as a lipid membrane structure by the ethanol dilution method, an aqueous solution of nucleic acid and an ethanol solution of the constituent components (lipids, etc.) of the lipid nanoparticles of the present invention are vigorously mixed using a vortex or microchannel, and then the mixture is diluted with an appropriate buffer. When forming liposomes as a lipid membrane structure by the simple hydration method, the constituent components (lipids, etc.) of the lipid nanoparticles of the present invention are dissolved in an appropriate organic solvent, the solution is placed in a glass container, and the solvent is removed by vacuum drying to obtain a lipid thin film. An aqueous solution of nucleic acid is then added and hydrated, and then sonicated with a sonicator.

[0114] One form of the lipid nanoparticles of the present invention containing nucleic acids is a lipid nanoparticle in which nucleic acids are encapsulated by forming an electrostatic complex between the nucleic acid and a cationic lipid. These lipid nanoparticles can be used as a drug delivery system for selectively delivering nucleic acids and the like into specific cells, and are useful, for example, for DNA vaccines and tumor gene therapies that introduce antigen genes into dendritic cells, and for nucleic acid drugs that suppress the expression of target genes using RNA interference.

[0115] The lipid nanoparticles of the present invention may contain cationic lipids. In this specification, "cationic lipid" means a lipid having an amino group that is cationized at physiological pH. The present invention is not particularly limited to cationic lipids, and known cationic lipids can be used. Only one type of cationic lipid may be used, or two or more types may be used in combination.

[0116] Examples of cationic lipids include those described in WO2010 / 054401, WO2013 / 073480, WO2016 / 027699, WO2016 / 121942, WO2017 / 075531, WO2018 / 119163, WO2019 / 188867, WO2020 / 219876, WO2021 / 055833, WO2021 / 055835, WO2021 / 095838, WO2021 / 193397, WO2023 / 190166, WO2024 / 203577, etc. Note that "cationic lipids" may be referred to as "ionizable amine lipids," "cationic / ionic lipids," "cationic and / or ionic lipids," or "ionic lipids" in some literature.

[0117] The cationic lipid is preferably DLin-M-C3-DMA as described in Figure 3 (1 of 2) of WO2010 / 054401. The structure of DLin-M-C3-DMA is as follows:

[0118]

[0119] When the lipid nanoparticles of the present invention contain cationic lipids, their content is preferably 20 to 75 mol%, more preferably 25 to 70 mol%, and even more preferably 35 to 65 mol%, relative to the total lipids, from the viewpoint of nucleic acid encapsulation rate, intracellular nucleic acid release efficiency, and stability of the lipid nanoparticles.

[0120] The lipid nanoparticles of the present invention may contain sterols or their derivatives as lipids. One sterol or its derivative may be used alone, or two or more may be used in combination. Examples of sterols or their derivatives include cholesterol, lanosterol, phytosterol, dimosterol, dimostenol, desmosterol, stigmastanol, dihydrolanosterol, and 7-dehydrocholesterol, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, etc. The sterol or its derivative is preferably cholesterol.

[0121] When the lipid nanoparticles of the present invention contain sterols or their derivatives (preferably cholesterol), the content thereof is preferably 10 to 70 mol%, more preferably 20 to 60 mol%, and even more preferably 25 to 55 mol%, relative to the total lipids, from the viewpoint of nucleic acid encapsulation rate, intracellular nucleic acid release efficiency, and stability of the lipid nanoparticles.

[0122] The lipid nanoparticles of the invention may contain phospholipids. Only one type of phospholipid may be used, or two or more types may be used in combination. There are no particular limitations on the phospholipids, and known phospholipids used in lipid nanoparticles can be used.

[0123] Examples of phospholipids include 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphoserine (PS), 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA), and their lyso compounds.

[0124] Specific examples of 1,2-diacyl-sn-glycero-3-phosphocholine (PC) include the following: 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimiristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-dielcoyl-sn-glycero-3-phosphocholine (DEPC), 1-Myristoyl-2-Palmitoyl-sn-Glycerol-3-Phosphocholine (MPPC), 1-Myristoyl-2-Stearoyl-sn-Glycerol-3-Phosphocholine (MSPC), 1-Palmitoyl-2-Myristoyl-sn-Glycerol-3-Phosphocholine (PMPC), 1-Palmitoyl-2-Stearoyl-sn-Glycerol-3-Phosphocholine (PSPC), 1-Palmitoyl-2-Oleoyl-sn-Glycerol-3-Phosphocholine (POPC), 1-Stearoyl-2-Oleoyl-sn-Glycerol-3-Phosphocholine (SOPC).

[0125] In this specification, phospholipids may be referred to by their abbreviations. For example, 1,2-diacyl-sn-glycero-3-phosphocholine may be referred to as PC, and 1,2-didecanoyl-sn-glycero-3-phosphocholine may be referred to as DDPC.

[0126] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoethanolamine (PE) include the following: 1,2-didecanoyl-sn-glycero-3-phosphoethanolamine (DDPE), 1,2-dilauroyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLoPE), 1,2-dielcoyl-sn-glycero-3-phosphoethanolamine (DEPE), 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphoethanolamine (MPPE), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (MSPE), 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphoethanolamine (PMPE), 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphoethanolamine (PSPE), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (SOPE).

[0127] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoserine (PS) include the following: 1,2-didecanoyl-sn-glycero-3-phosphoserine (DDPS), 1,2-dilauroyl-sn-glycero-3-phosphoserine (DLPS), 1,2-dimiristoyl-sn-glycero-3-phosphoserine (DMPS), 1,2-dipalmitoyl-sn-glycero-3-phosphoserine (DPPS), 1,2-distearoyl-sn-glycero-3-phosphoserine (DSPS), 1,2-dioleoyl-sn-glycero-3-phosphoserine (DOPS), 1,2-dilinoleoyl-sn-glycero-3-phosphoserine (DLoPS), 1,2-Diellcoil-sn-glycero-3-phosphoserine (DEPS), 1-Myristoyl-2-Palmitoyl-sn-glycero-3-phosphoserine (MPPS), 1-Myristoyl-2-Stearoyl-sn-glycero-3-phosphoserine (MSPS), 1-Palmitoyl-2-Myristoyl-sn-glycero-3-phosphoserine (PMPS), 1-Palmitoyl-2-Stearoyl-sn-glycero-3-phosphoserine (PSPS), 1-Palmitoyl-2-Oleoyl-sn-glycero-3-phosphoserine (POPS), 1-Stearoyl-2-Oleoyl-sn-glycero-3-phosphoserine (SOPS).

[0128] Specific examples of 1,2-diacyl-sn-glycero-3-phosphoglycerol (PG) include the following: 1,2-didecanoyl-sn-glycero-3-phosphoglycerol (DDPG), 1,2-dilauroyl-sn-glycero-3-phosphoglycerol (DLPG), 1,2-dimiristoyl-sn-glycero-3-phosphoglycerol (DMPG), 1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol (DPPG), 1,2-distearoyl-sn-glycero-3-phosphoglycerol (DSPG), 1,2-dioleoyl-sn-glycero-3-phosphoglycerol (DOPG), 1,2-dilinoleoyl-sn-glycero-3-phosphoglycerol (DLoPG), 1,2-Diellcoil-sn-glycero-3-phosphoglycerol (DEPG), 1-Myristoyl-2-Palmitoyl-sn-glycero-3-phosphoglycerol (MPPG), 1-Myristoyl-2-Stearoyl-sn-glycero-3-phosphoglycerol (MSPG), 1-Palmitoyl-2-Myristoyl-sn-glycero-3-phosphoglycerol (PMPG), 1-Palmitoyl-2-Stearoyl-sn-glycero-3-phosphoglycerol (PSPG), 1-Palmitoyl-2-Oleoyl-sn-glycero-3-phosphoglycerol (POPG), 1-Stearoyl-2-Oleoyl-sn-glycero-3-phosphoglycerol (SOPG).

[0129] Specific examples of 1,2-diacyl-sn-glycero-3-phosphatidic acid (PA) include the following: 1,2-didecanoyl-sn-glycero-3-phosphatidic acid (DDPA), 1,2-dilauroyl-sn-glycero-3-phosphatidic acid (DLPA), 1,2-dimiristoyl-sn-glycero-3-phosphatidic acid (DMPA), 1,2-dipalmitoyl-sn-glycero-3-phosphatidic acid (DPPA), 1,2-distearoyl-sn-glycero-3-phosphatidic acid (DSPA), 1,2-dioleoyl-sn-glycero-3-phosphatidic acid (DOPA), 1,2-dilinoleoyl-sn-glycero-3-phosphatidic acid (DLoPA), 1,2-Diellicyl-sn-glycero-3-phosphatidic acid (DEPA), 1-Myristoyl-2-palmitoyl-sn-glycero-3-phosphatidic acid (MPPA), 1-Myristoyl-2-stearoyl-sn-glycero-3-phosphatidic acid (MSPA), 1-Palmitoyl-2-myristoyl-sn-glycero-3-phosphatidic acid (PMPA), 1-Palmitoyl-2-stearoyl-sn-glycero-3-phosphatidic acid (PSPA), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (POPA), 1-Stearoyl-2-oleoyl-sn-glycero-3-phosphatidic acid (SOPA).

[0130] The phospholipid is preferably at least one selected from the group consisting of DOPC, DSPC, and DPPC, and more preferably DOPC.

[0131] When the lipid nanoparticles of the present invention contain phospholipids, the amount of phospholipids is preferably 2 to 50 mol%, more preferably 3 to 40 mol%, and even more preferably 5 to 30 mol%, relative to the total lipids, from the viewpoint of nucleic acid encapsulation rate, intracellular nucleic acid release efficiency, and stability of the lipid nanoparticles.

[0132] The particle size of the lipid nanoparticles of the present invention containing nucleic acids is not particularly limited, but is preferably 10 nm to 500 nm, and more preferably 30 nm to 400 nm. The particle size can be measured using a particle size distribution analyzer such as Zetasizer Nano (manufactured by Malvern). The particle size of the lipid nanoparticles can be appropriately adjusted by the method of preparing the lipid nanoparticles.

[0133] The surface potential (zeta potential) of the lipid nanoparticles of the present invention containing nucleic acids is not particularly limited, but is preferably -25 mV to +25 mV, and more preferably -20 mV to +20 mV. In conventional gene transfer, particles with a positively charged surface potential have been mainly used. This is useful as a method to promote electrostatic interaction with heparin sulfate on the negatively charged cell surface and promote uptake into cells, but a positive surface potential may suppress the release of nucleic acids from carriers through interaction with delivery nucleic acids within cells, and may also suppress protein synthesis through interaction between mRNA and delivery nucleic acids. This problem can be solved by adjusting the surface potential within the above range. Surface potential can be measured using a zeta potential measuring device such as Zetasizer Nano. The surface potential of lipid nanoparticles can be adjusted by the composition of their constituent components.

[0134] By bringing a nucleic acid delivery agent of the present invention (preferably lipid nanoparticles of the present invention containing nucleic acids) into contact with cells, the nucleic acids contained in the nucleic acid delivery agent can be introduced into the cells. Accordingly, the present invention also provides a method for introducing nucleic acids contained in the nucleic acid delivery agent into cells, which includes bringing the nucleic acid delivery agent of the present invention containing nucleic acids into contact with cells.

[0135] The type of cell is not particularly limited, and prokaryotes and eukaryotes can be used, but eukaryotes are preferred. The type of eukaryote is also not particularly limited, and examples include vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), plants, microorganisms (e.g., yeast, etc.). More preferably, the cells targeted in the present invention are animal or plant cells, and even more preferably mammalian cells. The cells may be cultured cell lines including cancer cells, cells isolated from individuals or tissues, or cells from tissues or tissue fragments. Furthermore, the cells may be adherent cells or non-adherent cells.

[0136] The process of bringing the lipid nanoparticles of the present invention, which contain nucleic acids, into contact with cells in vitro will be described in detail below.

[0137] The cells are suspended in a suitable culture medium and cultured under appropriate conditions several days before contact with the lipid nanoparticles. At the time of contact with the lipid nanoparticles, the cells may or may not be in the proliferation phase.

[0138] The culture medium used at the time of contact may be either a serum-containing medium or a serum-free medium, but the serum concentration in the medium is preferably 30% by weight or less, and more preferably 20% by weight or less. If the medium contains an excess of serum or other proteins, contact between the lipid nanoparticles and the cells may be inhibited.

[0139] The cell density at the time of contact is not particularly limited and can be set appropriately considering the type of cell, etc., but is usually 1 × 10⁻⁶ ４ ~1 x 10 ７ This is within the range of cells / mL.

[0140] A suspension of lipid nanoparticles of the present invention containing nucleic acids (i.e., nucleic acid delivery agent of the present invention containing nucleic acids) is added to the cells. The amount of the suspension added is not particularly limited and can be set appropriately considering the number of cells, etc. The concentration of lipid nanoparticles when brought into contact with the cells is not particularly limited as long as the introduction of the target nucleic acid into the cells can be achieved, but the lipid concentration is usually 1 to 100 nmol / mL, preferably 10 to 50 nmol / mL, and the nucleic acid concentration is usually 0.01 to 100 μg / mL, preferably 0.1 to 10 μg / mL.

[0141] After adding the above-mentioned suspension to the cells, the cells are cultured. The temperature, humidity, and CO2 levels during culture are controlled. ２ The concentration and other parameters should be set appropriately considering the type of cell. If the cells are of mammalian origin, the temperature should be approximately 37°C, the humidity approximately 95%, and CO2. ２ The concentration is approximately 5% by volume. The culture time can also be set appropriately considering conditions such as the type of cells used, but it is usually in the range of 0.1 to 76 hours, preferably in the range of 0.2 to 24 hours, and more preferably in the range of 0.5 to 12 hours. If the culture time is too short, nucleic acids will not be sufficiently introduced into the cells, and if the culture time is too long, the cells may weaken.

[0142] Through the culture described above, nucleic acids are introduced into the cells. Preferably, the culture medium is replaced with fresh medium, or fresh medium is added to the medium and the culture is continued. If the cells are of mammalian origin, the fresh medium preferably contains serum or trophic factors.

[0143] Furthermore, as described above, by using the nucleic acid delivery agent of the present invention which contains nucleic acids, it is possible to introduce nucleic acids into cells not only in vitro but also in vivo. That is, by administering the nucleic acid delivery agent of the present invention which contains nucleic acids to a living organism, the nucleic acids contained in the nucleic acid delivery agent can be introduced into target cells in the living organism. Accordingly, the present invention also provides a method for introducing nucleic acids contained in the nucleic acid delivery agent into target cells in the living organism, which includes administering the nucleic acid delivery agent of the present invention which contains nucleic acids to a living organism.

[0144] The organisms to which the nucleic acid-containing nucleic acid-introducing agent of the present invention can be administered are not particularly limited, and include, for example, vertebrates such as mammals (e.g., humans, monkeys, mice, rats, hamsters, cows, etc.), birds (e.g., chickens, ostriches, etc.), amphibians (e.g., frogs, etc.), fish (e.g., zebrafish, medaka, etc.), invertebrates such as insects (e.g., silkworms, moths, fruit flies, etc.), and plants. The organism to which the nucleic acid-containing nucleic acid-introducing agent of the present invention can be administered is preferably a human or other mammal.

[0145] The type of target cell is not particularly limited, and by using the nucleic acid delivery agent of the present invention, which contains nucleic acids, it is possible to introduce nucleic acids into cells in various tissues (for example, liver, kidney, pancreas, lung, spleen, heart, blood, muscle, bone, brain, stomach, small intestine, large intestine, skin, adipose tissue, lymph nodes, tumors, etc.).

[0146] The method of administering the nucleic acid-containing nucleic acid-transfer agent of the present invention to a living organism is not particularly limited, and known administration methods (for example, oral administration, parenteral administration (for example, intravenous administration, intramuscular administration, local administration, transdermal administration, subcutaneous administration, intraperitoneal administration, spray, etc.)) can be appropriately selected. The dosage of the nucleic acid-containing nucleic acid-transfer agent of the present invention is not particularly limited, and can be appropriately selected considering the type of living organism to be administered to, the method of administration, the type and site of target cells, etc.

[0147] When the lipid nanoparticles of the present invention are used as nucleic acid delivery agents, they can be formulated according to conventional methods.

[0148] When the nucleic acid delivery agent of the present invention, which is a lipid nanoparticle of the present invention, is provided as a research reagent, it may be provided as is, or as a sterile solution or suspension of the lipid nanoparticle of the present invention with, for example, water or other physiologically acceptable liquid (e.g., water-soluble solvent (e.g., malic acid buffer), organic solvent (e.g., ethanol, methanol, dimethyl sulfoxide (DMSO), tert-butanol, etc.), or a mixture of a water-soluble solvent and an organic solvent). The nucleic acid delivery agent of the present invention may optionally contain physiologically acceptable additives known to the present (e.g., excipients, vehicles, preservatives, stabilizers, binders, etc.).

[0149] Furthermore, when the nucleic acid delivery agent of the present invention, which is a lipid nanoparticle of the present invention, is provided as a pharmaceutical product, the nucleic acid delivery agent of the present invention can be produced as an oral preparation (e.g., a tablet, a capsule, etc.) or a parenteral preparation (e.g., an injection, a spray, etc.), preferably as a parenteral preparation (more preferably as an injection), by using the lipid nanoparticles of the present invention as they are, or by using the lipid nanoparticles of the present invention together with pharmaceutically acceptable known additives (e.g., carriers, flavoring agents, excipients, vehicles, preservatives, stabilizers, binders, etc.) and mixing them in a unit dose form required for generally accepted formulation.

[0150] The nucleic acid delivery agent of the present invention can also be provided in the form of a kit. The kit may include reagents used for nucleic acid delivery in addition to the PEG lipid or lipid nanoparticles of the present invention. In one embodiment, the nucleic acid delivery agent (or kit) of the present invention further comprises a polycation (e.g., protamine). By using the nucleic acid delivery agent (or kit) of the present invention in this embodiment, an electrostatic complex between nucleic acid and polycation (e.g., protamine) can be easily encapsulated in the lipid nanoparticles of the present invention, and the nucleic acid can be delivered into cells.

[0151] The present invention will be described in more detail below using examples and test examples, but the present invention is not limited thereto. In the following examples and test examples, "%" means "weight percent" unless otherwise specified.

[0152] The meanings of the abbreviations used in the description of the examples are as follows: BSA: Bovine serum albumin Chol: Cholesterol COP: 2-chloro-2-oxo-1,3,2-dioxaphosphoran DCC: N,N'-dicyclohexylcarbodiimide DIPA: Diisopropylamine DMAP: 4-dimethylaminopyridine DOPC: 1,2-dioleoyl-sn-glycero-3-phosphocholine EDC: 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride GAn: Glutaric anhydride LNP: Lipid nanoparticles MNBA: 2-methyl-6-nitrobenzoic anhydride mRNA: Messenger RNA MyA: Myristic acid NMe ３ : Trimethylamine PBS: Phosphate-buffered saline

[0153] Table 1 shows the names and structures of the PEG lipids produced in the examples. Table 2 shows the structures of the comparative PEG lipids. Comparative compounds 1 and 2 were "SUNBRIGHT GS-020" and "SUNBRIGHT GM-020" manufactured by NOF Corporation, respectively, and comparative compound 3 was intermediate 1, described below. The values ​​of n in compounds 1 to 5 and comparative compounds 1 to 3 below are values ​​calculated from the number-average molecular weight of each compound. The number-average molecular weights were all calculated using matrix-assisted laser desorption / ionization-time of flight mass spectrometry (MALDI-TOF MS).

[0154]

[0155]

[0156] [Example 1-1] Synthesis of Compound 1 Compound 1 was synthesized by the following synthesis route, but the present invention is not limited to this synthesis route. In the following formula, n is 41.

[0157]

[0158] Intermediate 1 (3.0 g, 1.19 mmol), synthesized by the same method as described in WO2022 / 065263, was dissolved in acetonitrile and cooled with ice water. DIPA (242 mg, 2.39 mmol) and COP (255 mg, 1.79 mmol) were added to the mixture, and the mixture was stirred for 1 hour. After filtering off impurities produced in the reaction, 2 M NMe was added to the resulting filtrate. ３ The mixture was reacted with an acetonitrile solution at 50°C for 2 hours. The mixture was purified by column chromatography (silica gel particle size: 30 μm, chloroform:methanol:water = 79:20:1 (volume ratio)) to obtain a white compound 1 (950 mg). 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 60H), 1.57-1.65 (m, 4H), 2.26-2.34 (m, 4H), 2.83 (s, 9H), 3.57-3.73 (m, 178H), 3.75-3.98 (m, 2H), 4.15 (dd, 1H), 4.25 (d, 3H), 4.34(dd, 1H), 5.18-5.23 (m, 1H), 9.35-9.40 (brds, 1H) Number average molecular weight: 2836

[0159] [Examples 1-2] Synthesis of Compound 2 Compound 2 was synthesized by the following synthesis route, but the present invention is not limited to this synthesis route. In the following formula, n is 41.

[0160]

[0161] (Synthesis of Intermediate 2) Intermediate 1 (1.50 g, 0.58 mmol) and sodium acetate (14 mg, 0.12 mmol) were dissolved in toluene at room temperature. GAn (133 mg, 1.17 mmol) was added to this mixture and the mixture was reacted at 70°C for 3 hours. The reaction mixture was washed with 25% saline solution and dehydrated with magnesium sulfate. After filtering off the solid, the filtrate was concentrated to obtain Intermediate 2 (1.46 g). 1H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 60H), 1.57-1.65 (m, 4H), 1.97 (quin, 2H), 2.26-2.34 (m, 4H), 2.40 (t, 2H), 2.44 (t, 2H), 3.57-3.70 (m, 180H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34(dd, 1H), 5.18-5.23 (m, 1H) Number average molecular weight: 2787

[0162] (Synthesis of Compound 2) Intermediate 2 (400 mg, 0.15 mmol), DMAP (54 mg, 0.45 mmol), and (2-hydroxyethyl)dimethyl(3-sulfopropyl)ammonium hydroxide (48.2 mg, 0.228 mmol) were dissolved in chloroform at room temperature. MNBA (92.0 mg, 0.53 mmol) was added to this mixture and the mixture was reacted at room temperature for 24 hours. After filtering off impurities produced in the reaction, the filtrate was washed with deionized water, 5% sodium dihydrogen phosphate aqueous solution, 5% sodium bicarbonate solution, and 25% saline solution, and then dehydrated with sodium sulfate. After filtering off the solid, the filtrate was concentrated to obtain a white compound 2 (230 mg). 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 60H), 1.57-1.65 (m, 4H), 1.97 (quin, 2H), 2.26-2.34 (m, 6H), 2.39-2.41 (m, 4H), 3.24 (s, 9H), 3.52 (t, 1H), 3.57-3.73 (m, 186H), 3.76 (t, 1H), 3.81 (s, 1H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34(dd, 1H), 4.57 (brds, 2H), 5.18-5.23 (m, 1H) Number average molecular weight: 2980

[0163] [Examples 1-3] Synthesis of Compound 3 Compound 3 was synthesized by the following synthesis route, but the present invention is not limited to this synthesis route. In the following formula, n is 41.

[0164]

[0165] (Synthesis of Intermediate 3) 2-hydroxyethyldimethylamine (57.9 mg, 0.65 mmol) was dissolved in toluene, and then GAn (74.2 mg, 0.65 mmol) was added and the mixture was stirred at room temperature for 1 hour. EDC (302 mg, 1.95 mmol), DMAP (15.0 mg, 0.13 mmol), and Intermediate 1 (1.5 g, 0.65 mmol) were added to this mixture and the mixture was stirred for 1 hour. The reaction mixture was washed with 25% saline solution and dehydrated with magnesium sulfate. After filtering off the solid, the filtrate was concentrated to obtain Intermediate 3 (1.4 g). 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 60H), 1.57-1.65 (m, 4H), 2.26-2.34 (m, 2H), 2.39-2.41 (m, 4H), 2.93 (t, 2H), 3.47 (s, 6H), 3.52 (t, 1H), 3.57-3.73 (m, 188H), 3.76 (t, 1H), 3.81 (s, 1H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34(dd, 1H), 4.57 (brds, 2H), 5.18-5.23 (m, 1H) Number average molecular weight: 2742

[0166] (Synthesis of Compound 3) Intermediate 3 (400 mg, 0.15 mmol) was dissolved in acetonitrile at room temperature. 3-chloropropionic acid (57.5 mg, 0.53 mmol) was added to this mixture and the mixture was reacted at 70°C for 24 hours. The reaction mixture was washed with 25% saline solution and dehydrated with sodium sulfate. After filtering off the solid, the filtrate was concentrated to obtain white compound 3 (304 mg). 1H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 60H), 1.57-1.65 (m, 4H), 2.26-2.34 (m, 6H), 2.39-2.41 (m, 4H), 2.93 (t, 2H), 3.26 (s, 6H), 3.52 (t, 1H), 3.57-3.73 (m, 186H), 3.76 (t, 1H), 3.81 (s, 1H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34(dd, 1H), 4.57 (brds, 2H), 5.18-5.23 (m, 1H) Number average molecular weight: 2814

[0167] [Examples 1-4] Synthesis of Compound 4 Compound 4 was synthesized by the following synthesis route, but the present invention is not limited to this synthesis route. In the following formula, n is 41.

[0168]

[0169] (Synthesis of Intermediate 5) Intermediate 4 (24.5 g, 11.3 mmol), synthesized by the same method as described in WO2022 / 065263, was dissolved in toluene. Then, DCC (9.32 g, 45.2 mmol), DMAP (0.55 g, 0.45 mmol), and MyA (10.3 g, 45.1 mmol) were added and the mixture was stirred for 1 hour. The reaction mixture was washed with 25% saline solution and dehydrated with magnesium sulfate. After filtering off the solid, intermediate 5 (27.6 g) was obtained by concentrating the filtrate. 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 40H), 1.57-1.65 (m, 4H), 1.94 (s, 2H), 2.26-2.34 (m, 4H), 3.57-3.70 (m, 180H), 4.15 (dd, 1H), 4.34(dd, 1H), 4.57 (s, 2H), 5.18-5.23 (m, 1H), 7.27-7.29 (m, 1H), 7.35-7.32 (m, 4H) Number average molecular weight: 2696

[0170] (Synthesis of Intermediate 6) Intermediate 5 (27.6 g, 10.6 mmol) was dissolved in methanol, then 5% Pd / C, STD type (13.8 g) was added and the mixture was cooled with ice water. Cyclohexene (37.3 g, 45.4 mmol) was added to this mixture and the mixture was reacted at 55°C for 2 hours. The solid in the reaction mixture was removed by filtration using Kyoward 300 as a filter aid. Dibutylhydroxytoluene (27.6 mg) was added to the filtrate, and after concentration, it was dissolved in ethyl acetate (200 g). Magnesium sulfate was added to this solution to dehydrate it, and the solid was filtered off. MTBE (500 g) was added to the obtained filtrate and recrystallization was performed. This mixture was filtered to obtain intermediate 6 (24.2 g). 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 40H), 1.57-1.65 (m, 4H), 2.30 (q, 4H), 2.84 (t, 1H), 3.57-3.70 (m, 180H), 4.15 (dd, 1H), 4.34(dd, 1H), 5.18-5.23 (m, 1H) Number average molecular weight: 2531

[0171] (Synthesis of Compound 4) Intermediate 6 (5.0 g, 1.92 mmol), DIPA (389 mg, 3.84 mmol), COP (410 mg, 2.88 mmol), and 2 M NMe ３ By performing the same synthesis as for compound 1 using an acetonitrile solution (9.0 mL, 18.0 mmol), a white compound 4 (1.7 g) was obtained. 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 40H), 1.57-1.65 (m, 4H), 2.26-2.34 (m, 4H), 2.83 (s, 9H), 3.57-3.73 (m, 184H), 3.75-3.98 (m, 2H), 4.15 (dd, 1H), 4.25 (d, 3H), 4.34(dd, 1H), 5.18-5.23 (m, 1H), 9.35-9.40 (brds, 1H) Number average molecular weight: 2530

[0172] [Examples 1-5] Synthesis of Compound 5 Compound 5 was synthesized by the following synthesis route, but the present invention is not limited to this synthesis route. In the following formula, n is 41.

[0173]

[0174] (Synthesis of Intermediate 7) Using intermediate 6 (15.0 g, 5.97 mmol), sodium acetate (147 mg, 1.79 mmol), and GAn (1.36 g, 11.9 mmol), the same synthesis as for intermediate 2 was carried out to obtain a white intermediate 7 (13.6 g). 1 H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 40H), 1.57-1.65 (m, 4H), 1.97 (quin, 2H), 2.26-2.34 (m, 4H), 2.40 (t, 2H), 2.44 (t, 2H), 3.57-3.70 (m, 180H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34(dd, 1H), 5.18-5.23 (m, 1H) Number average molecular weight: 2647

[0175] (Synthesis of Compound 5) Using intermediate 7 (400 mg, 0.15 mmol), DMAP (55.8 mg, 0.45 mmol), (2-hydroxyethyl)dimethyl(3-sulfopropyl)ammonium hydroxide (48.2 mg, 0.23 mmol), and MNBA (95.2 mg, 0.28 mmol), the same synthesis as for Compound 1 was carried out to obtain a white compound 5 (167 mg). 1H NMR (600MHz, CDCl3) δ 0.88 (t, 6H), 1.20-1.35 (m, 40H), 1.57-1.65 (m, 4H), 1.97 (quin, 2H), 2.26-2.34 (m, 6H), 2.39-2.41 (m, 4H), 2.93 (t, 2H), 3.26 (s, 6H), 3.52 (t, 1H), 3.57-3.73 (m, 186H), 3.76 (t, 1H), 3.81 (s, 1H), 4.15 (dd, 1H), 4.20-4.27 (m, 2H), 4.34 (dd, 1H), 4.57 (brds, 2H), 5.18-5.23 (m, 1H) Number average molecular weight: 2840

[0176] [Example 2-1, Comparative Example 1, and Comparative Example 2] Preparation of mRNA-encapsulated LNPs Using one of compound 5, comparative compound 1, or comparative compound 2 as the PEG lipid, mRNA-encapsulated LNPs for Example 2-1, Comparative Example 1, and Comparative Example 2 were prepared as follows. DLin-M-C3-DMA, represented by the following formula, was used as the cationic lipid. DLin-M-C3-DMA is the cationic lipid described in Figure 3 (1 of 2) of WO2010 / 054401.

[0177]

[0178] (1) Preparation of lipid ethanol solution A 10 mM ethanol solution of cationic lipid, a 10 mM ethanol solution of DOPC, a 10 mM ethanol solution of chol, and a 1 mM ethanol solution of PEG lipid were mixed in an Eppendorf tube in the desired proportions (catenic lipid:DOPC:chol:PEG lipid = 50:10:38.5:1.5 (molar ratio)) so that the total amount of lipid was 720 nmol. Ethanol was added to the resulting mixture to prepare a lipid ethanol solution (total volume: 360 μL).

[0179] (2) Preparation of mRNA-encapsulated LNPs using a microfluidic system Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by PNI), sodium acetate buffer (buffer concentration: 25 mM, pH: 4.0) containing 0.044 mg / mL of ovalbumin-encoding mRNA was mixed at a flow rate of 6.75 mL / min, and a lipid ethanol solution was mixed at a flow rate of 2.25 mL / min for 3 seconds at 25°C to obtain a 0.45 mL mRNA-encapsulated LNP suspension. To the obtained mRNA-encapsulated LNP suspension (0.45 mL), 1.35 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was quickly added under stirring with a vortex mixer. The resulting mixture was ultrafiltered using Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrated mRNA-encapsulated LNP suspension was diluted with 3 mL of phosphate-buffered saline, and the diluted mRNA-encapsulated LNP suspension was concentrated again by ultrafiltration. The concentrated mRNA-encapsulated LNP suspension was collected so that the total lipid concentration (i.e., the total amount of cationic lipids, DOPC, chlor, and PEG lipids per 200 μL of mRNA-encapsulated LNP suspension) was 0.018 nmol / 200 μL.

[0180] [Test Example 1] Measurement of particle size, zeta potential, PdI, and mRNA encapsulation rate of mRNA-encapsulated LNPs. The particle size, zeta potential, and PdI (polydispersity index) of each mRNA-encapsulated LNP from Example 2-1, Comparative Example 1, and Comparative Example 2 were measured using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Nano (Malvern). The mRNA encapsulation rate of each mRNA-encapsulated LNP was also measured using the Ribogreen® assay. The results are shown in Table 3.

[0181]

[0182] As shown in Table 3, the particle size of all mRNA-encapsulated LNPs was within the preferred range (10 nm to 500 nm), and the charge (zeta potential) at physiological pH was also within the preferred range (-25 mV to +25 mV).

[0183] [Test Example 2] Evaluation of Anti-PEG Antibody Production (1) LNP Administration to Animals and Serum Sample Collection 200 μL of each mRNA-encapsulated LNP suspension (total lipid concentration: 0.018 nmol / 200 μL) from Example 2-1, Comparative Example 1, and Comparative Example 2 was administered via tail vein to 10-week-old female BALB / c mice. Seven days after administration, the mice were euthanized and blood was collected from the inferior vena cava. The blood was allowed to stand at 25°C for 2 hours, then centrifuged at 25°C and 500 G for 20 minutes to separate the serum and blood clot, and the serum sample was collected. The serum sample was stored at -80°C until measurement.

[0184] (2) ELISA evaluation of anti-PEG antibody Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0 to prepare a washing solution. In addition, Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0. BSA was then added to this solution to a concentration of 5% w / v to prepare a blocking solution.

[0185] To quantify the anti-PEG antibody, comparative compound 1, a PEG lipid, was used. Specifically, 50 μL of ethanol solution containing 20 nmol of comparative compound 1 was added to each well of a 96-well plate (Corning). After sealing, the ethanol solution in each well was stirred at 300 rpm for 16 hours. The 96-well plate was then air-dried overnight in a clean bench. 200 μL of blocking solution was added to each well, and the mixture in each well was allowed to stand at 37°C for 2 hours. The solution portion in each well was discarded, and each well was washed three times with 300 μL of washing solution. Next, 50 μL of washing solution was added to each well. Each serum sample recovered in (1) above was diluted 20-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. This procedure resulted in a final dilution ratio of 40-fold for the serum samples in each well.

[0186] After stirring serum samples at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. Then, 50 μL of washing solution was added to each well. Millipore's "Goat Anti-Mouse IgM Antibody, μ-chain, HRP conjugate" was diluted 2000-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. After stirring the dilution in each well at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. 100 μL of Millipore's "TMB / E solution" was added to each well, and the mixture was allowed to stand at 25°C for 30 minutes in the dark. 100 μL of 2 M sulfuric acid was added to each well, and the absorbance at 450 nm was measured within 15 minutes. This absorbance corresponds to the amount of anti-PEG antibody produced. The results are shown in Table 4.

[0187]

[0188] As shown in Table 4, the mRNA-encapsulated LNP suspension of Example 2-1, which contained compound 5 as the PEG lipid, produced a lower amount of anti-PEG antibody compared to the mRNA-encapsulated LNP suspensions of Comparative Examples 1 or 2, which contained comparative compound 1 or comparative compound 2.

[0189] [Examples 2-2, 2-3, Comparative Example 3, and Comparative Example 4] Preparation of mRNA-encapsulated LNPs Using one of Compound 1, Compound 2, Comparative Compound 1, and Comparative Compound 3 as the PEG lipid, mRNA-encapsulated LNPs for Examples 2-2, 2-3, Comparative Example 3, and Comparative Example 4 were prepared as follows. In addition, the above-mentioned DLin-M-C3-DMA was used as the cationic lipid.

[0190] (1) Preparation of lipid ethanol solution A 10 mM ethanol solution of cationic lipid, a 10 mM ethanol solution of DOPC, a 10 mM ethanol solution of chol, and a 1 mM solution of PEG lipid were mixed in an Eppendorf tube in the desired proportions (catenic lipid:DOPC:chol:PEG lipid = 50:10:38.5:1.5 (molar ratio)) so that the total amount of lipid was 720 nmol. Ethanol was added to the resulting mixture to prepare a lipid ethanol solution (total volume: 360 μL).

[0191] (2) Preparation of mRNA-encapsulated LNPs using a microfluidic system Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by PNI), sodium acetate buffer (buffer concentration: 25 mM, pH: 4.0) containing 0.044 mg / mL of ovalbumin-encoding mRNA was mixed at a flow rate of 6.75 mL / min, and a lipid ethanol solution was mixed at a flow rate of 2.25 mL / min for 3 seconds at 25°C to obtain a 0.45 mL mRNA-encapsulated LNP suspension. To the obtained mRNA-encapsulated LNP suspension (0.45 mL), 1.35 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was quickly added under stirring with a vortex mixer. The resulting mixture was ultrafiltered using Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrated mRNA-encapsulated LNP suspension was diluted with 3 mL of phosphate-buffered saline, and the diluted mRNA-encapsulated LNP suspension was concentrated again by ultrafiltration. The concentrated mRNA-encapsulated LNP suspension was collected so that the total lipid concentration (i.e., the total amount of cationic lipids, DOPC, chlor, and PEG lipids per 200 μL of mRNA-encapsulated LNP suspension) was 0.018 nmol / 200 μL.

[0192] [Test Example 3] Measurement of particle size, zeta potential, PdI, and mRNA encapsulation rate of mRNA-encapsulated LNPs. The particle size, zeta potential, and PdI (polydispersity index) of each mRNA-encapsulated LNP from Examples 2-2, 2-3, Comparative Example 3, and Comparative Example 4 were measured using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Nano (Malvern). The mRNA encapsulation rate of each mRNA-encapsulated LNP was also measured using the Ribogreen® assay. The results are shown in Table 5.

[0193]

[0194] As shown in Table 5, the particle size of all mRNA-encapsulated LNPs was within the preferred range (10 nm to 500 nm), and the charge (zeta potential) at physiological pH was also within the preferred range (-25 mV to +25 mV).

[0195] [Test Example 4] Evaluation of Anti-PEG Antibody Production (1) LNP Administration to Animals and Serum Sample Collection 200 μL of mRNA-encapsulated LNP suspension (total lipid concentration: 0.018 nmol / 200 μL) from each of Examples 2-2, 2-3, Comparative Example 3, and Comparative Example 4 was administered via tail vein to 10-week-old female BALB / c mice. Seven days after administration, the mice were euthanized and blood was collected from the inferior vena cava. The blood was allowed to stand at 25°C for 2 hours, then centrifuged at 25°C and 500G for 20 minutes to separate the serum and blood clot, and the serum sample was collected. The serum sample was stored at -80°C until measurement.

[0196] (2) ELISA evaluation of anti-PEG antibody Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0 to prepare a washing solution. In addition, Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0. BSA was then added to this solution to a concentration of 5% w / v to prepare a blocking solution.

[0197] To quantify the anti-PEG antibody, comparative compound 1, a PEG lipid, was used. Specifically, 50 μL of ethanol solution containing 20 nmol of comparative compound 1 was added to each well of a 96-well plate (Corning). After sealing, the ethanol solution in each well was stirred at 300 rpm for 16 hours. The 96-well plate was then air-dried overnight in a clean bench. 200 μL of blocking solution was added to each well, and the mixture in each well was allowed to stand at 37°C for 2 hours. The solution portion in each well was discarded, and each well was washed three times with 300 μL of washing solution. Next, 50 μL of washing solution was added to each well. Each serum sample recovered in (1) above was diluted 80-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. This procedure resulted in a final dilution ratio of 160-fold for the serum sample in each well.

[0198] After stirring serum samples at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. Then, 50 μL of washing solution was added to each well. Millipore's "Goat Anti-Mouse IgM Antibody, μ-chain, HRP conjugate" was diluted 2000-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. After stirring the dilution in each well at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. 100 μL of Millipore's "TMB / E solution" was added to each well, and the mixture was allowed to stand at 25°C for 30 minutes in the dark. 100 μL of 2 M sulfuric acid was added to each well, and the absorbance at 450 nm was measured within 15 minutes. This absorbance corresponds to the amount of anti-PEG antibody produced. The results are shown in Table 6.

[0199]

[0200] As shown in Table 6, the mRNA-encapsulated LNP suspensions of Example 2-2 or Example 2-3, which contained compound 1 or compound 2 as the PEG lipid, produced a lower amount of anti-PEG antibody compared to the mRNA-encapsulated LNP suspensions of Comparative Example 3 or Comparative Example 4, which contained comparative compound 1 or comparative compound 3.

[0201] [Examples 2-4 and Comparative Example 5] Preparation of mRNA-encapsulated LNPs Using either compound 1 or comparative compound 1 as the PEG lipid, mRNA-encapsulated LNPs for Examples 2-4 and Comparative Example 5 were prepared as follows. In addition, the above-mentioned DLin-M-C3-DMA was used as the cationic lipid.

[0202] (1) Preparation of lipid ethanol solution A 10 mM ethanol solution of cationic lipid, a 10 mM ethanol solution of DOPC, a 10 mM ethanol solution of chol, and a 1 mM ethanol solution of PEG lipid were mixed in an Eppendorf tube in the desired proportions (catenic lipid:DOPC:chol:PEG lipid = 50:10:38.5:1.5 (molar ratio)) so that the total amount of lipid was 720 nmol. Ethanol was added to the resulting mixture to prepare a lipid ethanol solution (total volume: 360 μL).

[0203] (2) Preparation of mRNA-encapsulated LNPs using a microfluidic system Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by PNI), sodium acetate buffer (buffer concentration: 25 mM, pH: 4.0) containing 0.044 mg / mL of ovalbumin-encoding mRNA was mixed at a flow rate of 6.75 mL / min, and a lipid ethanol solution was mixed at a flow rate of 2.25 mL / min for 3 seconds at 25°C to obtain a 0.45 mL mRNA-encapsulated LNP suspension. To the obtained mRNA-encapsulated LNP suspension (0.45 mL), 1.35 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was quickly added under stirring with a vortex mixer. The resulting mixture was ultrafiltered using Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrated mRNA-encapsulated LNP suspension was diluted with 3 mL of phosphate-buffered saline, and the diluted mRNA-encapsulated LNP suspension was subjected to ultrafiltration again.

[0204] In Comparative Example 5, the concentrated mRNA-encapsulated LNP suspension containing comparative compound 1 was recovered so that the total lipid concentration (i.e., the total amount of cationic lipids, DOPC, chol, and PEG lipids per 200 μL of mRNA-encapsulated LNP suspension (nmol)) was 0.18 nmol / 200 μL.

[0205] In Examples 2-4, concentrated mRNA-encapsulated LNP suspensions containing compound 1 were collected to a total lipid concentration (i.e., the total amount of cationic lipids, DOPC, chol, and PEG lipids in nmols per 200 μL of mRNA-encapsulated LNP suspension) of 180 nmol / 200 μL. Furthermore, each mRNA-encapsulated LNP suspension was prepared by diluting it with phosphate-buffered saline to a total lipid concentration of 18 nmol / 200 μL, 1.8 nmol / 200 μL, or 0.18 nmol / 200 μL.

[0206] [Test Example 5] Measurement of particle size, zeta potential, PdI, and mRNA encapsulation rate of mRNA-encapsulated LNPs. The particle size, zeta potential, and PdI (polydispersity index) of each mRNA-encapsulated LNP in Examples 2-4 and Comparative Example 5 were measured using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Nano (Malvern). The mRNA encapsulation rate of each mRNA-encapsulated LNP was measured using the Ribogreen® assay. The results are shown in Table 7. For these measurements, a suspension with a total lipid concentration of 180 nmol / 200 μL was used from the mRNA-encapsulated LNP suspension obtained in Example 2-4.

[0207]

[0208] As shown in Table 7, the particle size of all mRNA-encapsulated LNPs was within the preferred range (10 nm to 500 nm), and the charge (zeta potential) at physiological pH was also within the preferred range (-25 mV to +25 mV).

[0209] [Test Example 6] Evaluation of Anti-PEG Antibody Production (1) LNP Administration to Animals and Serum Sample Collection Ten-week-old female BALB / c mice were administered concentrated mRNA-encapsulated LNP suspensions from Examples 2-4 and Comparative Example 5 via the tail vein. In Comparative Example 5, 200 μL of concentrated RNA-encapsulated LNP suspension containing comparative compound 1 (total lipid concentration: 0.18 nmol / 200 μL) was administered via the tail vein. In Examples 2-4, 200 μL of each concentrated RNA-encapsulated LNP suspension containing compound 1 (total lipid concentration: 180 nmol / 200 μL, 18 nmol / 200 μL, 1.8 nmol / 200 μL, or 0.18 nmol / 200 μL) was administered via the tail vein. Seven days after administration, the mice were euthanized and blood was collected from the inferior vena cava. Blood was allowed to stand at 25°C for 2 hours, then centrifuged at 25°C and 500G for 20 minutes to separate the serum from the blood clot, and the serum sample was collected. The serum sample was stored at -80°C until measurement.

[0210] (2) ELISA evaluation of anti-PEG antibody Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0 to prepare a washing solution. In addition, Takara Bio's "Tris Buffered Saline with Tween(registered trademark) 20 (TBS-T) Tablets, pH 7.6" was dissolved in water to a concentration of 50 mM, and the pH of the resulting aqueous solution was adjusted to 8.0. BSA was then added to this solution to a concentration of 5% w / v to prepare a blocking solution.

[0211] To quantify the anti-PEG antibody, comparative compound 1, a PEG lipid, was used. Specifically, 50 μL of ethanol solution containing 20 nmol of comparative compound 1 was added to each well of a 96-well plate (Corning). A sealer was applied and the plate was stirred at 300 rpm for 16 hours. The 96-well plate was then air-dried overnight in a clean bench. 200 μL of blocking solution was added to each well, and the mixture in each well was allowed to stand at 37°C for 2 hours. The solution portion in each well was discarded, and each well was washed three times with 300 μL of washing solution. Next, 50 μL of washing solution was added to each well. Each serum sample recovered in (1) above was diluted 20-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. This procedure resulted in a final dilution ratio of 40-fold for the serum samples in each well.

[0212] After stirring the serum samples at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. Then, 50 μL of washing solution was added to each well. Millipore's "Goat Anti-Mouse IgM Antibody, μ-chain, HRP conjugate" was diluted 2000-fold with the washing solution, and 50 μL of the resulting dilution was added to each well. After stirring the dilution in each well at 25°C and 300 rpm for 1 hour, the solution in each well was discarded, and each well was washed five times with 300 μL of washing solution. 100 μL of Millipore's "TMB / E solution" was added to each well, and the mixture was allowed to stand at 25°C for 30 minutes in the dark. 100 μL of 2 M sulfuric acid was added to each well, and the absorbance at 450 nm was measured within 15 minutes. This absorbance corresponds to the amount of anti-PEG antibody produced. The results are shown in Table 8.

[0213]

[0214] As shown in Table 8, the mRNA-encapsulated LNP suspensions of Examples 2-4, which contained compound 1 as the PEG lipid, produced a lower amount of anti-PEG antibody compared to the mRNA-encapsulated LNP suspension of Comparative Example 5, which contained comparative compound 1, regardless of the amount of PEG lipid administered.

[0215] [Examples 2-5 and Comparative Example 6] Preparation of mRNA-free LNPs Using either compound 1 or comparative compound 1 as the PEG lipid, mRNA-free LNPs for Examples 2-4 and Comparative Example 5 were prepared as follows. DLin-M-C3-DMA, as described above, was used as the cationic lipid.

[0216] (1) Preparation of lipid ethanol solution A 10 mM ethanol solution of cationic lipid, a 10 mM ethanol solution of DOPC, a 10 mM ethanol solution of chol, and a 1 mM ethanol solution of PEG lipid were mixed in an Eppendorf tube in the desired proportions (cationic lipid:DOPC:chol:PEG lipid = 50:10:38.5:1.5 (molar ratio)) so that the total amount of lipids was 720 nmol. DiD was added to the resulting mixture in an amount of 0.5 mol per 100 mol of the total amount of cationic lipid, DOPC, chol, and PEG lipid. Ethanol was added to the resulting mixture to prepare a lipid ethanol solution (total amount: 360 μL).

[0217] (2) Preparation of mRNA-free LNPs using a microfluidic system Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by PNI), mRNA-free sodium acetate buffer (buffer concentration: 25 mM, pH: 4.0) was mixed at a flow rate of 6.75 mL / min and a lipid ethanol solution at a flow rate of 2.25 mL / min for 6.67 seconds at 25°C to obtain a 1.0 mL mRNA-free LNP suspension. To the obtained LNP suspension (0.45 mL), 1.35 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was quickly added under stirring with a vortex mixer. The resulting mixture was ultrafiltered using Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrated LNP suspension was diluted with 3 mL of phosphate-buffered saline, and the obtained diluted LNP suspension was ultrafiltered again. The concentrated LNP suspension was collected so that the total lipid concentration (i.e., the total amount of cationic lipids, DOPC, chol, and PEG lipids per 1000 μL of mRNA-encapsulated LNP suspension) was 1000 nmol / 1000 μL.

[0218] [Test Example 7] Measurement of LNP particle size, zeta potential, and PdI The particle size, zeta potential, and PdI (polydispersity index) of each LNP in Examples 2-4 and Comparative Example 5 were measured using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Nano (Malvern). The results are shown in Table 9.

[0219]

[0220] As shown in Table 9, the particle size of all LNPs was within the preferred range (10 nm to 500 nm), and the charge (zeta potential) at physiological pH was also within the preferred range (-25 mV to +25 mV).

[0221] [Test Example 8] Evaluation of blood retention: 200 μL each of the LNP suspensions (total lipid concentration: 1000 nmol / 1000 μL) from Examples 2-4 and Comparative Example 5 were administered via tail vein to 6-week-old female BALB / c mice. 24 hours after administration, blood (12.5 μL) was collected from the tail vein and mixed with 1% w / v sodium dodecyl sulfate aqueous solution (237.5 μL) to prepare the measurement sample. Mice that had not been administered the LNP suspension were euthanized, and blood was collected from the inferior vena cava. The collected blood (12.5 μL) was mixed with 1% w / v sodium dodecyl sulfate aqueous solution (237.5 μL), and then the LNP suspensions administered to the mice were added in amounts of 2.5 μL, 2.0 μL, 1.5 μL, 1.0 μL, or 0.5 μL, respectively, to prepare calibration curve samples and create a calibration curve. The measurement sample (200 μL) was transferred to a black plate, and fluorescence was measured (excitation wavelength 730 nm, fluorescence wavelength 780 nm).

[0222] By comparing the fluorescence values ​​of the measurement samples with the calibration curve, the amount of LNP remaining in the blood (%ID / mL Blood) relative to the total LNP administered to the mice from which the measurement samples were collected was calculated. The results are shown in Table 10. The amount of LNP remaining in the blood is calculated using the following formula: Amount of LNP remaining in the blood (%ID / mL Blood) = 100 × (Amount of LNP remaining per 1 mL of blood) / (Total amount of LNP administered per 1 mL of blood)

[0223]

[0224] As shown in Table 10, the LNP suspensions of Examples 2-5, which contained compound 1 as the PEG lipid, showed superior blood retention compared to the LNP suspension of Comparative Example 6, which contained comparative compound 1.

[0225] [Examples 2-6, 2-7, and Comparative Example 7] Preparation of mRNA-encapsulated LNPs Using one of compound 4, compound 5, or comparative compound 2 as the PEG lipid, mRNA-encapsulated LNPs for Examples 2-6, 2-7, and Comparative Example 7 were prepared as follows. In addition, the above-mentioned DLin-M-C3-DMA was used as the cationic lipid.

[0226] (1) Preparation of lipid ethanol solution A 10 mM ethanol solution of cationic lipid, a 10 mM ethanol solution of DOPC, a 10 mM ethanol solution of chol, and a 1 mM ethanol solution of PEG lipid were mixed in an Eppendorf tube in the desired proportions (cationic lipid:DOPC:chol = 50:10:38.5 (molar ratio)) so that the total amount of lipid was 720 nmol. Ethanol was added to the resulting mixture to prepare a lipid ethanol solution (total volume: 360 μL).

[0227] (2) Preparation of mRNA-encapsulated LNPs using a microfluidic system Using the nanoparticle manufacturing device "NanoAssemblr Ignite" (manufactured by PNI), sodium acetate buffer (buffer concentration: 25 mM, pH: 4.0) containing 0.044 mg / mL of luciferase-encoding mRNA was mixed at a flow rate of 6.75 mL / min, and a lipid ethanol solution was mixed at a flow rate of 2.25 mL / min for 3 seconds at 25°C to obtain a 0.45 mL mRNA-encapsulated LNP suspension. To the obtained mRNA-encapsulated LNP suspension (0.45 mL), 1.35 mL of 2-morpholinoethanesulfonic acid buffer (buffer concentration: 20 mM, pH: 5.5) was quickly added under stirring with a vortex mixer. The resulting mixture was ultrafiltered using Amicon Ultra-4 (MWCO: 100 kDa). After concentration by ultrafiltration, the concentrated mRNA-encapsulated LNP suspension was diluted with 3 mL of phosphate-buffered saline, and the diluted mRNA-encapsulated LNP suspension was concentrated again by ultrafiltration. The concentrated mRNA-encapsulated LNP suspension was collected so that the mRNA concentration (i.e., the amount of mRNA per 200 μL of mRNA-encapsulated LNP suspension (μg)) was 1 μg / 200 μL.

[0228] [Test Example 9] Measurement of particle size, zeta potential, PdI, and mRNA encapsulation rate of mRNA-encapsulated LNPs. The particle size, zeta potential, and PdI (polydispersity index) of each mRNA-encapsulated LNP from Examples 2-2, 2-3, Comparative Example 3, and Comparative Example 4 were measured using dynamic light scattering and laser Doppler electrophoresis with a Zetasizer Nano (Malvern). The mRNA encapsulation rate of each mRNA-encapsulated LNP was also measured using the Ribogreen® assay. The results are shown in Table 11.

[0229]

[0230] [Test Example 10] Evaluation of gene expression activity in each organ Six-week-old female BALB / c mice were administered 200 μL of each mRNA-encapsulated LNP suspension (mRNA concentration: 1 μg / 200 μL) from Examples 2-6, 2-7, and Comparative Example 7 via tail vein. Five hours and 55 minutes after administration, 200 μL of the resulting aqueous solution of "D-luciferin Potassium Salt" from Syd Labs, Inc. dissolved in phosphate saline (30 mg / mL) was administered intraperitoneally. Five minutes after administration, the mice were euthanized, and the liver, kidney, and lung were removed. Luminescence from each organ was measured using an in vivo imaging system (IVIS). The amount of luminescence serves as an indicator reflecting the gene expression activity (i.e., the ability to introduce the mRNA encoding luciferase into each organ) of each mRNA-encapsulated LNP suspension. The results are shown in Table 12. Note that "E+07" etc. listed in Table 12 are "×10 ７ This represents things like ".

[0231]

[0232] As shown in Table 12, the mRNA-encapsulated LNP suspension of Examples 2-6, containing compound 4 as the PEG lipid, showed superior gene expression activity in the spleen compared to the mRNA-encapsulated LNP suspension of Comparative Example 7, containing comparative compound 2. Furthermore, the mRNA-encapsulated LNP suspension of Example 2-7, containing compound 5 as the PEG lipid, showed higher gene expression activity in all organs compared to the mRNA-encapsulated LNP suspension of Comparative Example 7, containing comparative compound 2.

[0233] From the PEG lipids of the present invention, LNPs can be prepared that suppress the production of anti-PEG antibodies in vivo.

[0234] This application is based on Japanese Patent Application No. 2025-053637, which is entirely contained herein.

Claims

1. PEG lipid for the preparation of lipid nanoparticles, wherein formula (I): R １ -L １ -R ２ (I) (In equation (I), R １ L represents a zwitterionic moiety that has both positive and negative charges. １ represents the polyethylene glycol chain, and R ２ ) represents the lipid portion. PEG lipids are indicated by ).

2. Formula (Ia): (In Formula (Ia), R １ is as defined above; n represents a number from 10 to 300; and R ２ａ is any of Formulas (1) to (6): (In Formulas (1) to (6), * represents a bonding site; R ３ to R １３ each independently represent an aliphatic hydrocarbon group having 5 to 30 carbon atoms; L ２ , L ５ , and L ６ each independently represent an alkylene group having 1 to 3 carbon atoms; and L ３ and L ４ each independently represent a single bond or an amide bond.) and represents a group represented by any one of the above.) The PEG lipid according to claim 1, which is represented by said formula.

3. R １ However, equations (7) to (9): (In equations (7) to (9), * indicates the bonding position, and L ７ ~L ９ Each of these independently represents an alkylene group having 1 to 3 carbon atoms.) The PEG lipid according to claim 2, wherein the group is represented by any of the following.

4. Formula (Ib) to Formula (Ii): (In formulas (Ib) to (Ii), R １ , R ３ ~R １３ The PEG lipid according to claim 2, wherein n is as described above, and n is at least one selected from the group consisting of PEG lipids represented by any of the above.

5. Lipid nanoparticles containing the PEG lipid described in any one of claims 1 to 4.

6. A nucleic acid delivery agent containing a PEG lipid according to any one of claims 1 to 4.

7. The nucleic acid delivery agent according to claim 6, further comprising nucleic acid.

8. A method for introducing nucleic acids contained in the nucleic acid introduction agent into cells, comprising contacting the nucleic acid introduction agent described in claim 7 with cells in vitro.

9. A method for introducing nucleic acids contained in a nucleic acid delivery agent into target cells in a living organism, comprising administering the nucleic acid delivery agent described in claim 7 to a living organism.

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