Lipid particle

Abstract

Provided is a lipid particle which includes a drug and enables more efficient development of the function of the drug in cells. The lipid particle contains, in the polar head thereof, a modified ionized lipid having a disulfide unit and / or a modified polynucleotide having a disulfide unit.

Description

lipid particles

[0001] The present invention relates to lipid particles and the like.

[0002] In the medical field, etc., development of technologies for introducing polynucleotides such as siRNA and mRNA into cells to express their functions is progressing. Polynucleotides are negatively charged due to the presence of phosphate groups, and therefore have low permeability through the negatively charged cell membrane. For this reason, when introducing polynucleotides into cells, the introduction efficiency has been improved by forming polynucleotides into complex particles with cationic lipids and introducing these into cells (Patent Document 1). Furthermore, lipid nanoparticles (LNPs) have recently attracted attention as a delivery system for polynucleotide delivery.

[0003] Patent Publication No. 2002-529439 International Publication No. 2023 / 022055

[0004] Even if the lipid particles described above are introduced into cells, the function of the drug contained in the lipid particles is not necessarily expressed efficiently. For example, although the lipid particles are introduced into cells by endocytosis, the endosome is subject to degradation by lysosomes, and therefore the drug needs to escape from the endosome.

[0005] An object of the present invention is to provide lipid particles containing a drug, which are capable of more efficiently expressing the function of the drug within cells.

[0006] In view of the above-mentioned problems, the present inventors have conducted extensive research and found that the above-mentioned problems can be solved by lipid particles containing modified ionized lipids having disulfide units in their polar head groups and / or modified polynucleotides having disulfide units. Based on this finding, the present inventors have conducted further research and have completed the present invention. That is, the present invention encompasses the following aspects.

[0007] Item 1. A lipid particle comprising a modified ionizable lipid having a disulfide unit in its polar head group and / or a modified polynucleotide having a disulfide unit.

[0008] Item 2. The lipid particle according to Item 1, wherein the modified ionizable lipid has a tertiary amine structure.

[0009] Item 3. The modified ionizable lipid is represented by general formula (2):

[0010]

[0011] [In the formula: R a Ha-L 1a -R 31a -L 2a -R 32a (L 1a and L 2a are the same or different and represent a single bond or a linker; R 31a indicates a bond between reactive groups, and R 32a represents a monovalent group containing -S-S- or -Se-Se-. b and R c and the formula (I) is the same or different and represents a chain structure containing a hydrocarbon chain.

[0012] Item 4. The chain structure is —(CH2) s -R d -R e (In the formula: R d represents -O-C(=O)- or -C(=O)-O-, and R e represents a hydrocarbon chain; and s represents 1 to 10.

[0013] Item 5. The lipid particle according to Item 2, having a pKa of 5 to 7.

[0014] Item 6. The lipid particle according to Item 2, which encapsulates a polynucleotide.

[0015] Item 7. The modified polynucleotide is represented by the general formula (1):

[0016]

[0017] [In the formula: R 1 is a single bond, -(CH2) t - (t is an integer of 1 to 4), or -CH(-R 11 )-(R 11 represents an alkyl group). 2represents a hydrogen atom or a hydrocarbon group. 3 Ha-L 1 -R 31 -L 2 -R 32 (L 1 and L 2 are the same or different and represent a single bond or a linker; R 31 indicates a bond between reactive groups, and R 32 represents a monovalent group containing -S-S- or -Se-Se-), or represents a hydrocarbon group (provided that at least one R 3 Ha-L 1 -R 31 -L 2 -R 32 (Indicates R 4 is a hydrogen atom, or -L 1 -R 31 -L 2 -R 32 or R 3 R 5 is a hydrogen atom, -L 1 -R 31 -L 2 -R 32 , or -R 51 -R 52 (R 51 is a single bond or -P(=O)(-OR 511 )-O-(R 511 represents a hydrogen atom or a hydrocarbon group), and R 52 represents a hydrocarbon group which may be substituted with a hydroxyl group). 6 and R 7 are the same or different and represent a hydrogen atom or a hydrocarbon group. n represents a natural number.] is linked to the end of a polynucleotide directly or via a spacer.

[0018] Item 8. The lipid particle according to Item 7, wherein the spacer is an alkyl chain or a polyalkylene glycol chain.

[0019] Item 9. The structure is represented by general formula (1a):

[0020]

[0021] [In the formula: R8 , R 9 and R 10 are the same or different, -L 1b -R 31 -L 2b -R 32 Item 8. The lipid particle according to Item 7, wherein u, v, w, and x are the same or different and represent an integer of 1 to 8.

[0022] Item 10. A pharmaceutical comprising the lipid particle according to any one of Items 1 to 9.

[0023] Item 11. General formula (2):

[0024]

[0025] [In the formula: R a Ha-L 1a -R 31 -L 2a -R 32 (L 1a and L 2a are the same or different and represent a single bond or a linker; R 31 indicates a bond between reactive groups, and R 32 represents a monovalent group containing -S-S- or -Se-Se-. b and R c and the formula (I) may be the same or different and represent a chain structure containing a hydrocarbon chain.

[0026] Item 12. General formula (1a):

[0027]

[0028] [In the formula: R 8 , R 9 and R 10 are the same or different, -L 1b --R 31 -L 2b -R 32 and u, v, w, and x are the same or different and represent an integer of 1 to 8.] is linked to the end of a polynucleotide directly or via a spacer.

[0029] According to the present invention, it is possible to provide lipid particles containing a drug that can more efficiently express the function of the drug within cells. Such lipid particles can promote the escape of the drug from endosomes. Furthermore, it is possible to provide modified ionized lipids and modified polynucleotides that are suitable for expressing the function.

[0030] The graph shows the cell viability 24 hours after the addition of LNP containing disulfide-modified lipids (Test Example 1-2). A indicates the case where MC3 (Tables 3 and 4) was used as the lipid, and B indicates the case where SM102 (Tables 3 and 5) was used as the lipid. The horizontal axis indicates the lipid used. "CD-L" indicates cyclic disulfide lipid. The numbers correspond to the lipid numbers in Tables 1 and 2. N=3. The graph shows the NLuc expression level 24 hours after the addition of LNP containing disulfide-modified lipids (Test Example 1-2). C indicates the case where MC3 (Tables 3 and 4) was used as the lipid, and D indicates the case where SM102 (Tables 3 and 5) was used as the lipid. The horizontal axis indicates the lipid used. "CD-L" indicates cyclic disulfide lipid. The numbers correspond to the lipid numbers in Tables 1 and 2. N=3. The figures show the cellular uptake 6 hours after addition of LNP containing disulfide-modified lipids (Test Example 1-2). E indicates the case where MC3 (Tables 3 and 4) was used as the lipid, and F indicates the case where SM102 (Tables 3 and 5) was used as the lipid. The horizontal axis indicates the lipid used. The numbers correspond to the lipid numbers in Tables 1 and 2. N = 3. The figures show synthesis data (LC-MS) for disulfide-modified polynucleotides. The relative amount of RLU after addition of cationic lipid complex particles containing disulfide-modified polynucleotides is shown. The horizontal axis indicates the polynucleotide used. Neg indicates a control without polynucleotide. (A) A representative image of lysosomal localization of LNPs is shown, and (B) a graph showing the measurement results of the lysosomal colocalization rate is shown. The lipid used is shown on the left side of the image in (A) and below the graph in (B). "CDL" indicates cyclic disulfide lipid. The numbers correspond to the lipid numbers in Tables 1 and 2. The staining target is indicated above the image in (A). The results of an in vivo NLuc-mRNA delivery test are shown. A: A representative image of a mouse injected with 4 μg of LNP-formulated Nluc mRNA via the subcutaneous administration route is shown. B: The quantification results of the luminescence intensity in A (). The horizontal axis indicates the lipids used. "CDL" stands for cyclic disulfide lipid.The numbers correspond to the lipid numbers in Tables 1 and 2. The image information indicates the stained objects. N=3-4.

[0031] In this specification, the expressions "contain" and "comprise" include the concepts of "contain," "comprise," "consist essentially of," and "consist only of."

[0032] In one aspect, the present invention relates to lipid particles (sometimes referred to herein as "lipid particles of the present invention") comprising a modified ionizable lipid having a disulfide unit in its polar head group and / or a modified polynucleotide having a disulfide unit. This will be described below.

[0033] The disulfide unit is a monovalent group containing -S-S- or -Se-Se- as a partial structure, and is not particularly limited insofar as such a group is a ring-derived disulfide unit.

[0034] The ring-derived group is a monovalent group obtained by removing one hydrogen atom from a ring, and is not particularly limited within this scope. The ring is not particularly limited, and is preferably monocyclic or bicyclic, and more preferably monocyclic. The ring-constituting atoms other than S or Se are not particularly limited, and examples thereof include a ring consisting of only carbon atoms, or a ring consisting of carbon atoms and heteroatoms (e.g., nitrogen atoms, sulfur atoms, oxygen atoms, etc.). The number of ring-constituting atoms is not particularly limited, and is, for example, 3 to 20, preferably 3 to 12, more preferably 4 to 8, and even more preferably 5 to 7. Specific examples of rings having -S-S- or -Se-Se- in the ring structure include rings in which one -C-C- in a cycloalkane or cycloalkene (preferably a cycloalkane) is replaced with -S-S-. Particularly preferred ring-derived groups are those of the following formula:

[0035]

[0036] [wherein both Y's are S or Se] Among these, groups represented by formula p or formula q are preferred, and groups represented by formula p are more preferred.

[0037] The monovalent group containing -S-S- or -Se-Se- may have a chain structure. Examples of the group having a chain structure include -Y-Y-R 32x and both Y's are S or Se, and R 32x represents a protecting group. The protecting group is not particularly limited as long as it has the function of protecting the disulfide bond, and examples thereof include an alkyl group and an aryl group.

[0038] R 32x The alkyl group represented by the formula (I) includes both linear and branched alkyl groups. The alkyl group is preferably a branched alkyl group. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 6, preferably 2 to 5, more preferably 3 to 5, and even more preferably 4. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group. Of these, a tert-butyl group is preferred.

[0039] R 32x The aryl group represented by the formula (I) is not particularly limited, but preferably has 6 to 12 carbon atoms, more preferably 6 to 12 carbon atoms, and even more preferably 6 to 8 carbon atoms. The aryl group may be either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, a naphthyl group, a biphenyl group, a pentalenyl group, an indenyl group, an anthranyl group, a tetracenyl group, a pentacenyl group, a pyrenyl group, a perylenyl group, a fluorenyl group, and a phenanthryl group. Of these, a phenyl group is preferred.

[0040] The modified ionizable lipid has a disulfide unit in the polar head portion. The polar head portion is the portion of the ionizable lipid other than the hydrophobic tail portion (usually a long hydrocarbon chain (e.g., 6-30, 8-30, 10-30, or 12-30 carbon atoms)). For example, if the ionizable lipid has a tertiary amine structure, the polar head portion is the structural portion.

[0041] The ionizable lipid is not particularly limited as long as it can constitute lipid particles and is a lipid that exhibits a positive charge within the lipid particles. Examples of cationic lipids include [4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-K-C2-DMA; "XTC2"), 2,2-dilinoleyl-4-(3-dimethylaminopropyl)-[1,3]-dioxolane (DLin-K-C3-DMA"; "XTC2"). ), 2,2-Dilinoleyl-4-(4-dimethylaminobutyl)-[1,3]-dioxolane (DLin-K-C4-DMA), 2,2-Dilinoleyl-5-dimethylaminomethyl-[1,3]-dioxane (DLin-K6-DMA), 2,2-Dilinoleyl-4-N-methylpepiazino-[1,3]-dioxolane (DLin-K-MPZ), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-dilinoleoyl-3-dimethylaminopropane (DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanedio(propanedio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-distearyloxy-N,N-dimethylaminopropane (DSDMA), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dis Tearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2(spermine-carboxamido)ethyl]-N,N-dimethyl-1-propanaminium trifluoroacetate (DOSPA), dioctadecylamidoglycylspermine (DOGS), 3-dimethylamino-2-(cholest-5-ene-3-β-oxybutane-4-oxy)-1-(cis,cis-9,12- Examples include 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-ene-3-β-oxy)-3'-oxapentoxy]-3-dimethyl-1-(cis,cis-9',1-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), and 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP).

[0042] From the viewpoint of more efficiently exhibiting the effects of the present invention, the modified ionized lipid is particularly preferably a lipid represented by the general formula (2):

[0043]

[0044] It is a modified ionized lipid represented by the formula:

[0045] R a Ha-L 1a -R 31a -L 2a -R 32a (L 1a and L 2a are the same or different and represent a single bond or a linker; R 31a indicates a bond between reactive groups, and R 32a represents a monovalent group containing -S-S- or -Se-Se-.

[0046] L 1a or L 2a Examples of linkers represented by the formula (I) include alkylene groups, alkenylene groups, and heteroalkylene groups (for example, heteroalkylene groups containing -NH-COO-, -O-, etc. in the main chain). Linkers may be either linear or branched, and are preferably linear. The number of atoms constituting the main chain of the linker (the number of carbon atoms in the case of alkylene groups and alkenylene groups) is not particularly limited and is, for example, 1 to 10, preferably 2 to 6, and more preferably 2 to 4. Preferred examples of linkers include alkylene groups, more preferably alkylene groups having 2 to 6 carbon atoms, even more preferably alkylene groups having 2 to 5 carbon atoms, and even more preferably linear alkylene groups having 2 to 4 carbon atoms.

[0047] R 31a The bond between reactive groups represented by the formula (I) is not particularly limited as long as it is a bond formed by the reaction of two identical or different reactive groups. Examples of the reactive group include an amino group, a carboxy group, a hydroxy group, a ketone group, an ethynyl group, a vinyl group, an azide group, an epoxy group, an aldehyde group, an oxylamino group, a thiol group, an isocyanate group, and an isothiocyanate group.

[0048] Examples of reactions between reactive groups are as follows: Amino groups are known to react with carboxy groups (or groups obtained by esterifying carboxy groups with N-hydroxysuccinimide (NHS)) to form amide bonds. Ethynyl groups are known to undergo 1,3-dipolar cycloaddition with azide groups to form 1,2,3-triazole rings. Vinyl groups react with thiol groups to form bonds. Epoxy groups react with amino and thiol groups to form bonds. Aldehyde groups react with amino groups to form Schiff bases, which can be reduced to form bonds. Oxylamino groups react with ketone and aldehyde groups to form oximes. Azide groups are known to undergo 1,3-dipolar cycloaddition with ethynyl groups to form 1,2,3-triazole rings.

[0049] R 31a Specific examples of the bond between reactive groups represented by the formula (I) include an ester bond (-C(=O)-O-), an ether bond, an amide bond (-NH-COO-), and a 1,2,3-triazole ring.

[0050] R 32a For the monovalent group containing -S-S- or -Se-Se-, the same explanation as for the disulfide unit is applied.

[0051] R a In the above, particularly preferably, L 1a and L 2a are the same or different and are alkylene groups having 1 to 10 carbon atoms (preferably 2 to 6, more preferably 2 to 4), and R 31a is an ester bond, and R 32a is a ring-derived group (preferably a group represented by formula p, q, r, or s).

[0052] R b and R c are the same or different and represent a chain structure containing a hydrocarbon chain, in which the hydrocarbon chain is located at the end.

[0053] The hydrocarbon chain is a monovalent chain hydrocarbon group, and is not particularly limited as long as it is. Chain hydrocarbon groups include both linear and branched chains, with linear chains being particularly preferred. The number of carbon atoms in the chain hydrocarbon group is not particularly limited as long as it is a number that allows the formation of lipid particles, and is, for example, 8 to 50, preferably 10 to 40, more preferably 12 to 30, even more preferably 13 to 26, still more preferably 14 to 22, and particularly preferably 14 to 20. The number of double bonds contained in the chain hydrocarbon group is not particularly limited as long as it is a number that allows the formation of lipid particles, and is, for example, 0 to 6, preferably 0 to 4, more preferably 0 to 3, and even more preferably 0 to 2.

[0054] The chain structure may contain a linker (defined as L 1a / L 2a The definition of the linker represented by R 31a The definition of the reactive group bond represented by the formula (I) is used herein.

[0055] The chain structure is particularly preferably —(CH2) s -R d -R e (In the formula: R d represents -O-C(=O)- or -C(=O)-O-, and R e represents a hydrocarbon chain. s represents 1 to 10.)

[0056] s is preferably 2 to 6, more preferably 2 to 4.

[0057] The modified ionizable lipid may be one type alone or two or more types in combination.

[0058] The modified ionized lipid can be synthesized according to or in accordance with a known method, for example, by referring to the synthesis scheme of Test Example 1-1 described below.

[0059] The modified polynucleotide has disulfide units, preferably at the termini.

[0060] The modified polynucleotide preferably has the general formula (1):

[0061]

[0062] is a modified polynucleotide in which a structure represented by the following formula (I) is linked to the end of a polynucleotide directly or via a spacer.

[0063] R 1 is a single bond, -(CH2) t - (t is an integer of 1 to 4), or -CH(-R 11 )-(R 11 represents an alkyl group). 1 is preferably —CH— or —CH(—R 11 )-.

[0064] R 11 The alkyl group represented by the formula (I) includes both linear and branched alkyl groups. The alkyl group is preferably a linear alkyl group. The number of carbon atoms in the alkyl group is not particularly limited, and is, for example, 1 to 6, preferably 1 to 4, more preferably 1 to 2, and even more preferably 1. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, and a 3-methylpentyl group.

[0065] R 2 represents a hydrogen atom or a hydrocarbon group. 2 In one embodiment of the present invention, R is preferably a hydrogen atom. 2 is a hydrogen atom, it forms a hydroxy group with the adjacent oxygen atom, but the hydrogen atom is abstracted to form -O - In this case, it is also included in the modified polynucleotide.

[0066] R 2 The hydrocarbon group represented by is not particularly limited, and examples thereof include alkyl groups, aryl groups, and groups formed by any combination thereof (e.g., aralkyl groups, alkylaryl groups, alkylaralkyl groups), etc. Among these, alkyl groups are preferred.

[0067] R 2The alkyl group represented by the formula (I) includes any of linear, branched, and cyclic alkyl groups. The alkyl group is preferably a linear alkyl group. The number of carbon atoms in the alkyl group (when linear or branched) is not particularly limited and is, for example, 1 to 30. In one embodiment of the present invention, the number of carbon atoms is preferably 3 to 20, more preferably 3 to 15. In one embodiment of the present invention, the number of carbon atoms is preferably 3 to 10, more preferably 3 to 6, and in another embodiment, preferably 6 to 20, more preferably 8 to 20, and even more preferably 10 to 20. The number of carbon atoms in the alkyl group (when cyclic) is not particularly limited and is, for example, 3 to 7, preferably 4 to 6. Specific examples of the alkyl group include a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group, a tert-butyl group, a sec-butyl group, an n-pentyl group, a neopentyl group, an n-hexyl group, a 3-methylpentyl group, an n-heptyl group, and an n-octyl group.

[0068] R 2 The aryl group represented by the formula (I) is not particularly limited, but preferably has 6 to 12 carbon atoms, and more preferably 6 to 8 carbon atoms. The aryl group may be either monocyclic or polycyclic (e.g., bicyclic, tricyclic, etc.), but is preferably monocyclic. Specific examples of the aryl group include a phenyl group, naphthyl group, biphenyl group, pentalenyl group, indenyl group, anthranyl group, tetracenyl group, pentacenyl group, pyrenyl group, perylenyl group, fluorenyl group, and phenanthryl group, and a preferred example is a phenyl group.

[0069] R 2 The aralkyl group represented by the formula (I) is not particularly limited, and examples thereof include aralkyl groups in which a hydrogen atom (for example, 1 to 3, preferably 1 hydrogen atom) of a linear or branched alkyl group having 1 to 6 carbon atoms (preferably 1 to 3) is substituted with the above-mentioned aryl group. Specific examples of such aralkyl groups include a benzyl group and a phenethyl group.

[0070] The alkylaryl group is not particularly limited, and examples thereof include alkylaryl groups in which hydrogen atoms (for example, 1 to 3, preferably 1 hydrogen atom) of the aryl group are substituted with linear or branched alkyl groups having 1 to 6 carbon atoms (preferably 1 to 2). Specific examples of such alkylaryl groups include tolyl and xylyl groups.

[0071] R 2 The alkylaralkyl group represented by the formula (I) is not particularly limited, and examples thereof include alkylaralkyl groups in which hydrogen atoms (e.g., 1 to 3, preferably 1 hydrogen atom) on the aromatic ring of the aralkyl group are substituted with linear or branched alkyl groups having 1 to 6 carbon atoms (preferably 1 or 2).

[0072] R 3 Ha-L 1 -R 31 -L 2 -R 32 (L 1 and L 2 are the same or different and represent a single bond or a linker; R 31 indicates a bond between reactive groups, and R 32 represents a monovalent group containing -S-S- or -Se-Se-), or represents a hydrocarbon group (provided that at least one R 3 Ha-L 1 -R 31 -L 2 -R 32 indicates).

[0073] L 1 or L 2 Examples of linkers represented by the formula (I) include alkylene groups, alkenylene groups, and heteroalkylene groups (for example, heteroalkylene groups containing -NH-COO-, -O-, etc. in the main chain). Linkers may be either linear or branched, and are preferably linear. The number of atoms constituting the main chain of the linker (the number of carbon atoms in the case of alkylene and alkenylene groups) is not particularly limited and is, for example, 1 to 10, preferably 2 to 6, and more preferably 3 to 5.

[0074] R 31 For the bond between reactive groups represented by R 31aThe definition of the bond between reactive groups represented by the formula:

[0075] R 32 For the monovalent group containing -S-S- or -Se-Se-, the same explanation as for the disulfide unit is applied.

[0076] R 3 For hydrocarbon groups represented by R 2 The above description of the hydrocarbon group represented by the formula: is incorporated herein by reference.

[0077] R 4 is a hydrogen atom, or -L 1 -R 31 -L 2 -R 32 or R 3 R 3 To form a ring by connecting to, for example, R 3 It means that the ring structure is formed by bonding to any of the atoms constituting R. 3 and R 3 Specifically, for example, formula t:

[0078]

[0079] [wherein k represents an integer of 1 to 5 (preferably 1 to 4, more preferably 2 to 3, and even more preferably 2), L 1 , R 31 , L 2 , and R 32 is the same as above. In this case, L 1 is preferably an alkylene group having 2 to 3 carbon atoms (more preferably 2), and R 31 is preferably >N—COO—.

[0080] R 5 is a hydrogen atom, -L 1 -R 31 -L 2 -R 32 , or -R 51 -R 52 (R 51 is a single bond or -P(=O)(-OR 511 )-O-(R 511 represents a hydrogen atom or a hydrocarbon group), and R 52represents a hydrocarbon group which may be substituted with a hydroxyl group).

[0081] R 511 For hydrocarbon groups represented by R 2 The above description of the hydrocarbon group represented by the formula: is incorporated herein by reference.

[0082] R 52 For hydrocarbon groups represented by R 2 The above description of the hydrocarbon group represented by the formula (I) is incorporated herein by reference. The hydrocarbon group may be substituted with a hydroxyl group. The number of hydroxyl groups substituted in the hydrocarbon group is, for example, 0 to 3, 0 to 2, 0 to 1, or 0, or 1 to 3, 1 to 2, or 1.

[0083] R 6 and R 7 are the same or different and represent a hydrogen atom or a hydrocarbon group. 6 and R 7 is preferably a hydrogen atom.

[0084] R 6 or R 7 For hydrocarbon groups represented by R 2 The above description of the hydrocarbon group represented by the formula: is incorporated herein by reference.

[0085] n represents a natural number, for example, 1 or more, preferably 2 or more, more preferably 3 or more, even more preferably 5 or more, and still more preferably 10 or more. The upper limit of n is not particularly limited and is, for example, 50, 30, 20, or 15.

[0086] The spacer is not particularly limited as long as it is a structure that can appropriately separate the structure represented by general formula (1) from the polynucleotide. Examples of the spacer include the above-mentioned linkers, more specifically, alkyl chains, polyalkylene glycol chains, etc.

[0087] The structure represented by general formula (1) is particularly preferably represented by general formula (1a):

[0088]

[0089] The structure is expressed as follows.

[0090] R 8 , R 9 and R 10 are the same or different, -L 1b -R 31 -L 2b -R 32 Shows.

[0091] L 1b , L 2b Regarding L 1a , L 2a The definition of

[0092] R 8 , R 9 and R 10 In the above, particularly preferably, L 1b and L 2b are the same or different and are alkylene groups having 1 to 10 carbon atoms (preferably 2 to 6, more preferably 2 to 4), and R 31 is an amide bond or an ester bond, and R 32 is a ring-derived group (preferably a group represented by formula p, q, r, or s).

[0093] u, v, w, and x are the same or different and represent an integer of 1 to 8. u, v, w, and x are preferably 1 to 6, more preferably 1 to 4, even more preferably 1 or 2, and particularly preferably 1. u, v, w, and x are preferably the same.

[0094] The polynucleotide to be modified is not particularly limited, and may be DNA, RNA, or any other known chemical modification, as exemplified below. To prevent degradation by hydrolases such as nucleases, the phosphate residue of each nucleotide may be substituted with a chemically modified phosphate residue, such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate. The hydroxyl group at the 2-position of the sugar (ribose) of each ribonucleotide may also be substituted with -OR (where R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN). Furthermore, the base moiety (pyrimidine or purine) may be chemically modified, such as by introducing a methyl group or a cationic functional group into the 5-position of the pyrimidine base, or by substituting a thiocarbonyl group for the carbonyl group at the 2-position. Further examples include, but are not limited to, those in which the phosphate moiety or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, an acetyl group, etc. Also preferably used are BNA (LNA), in which the conformation of the sugar moiety is fixed to N-type by bridging the 2' oxygen and 4' carbon of the sugar moiety of the nucleotide.

[0095] The polynucleotide to be modified may be linked to another molecule. Examples of the other molecule include fluorescent labels. Examples of fluorescent labels include fluorescein, rhodamine, Texas Red, tetramethylrhodamine, carboxyrhodamine, phycoerythrin, 6-FAM™, Cy®3, Cy®5, and the Alexa Fluor® series.

[0096] The base length of the polynucleotide to be modified is not particularly limited, and is, for example, 1000 bases or less, preferably 500 bases or less, more preferably 200 bases or less, even more preferably 100 bases or less, and even more preferably 50 bases or less. The lower limit is not particularly limited, and is, for example, 5 bases, 10 bases, or 15 bases.

[0097] The polynucleotide to be modified is preferably one primarily intended for use after introduction into cells, and examples of such polynucleotides include antisense polynucleotides, siRNAs, miRNAs, miRNA precursors, aptamers, guide RNAs, and mRNAs.

[0098] The modified polynucleotide may be one type alone or a combination of two or more types.

[0099] The modified polynucleotide can be synthesized according to or in accordance with a known method, for example, by referring to Patent Document 2.

[0100] The lipid particles of the present invention are not particularly limited as long as they are particles formed from lipids, and examples thereof include lipid nanoparticles (LNPs, solid lipid nanoparticles: particles consisting of an outer layer of a lipid monolayer and an inner layer containing a lipid-soluble substance), liposomes (particles consisting of an outer layer of a lipid bilayer and an inner layer of an aqueous substance), and complexes of cationic lipids and polynucleotides.

[0101] The particle size of the lipid particles is preferably nano-sized, specifically, for example, 10 to 700 nm, preferably 20 to 500 nm, more preferably 40 to 300 nm, and even more preferably 60 to 200 nm.

[0102] The lipid particles of the present invention contain modified ionizable lipids as particle-constituting lipids and / or contain modified polynucleotides.

[0103] When the lipid particles of the present invention contain a modified ionized lipid as a particle-constituting lipid (Aspect 1), the content of the modified ionized lipid is, for example, 2 to 100 mol %, preferably 5 to 60 mol %, more preferably 10 to 40 mol %, even more preferably 15 to 30 mol %, and particularly preferably 15 to 25 mol %, relative to 100 mol % of the lipids constituting the lipid particles of the present invention.

[0104] In embodiment 1, the lipid particles of the present invention are particularly preferably lipid nanoparticles (LNPs, solid lipid nanoparticles: particles consisting of an outer layer of a lipid monolayer and an inner layer containing a lipid-soluble substance).

[0105] The particle size of the LNP is, for example, 10 to 700 nm, preferably 20 to 500 nm, more preferably 40 to 300 nm, and even more preferably 60 to 200 nm.

[0106] LNPs typically contain, as lipids, ionizable lipids (including modified ionizable lipids), phospholipids, and sterols. LNPs preferably further contain a water-soluble polymer-modified lipid.

[0107] The ionizable lipid is as defined above.

[0108] The ionizable lipid may be one type alone or a combination of two or more types.

[0109] The content of ionizable lipid in LNP is preferably 35 to 70 mol%, more preferably 40 to 65 mol%, even more preferably 45 to 60 mol%, and even more preferably 45 to 55 mol%, relative to 100 mol% of lipids constituting the lipid particles of the present invention.

[0110] The content of the modified ionized lipid in the LNP is, for example, 2 to 50 mol%, preferably 5 to 40 mol%, more preferably 10 to 30 mol%, even more preferably 15 to 30 mol%, and particularly preferably 15 to 25 mol%, relative to 100 mol% of the lipids constituting the lipid particles of the present invention.

[0111] Specific examples of phospholipids include phosphatidylcholines such as dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, myristoylpalmitoylphosphatidylcholine, myristoylstearoylphosphatidylcholine, and palmitoylstearoylphosphatidylcholine; dilauroylphosphatidylglycerol, dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, dioleoylphosphatidylglycerol, dilinoleoylphosphatidylglycerol, myristoylpalmitoylphosphatidylglycerol, and myristoylstearoylphosphatidyl Examples include phosphatidylglycerols such as glycerol and palmitoylstearoylphosphatidylglycerol; phosphatidylethanolamines such as dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine, dilinoleoylphosphatidylethanolamine, myristoylpalmitoylphosphatidylethanolamine, myristoylstearoylphosphatidylethanolamine, and palmitoylstearoylphosphatidylethanolamine; phosphatidylserine; phosphatidic acid; phosphatidylinositol; sphingomyelin; cardiolipin; egg yolk lecithin; soybean lecithin; and hydrogenated products thereof.

[0112] The phospholipids may be one type alone or two or more types in combination.

[0113] The phospholipid content in LNP is preferably 4 to 25 mol%, more preferably 4 to 20 mol%, even more preferably 4 to 15 mol%, even more preferably 6 to 12 mol%, and particularly preferably 8 to 12 mol%, relative to 100 mol% of lipids constituting the lipid particles of the present invention.

[0114] Specific examples of sterols include cholesterol, cholesteryl hemisuccinate, lanosterol, dihydrolanosterol, desmosterol, dihydrocholesterol, phytosterol, stigmasterol, zymosterol, ergosterol, sitosterol, campesterol, brassicasterol, etc. In particular, since the sterols have the effect of stabilizing the lipid particle membrane and regulating the fluidity of the lipid particle membrane, it is desirable that they be contained as constituent lipids of the lipid particle membrane.

[0115] The sterol may be one type alone or a combination of two or more types.

[0116] The sterol content in LNP is preferably 15 to 55 mol%, more preferably 25 to 50 mol%, even more preferably 30 to 45 mol%, and even more preferably 35 to 45 mol%, relative to 100 mol% of lipids constituting the lipid particles of the present invention.

[0117] The water-soluble polymer-modified lipid is a lipid to which a water-soluble polymer has been added, and is not particularly limited thereto. The water-soluble polymer is not particularly limited, but an example thereof is a polyethylene glycol (PEG) chain. The molecular weight of the water-soluble polymer is not particularly limited, but is, for example, 200 to 10,000, preferably 500 to 7,000, more preferably 500 to 4,000, even more preferably 1,000 to 3,000, and even more preferably 1,500 to 2,500. The lipid to be modified with the water-soluble polymer is preferably an amphipathic lipid, more preferably a phospholipid.

[0118] The water-soluble polymer-modified lipid may be one type alone or two or more types in combination.

[0119] The content of water-soluble polymer-modified lipid in LNP is, for example, 0 to 10 mol%, preferably 0.1 to 7 mol%, more preferably 0.2 to 5 mol%, and even more preferably 0.5 to 3 mol%, relative to 100 mol% of lipids constituting the lipid particles of the present invention.

[0120] In embodiment 1, the pKa of the lipid particles of the present invention is preferably 5-7.

[0121] When the lipid particles of the present invention contain a modified polynucleotide (Aspect 2), the modified polynucleotide is encapsulated in the lipid particle or forms a complex with the lipid (for example, a complex formed by electrostatic interaction with a cationic lipid).

[0122] In one embodiment of embodiment 2, the lipid particles of the present invention are lipid nanoparticles (LNPs, solid lipid nanoparticles: particles consisting of an outer layer of a lipid monolayer and an inner layer containing a lipid-soluble substance). The lipid nanoparticles are the same as those described above.

[0123] The lipid particles of the present invention can contain a drug that expresses its function in cells. Examples of the drug include polynucleotides, proteins, and low-molecular-weight compounds (with a molecular weight of, for example, 1000 or less, 50 to 700, or 100 to 500). In embodiment 2, the drug can be a modified polynucleotide.

[0124] The polynucleotide used as a drug is the same as the polynucleotide to be modified by the modified polynucleotide. Note that, in embodiment 1, relatively long polynucleotides are suitable for use. In this case, the upper limit of the base length of the polynucleotide can be, for example, 5,000 bases, 3,000 bases, 2,000 bases, or 1,000 bases.

[0125] The lipid particles of the present invention are usually formed in an aqueous solution, such as various buffer solutions (e.g., acetate buffer, phosphate buffer, formate buffer, histidine buffer, etc.).

[0126] The lipid particles of the present invention can be produced according to or in accordance with a known method for producing lipid particles, preferably by a method including a step (Step 1) of mixing an alcohol solution containing a lipid with an aqueous solution.

[0127] The alcohol used as the solvent for the alcohol solution is not particularly limited as long as it is an alcohol capable of dissolving lipids. Preferred examples of the alcohol include ethanol.

[0128] The lipid concentration in the alcohol solution is, for example, 0.1 to 20% by mass.

[0129] The mixing mode is not particularly limited as long as it allows the formation of lipid particles, but is usually a mode in which the mixture is vigorously stirred with a vortex mixer, etc. Alternatively, when the mixture is mixed in a reaction system using a microchannel, the mixture is mixed within the reaction system.

[0130] Step 1 is usually carried out at room temperature or under warming.

[0131] The lipid particles of the present invention can be used for various purposes aimed at introducing drugs into cells or causing them to function within cells, such as intracellular introduction agents, medicines, reagents, etc. (hereinafter, these may be collectively referred to as "agents of the present invention").

[0132] The pharmaceutical is one used for administration to animals, and is not particularly limited insofar as such. The pharmaceutical can be a pharmaceutical composition containing other components described below. The reagent is one used for use in experiments (particularly, in the present invention, one used for introduction into cells), and is not particularly limited insofar as such. The reagent can be a reagent composition containing other components described below.

[0133] The agent of the present invention is not particularly limited as long as it contains the lipid particles of the present invention, and may further contain other components as necessary. The other components are not particularly limited as long as they are pharmaceutically acceptable components, and examples thereof include bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, moisturizers, colorants, fragrances, chelating agents, etc.

[0134] The mode of use of the agent of the present invention is not particularly limited, and an appropriate mode of use can be adopted depending on the type of agent. For example, the agent of the present invention can be used in vitro (e.g., added to a culture medium for cultured cells) or in vivo (e.g., administered to an animal).

[0135] The target of application of the agent of the present invention is not particularly limited, and examples thereof include various mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; animal cells, etc. The type of cell is also not particularly limited, and examples thereof include blood cells, hematopoietic stem cells / progenitor cells, gametes (sperm, eggs), fibroblasts, epithelial cells, vascular endothelial cells, nerve cells, hepatocytes, keratinocytes, muscle cells, epidermal cells, endocrine cells, ES cells, iPS cells, tissue stem cells, cancer cells, etc.

[0136] The dosage form of the agent of the present invention is not particularly limited, and can be an appropriate dosage form depending on the mode of use.For example, when administered to animals, oral preparations such as tablets, capsules, granules, powders, fine granules, syrups, enteric-coated preparations, sustained-release capsules, chewable tablets, drops, pills, oral liquid preparations, confectionery tablets, sustained-release preparations, and sustained-release granules can be mentioned; external preparations such as nasal drops, inhalants, rectal suppositories, inserts, enemas, and jellies can be mentioned.In addition, the agent of the present invention can be any of solid preparations, semisolid preparations, and liquid preparations.

[0137] The content of the lipid particles of the present invention in the agent of the present invention depends on the mode of use, the subject to which it is applied, the condition of the subject to which it is applied, etc., and is not limited thereto, but can be, for example, 0.0001 to 100% by weight, preferably 0.001 to 50% by weight.

[0138] The dosage of the agent of the present invention when administered to an animal is not particularly limited as long as it is an effective amount that exhibits medicinal efficacy, and is typically 0.1 to 1000 mg / kg body weight per day in oral administration and 0.01 to 100 mg / kg body weight per day in parenteral administration, in terms of the weight of the lipid particles of the present invention as an active ingredient. The dosage is preferably administered once a day or in 2 to 3 divided doses per day, and can be increased or decreased as appropriate depending on the age, pathological condition, and symptoms.

[0139] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0140] Test Example 1. Evaluation test of lipid particles containing modified ionized lipids having disulfide units Unless otherwise specified, first- or special-grade reagents and solvents purchased from Wako Pure Chemical Industries, Ltd., Kanto Chemical Co., Ltd., Sigma Aldrich, Nacalai Tesque, and Tokyo Chemical Industry Co., Ltd. were used in the experiment. TLC was performed using Silicagel 70 F 254 TLC Plate Wako was used. NMR spectra were measured using a 400 MHz NMR ECS400 (JEOL Delta) and a 600 MHz NMR ECA600 (JEOL Delta). For the chemical shift values ​​of 1H NMR spectra, residual protons in the solvent were used as the internal standard when CDCl3 was used as the measurement solvent. For the chemical shift values ​​of 13C NMR spectra, CHCl3 (77.0 ppm) was used as the internal standard. Signal multiplicities are indicated using the abbreviations s: singlet, t: triplet, q: quartet, and m: multiplet, respectively. ESI-MS was measured using a compact (Bruker). ESI Tuning Mix (Agilent Technologies) was used for calibration when using the Compact (Bruker).

[0141] Test Example 1-1. Synthesis of modified ionized lipids with disulfide units The lipid synthesis scheme is shown below. Synthesis was carried out in a two-step reaction. Commercially available triethanolamine was used as the starting material, and a total of 11 fatty acids were used, differing in structure in terms of carbon chain length, number of unsaturated bonds, and whether or not they were branched. Compound 1 was synthesized by condensation reaction with each fatty acid using N,N'-dicyclohexylcarbodiimide (DCC) and N,N-dimethyl-4-aminopyridine (DMAP). Similarly, DCC and DMAP were used to condense α-lipoic acid with compound 1, synthesizing target compound 2.

[0142]

[0143] The synthesis protocol and compound data for each synthetic lipid are shown below. The numbers in parentheses indicate the library serial numbers.

[0144]

[0145] C18-2-A' Thionyl chloride (30 μL, 0.42 mmol, 1.2 eq) was dissolved in 2 mL of DCM, and the solution was stirred in an ice bath. Linoleic acid (110 μL, 0.35 mmol, 1.0 eq) was dissolved in 1.0 mL of DCM and added dropwise to the mixture. After stirring for 3 hours, the reaction was confirmed to be complete by TLC. The mixture was completely evaporated.

[0146] The starting material was dissolved in 2 mL of DCM. 48 μL (0.35 mmol, 1.0 eq) of triethylamine and 23 μL (0.18 mmol, 0.5 eq) of triethanolamine were added to the solution, and the mixture was stirred overnight at room temperature. After confirming the completion of the reaction by TLC, 5 mL of MQ was added and the mixture was extracted with DCM. The organic phase was washed twice with brine, dried over Na2SO4, and evaporated. The product was purified by column chromatography (DCM / MeOH = 19 / 1 to 12 / 1). The desired compound was obtained (24 mg, 20% yield).

[0147] 1 H-NMR 400 MHz CDCl3 0.866-0.902 (6H, q), 1.280-1.302 (28 H, m), 1.607 (4H, s), 2.017-2.07 (8H, q), 2.28-2.34 (4H, m), 2.72-2.84 (10H, m), 3.60-3.63 (2H, t, J=4.0), 4.12-4.20 (4H, m), 5.30-5.38 (8H, m) 13 C-NMR 400 MHz CDCl3 14.057, 22.555, 24.854, 25.611, 27.182, 29.108, 29.328, 29.597, 31.503, 34.233, 56.699, 59.708, 127.891, 128.025, 130.027, 130.209 HRMS(ESI) calcd. for C 42 H 75 NO5 [M+H] + 674.5645 found 674.5725. ​

[0148]

[0149] C18-2-A (8) C18-2-A' (100 mg, 0.14 mmol, 1.0 eq) was dissolved in 10 mL of DCM. DMAP (17 mg, 0.14 mmol, 1.0 eq) and lipoic acid (29 mg, 0.14 mmol, 1.0 eq) were added to the solution. After stirring in an ice bath, DCC (29 mg, 0.14 mmol, 1.0 eq) dissolved in 1.0 mL of DCM was added dropwise. The reaction mixture was stirred overnight at room temperature. After TLC showed the reaction was complete, the reaction mixture was filtered and evaporated. The crude product was purified by column chromatography (hexane / EtOAc = 5 / 1) to give the desired compound (72 mg, 60% yield).

[0150] 1 H-NMR 400 MHz CDCl3 0.899-0.894 (6H, m), 1.247-1.271(30H, m), 1.288-1.294(3H,m), 1.577-1.660 (6H, m), 1.872-1.939 (1H, q), 2.009-2.061(8H, m), 2.261-2.306 (6H, m), 2.412-2.491 (1H, m), 2.741-2.836 (9H, m), 3.070-3.201(2H, m), 3.522-3.590(1H, m), 4.083-4.136 (6H, m), 5.288-5.390 (8H, m) 13 C-NMR 400 MHz CDCl3 14.019, 14.067, 22.517, 24.854, 25.563, 27.134, 29.079, 29.146, 29.290, 29.558, 53.260, 56.259, 60.321, 62.342, 62.448, 127.833, 127.986, 129.663, 129.960, 130.142, 173.253, 173.608 HRMS(ESI) calcd. for C 50 H 87 NO6S2 [M+Na] + ​884.5872 found 884.6091.

[0151]

[0152] Control lipid (no cyclic disulfide) C18-2-A' (0.2 g, 0.28 mmol, 1.0 eq) was dissolved in 10 mL of DCM. DMAP (34 mg, 0.28 mmol, 1.0 eq) and hexanoic acid (32 mg, 0.28 mmol, 1.0 eq) were added to the solution. While stirring in an ice bath, DCC (58 mg, 0.28 mmol, 1.0 eq) dissolved in 1.0 mL of DCM was added dropwise. The reaction mixture was stirred overnight at room temperature. After TLC showed the reaction was complete, the reaction mixture was filtered and evaporated. The crude product was purified by column chromatography (hexane / EtOAc = 5 / 1) to give the desired compound (0.15 g, 70% yield).

[0153] 1 H-NMR 400 MHz CDCl3 0.887-0.898(9H, d), 1.310-1.480(30H, m), 1.593-1.614(9H, t, J=6.0), 1.948-2.046(8H, d), 2.281-2.308(6H, m), 2.776-2.915(9H, m), 4.111-4.135(6H, t, J=4.0), 5.323-5.396(8H, m) 13 C-NMR 400 MHz CDCl3 13.894, 14.057, 22.306, 22.565, 24.596, 24.902, 25.621, 27.192, 29.127, 29.194, 29.338, 29.606, 31.302, 31.513, 34.243, 53.298, 62.409, 127.891, 128.034, 130.027, 130.209, 173.694 HRMS(ESI) calcd. for C 48 H 85 NO6 [M+Na] + 772.6385 found 772.6707.

[0154] ​

[0155] C16-1-A' palmitoleic acid (0.10 g, 0.39 mmol, 2.0 eq) was dissolved in 4 mL of DCM. Triethanolamine (58 mg, 0.20 mmol, 1.0 eq) and DMAP (47 mg, 0.39 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (80 mg, 0.39 mmol, 2.0 eq) was dissolved in 2 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (43 mg, 35% yield) was obtained.

[0156] 1 H-NMR 400 MHz CDCl3 0.876-0.893 (6H, m), 1.116-1.299(20H, m), 1.62-1.71 (16H, m), 1.91-2.01(10H, m), 2.28-2.34 (4H, m), 2.72-2.84 (4H, m), 3.48-3.53 (2H, m), 4.12-4.15 (4H,m), 5.30-5.34 (4H, q) 13 C-NMR 400 MHz CDCl3 14.310, 22.656, 24.916, 25.593, 27.214, 28.979, 29.122, 29.694, 33.919, 46.871, 65.059, 65.689, 129.742, 129.990, 165.384 HRMS(ESI) calcd. for C 38 H 71 NO5 [M+H] + 621.5322 found 621.5320.

[0157]

[0158] ​C16-1-A (3) C16-1-A' (43 mg, 0.07 mmol, 1.0 eq) was dissolved in 2 mL of DCM, and α-lipoic acid (25 mg, 0.12 mmol, 1.5 eq) and DMAP (15 mg, 0.12 mmol, 1.5 eq) were added. The mixture was then stirred at 0 °C. DCC (25 mg, 0.12 mmol, 1.5 eq) was dissolved in 1 mL of DCM and added dropwise to the solution. The mixture was stirred in an ice bath for 3 h and overnight at rt. After TLC showed the reaction was complete, the residue was filtered off. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.12 g, yield 98%) was obtained.

[0159] 1 H-NMR 400 MHz CDCl3 0.86-0.90 (6H, m), 1.25-1.34(26H, m), 1.42-1.51 (1H, m), 1.53-1.75 (16H, m), 1.98-2.06 (8H, m), 2.27-2.34 (6H, m), 2.41-2.50 (1H, m), 2.82-2.85 (6H, t, J=6.0), 2.82-2.85 (6H,t, J=6), 3.08-3.17(2H,m), 4.09-4.15 (6H,m), 5.30-5.39 (4H, m) 13 C-NMR 400 MHz CDCl3 14.201, 22.747, 24.721, 25.007, 27.258, 27.306, 28.860, 29.070, 29.232, 29.289, 29.814, 31.864, 34.087, 34.344, 34.697,38.569, 53.390, 62.489, 62.604, 129.824, 130.091, 173.801 HRMS(ESI) calcd. for C 46 H 83 NO6S2 [M+H] + 810.5742 found 810.5752.

[0160]

[0161] ​C16-0-A' stearic acid (0.76 g, 2.68 mmol, 2.0 eq) was dissolved in 18 mL of DCM. Triethanolamine (0.20 g, 1.34 mmol, 1.0 eq) and DMAP (0.33 g, 2.68 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.55 g, 2.68 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.59 g, 65% yield) was obtained.

[0162] 1 H-NMR 400 MHz CDCl3 0.87-0.90 (6H, t, J=6.8), 1.25-1.29(46H, m),1.61-1.78 (6H, m), 1.94-2.01(4H,m), 2.29-2.34(4H,m), 2.73-2.84(6H,m), 3.54-3.63(2H,m), 4.13-4.18(4H,m) 13 C-NMR 400 MHz CDCl3 14.110, 22.684, 24.878, 27.205, 29.170, 29.313, 29.5484, 29.694, 31.916, 34.225, 52.947, 56.523, 58.841, 62.045, 129.723, 129.981, 173.824 HRMS(ESI) calcd. for C 38 H 75 NO5 [M+H] + 626.5653 found 626.5677.

[0163]

[0164] ​C16-0-A (1) C16-0-A' (0.30 g, 0.48 mmol, 1.0 eq) was dissolved in 8 mL of DCM. α-Lipoic acid (0.12 g, 0.58 mmol, 1.2 eq) and DMAP (70 mg, 0.58 mmol, 1.2 eq) were added. The mixture was then stirred at 0 °C. DCC (0.12 g, 0.58 mmol, 1.2 eq) was dissolved in 4 mL of DCM and added dropwise to the solution. After confirming the completion of the reaction by TLC, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.35 g, 89% yield) was obtained.

[0165] 1 H-NMR 400 MHz CDCl3 0.86-0.90 (6H, t, J=6.8), 1.25-1.29(46H, m),1.57-1.73 (16H, m), 1.83-1.97(1H,m), 2.27-2.32 (6H,m), 2.82-2.85(6H,t,J=6.0), 4.10-4.14 (6H,t,J=6.0), 13 C-NMR 400 MHz CDCl3 14.211, 22.785, 25.026, 29.280, 29.394, 29.451, 29.594, 29.795, 32.017, 34.373, 53.390, 62.480, 173.839 HRMS(ESI) calcd. for C 46 H 87 NO6S2 [M+Na] + 836.5872 found 836.5860.

[0166]

[0167] ​C18-1-A' oleic acid (0.76 g, 2.68 mmol, 2.0 eq) was dissolved in 17 mL of DCM. Triethanolamine (0.2 g, 1.34 mmol, 1.0 eq) and DMAP (0.33 g, 2.68 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.55 g, 2.68 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.89 g, 96% yield) was obtained.

[0168] 1 H-NMR 400 MHz CDCl3 0.85-0.93(6H,m), 1.26-1.29 (30H, m), 1.59-1.65 (6H,m), 1.99-2.01(8H,d), 2.13-2.18(8H,m), 2.28-2.32(4H,t,J=2.0), 2.71-2.74(4H,m), 2.81-2.88(4H,m), 3.51-3.66(2H, m), 4.10-4.19(4H,m), 5.29-5.37(4H,m) 13 C-NMR 400 MHz CDCl3 14.100, 22.665, 24.687, 24.849, 27.167, 29.112, 29.294, 29.503, 29.742, 30.925, 31.888, 34.196, 52.927, 56.533, 56.657, 62.045, 129.714, 129.971, 173.786 HRMS(ESI) calcd. for C 42 H 79 NO5 [M+H] + 677.5995 found 677.5990.

[0169]

[0170] ​C18-1-A (7) C18-1-A' (0.33 g, 0.48 mmol, 1.0 eq) was dissolved in 10 mL of DCM. α-Lipoic acid (0.12 g, 0.58 mmol, 1.2 eq) and DMAP (70 mg, 0.58 mmol, 1.2 eq) were added. The mixture was then stirred at 0 °C. DCC (0.12 g, 0.58 mmol, 1.2 eq) was dissolved in 4 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.37 g, yield 90%) was obtained.

[0171] 1 H-NMR 400 MHz CDCl3 0.860-0.877 (6H,t, J=6.8), 1.164-1.299 (42H, m), 1.394-1.68 (1H,m), 1.605-1.693 (9H,m), 1.704-1.882(1H,m), 1.980-2.014 (6H, m), 2.269-2.336(6H, m), 2.387-2.502(1H, m), 2.814-2.846(6H, t, J=6.4), 3.080-3.212 (2H,m), 3.531-3.601 (1H, m), 4.093-4.129 (6H, m), 5.298-5.384 (4H, m), 13 C-NMR 400 MHz CDCl3 14.201, 22.766, 25.007, 27.267, 27.306, 29.232, 29.404, 29.614, 29.804, 29.852, 31.988, 34.344, 53.400, 56.414, 62.480, 62.604, 129.814, 130.091, 173.782 HRMS(ESI) calcd. for C 50 H 91 NO6S2 [M+H] + 866.6296 found 866.6322.

[0172]

[0173] ​C18-0-A (5) Palmitic acid (0.68 g, 2.68 mmol, 2.0 eq) was dissolved in 15 mL of DCM. Triethanolamine (0.2 g, 1.34 mmol, 1.0 eq) and DMAP (0.33 g, 2.68 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.55 g, 2.68 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After completion of the reaction was confirmed by TLC, the residue was removed by filtration and completely evaporated. The reaction mixture (0.10 g, 0.14 mmol, 1.0 eq) was dissolved in 1 mL of DCM. α-Lipoic acid (31 mg, 0.15 mmol, 1.1 eq) and DMAP (18 mg, 0.15 mmol, 1.1 eq) were added. The mixture was then stirred at 0°C. DCC (31 mg, 0.15 mmol, 1.1 eq) was dissolved in 1 mL of DCM and added dropwise to the solution. After confirming the completion of the reaction by TLC, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (91 mg, yield 75%) was obtained.

[0174] 1 H-NMR 400MHz CDCl3 0.854-0.872 (6H, m), 1.248-1.287 (22H, m), 1.569-1.672 (9H, m), 1.867 -1.987 (1H, m), 2.300-2.334(3H, m), 2.425-2.489(1H, m), 2.729-2.855(6H, m), 3.100-3.186(2H, m), 3.526-3.604(2H, m), 4.101-4.167(4H, m), 13 ​C-NMR 400MHz CDCl3 13.665, 14.076, 22.660, 27.422, 22.470, 29.223, 29.261, 29.434, 31.676, 31.848, 32.384, 38.478, 40.212, 45.739, 52.848, 53.231, 56.316, 56.421, 58.797, 61.834, 62.170, 173.416, 176.549 HRMS(ESI) calcd. for C 50 H 95 NO6S2 [M+Na] + 892.6498 found 892.6477.

[0175]

[0176] C20-2-A (12) 11,14-Eicosadienoic acid (0.68 g, 2.68 mmol, 2.0 eq) was dissolved in 15 mL of DCM. Triethanolamine (0.20 g, 1.34 mmol, 1.0 eq) and DMAP (0.33 g, 2.68 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.55 g, 2.68 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After completion of the reaction by TLC, the residue was removed by filtration and completely evaporated. The reaction mixture (0.11 g, 0.14 mmol, 1.0 eq) was dissolved in 1 mL of DCM, and α-lipoic acid (30 mg, 0.15 mmol, 1.1 eq) and DMAP (18 mg, 0.15 mmol, 1.1 eq) were added. The mixture was then stirred at 0 °C. DCC (31 mg, 0.15 mmol, 1.1 eq) was dissolved in 1 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (61 mg, 46% yield) was obtained.

[0177] 1 ​H-NMR 400MHz CDCl3 0.869-0.905(6H, t, J=7.2), 1.121-1.336(42H, m), 1.602-1.693(8H, m), 1.882-1.950(1H, m), 2.019-2.072(6H, m), 2.386-2.502(1H, m), 2.754-2.770(4H, t, J=6.4), 2.830-2.846(4H, t, J=6.4), 3.108-3.183(2H, m), 3.547-3.692(1H, m), 4,110-4.114(6H, d), 5.310-5.369(8H, m) 13 C-NMR 400MHz CDCl3 14.062, 22.560, 24.916, 25.612, 27.186, 27.224, 29.170, 29.294, 29.332, 29.446, 29.503, 29.666, 31.516, 34.253, 53.299, 62.379, 127.911, 127.959, 130.114, 130.181, 173.710 HRMS(ESI) calcd. for C 54 H 95 NO6S2 [M+Na] + 940.6498 found 940.6501.

[0178]

[0179] C20-1-A' Cis-11-Eicosenoic acid (0.10 g, 0.32 mmol, 2.0 eq) was dissolved in 10 mL of DCM. Triethanolamine (24 mg, 0.16 mmol, 1.0 eq) and DMAP (39 mg, 0.16 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (66 mg, 0.16 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.10 g, 85% yield) was obtained.

[0180] 1 ​H-NMR 400MHz CDCl3 0.862-0.895(6H, t, J=6.4), 1.271(44H, m), 1.563-1.608(10H, m), 2.003-2.016(8H, d), 2.268-2.322(4H, m), 2.717-2.743(1H, t, J=5.2), 2.815-2.844(4H, t, J=6.0), 3.537(s, 1H), 4.096-4.152(4H, m), 5.304-5.357(4H, m) 13 C-NMR 400MHz CDCl3 14.105, 22.690, 27.211, 29.309, 29.510, 29.769, 31.905, 34.224, 129.835, 129.931, 173.809 HRMS(ESI) calcd. for C 46 H 87 NO5 [M+H] + 734.6592 found 734.6590.

[0181]

[0182] C20-1-A (11) C20-1-A' (0.10 g, 0.14 mmol, 1.0 eq) was dissolved in 10 mL of DCM, and α-lipoic acid (35 mg, 0.17 mmol, 1.2 eq) and DMAP (22 mg, 0.17 mmol, 1.2 eq) were added. The mixture was then stirred at 0 °C. DCC (35 mg, 0.17 mmol, 1.2 eq) was dissolved in 4 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (61 mg, 47% yield) was obtained.

[0183] 1 ​H-NMR 400MHz CDCl3 0.860-0.895(6H, t, J=7.2), 1.269-1.316(46H, m), 1.583-1.693(10H, m), 1.983-2.031(9H, m), 2.268-2.316(6H, m), 2.405-2.503(1H, m), 2.814-2.846(6H, t, J=6.4), 3.080-3.212(2H, m), 3.531-3.601(1H, m), 4.094-4.125(6H, t, J=6.4), 5.299-5.384(5H, m) 13 C-NMR 400MHz CDCl3 14.110, 22.675, 24.925, 27.205, 29.179, 29.313, 29.465, 29.523, 29.771, 31.897, 34.272, 53.309, 62.389, 129.828, 129.933, 173.729 HRMS(ESI) calcd. for C 54 H 99 NO6S2 [M+Na] + 944.6811 found 944.6799.

[0184]

[0185] C20-0-A' arachidic acid (0.30 g, 0.96 mmol, 2.0 eq) was dissolved in 8 mL of DCM. Triethanolamine (69 mg, 0.48 mmol, 1.0 eq) and DMAP (119 mg, 0.96 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.20 g, 0.96 mmol, 2.0 eq) was dissolved in 6 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.48 g, 64% yield) was obtained.

[0186] 1 ​H-NMR 400MHz CDCl3 0.858-0.892(6H, t, J=7.2), 1.161-1.363(62H, m), 1.585-1.676(16H, m), 2.171(1H, s), 2.283-2.308(4H, m), 2.714-2.740(2H, t, J=5.2), 2.812-2.841(5H, m), 3.481-3.555(2H, m), 4.109-4.150(4H, m) 13 C-NMR 400MHz CDCl3 14.120, 22.684, 24.887, 29.170, 29.294, 29.360, 29.484, 29.704, 31.926, 34.234, 52.947, 56.514, 58.841, 62.055, 173.853 HRMS(ESI) calcd. for C 46 H 91 NO5 [M+H] + 738.6905 found 738.6905.

[0187]

[0188] C20-0-A (9) C20-0-A' (0.10 g, 0.14 mmol, 1.0 eq) was dissolved in 9 mL of DCM, and α-lipoic acid (35 mg, 0.17 mmol, 1.2 eq) and DMAP (22 mg, 0.17 mmol, 1.2 eq) were added. The mixture was then stirred at 0 °C. DCC (35 mg, 0.17 mmol, 1.2 eq) was dissolved in 4 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (97 mg, 75% yield) was obtained.

[0189] 1 ​H-NMR 400MHz CDCl3 0.872-0.889(6H, t, J=4.8), 1.261-1.366(61H, m), 1.586-1.689(13H, m), 1.946(2H, s), 2.298-2.317(6H, m), 2.409-2.482(1H, m), 2.767-2.843(7H, m), 3.109-3.195(1H, m), 4.111-4.121(6H, d) 13 C-NMR 400MHz CDCl3 13.910, 22.474, 24.420, 24.725, 28.559, 28.969, 29.093, 29.141, 29.284, 29.494, 29.637, 31.707, 33.738, 33.776, 34.062, 40.004, 53.090, 62.179, 137.963, 173.529 HRMS(ESI) calcd. for C 54 H 103 NO6S2 [M+Na] + 948.7124 found 948.7113.

[0190]

[0191] C18-0-B' 2-Hexadecylooctadecanoic acid (0.20 g, 0.39 mmol, 2.0 eq) was dissolved in 10 mL of DCM. Triethanolamine (28 mg, 0.20 mmol, 1.0 eq) and DMAP (48 mg, 0.39 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (80 mg, 0.39 mmol, 2.0 eq) was dissolved in 5 mL of DCM and added dropwise to the solution. The mixture was stirred overnight. After TLC showed the reaction was complete, the residue was removed by filtration. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.28 g, 64% yield) was obtained.

[0192] 1 ​H-NMR 400 MHz CDCl3 0.827-0.861(12H, t, J=7.2), 1.118-1.285(107H, m), 1.338-1.411(2H, m), 1.547-1.585(8H, m), 1.676-1.778(4H, m), 1.870-1.956(4H, m), 2.269-2.313(1H, m), 2.704-2.731(1H, t, J=5.6), 2.782-2.827(3H, m) 13 C-NMR 400 MHz CDCl3 14.024, 22.617, 24.649, 27.415, 27.606, 29.303, 29.473, 29.637, 29.732, 31.029, 31.859, 32.327, 32.718, 33.318, 44.897, 45.669, 52.842, 61.788, 153.920, 176.361 HRMS(ESI) calcd. for C 74 H 147 NO2 [M+Na] + 1153.1176 found 1153.1172.

[0193]

[0194] C18-0-B (6) C18-0-B' (0.28 g, 0.25 mmol, 1.0 eq) was dissolved in 7 mL of DCM, and α-lipoic acid (62 mg, 0.30 mmol, 1.2 eq) and DMAP (43 mg, 0.30 mmol, 1.2 eq) were added. The mixture was then stirred at 0 °C. DCC (62 mg, 0.30 mmol, 1.2 eq) was dissolved in 2 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (96 mg, 29% yield) was obtained.

[0195] 1 ​H-NMR 400 MHz CDCl3 0.849-0.884(12H, t, J=7.2), 1.146-1.287(108H, m), 1.389-1.420(4H, m), 1.571-1.775(16H, m), 1.901-2.030(3H, m), 2.273-2.472(5H, m), 2.811-2.842(4H, t, J=6.0), 3.080-3.167(1H, m), 3.534-3.686(2H, m), 4.093-4.124(4H, t, J=6.4) 13 C-NMR 400 MHz CDCl3 14.072, 22.656, 24.678, 27.453, 27.653, 29.332, 29.465, 29.494, 29.666, 29.771, 31.077, 31.897, 32.765, 33.366, 38.421, 40.176, 44.954, 45.708, 49.837, 53.166, 53.328, 56.526, 62.179, 62.513, 153.958, 173.214, 176.352 HRMS(ESI) calcd. for C 82 H 159 NO6S2 [M+H] + 1319.1689 found 1319.1734

[0196]

[0197] C20-0-B' 2-octadecyleicosanoic acid (0.30 g, 0.53 mmol, 2.0 eq) was dissolved in 12 mL of DCM. Triethanolamine (37 mg, 0.26 mmol, 1.0 eq) and DMAP (65 mg, 0.53 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (0.11 g, 0.53 mmol, 2.0 eq) was dissolved in 5 mL of DCM and added dropwise to the solution. The mixture was stirred overnight. After TLC showed the reaction was complete, the residue was filtered off. The mixture was then evaporated and purified by column chromatography (DCM / MeOH = 15 / 1). The desired product (0.38 g, 58% yield) was obtained.

[0198] ​1 H-NMR 400 MHz CDCl3 0.856-0.890(12H, t, J=6.8), 1.110-1.291(105H, m), 1.313-1.425(7H, m), 1.567-1.622(7H, m), 1.728-1.781(8H, m), 1.900-2.038(6H, m), 2.315-2.390(3H, m), 2.486-2.501(1H, d), 2.733-2.759(2H, t, J=5.2), 2.825-2.854(3H, t, J=6.0), 3.165-3.215(1H, t). 3.482(1H, s), 3.543(1H, d), 3.659-3.691(1H, d), 4.088-4.159(4H, m) 13 C-NMR 400 MHz CDCl3 14.100, 22.675, 24.687, 27.472, 29.351, 29.494, 29.694, 29.790, 31.907, 42.799, 44.983, 45.736, 52.823, 53.395, 55.731, 58.831, 61.806, 153.977, 176.476C82H163NO5 1242.2531 HRMS(ESI) calcd. for C 82 H 163 NO5 [M+H] + 1243.2539 found 1243.2469.

[0199]

[0200] C20-0-B (10) C20-0-B' (0.10 g, 0.08 mmol, 1.0 eq) was dissolved in 3 mL of DCM, and α-lipoic acid (31 mg, 0.16 mmol, 2.0 eq) and DMAP (23 mg, 0.16 mmol, 2.0 eq) were added. The mixture was then stirred at 0 °C. DCC (31 mg, 0.16 mmol, 2.0 eq) was dissolved in 1 mL of DCM and added dropwise to the solution. After TLC confirmed the completion of the reaction, the residue was removed by filtration. The product was then evaporated and purified by column chromatography (DCM / MeOH = 10 / 1). The desired product (68 mg, 59% yield) was obtained.

[0201] 1 H-NMR 400 MHz CDCl3 0.849-0.884(12H, t, J=7.2), 1.146-1.287(124H, m), 1.389-1.420(4H, m), 1.571-1.775(16H, m), 1.901-2.030(3H, m), 2.273-2.472(5H, m), 2.811-2.842(4H, t, J=6.0), 3.080-3.167(1H, m), 3.534-3.686(2H, m), 4.093-4.124(4H, t, J=6.4) 13 C-NMR 400 MHz CDCl3 14.081, 22.655, 24.601, 24.678, 25.355, 25.450, 27.462, 27.663, 29.341, 29.475, 29.503, 29.675, 29.771, 31.087, 31.897, 32.384, 32.775, 33.376, 33.948, 38.431, 40.176, 44.964, 45.708, 53.175, 53.338, 56.266, 62.179, 62.522, 153.967, 173.214, 176.352 HRMS(ESI) calcd. for C 90 H 175 NO6S2 [M+Na] + 1454.2758 found 14.2786.

[0202] The general formula of the synthesized disulfide lipid is as follows:

[0203]

[0204] The structure, molecular weight and yield of the synthesized disulfide lipid are as follows:

[0205]

[0206]

[0207] ​Test Example 1-2. Preparation and evaluation of lipid particles using modified ionized lipids with disulfide units [Preparation of lipid nanoparticles (LNP)] The lipid composition of LNP and the encapsulated mRNA are as follows. DiPure NLuc was used as previously reported (Nat Commun. 2023 May 11;14(1):2657. doi: 10.1038 / s41467-023-38244-8. PMID: 37169757; PMCID: PMC10175277.).

[0208]

[0209] In the following experiments, unless otherwise specified, LNPs were prepared as follows. A lipid ethanol solution (total lipid amount: 600 nmol) with the desired lipid composition was prepared in a test tube. 200 μL of mRNA solution (1 mM citrate buffer, pH 4, 10 μg of mRNA (0.045 nmol)) was added to the test tube containing the lipid ethanol solution under vortexing, followed by 1 mL of acidic buffer (1 mM citrate buffer, pH 4). The LNP suspension in the test tube was then transferred to a centrifugal ultrafiltration device (product name: "Amicon Ultra (100 kDa)", Merck) and ultrafiltered by centrifugation at 1500 × g for 30 minutes. The resulting PBS suspension was then recovered in 400 μL of phosphate buffer (PBS, pH 7.4). The recovered PBS suspension was used as the LNP suspension.

[0210] [Measurement of particle size and zeta potential of LNP] The particle size and zeta potential of LNP were measured using a dynamic light scattering DLS device (product name: "Zetasizer", manufactured by Malvern Panalytical).

[0211] [Evaluation of mRNA Recovery Rate and Encapsulation Rate in LNP] The mRNA recovery rate and entrapment rate in LNP were evaluated using an RNA quantification reagent (product name: "RiboGreen," Thermo Fisher Scientific). The LNP suspension was diluted 50-100 times with TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) to prepare the measurement sample. Equal volumes of the measurement sample and the RiboGreen diluted solution and the RiboGreen diluted solution containing Triron X-100 were mixed to prepare the analytical sample. The fluorescence of this analytical sample was measured using a plate reader. Based on a calibration curve prepared using an mRNA solution of known concentration, the amount of mRNA after LNP preparation and the amount of mRNA not encapsulated in LNP were calculated, and the recovery rate and entrapment rate were calculated using the following formula.

[0212]

[0213] [Measurement of LNP lipid membrane pKa] TNS is a fluorescent substance with negative charges and hydrophobic sites. In aqueous solution, the hydrophobic sites are hydrated, resulting in almost no fluorescence. However, it interacts electrostatically with cationic lipid membranes, and the hydrophobic sites migrate into the lipid membrane, resulting in strong fluorescence. LNP and TNS are diluted and mixed with buffer solutions adjusted to various pH levels, and the fluorescence intensity of the resulting solutions is measured. The relationship between pH and fluorescence intensity is plotted, allowing the in situ measurement of the lipid membrane pKa of LNP (TNS assay).

[0214] Buffers of various pH values ​​were prepared: pH 3.5-5.5: 20 mM citrate buffer + 130 mM NaCl; pH 6.0-8.0: 20 mM phosphate buffer + 130 mM NaCl; and pH 8.5-9.5: 20 mM Tris-HCl buffer + 130 mM NaCl.

[0215] 396 μL of each pH buffer and 4 μL of 0.6 mM TNS were added to a 5 mL tube for a 100-fold dilution. The LNP solution was diluted with saline to a lipid concentration of 0.5 mM. 94 μL of each pH buffer and 6 μL of the LNP solution were mixed and applied to a 96-well black plate. Fluorescence intensity was measured using a plate reader.

[0216] [Transfection experiment into cultured cells] LNPs carrying mRNA encoding NanoLuciferase (NLuc) were added to HeLa cells, and the cytotoxicity and gene expression activity after 24 hours were evaluated as follows.

[0217] Cell culture was performed in DMEM medium supplemented with FBS (final concentration 10%), penicillin (100 U / mL), and streptomycin (100 ng / mL). To initiate the culture, cell stocks stored at -80°C were quickly thawed in a water bath and added to 9 mL of medium. The cells were then centrifuged at 4°C, 500 x g, and 5 min. After removing the supernatant, the cell pellet was suspended in 10 mL of medium and seeded onto a 10 cm dish. For subculture, upon reaching 80-90% confluency, cells were detached with 0.5 mL of 0.25% trypsin-0.02% EDTA solution and 1.5 mL of PBS(-), and 8 mL of medium was added. The cells were then centrifuged at rt, 500 x g, and 5 min. After removing the supernatant, the cell pellet was suspended in medium and counted using a hemocytometer before seeding at the appropriate concentration. The cells were cultured in a CO2 incubator (37°C, 5% CO2).

[0218] Twenty-four hours before the addition of LNP, HeLa cells were seeded in a 96-well plate at 6,000 cells / well in 100 μL. LNP diluted in medium to the desired concentration (1, 2.5, or 5 ng mRNA / well) was added and cultured for 24 hours. After removing the medium, 10-fold diluted CCK-8 solution was added at 100 μL / well. After incubation for 30 minutes at 37°C under 5% CO2, absorbance at 450 nm was measured using a plate reader. Cell viability (%) was calculated relative to the untreated group.

[0219] After measuring cell viability, the medium was removed, PBS was added at 50 μL / well, and NLuc substrate solution (Nano-Glo® Luciferase Assay System) was added at 50 μL / well. After shaking for 2 minutes, luminescence was measured using a plate reader.

[0220] [Cellular uptake measurement] Cell culture was performed as described above. HeLa cells were seeded in a 12-well plate at 100,000 cells / well / 1 mL and cultured for 24 hours. LNPs fluorescently labeled with DiD (0.5 mol% DiD of total lipid) were added at the desired amount, and after 6 hours, the cells were harvested by trypsinization. DiD fluorescence intensity was then measured using a flow cytometer (CytoFLEX).

[0221] [LNP lysosomal localization observation] 24 hours before the addition of DiD-labeled LNPs, HeLa cells were seeded at 10,000 cells / well in a 96-well plate (Keyence BZ-X800 for fluorescence microscopy) or 200,000 cells / dish in a 35 mm glass-bottom dish (Mica for confocal microscopy). DiD-labeled LNPs were added to each well. Four hours after LNP addition, nuclei were stained with Hoechst 33342 (Wako, Japan) and lysosomes with Lysosensor Green DND-189 (Thermo, USA). For semiquantitative analysis, the lysosomal colocalization rate was calculated using the following formula: lysosomal colocalization rate = colocalization area (yellow area indicating the confocal area between lysosomes and LNPs) / LNP area (red area indicating LNP localization). A lower rate indicates a higher rate of escape from lysosomes.

[0222] [In vivo NLuc-mRNA delivery] LNPs were subcutaneously injected into 4-6 week-old female ICR mice at a dose of 4 μg of mRNA per mouse. Mice were anesthetized with isoflurane, and 6 hours after LNP administration, 150 μl of fluorofurimazine (FFz) substrate solution (2.4 mM) was intraperitoneally injected into each mouse (0.48 μmol FFz / mouse). 10 minutes after substrate injection, mice were imaged using an IVIS imaging system (IVIS Lumina LT, PerkinElmer). Luminescence intensity was quantified using Living Image 4.0.

[0223] [Evaluation Results] The results of measuring the physical properties of LNP (average values ​​of N=3) are shown below.

[0224]

[0225]

[0226] The cell viability 24 hours after LNP addition is shown in Figure 1. The NLuc expression level 24 hours after LNP addition is shown in Figure 2. The cellular uptake level 6 hours after LNP addition is shown in Figure 3.

[0227] Representative images of lysosomal localization of LNPs are shown in Figure 6. The use of cyclic disulfide lipids reduced the lysosomal colocalization rate.

[0228] The results of the in vivo NLuc-mRNA delivery test are shown in Figure 7. The use of cyclic disulfide lipids improved the amount of protein expressed from mRNA in vivo.

[0229] Test Example 2. Evaluation test of lipid particles containing modified polynucleotides having disulfide units In this test example, the activity of antisense oligonucleotides was measured using cells containing a modified luciferase gene. Active luciferase is not expressed from the modified luciferase gene as is due to aberrant splicing, but introduction of the designed antisense oligonucleotide induces normal splicing, resulting in the expression of active luciferase.

[0230] Test Example 2-1. Synthesis of modified polynucleotide having disulfide unit A disulfide unit was added to an antisense oligonucleotide (ON-705: previously reported (Biochemistry, 1998, 37, 6236.)). The sequence of ON-705 (SEQ ID NO: 1) and the structure of the modified polynucleotide are as follows:

[0231]

[0232] [Synthesis of Trimeric MPON Sample] The total synthesis procedure for trimeric MPON (Membrane Permeable OligoNucleotide) includes the synthesis of a trimeric precursor using an automated DNA synthesizer, post-conjugation of α-lipoic acid-NHS, purification, and characterization.

[0233] Synthesis of Trimeric Precursors. Trimeric precursor oligonucleotides were synthesized according to standard synthetic protocols under optimized conditions. Trimeric MPON amidites were prepared as 75 mM acetonitrile solutions, and the coupling time was extended to 900 seconds. When incorporating a spacer linker, it was prepared and used according to the supplier's recommended conditions. Typically, deprotection and cleavage of oligonucleotides were performed by extruding them into 1.5 mL controlled-pore glass screw-cap tubes (Sarstedt, Germany) and treating them with 28% concentrated aqueous ammonia (aq.) appropriate for the unmodified MPON sequence at 55°C for 5 hours. The oligonucleotides were then concentrated using a centrifugal evaporator (CVE-3100, EYELA) and purified by reverse-phase HPLC.

[0234] For trimeric MPON, a modified procedure was used: the synthesized CPG was treated with 2 mL of a 10% diethylamine / acetonitrile solution for 5 min, washed with pure acetonitrile, and then purified directly on a column. The material was then extruded and deprotected using 28% aqueous ammonium hydroxide at 55 °C for 5 h. The resulting mixture was filtered and completely evaporated. To confirm the successful synthesis of the trimeric precursor at this step, it can be confirmed and characterized by Nanodrop, LC-MS, and HPLC before the next step.

[0235] Postconjugation: The resulting trimeric precursor oligonucleotide was adjusted to an appropriate concentration in 2 M phosphate buffer (pH 8.46) and mixed with an excess amount of (R)-α-lipoic acid-NHS solution (dissolved in DMF) and briefly vortexed. After shaking at room temperature for 5 hours, the reaction was terminated. The nucleic acid was isolated by ethanol precipitation (using a 3 M NaOAc / ethanol = 1:10 (v / v) solution) at -30 °C. After centrifugation, the resulting crude product was analyzed by LC-MS (Figure 4) and purified by HPLC. Finally, the isolated product was evaporated to dryness using a nitrogen purge to avoid oxidation of disulfide groups during concentration. The isolated yield of the desired product was quantified by nanotitration.

[0236] Test Example 2-2. Preparation and evaluation of lipid particles containing modified polynucleotides having disulfide units [Cationic lipid complex particles] Cationic lipid complex particles were obtained using modified polynucleotides and lipofectamine, and the antisense activity was evaluated by a splicing correction assay (HeLa 705: previously reported (Biochemistry, 1998, 37, 6236.)).

[0237] HeLa 705 cells were seeded at a density of 30,000 cells / well in 24-well plates 24 hours prior to treatment. The medium was removed and the cells were treated with 0.2 mM OPTI-MEM containing the synthesized oligonucleotides for 6 hours. Transfection was performed using the Lipofectamine® 3000 Transfection Kit (Invitrogen) according to the manufacturer's instructions (addition volume: 0.1 μL per well, incubation time: approximately 10 minutes). The medium was then replaced with D-MEM containing 10% FBS up to 300 mL per well and the cells were further cultured at 37°C under 5% CO2 for 18 hours. The medium was then removed and 100 μL of Glo Lysis Buffer 1x (Promega) was added to lyse the cells. The cell lysate was collected in a microtube, incubated at room temperature for 15 minutes, and centrifuged at 380 rpm.

[0238] Luciferase expression levels were assessed by mixing 40 μL of cell lysate with 50 μL of luciferase assay reagent (One-Glo luciferase reporter, Promega) and measuring light production using a microplate reader (Tristar 5, Berthold Technologies, USA).

[0239] The antisense activity of each oligonucleotide was determined by the relative luminescence units (RLU) per μg of protein assessed using the bicinchoninic acid method (Thermo Fisher Scientific, MA). Absorbance at 560 nm was measured using a Tristar 5 (Berthold Technologies, US) according to standard procedures and protocols. Each RLU / μg protein value obtained was normalized to that of the negative control.

[0240] The results are shown in Figure 5. Compared to LNP + ASO (ON-705), the LNP + MPON (M, M-C4, M-P18) system was superior, with an induction effect of up to nearly three times greater.

Claims

1. Lipid particles comprising modified ionizable lipids having disulfide units in their polar head groups and / or modified polynucleotides having disulfide units.

2. The lipid particle of claim 1, wherein the modified ionizable lipid has a tertiary amine structure.

3. The modified ionizable lipid has the general formula (2): [In the formula: R a Ha-L 1a -R 31a -L 2a -R 32a (L 1a and L 2a are the same or different and represent a single bond or a linker; R 31a indicates a bond between reactive groups, and R 32a represents a monovalent group containing -S-S- or -Se-Se-. b and R c and each of the formula (I) and (II) is the same or different and represents a chain structure containing a hydrocarbon chain.

4. The chain structure is -(CH2) s -R d -R e (In the formula: R d represents -O-C(=O)- or -C(=O)-O-, and R e represents a hydrocarbon chain; and s represents 1 to 10. The lipid particle according to claim 3, wherein the lipid particle has a structure represented by the following formula:

5. The lipid particle of claim 2, having a pKa of 5 to 7.

6. The lipid particle of claim 2, which encapsulates a polynucleotide.

7. The modified polynucleotide has the general formula (1): [In the formula: R 1 is a single bond, -(CH2) t - (t is an integer of 1 to 4), or -CH(-R 11 )-(R 11 represents an alkyl group). 2 represents a hydrogen atom or a hydrocarbon group. 3 Ha-L 1 -R 31 -L 2 -R 32 (L 1 and L 2 are the same or different and represent a single bond or a linker; R 31 indicates a bond between reactive groups, and R 32 represents a monovalent group containing -S-S- or -Se-Se-), or represents a hydrocarbon group (provided that at least one R 3 Ha-L 1 -R 31 -L 2 -R 32 (Indicates R 4 is a hydrogen atom, or -L 1 -R 31 -L 2 -R 32 or R 3 R 5 is a hydrogen atom, -L 1 -R 31 -L 2 -R 32 , or -R 51 -R 52 (R 51 is a single bond or -P(=O)(-OR 511 )-O-(R 511 represents a hydrogen atom or a hydrocarbon group), and R 52 represents a hydrocarbon group which may be substituted with a hydroxyl group). 6 and R 7 are the same or different and represent a hydrogen atom or a hydrocarbon group. n represents a natural number.] is linked to the end of a polynucleotide directly or via a spacer.

8. The lipid particle of claim 7, wherein the spacer is an alkyl chain or a polyalkylene glycol chain.

9. The structure has the general formula (1a): [In the formula: R 8 , R 9 and R 10 are the same or different, -L 1b -R 31 -L 2b -R 32 wherein u, v, w and x are the same or different and represent an integer of 1 to 8. The lipid particle according to claim 7, wherein u, v, w and x are the same or different and represent an integer of 1 to 8.

10. A pharmaceutical comprising the lipid particles according to any one of claims 1 to 9.

11. General formula (2): [In the formula: R a Ha-L 1a -R 31 -L 2a -R 32 (L 1a and L 2a are the same or different and represent a single bond or a linker; R 31 indicates a bond between reactive groups, and R 32 represents a monovalent group containing -S-S- or -Se-Se-. b and R c and the formula (I) may be the same or different and represent a chain structure containing a hydrocarbon chain.

12. General formula (1a): [In the formula: R 8 , R 9 and R 10 are the same or different, -L 1b --R 31 -L 2b -R 32 and u, v, w, and x are the same or different and represent an integer of 1 to 8.] is linked to the end of a polynucleotide directly or via a spacer.

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