LIPID COMPOUND OR A DERIVATIVE THEREOF AND PHARMACEUTICAL COMPOSITION EMPLOYING THE SAME

Abstract

A lipid compound or a derivative thereof and a pharmaceutical composition employing the same are provided. The lipid compound has a structure represented by Formula (I):

##STR00001## wherein Z.sup.1, Z.sup.2, Z.sup.3 and Z.sup.4 are as disclosed in the specification.

Claims

1. A lipid compound or a derivative thereof, wherein the lipid compound has a structure represented by Formula (I): ##STR00068## wherein Z.sup.1 is O or S; Z.sup.2 is -Q.sup.1-A.sup.1-Q.sup.2-A.sup.2-X.sup.1; Z.sup.3 and Z.sup.2 are the same; Z.sup.4 is -Q.sup.3-A.sup.3-Q.sup.4-A.sup.4-X.sup.2; each of Q.sup.1 and Q.sup.3 is independently a single bond, O or NH; each of Q.sup.2 and Q.sup.4 is independently a single bond, O, NH, SS, ##STR00069## A.sup.1 is C1-C6 alkylene group; A.sup.3 is a single bond or a C1-C6 alkylene group; each of A.sup.2 and A.sup.4 is independently a single bond, a C1-C12 alkylene group, a C2-C12 alkenylene group or a C2-C12 alkynylene group; X.sup.1 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, NR.sup.1R2, ##STR00070## X.sup.2 is hydrogen, a C6-C12 aryl group, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, NR.sup.7R.sup.8, ##STR00071## R.sup.1 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.9-Q.sup.7-X.sup.5; R.sup.2 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.10-Q.sup.8-X.sup.6; each of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 is independently hydrogen or a C1-C6 alkyl group; R.sup.7 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.11-Q.sup.9-X.sup.7; R.sup.8 is hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.12-Q.sup.10-X.sup.8; each of A.sup.5, A.sup.6, A.sup.7, A.sup.8, A.sup.9, A.sup.10, A.sup.11 and A.sup.12 is independently a C1-C12 alkylene group; each of Q.sup.5, Q.sup.6, Q.sup.7, Q.sup.8, Q.sup.9 and Q.sup.10 is independently O, NH, SS, ##STR00072## and each of X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7 and X.sup.8 is independently a C1-C24 alkyl group.

2. The lipid compound or a derivative thereof as claimed in claim 1, wherein Q.sup.1 is a single bond, and Q.sup.2 is O, NH, SS ##STR00073##

3. The lipid compound or a derivative thereof as claimed in claim 1, wherein Q.sup.2 is a single bond, and Q.sup.1 is O or NH.

4. The lipid compound or a derivative thereof as claimed in claim 1, wherein Q.sup.3 is a single bond, and Q.sup.4 is O, NH, SS, ##STR00074##

5. The lipid compound or a derivative thereof as claimed in claim 1, wherein Q.sup.4 is a single bond, and then Q.sup.3 is O or NH.

6. The lipid compound or a derivative thereof as claimed in claim 1, wherein A.sup.3 is a single bond, and A.sup.4 is a C1-C12 alkylene group, a C2-C12 alkenylene group or a C2-C12 alkynylene group.

7. The lipid compound or a derivative thereof as claimed in claim 1, wherein A.sup.4 is a single bond, and A.sup.3 is a C1-C6 alkylene group.

8. The lipid compound or a derivative thereof as claimed in claim 1, wherein A.sup.3 is a single bond and A.sup.4 is a single bond, and then Q.sup.4 is a single bond and Q.sup.3 is O or NH.

9. The lipid compound or a derivative thereof as claimed in claim 1, wherein Z.sup.4 and Z.sup.2 are different.

10. The lipid compound or a derivative thereof as claimed in claim 1, wherein Z.sup.2, Z.sup.3 and Z.sup.4 are the same.

11. The lipid compound or a derivative thereof as claimed in claim 1, wherein the lipid compound is ##STR00075##

12. The lipid compound or a derivative thereof as claimed in claim 1, wherein the derivative of the lipid compound is a pharmaceutically acceptable salt of the lipid compound or a solvate of the lipid compound.

13. A pharmaceutical composition, comprising: the lipid compound or a derivative thereof as claimed in claim 1; and a sterol.

14. The pharmaceutical composition as claimed in claim 13, wherein the sterol is cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol or a combination thereof.

15. The pharmaceutical composition as claimed in claim 13, further comprising: a helper lipid, wherein the helper lipid is 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol)(sodium salt)(DMPG), sulfoquinovosyl diacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-1-serine (DOPS), L-a-phosphatidylcholine (EPC), phosphatidylcholine (PC) or a combination thereof.

16. The pharmaceutical composition as claimed in claim 13, further comprising: a polyethylene glycol (PEG)-ylated lipid, wherein the polyethylene glycol (PEG)-ylated lipid is a polyethylene glycol-modified phosphatidylethanolamine, a polyethylene glycol-modified phosphatidic acid, a polyethylene glycol-modified ceramide, a polyethylene glycol-modified dialkyl amine, a polyethylene glycol-modified diacylglycerol, a polyethylene glycol-modified dialkyl glycerol, a polyethylene glycol-modified sterol, a polyethylene glycol-modified phospholipid or a combination thereof.

17. The pharmaceutical composition as claimed in claim 16, wherein the polyethylene glycol (PEG)-ylated lipid is 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-Dipalmitoyl-sn-glycerol-3-succinate-polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DPPE-PEG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycerol-3-methoxy-polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-sn-glycerol-methoxy-polyethylene glycol (DPG-PEG), ?-[2-(ditetradecylamino)-2-oxoethyl]-?-methoxy-poly(oxy-1,2-ethanediyl) or a combination thereof.

18. The pharmaceutical composition as claimed in claim 13, further comprising: a nucleic acid, wherein the nucleic acid is deoxyribonucleic acid (DNA), plasmid DNA, messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), small activated ribonucleic acid (saRNA), circular ribonucleic acid (circular RNA) or a combination thereof.

Description

DETAILED DESCRIPTION

[0012] In the following detailed description, for purposes of explanation, numerous specific details are set forth in order to provide a thorough understanding of the disclosed embodiments. It will be apparent, however, that one or more embodiments may be practiced without these specific details. In other instances, well-known structures and devices are schematically shown in order to simplify the drawing.

[0013] The following is directed to the lipid compound mentioned in the present disclosure or a derivative thereof and a pharmaceutical composition employing the same. It should be understood that the following description provides many different embodiments or examples for implementing the present disclosure. The specific components and arrangements described below are merely descriptive of the present disclosure. Of course, these are only examples and not limitations of the present disclosure. In present disclosure, the word about means that the specified amount increases or decreases an amount that is generally recognized as reasonable by a person skilled in the art.

[0014] The present disclosure provides a lipid compound or a derivative thereof. According to one embodiment of the present disclosure, the lipid compound mentioned in the present disclosure is an ionizable cationic phospholipid with a specific structure of which the main core is pentavalent phosphorus and which may include 1 to 3 ionizable tertiary amine groups. The lipid compound mentioned in the present disclosure is easier to metabolize in vivo and has lower toxicity. The lipid compound or a derivative thereof mentioned in the present disclosure can be positively charged in an acidic environment, thereby adsorbing a negatively charged nucleic acid to form lipid nanoparticles (LNP). In addition, the present disclosure also provides a pharmaceutical composition comprising the lipid compound or a derivative thereof mentioned in the present disclosure. By having the lipid compound or a derivative thereof mentioned in the present disclosure, the pharmaceutical composition mentioned in the present disclosure can form lipid nanoparticles (LNPs) encapsulating ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), thereby achieving the purposes such as protection of ribonucleic acid (RNA) and/or deoxyribonucleic acid (DNA), slowing down its degradation rate, and improving cell transfection efficiency.

[0015] According to one embodiment of the present disclosure, the lipid compound provided by the present disclosure has a structure represented by Formula (I):

##STR00007##

wherein Z.sup.1 may be 0 or S; Z.sup.2 may be -Q.sup.1-A.sup.1-Q.sup.2-A.sup.2-X.sup.1; Z.sup.3 and Z.sup.2 are the same; Z.sup.4 may be -Q.sup.3-A.sup.3-Q.sup.4-A.sup.4-X.sup.2; each of Q.sup.1 and Q.sup.3 may be independently a single bond, O or NH; each of Q.sup.2 and Q.sup.4 may be independently a single bond, O, NH, SS,

##STR00008##

A.sup.1 may be C1-C6 alkylene group; A.sup.3 may be a single bond or a C1-C6 alkylene group; each of A.sup.2 and A.sup.4 may be independently a single bond, a C1-C12 alkylene group, a C2-C12 alkenylene group or a C2-C12 alkynylene group; X.sup.1 may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, NR.sup.1R.sup.2,

##STR00009##

X.sup.2 may be hydrogen, a C6-C12 aryl group, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group, NR.sup.7R.sup.8,

##STR00010##

R.sup.1 may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.9-Q.sup.7-X.sup.5; R.sup.2 may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.10-Q.sup.8-X.sup.6; each of R.sup.3, R.sup.4, R.sup.5, R.sup.6, R.sup.9, R.sup.10, R.sup.11 and R.sup.12 may be independently hydrogen or a C1-C6 alkyl group; R.sup.7 may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.11-Q.sup.9-X.sup.7; R.sup.8 may be hydrogen, a C1-C24 alkyl group, a C2-C24 alkenyl group, a C2-C24 alkynyl group, a C1-C24 alkanol group, a C2-C24 alkenol group, a C2-C24 alkynol group or -A.sup.12-Q.sup.10-X.sup.8; each of A.sup.5, A.sup.6, A.sup.7, A.sup.8, A.sup.9, A.sup.10, A.sup.11 and A.sup.12 may be independently a C1-C12 alkylene group; each of Q.sup.5, Q.sup.6, Q.sup.7, Q.sup.8, Q.sup.9 and Q.sup.10 may be independently O, NH, SS,

##STR00011##

and each of X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7 and X.sup.8 may be independently a C1-C24 alkyl group.

[0016] According to one embodiment of the present disclosure, the derivative of the lipid compound mentioned in the present disclosure may be a pharmaceutically acceptable salt of the lipid compound or a solvate of the lipid compound.

[0017] According to one embodiment of the present disclosure, the pharmaceutically acceptable salt of the lipid compound may be formed by reacting the lipid compound with a pharmaceutically acceptable acid. Here, the pharmaceutically acceptable salt includes a salt formed by the lipid compound and an inorganic acid, such as a hydrochloride, a phosphate, a diphosphate, a hydrobromide, a sulfate, a sulfinate, or a nitrate. According to one embodiment of the present disclosure, the pharmaceutically acceptable salt includes a salt formed by the lipid compound and an organic acid, such as lactate, oxalate, malate, maleate, fumarate, tartrate, succinate, citrate, lactate, sulfonate, p-toluenesulfonate, 2-hydroxyethanesulfonate, benzoate, salicylate, stearate, trifluoroacetate, amino acid salt, or acetate. In addition, pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium and ammonium.

[0018] According to one embodiment of the present disclosure, the solvate of the lipid compound includes the hydrate of the lipid compound or the alcoholate of the lipid compound.

[0019] According to one embodiment of the present disclosure, a single bond mentioned in the present disclosure refers to the absence of a single atom at the relevant site. For example, in the structure where Z.sup.2 is -Q.sup.1-A.sup.1-Q.sup.2-A.sup.2-X.sup.1, when Q.sup.2 is a single bond, there are no individual atoms at the site represented by Q.sup.2, and A.sup.1 and A.sup.2 are directly connected such that Z.sup.2 is a structure of -Q.sup.1-A.sup.1-A.sup.2-X.sup.1.

[0020] According to one embodiment of the present disclosure, the alkyl group mentioned in the present disclosure may be a linear or branched alkyl group. According to one embodiment of the present disclosure, the alkenyl group mentioned in the present disclosure may be a linear or branched alkenyl group and include at least one carbon-carbon double bond. According to one embodiment of the present disclosure, the alkynyl group mentioned in the present disclosure may be a linear or branched alkynyl group and include at least one carbon-carbon triple bond).

[0021] For example, C1-C24 alkyl may be methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl or an isomer thereof.

[0022] According to one embodiment of the present disclosure, the alkanol group mentioned in the present disclosure may be a linear or branched alkanol group. According to one embodiment of the present disclosure, the alkenol group mentioned in the present disclosure may be a linear or branched alkenol group and include at least one carbon-carbon double bond. According to one embodiment of the present disclosure, the alkynol group mentioned in the present disclosure may be a linear or branched alkynyl group and include at least one carbon-carbon triple bond. Herein, the term alkanol group refers to an alkyl group in which at least one of its hydrogen on carbon is replaced by a hydroxyl group. The term alkenol group refers to an alkenyl group in which at least one of its hydrogen on carbon is replaced by a hydroxyl group. The term alkynol group refers to an alkynyl group in which at least one of its hydrogen on carbon is replaced by a hydroxyl group. In addition, According to one embodiment of the present disclosure, the C6-C12 aryl group mentioned in the present disclosure may be phenyl, biphenyl or naphthyl.

[0023] According to one embodiment of the present disclosure, the alkylene group mentioned in the present disclosure may be a linear or branched alkylene group. According to one embodiment of the present disclosure, the alkenylene group mentioned in the present disclosure may be a linear or branched alkenylene group. According to one embodiment of the present disclosure, the alkynylene group mentioned in the present disclosure may be a linear or branched alkynylene group.

[0024] For example, C1-C24 alkylene group may be methylene group, ethylene group, propylene group, butylene group, pentylene group, hexylene group or an isomer thereof.

[0025] According to one embodiment of the present disclosure, for the lipid compound having a structure represented by Formula (I) mentioned in the present disclosure, Z.sup.4 is different from Z.sup.2 (i.e. Z.sup.4 is different from Z.sup.3).

[0026] According to one embodiment of the present disclosure, for the lipid compound having a structure represented by Formula (I) mentioned in the present disclosure, Z.sup.2, Z.sup.3 and Z.sup.4 are the same.

[0027] According to one embodiment of the present disclosure, for the lipid compound having a structure represented by Formula (I) mentioned in the present disclosure, Q.sup.1 and Q.sup.2 will not be single bonds at the same time. In other words, when Q.sup.1 is a single bond, Q.sup.2 is not a single bond; and when Q.sup.2 is a single bond, Q.sup.1 is not a single bond.

[0028] According to one embodiment of the present disclosure, Q.sup.1 is a single bond, and Q.sup.2 may be O, NH, SS,

##STR00012##

[0029] According to one embodiment of the present disclosure, Q.sup.2 is a single bond, and Q.sup.1 may be O or NH.

[0030] According to one embodiment of the present disclosure, Q.sup.1 may be O or NH,

##STR00013##

[0031] According to one embodiment of the present disclosure, for the lipid compound having a structure represented by Formula (I) mentioned in the present disclosure, Q.sup.3 and Q.sup.4 will not be single bonds at the same time. In other words, when Q.sup.3 is a single bond, Q.sup.4 is not a single bond; and when Q.sup.4 is a single bond, Q.sup.3 is not a single bond.

[0032] According to one embodiment of the present disclosure, Q.sup.3 is a single bond, and Q.sup.4 may be O, NH, SS,

##STR00014##

[0033] According to one embodiment of the present disclosure, Q.sup.4 is a single bond, and Q may be O or NH.

[0034] According to one embodiment of the present disclosure, Q.sup.3 may be O or NH, Q.sup.4 may be O, NH, SS,

##STR00015##

and A.sup.3 is not a single bond.

[0035] According to one embodiment of the present disclosure, when A.sup.2 is a C1-C12 alkylene group, a C2-C12 alkenylene group or a C2-C12 alkynylene group, Q.sup.2 is not a single bond.

[0036] According to one embodiment of the present disclosure, when A.sup.3 is a single bond, A.sup.4 is a C1-C12 alkylene group, a C2-C12 alkenylene group or a C2-C12 alkynylene group.

[0037] According to one embodiment of the present disclosure, when A.sup.4 is a single bond, A.sup.3 is a C1-C6 alkylene group.

[0038] According to one embodiment of the present disclosure, when A.sup.3 is a single bond and A.sup.4 is a single bond, Q.sup.3 is O or NH.

[0039] According to one embodiment of the present disclosure, the lipid compound having a structure represented by Formula (I) mentioned in the present disclosure may be

##STR00016##

wherein the definitions of Q.sup.1, Q.sup.2, Q.sup.3, Q.sup.4, A.sup.1, A.sup.2, A.sup.3, A.sup.4, X.sup.1 and X.sup.2 are the same as above.

[0040] According to one embodiment of the present disclosure, the present disclosure also provide a pharmaceutical composition, such as a lipid nanoparticle composition. According to one embodiment of the present disclosure, the pharmaceutical composition comprise the lipid compound or a derivative thereof mentioned in the present disclosure and a sterol.

[0041] According to one embodiment of the present disclosure, in the pharmaceutical composition, the lipid compound (and/or a derivative thereof) is 100 parts by mole, and the content of the sterol may be 50 to 300 parts by mole, such as 60 parts by mole, 80 parts by mole, 100 parts by mole, 120 parts by mole, 150 parts by mole, 180 parts by mole, 200 parts by mole, 230 parts by mole, 250 parts by mole or 280 parts by mole. According to one embodiment of the present disclosure, the sterol may be cholesterol, cholesterol hexasuccinate, ergosterol, lanosterol, or a combination thereof.

[0042] According to one embodiment of the present disclosure, the pharmaceutical composition may further comprise a helper lipid, wherein the helper lipid may be 5 to 200 parts by mole, such as 6 parts by mole, 8 parts by mole, 10 parts by mole, 20 parts by mole, 30 parts by mole, 50 parts by mole, 75 parts by mole, 100 parts by mole, 120 parts by mole, 150 parts by mole, 170 parts by mole or 190 parts by mole.

[0043] According to one embodiment of the present disclosure, the helper lipid may be 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl 2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), hydrogenated soy phosphatidylcholine (HSPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(1-rac-glycerol)(sodium salt)(DMPG), sulfoquinovosyl diacylglycerol (SQDG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1,2-dioleoyl-sn-glycero-3-phospho-1-serine (DOPS), L-?-phosphatidylcholine (EPC), phosphatidylcholine (PC) or a combination thereof.

[0044] According to one embodiment of the present disclosure, the pharmaceutical composition may further comprise a polyethylene glycol (PEG)-ylated lipid, wherein the content of the PEGylated lipid may be 1 to 30 parts by mole, such as 2 parts by mole, 3 parts by mole, 4 parts by mole, 5 parts by mole, 6 parts by mole, 7 parts by mole, 8 parts by mole, 9 parts by mole, 10 parts by mole, 11 parts by mole, 12 parts by mole, 13 parts by mole, 14 parts by mole, 15 parts by mole, 16 parts by mole, 17 parts by mole, 18 parts by mole, 19 parts by mole, 20 parts by mole, 21 parts by mole, 22 parts by mole, 23 parts by mole, 24 parts by mole, 25 parts by mole, 26 parts by mole, 27 parts by mole, 28 parts by mole or 29 parts by mole.

[0045] According to one embodiment of the present disclosure, the PEGylated lipid may be a polyethylene glycol-modified phosphatidylethanolamine, a polyethylene glycol-modified phosphatidic acid, a polyethylene glycol-modified ceramide, a polyethylene glycol-modified dialkyl amine, a polyethylene glycol-modified diacylglycerol, a polyethylene glycol-modified dialkyl glycerol, a polyethylene glycol-modified sterol, a polyethylene glycol-modified phospholipid or a combination thereof.

[0046] According to one embodiment of the present disclosure, the PEGylated lipid may be 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DSPE-PEG), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DMPE-PEG), 1,2-Dipalmitoyl-sn-glycerol-3-succinate-polyethylene glycol (DPGS-PEG), cholesteryl-polyethylene glycol, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DPPE-PEG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-polyethylene glycol (DOPE-PEG), 1,2-dimyristoyl-sn-glycerol-methoxypolyethylene glycol (DMG-PEG), 1,2-distearoyl-sn-glycerol-3-methoxy-polyethylene glycol (DSG-PEG), 1,2-dipalmitoyl-sn-glycerol-methoxy-polyethylene glycol (DPG-PEG), ?-[2-(ditetradecylamino)-2-oxoethyl]-?-methoxy-poly(oxy-1,2-ethanediyl) (ALC-0159) or a combination thereof.

[0047] According to one embodiment of the present disclosure, the pharmaceutical composition may further comprise a nucleic acid, wherein the content of the nucleic acid may be 0.1 to 30 parts by mole, such as 0.2 parts by mole, 0.5 parts by mole, 1 molar part, 1.5 parts by mole, 2 parts by mole, 3 parts by mole, 4 parts by mole, 5 parts by mole, 6 parts by mole, 7 parts by mole, 8 parts by mole, 9 parts by mole, 10 parts by mole, 11 parts by mole, 12 parts by mole, 13 parts by mole, 14 parts by mole, 15 parts by mole, 16 parts by mole, 17 moles parts by mole, 18 parts by mole, 19 parts by mole, 20 parts by mole, 21 parts by mole, 22 parts by mole, 23 parts by mole, 24 parts by mole, 25 parts by mole, 26 parts by mole, 27 parts by mole, 28 parts by mole or 29 parts by mole.

[0048] According to one embodiment of the present disclosure, the nucleic acid may be deoxyribonucleic acid (DNA), plasmid DNA, messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), small activated ribonucleic acid (saRNA), circular ribonucleic acid (circular RNA) or a combination thereof.

[0049] In order to make the above content and other objects, features, and advantages of the present disclosure more obvious and understandable, exemplary examples are shown below and described in detail, but the present disclosure is not limited thereto.

[0050] Lipid Compound

[0051] The lipid compounds mentioned in Examples of the present disclosure are listed in Table 1.

TABLE-US-00001 TABLE 1 Structure Ex- am- ple 1 [00017] Lipid compound (1) (Et: C.sub.2H.sub.5) Ex- am- ple 2 [00018] Lipid compound (2) Ex- am- ple 3 [00019] Lipid compound (3)(Et: C.sub.2H.sub.5) Ex- am- ple 4 [00020] Lipid compound (4) (Ph: phenyl group) Ex- am- ple 5 [00021] Lipid compound (5) Ex- am- ple 6 [00022] Lipid compound (6) Ex- am- ple 7 [00023] Lipid compound (7) Ex- am- ple 8 [00024] Lipid compound (8) Ex- am- ple 9 [00025] Lipid compound (9) Ex- am- ple 10 [00026] Lipid compound (10) Ex- am- ple 11 [00027] Lipid compound (11) Ex- am- ple 12 [00028] Lipid compound (12) Ex- am- ple 13 [00029] Lipid compound (13) Ex- am- ple 14 [00030] Lipid compound (14) Ex- am- ple 15 [00031] Lipid compound (15) Ex- am- ple 16 [00032] Lipid compound (16) Ex- am- ple 17 [00033] Lipid compound (17) Ex- am- ple 18 [00034] Lipid compound (18) Ex- am- ple 19 [00035] Lipid compound (19) Ex- am- ple 20 [00036] Lipid compound (20) Ex- am- ple 21 [00037] Lipid compound (21) Ex- am- ple 22 [00038] Lipid compound (22) Ex- am- ple 23 [00039] Lipid compound (23) Ex- am- ple 24 [00040] Lipid compound (24) Ex- am- ple 25 [00041] Lipid compound (25) (Et: C.sub.2H.sub.5) Ex- am- ple 26 [00042] Lipid compound (26) Ex- am- ple 27 [00043] Lipid compound (27) Ex- am- ple 28 [00044] Lipid compound (28) Ex- am- ple 29 [00045] Lipid compound (29) (Et: C.sub.2H.sub.5) Ex- am- ple 30 [00046] Lipid compound (30) (Et: C.sub.2H.sub.5) Ex- am- ple 31 [00047] Lipid compound (31) Ex- am- ple 32 [00048] Lipid compound (32) Ex- am- ple 33 [00049] Lipid compound (33) Ex- am- ple 34 [00050] Lipid compound (34)

[0052] In order to further illustrate the preparation methods of the lipid compounds mentioned in the present disclosure, the preparation processes of the lipid compounds mentioned in Examples 1-3, 6-15, 18 and 19 are shown below to illustrate.

Example 1

[0053] Compound (1) (2 equivalents) and tetrahydrofuran (THF) were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran; the concentration was 1.0 M; LiHMDS was 3 equivalents) was added to the reaction flask. After stirring at room temperature for reacting for 1 hour, Compound (2) (1 equivalent) was added dropwise to the reaction flask. After the reaction was completed, the resultant was concentrated and extracted with n-hexane and brine. After collecting the organic phase, removing water with sodium sulfate (Na.sub.2SO.sub.4) and removing the solvent, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (1). The reaction formula of the reaction mentioned above is as follows:

##STR00051##

[0054] Next, Lipid compound (1) mentioned in Example 1 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 4.48 (s, 4H), 4.18 (t, J=7.0, 7.5 Hz, 2H), 3.40 (s, 4H), 3.09 (m, 9H), 1.68 (s, 9H), 1.36-1.26 (m, 55H), 0.87 (t, J=7.0, 7.0 Hz, 12H). After that, Lipid compound (1) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=718.7.

Example 2

[0055] Compound (1) (2.5 equivalents) and tetrahydrofuran (THF) were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran; the concentration was 1.0 M; LiHMDS was 3 equivalents) was added to the reaction flask. After stirring at room temperature for reacting for 1 hour, Compound (3) (1 equivalent) was added dropwise to the reaction flask. After the reaction was completed, the resultant was concentrated and extracted with n-hexane and brine. After collecting the organic phase, removing water with sodium sulfate (Na.sub.2SO.sub.4) and removing the solvent, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (2). The reaction formula of the reaction mentioned above is as follows:

##STR00052##

[0056] Next, Lipid compound (2) mentioned in Example 2 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 4.42 (s, 2H), 4.29 (s, 2H), 4.10-4.09 (m, 2H), 3.73 (s, 4H), 3.47-3.43 (m, 2H), 3.32-3.30 (m, 4H), 3.11-3.04 (m, 8H), 1.67 (s, 12H), 1.32-1.25 (m, 77H), 0.88 (t, J=7.5, 7.5 Hz, 18H). After that, Lipid compound (2) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=986.1.

Example 3

[0057] Compound (4) (2 equivalents) and tetrahydrofuran (THF) were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.1 M). Next, a lithium bis(trimethylsilyl)amide (LiHMDS) solution (dissolved in tetrahydrofuran; the concentration was 1.0 M; LiHMDS was 3 equivalents) was added to the reaction flask. After stirring at room temperature for reacting for 1 hour, Compound (2) (1 equivalent) was added dropwise to the reaction flask. After the reaction was completed, the resultant was concentrated and extracted with n-hexane and brine. After collecting the organic phase, removing water with sodium sulfate (Na.sub.2SO.sub.4) and removing the solvent, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (3). The reaction formula of the reaction mentioned above is as follows:

##STR00053##

[0058] Next, Lipid compound (3) mentioned in Example 3 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): 4.13 (t, J=6.5, 7.5 Hz, 6H), 3.19 (s, 4H), 3.03-3.02 (m, 8H), 2.17-2.14 (m, 11H), 1.67 (s, 8H), 1.35-1.26 (m, 52H), 0.87 (t, J=7.0, 6.5 Hz, 12H). After that, Lipid compound (3) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=746.5.

Example 6

[0059] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (6) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (6). The reaction formula of the reaction mentioned above is as follows:

##STR00054##

[0060] Next, Lipid compound (6) mentioned in Example 6 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 10.62 (s, 1H), 8.73 (s, 1H), 3.60 (s, 3H), 3.22 (s, 3H), 3.09-3.06 (m, 4H), 2.54-2.56 (m, 2H), 2.11-2.13 (m, 4H), 1.63 (s, 12H), 1.32-1.26 (m, 77H), 0.88 (t, J=7.5, 7.5 Hz, 18H). After that, Lipid compound (6) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1151.3.

Example 7

[0061] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (7) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (7). The reaction formula of the reaction mentioned above is as follows:

##STR00055##

[0062] Next, Lipid compound (7) mentioned in Example 7 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 7.98 (s, 3H), 4.49 (s, 7H), 3.21 (s, 6H), 3.10-2.8 (m, 18H), 2.48 (s, 5H), 2.07 (s, 5H), 1.80-1.40 (m, 26H), 1.40-1.10 (m, 78H), 0.84 (t, J=6.0, 7.0 Hz, 18H). After that, Lipid compound (7) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1235.5.

Example 8

[0063] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (8) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (8). The reaction formula of the reaction mentioned above is as follows:

##STR00056##

[0064] Next, Lipid compound (8) mentioned in Example 8 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 11.52 (s, 1H), 5.57 (s, 1H), 3.18 (s, 7H), 2.30 (s, 10H), 2.50 (s, 3H), 2.06 (s, 4H), 1.67-1.65 (m, 11H), 1.34-1.25 (m, 51H), 0.87 (t, J=2.0, 5.5 Hz, 9H). After that, Lipid compound (8) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1319.6.

Example 9

[0065] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Next, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (7) (4 equivalents) and Compound (9) (1 equivalent) were added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (9). The reaction formula of the reaction mentioned above is as follows:

##STR00057##

[0066] Next, Lipid compound (9) mentioned in Example 9 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 10.84 (s, 1H), 8.03 (s, 2H), 3.66 (s, 2H), 3.33-3.24 (m, 6H), 3.04-2.99 (m, 16H), 2.81 (s, 1H), 2.53 (s, 4H), 2.11 (s, 4H), 1.76-1.56 (m, 12H), 1.33-1.27 (m, 38H), 0.88 (t, J=6.5, 7.5 Hz, 9H). After that, Lipid compound (9) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=955.8.

Example 10

[0067] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (10) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (10). The reaction formula of the reaction mentioned above is as follows:

##STR00058##

[0068] Next, Lipid compound (10) mentioned in Example 10 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J=7.0, 7.5 Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J=6.5, 7.0 Hz, 9H). After that, Lipid compound (10) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1024.8.

Example 11

[0069] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Next, diisopropylethylamine (DIPEA) (3 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (3 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (6) (4 equivalents) and Compound (9) (1 equivalent) were added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (11). The reaction formula of the reaction mentioned above is as follows:

##STR00059##

[0070] Next, Lipid compound (11) mentioned in Example 11 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 8.79 (s, 1H), 3.70-3.68 (m, 3H), 3.54 (s, 2H), 3.38 (s, 2H), 3.23-3.03 (m, 9H), 2.60-2.57 (m, 16H), 2.16-2.02 (m, 5H), 1.63 (s, 6H), 1.50 (t, J=7.0, 7.5 Hz, 1H), 1.33-1.27 (m, 39H), 0.89 (t, J=6.5, 7.0 Hz, 9H). After that, Lipid compound (11) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=899.7.

Example 12

[0071] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (11) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (12). The reaction formula of the reaction mentioned above is as follows:

##STR00060##

[0072] Next, Lipid compound (12) mentioned in Example 12 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 8.50 (s, 2H), 6.46 (s, 5H), 4.10-3.90 (m, 3H), 3.90-3.0 (m, 13H), 2.55 (s, 3H), 2.15 (s, 3H), 1.60-1.20 (m, 61H), 0.88 (t, J=7.0, 7.5 Hz, 10H). After that, Lipid compound (12) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1499.4.

Example 13

[0073] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (12) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (13). The reaction formula of the reaction mentioned above is as follows:

##STR00061##

[0074] Next, Lipid compound (13) mentioned in Example 13 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 10.68 (s, 2H), 8.78 (s, 3H), 3.59 (d, J=4.0 Hz, 5H), 3.45-3.20 (m, 12H), 3.20-3.0 (m, 11H), 2.60-2.40 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.45 (m, 10H), 1.40-1.20 (m, 32H), 0.88 (t, J=6.5 Hz, 15H). After that, Lipid compound (13) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=898.8.

Example 14

[0075] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (13) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (14). The reaction formula of the reaction mentioned above is as follows:

##STR00062##

[0076] Next, Lipid compound (14) mentioned in Example 14 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 10.67 (s, 2H), 8.75 (s, 2H), 4.25-4.0 (m, 5H), 3.62 (d, J=4.5 Hz, 4H), 3.25-3.20 (m, 4H), 3.20-3.0 (m, 9H), 2.65-2.45 (m, 4H), 2.25-2.15 (m, 4H), 1.80-1.60 (m, 9H), 1.45-1.20 (m, 44H), 0.89 (t, J=6.0, 7.5 Hz, 12H). After that, Lipid compound (14) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1067.

Example 15

[0077] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (14) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (15). The reaction formula of the reaction mentioned above is as follows:

##STR00063##

[0078] Next, Lipid compound (15) mentioned in Example 15 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 10.77 (s, 2H), 8.80 (s, 2H), 3.62-3.50 (m, 7H), 3.30-3.20 (m, 5H), 3.20-3.0 (m, 11H), 2.60-2.50 (m, 5H), 2.20-2.0 (m, 5H), 1.80-1.50 (m, 11H), 1.50-1.20 (m, 40H), 0.89 (t, J=5.0, 7.0 Hz, 14H). After that, Lipid compound (15) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=982.8.

Example 18

[0079] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (15) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (18). The reaction formula of the reaction mentioned above is as follows:

##STR00064##

[0080] Next, Lipid compound (18) mentioned in Example 18 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 8.66 (s, 2H), 4.60-3.90 (m, 13H), 3.90-3.0 (m, 9H), 2.70-2.40 (m, 4H), 2.30-2.0 (m, 4H), 1.70-1.20 (m, 45H), 0.89 (t, J=6.0, 7.0 Hz, 13H). After that, Lipid compound (18) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=864.3.

Example 19

[0081] Compound (5) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.04 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (16) (4 equivalents) was added dropwise to the reaction flask. After reacting for 8 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (19). The reaction formula of the reaction mentioned above is as follows:

##STR00065##

[0082] Next, Lipid compound (19) mentioned in Example 19 was analyzed by nuclear magnetic resonance spectroscopy. The obtained spectral information is shown in the following: .sup.1H-NMR (500 MHz, CDCl.sub.3): ? 8.63 (s, 2H), 4.70-3.90 (m, 12H), 3.90-3.0 (m, 17H), 2.70-2.50 (m, 4H), 2.30-2.10 (m, 4H), 1.60-1.20 (m, 48H), 0.88 (t, J=6.5, 7.5 Hz, 11H). After that, Lipid compound (19) was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1247.1.

Example 23

[0083] Compound (23a) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (17) (2.5 equivalents) was added dropwise to the reaction flask. After reacting for 12 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (23). The reaction formula of the reaction mentioned above is as follows:

##STR00066##

[0084] Next, Lipid compound (23) mentioned in Example 23 was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1147.6.

Example 24

[0085] Compound (24a) (1 equivalent) and dichloromethane were added into a reaction flask under nitrogen to obtain a solution (concentration: 0.05 M). Next, diisopropylethylamine (DIPEA) (4 equivalents) and N-[(dimethylamino)-1H-1,2,3-triazolo-[4,5-b]pyridine-1-yl-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU) (4 equivalents) were added to the reaction flask. After stirring at room temperature for reacting for 10 minutes, Compound (18) (2.5 equivalents) was added dropwise to the reaction flask. After reacting for 16 hours, the resultant was purified with high-performance liquid chromatography (HPLC) (using acetonitrile and trifluoroacetic acid (TFA) aqueous solution (the concentration was 0.1%) as eluent) to obtain Lipid compound (24). The reaction formula of the reaction mentioned above is as follows:

##STR00067##

[0086] Next, Lipid compound (24) mentioned in Example 24 was analyzed by liquid chromatography-mass spectrometry (LC-MS) and M/Z was measured: [M+H].sup.+=1175.7.

[0087] Preparation of Compositions

Example 35

[0088] Luciferase mRNA (product number R1018, purchased from APExBIO) was dissolved in an acidic buffer (product number J63669, purchased from Alfa Aesar) (pH value 4.5) to obtain a nucleic acid aqueous solution (concentration: 0.1 mg/mL).

[0089] Lipid compound (1) prepared in Example 1, cholesterol, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and DMG-PEG lipid (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol)(product number: DMG-PEG-2K, purchased from Nippon Fine Chemical) were dissolved in ethanol to obtain a lipid solution (concentration: 10-15 mg/mL), wherein the contents of Lipid compound (1), cholesterol, DSPC and DMG-PEG lipid contents are shown in Table 2.

[0090] Next, the nucleic acid aqueous solution and the lipid solution were mixed through a microfluidic system (NanoAssemblr Ignite system, purchased from Precision NanoSystem Inc.), and the volumes of the nucleic acid aqueous solution and the lipid solution were adjusted so that the ratio of nitrogen atoms (N) of Lipid compound (1) to phosphorus atoms (P) derived from luciferase mRNA (N/P) was 12. Then, after ultrafiltration or dialysis, the obtained composition was self-assembled to form Nucleic acid-lipid nanoparticle carrier (1).

Examples 36-43

[0091] Nucleic acid-lipid nanoparticle carriers (2)-(9) were obtained by performing the same manner as in Example 35 but changing the ingredients and/or contents in the lipid solution and the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) according to Table 1.

Comparative Example 1

[0092] Nucleic acid-lipid nanoparticle carrier (10) was obtained by performing the same manner as in Example 35 but replacing Lipid compound (1) with (6Z,9Z,28Z,31Z)-heptatriacont-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA) and adjusting the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution).

Comparative Example 2

[0093] Nucleic acid-lipid nanoparticle carrier (11) was obtained by performing the same manner as in Example 35 but replacing Lipid compound (1) with heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate (SM-102) and adjusting the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution).

TABLE-US-00002 TABLE 2 Lipid DMG- compound PEG (1) (parts DSPC Cholesterol (parts by by (parts by (parts by N/P mole) mole) mole) mole) Example Nucleic 12 100 3 20 77 35 acid-lipid nanoparticle carrier (1) Example Nucleic 12 100 2.5 0 64.17 36 acid-lipid nanoparticle carrier (2) Example Nucleic 12 100 3.75 50 96.25 37 acid-lipid nanoparticle carrier (3) Example Nucleic 12 100 7.15 45.71 132.86 38 acid-lipid nanoparticle carrier (4) Lipid DMG- compound PEG (2) (parts DSPC Cholesterol (parts by by (parts by (parts by N/P mole) mole) mole) mole) Example Nucleic 18 100 3.75 50 96.25 39 acid-lipid nanoparticle carrier (5) Example Nucleic 18 100 7.15 45.71 132.86 40 acid-lipid nanoparticle carrier (6) Example Nucleic 9 100 4.55 150 200 41 acid-lipid nanoparticle carrier (7) Lipid compound (1)(parts by DMG- mole)/Lipid PEG compound (parts DSPC Cholesterol (2)(parts by by (parts by (parts by N/P mole) mole) mole) mole) Example Nucleic 16.5 75/25 3.75 50 96.25 42 acid-lipid nanoparticle carrier (8) Example Nucleic 17.3 87.5/12.5 3.75 50 96.25 43 acid-lipid nanoparticle carrier (9) DMG- PEG DSPC Cholesterol DLin-MC3- (parts by (parts by (parts by N/P DMA mole) mole) mole) Compar- Nucleic 6 100 3 20 77 ative acid-lipid example nanoparticle 1 carrier (10) N/P SM-102 DMG- DSPC Cholesterol PEG (parts by (parts by (parts by mole) mole) mole) Compar- Nucleic 6 100 3 20 77 ative acid-lipid example nanoparticle 2 carrier (11)

Examples 44-52

[0094] Nucleic acid-lipid nanoparticle carriers (12)-(20) were obtained by performing the same manner as in Example 35 but changing the ingredients and/or contents in the lipid solution and the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) according to Table 3.

TABLE-US-00003 TABLE 3 Lipid DMG- compound PEG DSPC Cholesterol (3) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 12 100 3.75 50 96.25 44 lipid nanoparticle carrier (12) Example Nucleic acid- 12 100 7.15 45.71 132.86 45 lipid nanoparticle carrier (13) Example Nucleic acid- 16 100 7.15 45.71 132.86 46 lipid nanoparticle carrier (14) Lipid DMG- compound PEG DSPC Cholesterol (6) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 18 100 3 20 77 47 lipid nanoparticle carrier (15) Example Nucleic acid- 18 100 5 100 128.33 48 lipid nanoparticle carrier (16) Example Nucleic acid- 18 100 7.15 45.71 132.86 49 lipid nanoparticle carrier (17) Lipid DMG- DSPC Cholesterol compound PEG (parts by (parts by (7) (parts by (parts by N/P mole) mole) mole) mole) Example Nucleic acid- 18 100 3 20 77 50 lipid nanoparticle carrier (18) Example Nucleic acid- 18 100 5 100 128.33 51 lipid nanoparticle carrier (19) Example Nucleic acid- 18 100 7.15 45.71 132.86 52 lipid nanoparticle carrier (20)

Examples 53-61

[0095] Nucleic acid-lipid nanoparticle carriers (21)-(29) were obtained by performing the same manner as in Example 35 but changing the ingredients and/or contents in the lipid solution and the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) according to Table 4.

TABLE-US-00004 TABLE 4 Lipid DMG- compound PEG DSPC Cholesterol (8) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 18 100 3 20 77 53 lipid nanoparticle carrier (21) Example Nucleic acid- 18 100 5 100 128.33 lipid nanoparticle 54 carrier (22) Example Nucleic acid- 18 100 7.15 45.71 132.86 lipid nanoparticle 55 carrier (23) Example Nucleic acid- 6 100 3 20 77 lipid nanoparticle 56 carrier (24) Example Nucleic acid- 6 100 5 100 128.33 lipid nanoparticle 57 carrier (25) Example Nucleic acid- 6 100 7.15 45.71 132.86 58 lipid nanoparticle carrier (26) Lipid DMG- compound PEG DSPC Cholesterol (9) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 12 100 3 20 77 59 lipid nanoparticle carrier (27) Example Nucleic acid- 12 100 5 100 128.33 60 lipid nanoparticle carrier (28) Example Nucleic acid- 12 100 7.15 45.71 132.86 61 lipid nanoparticle carrier (29)

Examples 62-70

[0096] Nucleic acid-lipid nanoparticle carriers (30)-(38) were obtained by performing the same manner as in Example 35 but changing the ingredients and/or contents in the lipid solution and the N/P value (adjusted by the volume ratio of the nucleic acid aqueous solution and the lipid solution) according to Table 5.

TABLE-US-00005 TABLE 5 Lipid DMG- compound PEG DSPC Cholesterol (9) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 6 100 3 20 77 62 lipid nanoparticle carrier (30) Example Nucleic acid- 6 100 5 100 128.33 lipid 63 nanoparticle carrier (31) Example Nucleic acid- 6 100 7.15 45.71 132.86 64 lipid nanoparticle carrier (32) Lipid DMG- compound PEG DSPC Cholesterol (10) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 18 100 3 20 77 65 lipid nanoparticle carrier (33) Example Nucleic acid- 18 100 5 100 128.33 66 lipid nanoparticle carrier (34) Example Nucleic acid- 18 100 7.15 45.71 132.86 67 lipid nanoparticle carrier (35) Lipid DMG- compound PEG DSPC Cholesterol (11) (parts (parts (parts (parts by by by by N/P mole) mole) mole) mole) Example Nucleic acid- 12 100 3 20 77 68 lipid nanoparticle carrier (36) Example Nucleic acid- 12 100 5 100 128.33 69 lipid nanoparticle carrier (37) Example Nucleic acid- 12 100 7.15 45.71 132.86 70 lipid nanoparticle carrier (38)

[0097] Property Evaluation of Pharmaceutical Compositions

[0098] Nucleic acid-lipid nanoparticle carriers (1)-(9) obtained in Examples 35-43 and Nucleic acid-lipid nanoparticle carriers (10) and (11) obtained in Comparative examples 1 and 2 were evaluated for particle size, polydispersity index (PdI), Zeta potential, ribonucleic acid recovery rate (mRNA recovery), encapsulation efficiency (EE) for ribonucleic acid and acid dissociation constant (pKa). The results are shown in Table 6.

[0099] The particle size, polydispersity index (PdI) and Zeta potential of nucleic acid-lipid nanoparticle carriers are evaluated as follows. 20 ?L of the sample was taken to add to 330 ?L of Dulbecco's Phosphate-Buffered Saline (DPBS) and shaken evenly, and then measured for particle size and polydispersity index (PdI) of liposomes with a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical). In addition, 20 ?L of the sample was taken to add to 680 ?L of sodium chloride aqueous solution (concentration: 10 mM), shaken evenly, and then measured for Zeta potential of liposomes with a dynamic light scattering instrument (Zetasizer Nano-ZS, Malvern Panalytical).

[0100] The ribonucleic acid recovery rate (mRNA recovery) is evaluated as follows. After sterile filtration of the nucleic acid lipid nanoparticle carriers, 100 ?L of sample was taken to add to 25 ?L of 5% Triton X-100. Next, after incubating at 37? C. for 10 minutes, 100 ?L of chloroform was added thereto. After high-speed shaking (rotation speed: 2000 rpm) for 10 minutes and centrifugation (centrifugal force: 14000?g) for 30 minutes, 80 ?L of the supernatant was taken and its absorbance was analyzed by a UV-visible spectrophotometer (UV7, Mettler Toledo). After that, the concentration of ribonucleic acid was calculated using Formula (1) listed below, and the recovery rate was calculated using Formula (2) listed below, wherein C is the concentration of ribonucleic acid, A.sub.260 nm is the absorbance value of ribonucleic acid at a wavelength of 260 nm, Vf is the volume of supernatant (?L), and W is the initial weight of ribonucleic acid (ng).

C=(A.sub.260 nm?40)/(100/125)Formula (1)

Recovery rate (%)=[(C?Vf)/W]?100%Formula (2)

[0101] The encapsulation efficiency (EE) for ribonucleic acid was measured by the Quant-iT RiboGreen RNA quantitative detection kit (RNA Assay kit) (purchased from Invitrogen). Namely, the mRNA concentration in the dispersion containing nucleic acid-lipid particles was quantified in the presence and absence of 0.2% Triton X-100 surfactant, and the encapsulation efficiency (EE) for ribonucleic acid was calculated according to Formula (3) listed below, wherein EE is the encapsulation efficiency for ribonucleic acid, C.sub.Free is the non-encapsulated ribonucleic acid concentration, and C.sub.All is the total ribonucleic acid concentration.

EE(%)=(1?C.sub.Free/C.sub.All)?100%Formula (3)

[0102] For the acid dissociation constant (pKa), a titration method based on 6-p-Toluidino-2-naphthalenesulfonic acid (TNS) was used with a microdisk fluorescence analyzer (Infinite F200 Pro, Tecan, excitation wavelength: 320 nm, emission wavelength: 448 nm) to analyze the fluorescence intensity titration curve with a pH value of 2.5-11.0 and a total of 18 points of the sample in the buffer. The titration curve was sigmoidal fitted, and the calculated acid dissociation constant (pKa) was the pH value when the nucleic acid-lipid nanoparticle carrier reaches half of the maximum fluorescence intensity.

TABLE-US-00006 TABLE 6 Ribonucleic Encapsulation Acid Particle Zeta acid efficiency for dissociation size Polydispersity potential recovery ribonucleic constant (nm) index (PdI) (mV) rate (%) acid (%) (pKa) Nucleic 88.5 0.33 ?6.16 75.8 44.8 NA acid-lipid nanoparticle carrier (1) Nucleic 92 0.18 ?7.35 33.9 5.1 5.02 acid-lipid nanoparticle carrier (2) Nucleic 66 0.15 ?3.44 46.3 76.2 5.36 acid-lipid nanoparticle carrier (3) Nucleic 63.9 0.13 ?11.7 52.3 81.1 5.41 acid-lipid nanoparticle carrier (4) Nucleic 63.9 0.16 ?4.42 86.7 57.6 7 acid-lipid nanoparticle carrier (5) Nucleic 60.1 0.25 ?3.64 85 59.9 7.61 acid-lipid nanoparticle carrier (6) Nucleic 78.2 0.12 ?4.38 71.8 83.2 4.93 acid-lipid nanoparticle carrier (7) Nucleic 58.4 0.14 ?6.93 86.6 55 6.64 acid-lipid nanoparticle carrier (8) Nucleic 58.6 0.11 ?2.42 80 58.8 6.76 acid-lipid nanoparticle carrier (9) Nucleic 75.1 0.07 ?6.33 82.4 97.2 6.45 acid-lipid nanoparticle carrier (10) Nucleic 99.3 0.09 ?4.54 61.5 94.8 6.74 acid-lipid nanoparticle carrier (11)

[0103] Nucleic acid-lipid nanoparticle carriers (12)-(20) obtained in Examples 44-52 were evaluated for particle size, polydispersity index (PdI), Zeta potential, ribonucleic acid recovery rate (mRNA recovery), encapsulation efficiency (EE) for ribonucleic acid and acid dissociation constant (pKa). The results are shown in Table 7.

TABLE-US-00007 TABLE 7 Ribonucleic Encapsulation Acid Zeta acid efficiency for dissociation Particle Polydispersity potential recovery ribonucleic constant size(nm) index (PdI) (mV) rate (%) acid (%) (pKa) Nucleic acid- 64.3 0.15 ?3.04 70 55.2 5.96 lipid nanoparticle carrier (12) Nucleic acid- 59.3 0.26 ?2.24 84.2 79.7 6.04 lipid nanoparticle carrier (13) Nucleic acid- 57.9 0.19 ?3.19 73.1 72.8 6.06 lipid nanoparticle carrier (14) Nucleic acid- 138.8 0.11 ?5.85 63.2 44.2 7.22 lipid nanoparticle carrier (15) Nucleic acid- 118.8 0.05 ?4.59 68.8 40.6 6.94 lipid nanoparticle carrier (16) Nucleic acid- 98.1 0.08 ?3.65 64.7 45 7.06 lipid nanoparticle carrier (17) Nucleic acid- 181.2 0.14 ?4.02 41 41 7.73 lipid nanoparticle carrier (18) Nucleic acid- 133.4 0.11 ?5.6 50.2 53.7 7.46 lipid nanoparticle carrier (19) Nucleic acid- 110.9 0.15 ?4.85 47.3 47.2 7.47 lipid nanoparticle carrier (20)

[0104] Nucleic acid-lipid nanoparticle carriers (21)-(29) obtained in Examples 53-61 were evaluated for particle size, polydispersity index (PdI), Zeta potential, ribonucleic acid recovery rate (mRNA recovery), encapsulation efficiency (EE) for ribonucleic acid and acid dissociation constant (pKa). The results are shown in Table 8.

TABLE-US-00008 TABLE 8 Ribonucleic Encapsulation Acid Particle Zeta acid efficiency for dissociation size Polydispersity potential recovery ribonucleic constant (nm) index (PdI) (mV) rate (%) acid (%) (pKa) Nucleic acid- 147.1 0.09 ?5 40.9 19.7 NA lipid nanoparticle carrier (21) Nucleic acid- 129.3 0.06 ?5.68 54.3 37.3 NA lipid nanoparticle carrier (22) Nucleic acid- 104.7 0.08 ?6.64 56.8 40.1 NA lipid nanoparticle carrier (23) Nucleic acid- 151.3 0.1 ?8.32 33.5 9.2 6.82 lipid nanoparticle carrier (24) Nucleic acid- 123.4 0.09 ?5.54 64.4 55.4 6.4 lipid nanoparticle carrier (25) Nucleic acid- 102.6 0.08 ?11.6 84.9 39.5 6.81 lipid nanoparticle carrier (26) Nucleic acid- 158.1 0.11 ?0.23 26.5 61 7.6 lipid nanoparticle carrier (27) Nucleic acid- 243.6 0.16 0.79 31.3 30.6 8.12 lipid nanoparticle carrier (28) Nucleic acid- 194.3 0.14 0.04 20.3 38.8 7.77 lipid nanoparticle carrier (29)

[0105] Nucleic acid-lipid nanoparticle carriers (30)-(38) obtained in Examples 62-70 were evaluated for particle size, polydispersity index (PdI), Zeta potential, ribonucleic acid recovery rate (mRNA recovery), encapsulation efficiency (EE) for ribonucleic acid and acid dissociation constant (pKa). The results are shown in Table 9.

TABLE-US-00009 TABLE 9 Ribonucleic Encapsulation Acid Zeta acid efficiency for dissociation Particle Polydispersity potential recovery ribonucleic acid constant size (nm) index (PdI) (mV) rate (%) (%) (pKa) Nucleic 172 0.09 ?4.45 41.3 11.58 7.6 acid-lipid nanoparticle carrier (30) Nucleic 205.9 0.13 ?5.24 21 NA 8.12 acid-lipid nanoparticle carrier (31) Nucleic 188.3 0.07 ?4.77 40.8 17.71 7.77 acid-lipid nanoparticle carrier (32) Nucleic 98.3 0.08 ?2.87 72.7 24.9 7.55 acid-lipid nanoparticle carrier (33) Nucleic 120.6 0.08 ?6.93 77.2 30.4 7.16 acid-lipid nanoparticle carrier (34) Nucleic 66.5 0.03 ?4.78 72.2 29.5 7.05 acid-lipid nanoparticle carrier (35) Nucleic 110.7 0.07 ?6.41 74.9 40 7.55 acid-lipid nanoparticle carrier (36) Nucleic 223.6 0.16 ?6.49 62.7 19.9 7.57 acid-lipid nanoparticle carrier (37) Nucleic 97.5 0.11 ?4.1 74.2 41.8 7.51 acid-lipid nanoparticle carrier (38)

[0106] Based on Table 6 to Table 9, it is know that the particle sizes of the nucleic acid-lipid nanoparticle carriers formed by self-assembly of the pharmaceutical composition of the present disclosure could be about 50 nm to 250 nm, and the polydispersity index could be between 0.03 and 0.33. In some Examples, the ribonucleic acid recovery rate (%) of the nucleic acid-lipid nanoparticle carriers formed by self-assembly of some of the pharmaceutical compositions of the present disclosure could be higher than about 80%, and the acid dissociation constant (pKa) could range between 5 and 8.5.

[0107] Transfection Efficiency and Cell Viability Evaluation

[0108] HEK293 cells were seeded into a 96-well culture plate at 5?10.sup.4 cells/well for adherent culture for 24 hours. Next, 97 ?L of Opti-MEM medium and 3 ?L of lipofectamine reagent (purchased from Invitrogen) were evenly mixed and reacted at room temperature for 10 minutes to obtain a first solution. 1 ?L of luciferase mRNA (product number: R1018, purchased from APExBIO) (concentration: 1 mg/mL) and 99 ?L of Opti-MEM? medium were evenly mixed to obtain a second solution. After that the second solution was added to the first solution, mixed evenly, and reacted at room temperature for 5 minutes to obtain a transfection solution. Next, the lipid nanoparticle (LNP) solutions prepared in Examples 35-43 were respectively diluted with 1?PBS to obtain respectively diluted lipid nanoparticle solutions (volume: 200 ?L, concentration: 5 ?g/mL). Then, 800 ?L of cell culture medium (product number: MT-10-009-CVS, purchased from Corning) was added to the respective diluted lipid nanoparticle solutions and mixed with the transfection solution to obtain respective cell co-culture solutions (concentration of luciferase mRNA was 0.1 ?g/100 ?L). After that, 100 ?L of different cell co-culture solutions were respectively added to the 96-well culture plate and co-cultured with the cells (0.1 ?g mRNA/well). After 24 hours of culture, 0 ?L of supernatant was taken and added to 20 ?L of cell viability assay reagent (GF-AFC). After reacting at 37? C. for 35-40 minutes, the fluorescence value of the resultant product was measured by a fluorometer (Ex: 380 nm; Em: 540 nm). By dividing the fluorescence detection reading of the sample and the fluorescence value of the untreated negative control group, the cell viability in the presence of different lipid nanoparticle (LNP) solutions was calculated. The results are shown in Table 10.

TABLE-US-00010 TABLE 10 Cell viability Cell culture medium >99% Nucleic acid-lipid nanoparticle carrier (2) >99% Nucleic acid-lipid nanoparticle carrier (6) >99% Nucleic acid-lipid nanoparticle carrier (7) >99% Nucleic acid-lipid nanoparticle carrier (8) >99% Nucleic acid-lipid nanoparticle carrier (10) >99%

[0109] Based on the foregoing, it is known that the lipid compounds mentioned in the present disclosure hardly affect cell activity when performing in vitro cell experimental analysis.

[0110] Next, 100 ?L of ONE-Glo Reagent was added to each well of the culture plate, and the luminescence value was measured within 3 minutes to evaluate the transfection ability of different lipid nanoparticle (LNP) solutions. The results are shown in Table 11.

TABLE-US-00011 TABLE 11 Transfection ability Cell culture medium ? Nucleic acid-lipid nanoparticle carrier (2) ++ Nucleic acid-lipid nanoparticle carrier (6) +++ Nucleic acid-lipid nanoparticle carrier (7) +++ Nucleic acid-lipid nanoparticle carrier (8) +++ Nucleic acid-lipid nanoparticle carrier (10) +++ Transfection ability: ?represents: Normalized luminescence intensity (RLU) ? 1.2e+03 + represents: 1.2e+03 < Normalized luminescence intensity (RLU) < 1e+05 ++ represents: 1e+05 ? Normalized luminescence intensity (RLU) < 1e+06 +++ represents: Normalized luminescence intensity (RLU)) ? 1e+06

[0111] According to Table 11, it is known that the lipid compounds mentioned in the present disclosure can indeed improve the transfection efficiency of HEK293 cells.

[0112] It will be apparent to those skilled in the art that various modifications and variations can be made to the disclosed embodiments. It is intended that the specification and examples be considered as exemplary only, with a true scope of the disclosure being indicated by the following claims and their equivalents.