Novel ionizable lipid and lipid nanoparticle composition comprising the same

Abstract

The present invention relates to a novel ionized lipid and a lipid nanoparticle composition containing the same. The lipid nanoparticles formed by the novel ionized lipid compound according to the present invention are characterized by exhibiting excellent gene encapsulation rate and in vivo gene delivery rate, while also demonstrating excellent gene delivery function in a liver injury model, and thus can be usefully utilized as a drug delivery composition.

Description

Technology Field

[0001] The present invention relates to a novel ionized lipid and a lipid nanoparticle composition containing the same. Background Technology

[0002] Nucleic acid-based drugs are utilized not only as therapeutic agents but also as preventive agents that defend against diseases by injecting genes capable of expressing antigens for specific diseases. Among these, gene-based vaccines are classified into DNA vaccines, RNA vaccines, and viral vector vaccines. RNA vaccines, in particular, involve injecting mRNA encoding an antigen into the human body to induce antigen expression and antibody formation. They possess the advantages of being able to be rapidly developed, as they eliminate concerns about infection associated with viral vector-based vaccines and potential risks such as genetic mutations associated with DNA vaccines, and thus gained prominence as an effective countermeasure against COVID-19, which emerged in 2019.

[0003] However, genes have the disadvantage of being easily degraded by nucleic acid hydrolyzing enzymes within the human body and being negatively charged macromolecules that cannot be easily delivered into cells, so a method is needed to deliver them stably and efficiently to the desired location. Delivery techniques based on various materials, such as lipids, polymers, dendrimers, and inorganic metal materials, have been reported as gene delivery systems. Among these, lipid nanoparticle technology is recognized for its technical value as a clinically useful delivery system for nucleic acid drugs, such as siRNA and mRNA, after lipid nanoparticles were applied as lipid carriers to the development of patisiran, a novel siRNA drug that received FDA approval in 2018, as well as the mRNA vaccine for COVID-19 that received emergency use authorization in 2020.

[0004] Currently, lipid nanoparticles are generally used in the form of a mixture of four components in specific proportions: ionized lipids, phospholipids (helper lipids), cholesterol (structural maintenance lipids), and PEG-lipids. However, as the development of gene therapies for various indications gains momentum, unmet needs have been identified in existing lipid nanoparticle-based delivery systems in various aspects, such as improving gene delivery capabilities, maintaining gene delivery capabilities for specific diseases like liver disease, enhancing stability, improving targeting characteristics, ensuring ease of storage and distribution, mitigating side effects, reducing costs, and responding to breakthrough infections. Consequently, there is a continuous demand for the development of lipid nanoparticles using new ionized lipid delivery systems. Prior art literature

[0005] Republic of Korea Published Patent No. 10-2008-0094473 (Published on Oct. 23, 2008) The problem to be solved

[0006] The object of the present invention is to provide a novel ionized lipid compound.

[0007] Another objective of the present invention is to provide a lipid nanoparticle composition comprising the above compound.

[0008] Another objective of the present invention is to provide medical uses for the lipid nanoparticle composition. means of solving the problem

[0009] To achieve the above objective, the present invention provides a compound selected from a compound represented by the following formula I, a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof:

[0010] <Chemical Formula I>

[0011]

[0012] In the above chemical formula I, n is 0 or 1, and L is one of (C1-C10)alkylene, (C2-C10)alkenylene and (C2-C10)alkynylene, and one or more -CH2- in the alkylene, alkenylene and alkynylene are -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH- and Substituted or unsubstituted with one of -NHC(=O)O-, and R 1 is one of hydrogen, (C1-C20)alkyl, (C2-C20)alkenyl, and (C2-C20)alkynyl, and R 2 and R 3 Each may be the same or different, and is one of a 5 to 30-atom heteroaryl comprising one or more heteroatoms selected from the group consisting of (C5-C30)alkyl, (C5-C30)alkenyl, (C5-C30)alkynyl, (C3-C30)cycloalkyl, (C3-C30)cycloalkenyl, (C3-C30)cycloalkynyl, (C5-C30)aryl, and N, O, and S, wherein one or more -CH2- in the alkyl, alkenyl, and alkynyl are -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, It may be substituted or unsubstituted with one of -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-.

[0013] The present invention provides a lipid nanoparticle composition comprising the above-mentioned compound.

[0014] In addition, the present invention provides a drug delivery composition comprising the above lipid nanoparticle composition; and a preventive or therapeutic agent. Effects of the invention

[0015] The present invention relates to a novel ionized lipid compound and a lipid nanoparticle composition using the same. Since the lipid nanoparticles exhibit excellent gene encapsulation rate and in vivo gene delivery rate, and also demonstrate excellent gene delivery ability in a liver injury model, they can be usefully utilized in the development of gene therapies for various indications. Brief explanation of the drawing

[0016] Figure 1 shows the process of manufacturing compound 1 according to one embodiment of the present invention. Figure 2 shows the manufacturing process of compound 2. Figure 3 shows the manufacturing process of compound 3. Figure 4 shows the preparation process of compound 4. Figure 5 shows the manufacturing process of compound 5. Figure 6 shows the manufacturing process of compound 6. Specific details for implementing the invention

[0017] The present invention will be described in detail below.

[0019] The inventors synthesized a novel ionized lipid compound and completed the present invention by confirming that a lipid nanoparticle composition prepared using the same can deliver genes into the body with excellent efficiency.

[0021] The present invention provides a compound selected from a compound represented by the following chemical formula I, a pharmaceutically acceptable salt, tautomer, prodrug, or stereoisomer thereof:

[0022] <Chemical Formula I>

[0023]

[0024] In the above chemical formula I, n is 0 or 1, and

[0025] L is one of (C1-C10)alkylene, (C2-C10)alkenylene, and (C2-C10)alkynylene, and one or more -CH2- in the alkylene, alkenylene, and alkynylene are substituted or unsubstituted with -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-.

[0026] R 1 It is one of hydrogen, (C1-C20)alkyl, (C2-C20)alkenyl, and (C2-C20)alkynyl, and

[0027] R 2 and R 3 Each may be the same or different, and is one of a 5 to 30-atom heteroaryl comprising one or more heteroatoms selected from the group consisting of (C5-C30)alkyl, (C5-C30)alkenyl, (C5-C30)alkynyl, (C3-C30)cycloalkyl, (C3-C30)cycloalkenyl, (C3-C30)cycloalkynyl, (C5-C30)aryl, and N, O, and S, wherein one or more -CH2- in the alkyl, alkenyl, and alkynyl are -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, It may be substituted or unsubstituted with one of -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-.

[0028] Preferably, in the following chemical formula I, L is one of (C1-C6)alkylene, (C2-C6)alkenylene, and (C2-C6)alkynylene, and one or more -CH2- in the alkylene, alkenylene, and alkynylene are substituted with -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-. R 1 is hydrogen or (C1-C10)alkyl, and R 2 and R 3 Each of the above may be the same or different and is one of (C10-C30)alkyl, (C10-C30)alkenyl, and (C10-C30)alkynyl, and one or more -CH2- in the alkyl, alkenyl, and alkynyl may be substituted with -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -OP(=O)(OH)O-, -OC(=S)-, -C(=S)O-, -SC(=O)-, -C(=O)S-, -SC(=S)-, -C(=S)S-, -SC(=O)O-, -OC(=O)S-, -S-, -SS-, -C(=O)NH-, -NHC(=O)-, -OC(=O)NH-, and -NHC(=O)O-. there is.

[0029] Preferably, in the above formula I, L is a (C2-C6)alkylene, and one or more -CH2- groups in the alkylene are substituted with -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, and -OC(=O)O-, and R 1 is hydrogen or (C1-C5)alkyl, and R 2 and R 3Each may be the same or different and is one of (C10-C30)alkyl, (C10-C30)alkenyl and (C10-C30)alkynyl, and one or more -CH2- in the alkyl, alkenyl, and alkynyl may be substituted with one of -O-, -C(=O)-, -OC(=O)-, -C(=O)O-, -OC(=O)O-, -CH(OH)-, -S- and -SS-.

[0030] More preferably, in the above formula I, L is a (C2-C6)alkylene, one -CH2- in the alkylene is substituted with -OC(=O)- or -C(=O)O-, and R 1 is hydrogen or (C1-C5)alkyl, and R 2 and R 3 Each may be the same or different, and is a (C10-C30)alkyl, and one or two -CH2- in the alkyl may be substituted with one of -OC(=O)-, -C(=O)O-, -S- and -SS-.

[0031] Specifically, the compound may be selected from the group of compounds below:

[0032] (1) (pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0033] (2) bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate) [bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate)];

[0034] (3) bis(2-butyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate

[0035] (4) bis(2-hexyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate

[0036] (5) bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate

[0037] (6) (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis (hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0038] (7) bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate;

[0039] (8) bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate [bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate];

[0040] (9) ((3-((3-(pyridin-4-ylamino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0041] (10) ((3-((3-methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((3-((3-(methyl(pyridin-4-yl)amino)propanoyl)oxy)propyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0042] (11) ((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [((4-(2-(pyridin-4-ylamino)acetoxy)butyl)azanediyl) bis(hexane-6,1-diyl)bis(2-hexyldecanoate)];

[0043] (12) (((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(disulfandyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)(2-hexyldecanoate) [(((pyridin-4-ylazanediyl)bis(hexane-6,1-diyl))bis(disulfanediyl))bis(butane-4,1-diyl)bis(2-butyloctanoate)]; and

[0044] (13) bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridin-4-ylazanediyl) dihexanoate.

[0046] More specifically, the above (1) (pyridine-4-ylazanedyl)bis(hexane-6,1-dyl)bis(2-hexyl decanoate) (hereinafter, compound 1) has a structure as shown in Chemical Formula 1 below, and its molecular formula is C 49 H 90 It is N2O4 and its molecular weight is 771.25:

[0047] <Chemical Formula 1>

[0048]

[0050] The above (2) bis(2-butyloctyl)10,10'-(pyridine-4-ylazanedyl)bis(decanoate) (hereinafter, compound 2) has a structure as shown in the following chemical formula 2, and its molecular formula is C 49 H 90 It is N2O4, and its molecular weight is 771.25:

[0051] <Chemical Formula 2>

[0052]

[0054] The above (3) bis(2-butyloctyl)8,8'-(pyridine-4-ylazanedyl)dioctanoate (hereinafter, compound 3) has a structure as shown in the following chemical formula 3, and its molecular formula is C 45 H 82 It is N2O4, and its molecular weight is 715.14:

[0055] <Chemical Formula 3>

[0056]

[0058] The above (4) bis(2-hexyloctyl)8,8'-(pyridine-4-ylazanedyl)dioctanoate (hereinafter, compound 4) has a structure as shown in the following chemical formula 4, and its molecular formula is C 49 H 90 It is N2O4, and its molecular weight is 771.25:

[0059] <Chemical Formula 4>

[0060]

[0062] The above (5) bis(2-hexyldecyl)8,8'-(pyridine-4-ylazanedyl)dioctanoate (hereinafter, compound 5) has a structure as shown in the following chemical formula 5, and its molecular formula is C 53 H 98 It is N2O4, and its molecular weight is 827.36:

[0063] <Chemical Formula 5>

[0064]

[0066] The above (6) (((2-(pyridine-4-ylamino)ethoxy)carbonyl)azanedyl)bis(hexane-6,1-dyl)bis(2-hexyldecanoate) (hereinafter, compound 6) has a structure as shown in the following chemical formula 6, and its molecular formula is C 52 H 95 It is N3O6, and its molecular weight is 858.33:

[0067] <Chemical Formula 6>

[0068]

[0070] The above (7) bis(2-hexyldecyl)6,6'-(pyridine-4-ylazanedyl)dihexanoate (hereinafter, compound 7) has a structure as shown in the following chemical formula 7, and its molecular formula is C 49 H 90 It is N2O4, and its molecular weight is 771.25:

[0071] <Chemical Formula 7>

[0072]

[0074] The above (8) bis(2-hexyloctyl)6,6'-(pyridine-4-ylazanedyl)dihexanoate (hereinafter, compound 8) has a structure as shown in chemical formula 8, and its molecular formula is C 45 H 82 It is N2O4, and its molecular weight is 715.14:

[0075] <Chemical Formula 8>

[0076]

[0078] The above (9) ((3-((3-(pyridine-4-ylamino)propanoyl)oxy)propyl)azanedyl)bis(hexane-6,1-dyl)bis(2-hexyl decanoate) (hereinafter, compound 9) has a structure as shown in the following chemical formula 9, and its molecular formula is C 55 H 101 It is N3O6, and its molecular weight is 900.41:

[0079] <Chemical Formula 9>

[0080]

[0082] The above (10) ((3-((3-methyl(pyridine-4-yl)amino)propanoyl)oxy)propyl)azanedyl)bis(hexane-6,1-dyl)bis(2-hexyl decanoate) (hereinafter, compound 10) has a structure as shown in the following chemical formula 10, and its molecular formula is C 56 H 103 It is N3O6, and its molecular weight is 914.43:

[0083] <Chemical Formula 10>

[0084]

[0086] The above (11) ((4-(2-(pyridine-4-ylamino)acetoxy)butyl)azanedyl)bis(hexane-6,1-dyl)bis(2-hexyldecanoate) (hereinafter, compound 11)) has a structure as shown in the following chemical formula 11, and its molecular formula is C 55 H 101 It is N3O6, and its molecular weight is 900.41:

[0087] <Chemical Formula 11>

[0088]

[0090] The above (12) (((pyridine-4-ylazane-edyl)bis(hexane-6,1-dyl)bis(disulfandyl))bis(butane-4,1-dyl)bis(2-butyloctanoate)(2-hexyldecanoate) (hereinafter, compound 12)) has a structure as shown in the following chemical formula 12, and its molecular formula is C 49 H 90 It is N2O4S4, and its molecular weight is 899.51:

[0091] <Chemical Formula 12>

[0092]

[0094] The above (13) bis(2-(2-(hexyldisulfanyl)ethyl)octyl)6,6'-(pyridine-4-ylazanedyl)dihexanoate (hereinafter, compound 13) has a structure as shown in the following chemical formula 13, and its molecular formula is C 49 H 90 It is N2O4S4, and its molecular weight is 899.51:

[0095] <Chemical Formula 13>

[0096]

[0098] The present invention provides a lipid nanoparticle composition comprising a compound represented by the above chemical formula I, a pharmaceutically acceptable salt thereof, a tautomer, a prodrug, or a stereoisomer.

[0099] The above compound can act as an ionized lipid.

[0100] The above lipid nanoparticle composition may further include one or more selected from the group consisting of neutral lipids, steroids, and polymeric lipids.

[0101] The above neutral lipid can be selected from phospholipids or glycolipids. Specifically, the phospholipids are dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), and distearoylphosphatidylethanolamine (DSPE). The glycolipid may be one or more selected from the group consisting of phosphatidylethanolamine (PE) and dipalmitoylphosphatidylethanolamine, and the glycolipid may be one or more selected from the group consisting of glucosylceramide, galactosylceramide, glucosylsphingosine, galactosylsphingosine, and phosphoglycoceramide, but is not limited thereto.

[0102] The above steroid may be one or more selected from the group consisting of cholesterol, bile acid derivatives and choline acid derivatives, but is not limited thereto.

[0103] The above-mentioned polymer lipid may be a pegylated lipid having a structure in which a water-soluble polymer and a lipid are combined, and the pegylated lipid may be one or more selected from the group consisting of PEG combined to dialkyloxypropyl (PEG-DAA); PEG combined to diacylglycerol as PEG-c-DOMG and PEG-DMG; PEG combined to a phospholipid such as phosphatidylethanolamine as PEG-DLPE, PEG-DMPE and PEG-DSPE; PEG conjugated to ceramide (PEG-CER); PEG conjugated to cholesterol or a derivative thereof; PEG-modified phosphatidic acid; PEG-modified dialkylamine; and PEG-modified dialkylglycerol, but is not limited thereto. Additionally, the above-mentioned pegylated lipid may be a functionalized PEG in which a functional group is attached to the side not bound to the lipid. The functional groups available for use at this time may be one or more selected from the group consisting of succinyl, carboxylic acid, maleimide, n-hydroxysuccinimide, amine, biotin, cyanur, and folate, but are not limited thereto.

[0104] The above lipid nanoparticle composition may be prepared by including ionized lipids, neutral lipids, steroids, polymerized lipids, etc. For a composition suitable for the preparation of lipid nanoparticles, the lipid composition excluding active ingredients such as genes such as RNA and DNA may include 20 to 65 mol% of ionized lipids, 2.5 to 30 mol% of neutral lipids, 20 to 60 mol% of steroids, and 0.5 to 5 mol% of polymerized lipids.

[0105] For example, ionized lipids : neutral lipids : steroids : polymeric lipids are 60:5:33.5:1.5, 50:10:38.5:1.5, 40:15:43.5:1.5, 30:20:48.5:1.5, 25:25:48.5:1.5, 59.5:5:33.5:2.0, 49.5:10:38.5:2.0, 39.5:15:43.5:2.0, 29.5:20:48.5:2.0, 24.5:25:48.5:2.0, 59:5:33.5:2.5, 49:10:38.5:2.5, 39:15:43.5:2.5, It can be composed of one mol% ratio selected from the group consisting of 29:20:48.5:2.5, 24:25:48.5:2.5, 58.5:5:33.5:3.0, 48.5:10:38.5:3.0, 38.5:15:43.5:3.0, 28.5:20:48.5:3.0, 23.5:25:48.5:3.0, 58:5:33.5:3.5, 48:10:38.5:3.5, 38:15:43.5:3.5, 28:20:48.5:3.5, and 23:25:48.5:3.5.

[0106] The above lipid nanoparticle composition may further include a prophylactic or therapeutic agent, and the prophylactic or therapeutic agent may be a gene including RNA, DNA, or a mixture thereof, specifically, one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), and mixtures thereof, but is not limited thereto.

[0107] Alternatively, the active ingredient of the above composition may be various anionic peptides, protein drugs, protein-nucleic acid structures, or anionic biopolymer-drug conjugates such as hyaluronic acid-peptide conjugates, hyaluronic acid-protein conjugates, and antibodies.

[0108] The lipid nanoparticle composition of the present invention, including ionized lipids, neutral lipids, steroids, polymerized lipids, etc., can be prepared as a lipid solution by dissolving it in a solvent that is miscible with ethanol or water. Separately, the active ingredient can be prepared as an active ingredient solution by dissolving it in a citric acid or acetic acid buffer solution with a pH of 4.0 ± 1.0. In addition, the lipid solution and the active ingredient solution can be prepared as lipid nanoparticles by mixing them through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of about 10 to 15 mL / min in a volume ratio of 1:3.

[0110] In addition, the present invention provides a drug delivery composition comprising the lipid nanoparticle composition; and a preventive or therapeutic agent.

[0111] The above-mentioned prophylactic or therapeutic agent may be a gene containing RNA, DNA, or a mixture thereof, and specifically may be one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), and mixtures thereof, but is not limited thereto.

[0112] The lipid nanoparticles according to the present invention possess excellent safety and stability and can function as a carrier to enable genes containing RNA, DNA, or a mixture thereof as active ingredients to effectively exert their effects within cells. Through this, the lipid nanoparticles formed from the novel ionized lipids described above exhibit excellent gene encapsulation rates and in vivo gene delivery rates, and on the other hand, demonstrate excellent gene delivery capabilities even in a liver injury model, thereby making them useful as drug delivery compositions.

[0113] The above drug delivery composition may be administered to mammals, including humans, via various routes including parenteral administration, which may be applied intravenously, subcutaneously, intraperitoneally, or topically, and the dosage may be appropriately selected by a person skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0114] When formulating the above drug delivery composition according to one example, it is manufactured using diluents or excipients such as commonly used fillers, extenders, lyophilizers, binders, wetting agents, disintegrants, and surfactants.

[0115] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0116] Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0117] The drug delivery composition of the present invention may be administered while containing a pharmaceutically effective amount of a prophylactic or therapeutic agent. The effective dose level of said prophylactic or therapeutic agent may be determined according to factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant drugs, and other factors well known in the medical field. According to one example, said composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art. For example, said composition may be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0119] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0121] <Example 1> Preparation of Compound

[0122] 1-1. Preparation of Compound 1

[0123] As shown in Fig. 1, compound 1-b (9.74 g, 85.3 mmol) and compound 1-a (10.0 g, 85.3 mmol) were added to xylene (o-xylene) (100 mL), and the mixture was stirred at 140°C for 12 hours under nitrogen gas. The mixture was diluted with petroleum ether (PE) (200 mL) and purified on silica gel (methanol; MeOH) / dichloromethane (DCM) = 0 / 100 to 10 / 90 to obtain compound 1-c, a white solid. Compound 1-c (5.00 g, 21.6 mmol) was dissolved in tetrahydrofuran (THF) (50 mL), and dimethylsulfide borane (10 M, 8.7 mL) was added dropwise at 25°C and stirred at 70°C for 12 hours. MeOH (10 mL) was additionally added dropwise and stirred at 25°C for 0.5 hours. The mixture was concentrated under reduced pressure and purified on silica gel (MeOH / DCM = 0 / 100 to 15 / 85, containing 0.5% ammonia water) to obtain compound 1-d, a white solid. Compound 1-d (3.5 g, 16.1 mmol), compound 1-e (3.14 g, 20.9 mmol, hydrochloride), and N,N-diisopropylethylamine (DIEA) (10.4 g, 80.5 mmol) were added to dimethylformamide (DMF) (5 mL) and stirred at 100°C for 12 hours. After adding a saturated sodium bicarbonate solution (sat. NaHCO3) (50 mL) to the mixture, the extract was extracted with DCM (50 mL × 2 times) and washed with a saturated sodium chloride solution (brine) (50 mL × 2 times). After drying with sodium sulfate (Na2SO4) and concentrating under reduced pressure, the mixture was purified on silica gel (MeOH / DCM = 0 / 100 to 10 / 90, containing 0.1% ammonia water) to obtain compound 1-f, a red oil. Compound 1-f (400 mg, 1.1) in DMF (5 mL).36 mmol) was added, and 1 g of compound (1.05 g, 4.08 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (781 mg, 4.08 mmol), and dimethylaminopyridine (DMAP) (499 mg, 4.08 mmol) were dissolved and stirred at 25°C for 12 hours. In the mixture, sat. After adding Na2CO3 (30 mL), the liquid extracted with DCM (50 mL × 2 times) was washed with brine (50 mL × 2 times), dried with sodium sulfate (Na2SO4), and concentrated under reduced pressure. Subsequently, the mixture was purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97 and MeOH / DCM = 0 / 100 to 2 / 98, containing 0.1% ammonia water) to obtain Compound 1, which is a yellow oil. 1 The results of the H NMR analysis were as follows.

[0124] 1 ¹H NMR (400 MHz, CDCl₃) δ 8.17 (d, J = 6.4 Hz, 2H), 6.45 (d, J = 6.4 Hz, 2H), 4.08 (t, J = 6.4 Hz, 4H), 3.22 - 3.35 (m, 4H), 2.27 - 2.36 (m, 2H), 1.56 - 1.69 (m, 12H), 1.36 - 1.47 (m, 12H), 1.18 - 1.32 (m, 40H), 0.80 - 0.95 (m, 12H).

[0126] 1-2. Preparation of Compound 2

[0127] As shown in Fig. 2, compound 2-a (11.0 g, 39.5 mmol) and compound 2-b (3.0 g, 17.9 mmol, 2.70 mL) were added to DMF (50 mL), followed by the addition of potassium carbonate (K2CO3) (12.4 g, 89.7 mmol) and potassium iodide (KI) (7.45 g, 44.9 mmol), and the mixture was stirred at 80°C for 12 hours. Purified water (500 mL) was added to the mixture, and the extract obtained with ethyl acetate (EA) (200 mL × 2 times) was washed with brine (300 mL × 3 times). After drying with Na2SO4, the solution was concentrated under reduced pressure and purified on silica gel (100% DCM) to obtain compound 2-c, a yellow oil. Compound 2-c (7.2 g, 12.8 mmol) was added to 1,2-dichloroethane (DCE) (70 mL) at 0°C, followed by the addition of chloroethyl carbonochloridate (2.19 g, 15.3 mmol) and DIEA (3.30 g, 25.54 mmol), and the mixture was stirred at 15°C for 2 hours. The mixture was concentrated under reduced pressure, dissolved in ethyl alcohol (EtOH) (100 mL), stirred at 60°C for 0.5 hours, and then concentrated. The liquid was diluted with Sat. NaHCO3 (100 mL) and extracted with EA (100 mL × 2 times), dried with Na2SO4, concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 1 / 99) to obtain compound 2-d, a milky white solid. Compound 2-d (4.4 g, 10.6 mmol) and compound 2-e (3.99 g, 26.6 mmol hydrochloride), DavePhos (1.67 g, 4.25 mmol), tris(dibenzylideneacetone)dipalladium (Pd2(dba)3) (1.95 g, 2.13 mmol), and t-butoxysodium (t-BuONa) (5.62 g, 58.5 mmol) were added to toluene (40 mL) and stirred at 100°C for 15 hours. To the mixture, sat.Compound 2-f, a red oil, was obtained by adding NaHCO3 (100 mL), extracting with EA (100 mL × 2 times), drying with Na2SO4, concentrating under reduced pressure, and purifying on silica gel (MeOH / DCM = 0 / 100 to 1 / 99). Compound 2-f (800 mg, 1.63 mmol) was added to THF (6 mL), sodium hydroxide (326 mg, 8.15 mmol) dissolved in purified water (2 mL) was added, and the mixture was stirred at 15°C for 3 hours. The mixture was concentrated, the pH was adjusted to 4 to 5 using a 1 M hydrochloric acid solution, and the extract was extracted with DCM (50 mL × 3 times), drying with Na2SO4, and concentrating under reduced pressure to obtain Compound 2-g, a red solid. Compound 2-g (500 mg, 1.15 mmol), compound 2-h (643 mg, 3.45 mmol), EDCI (662 mg, 3.45 mmol), and DMAP (422 mg, 3.45 mmol) were dissolved in DMF (5 mL) and stirred at 10°C for 12 hours. After adding sat. Na2CO3 (30 mL) to the mixture, the extract was extracted with EA (50 mL × 2 times) and washed with brine (50 mL × 3 times). Compound 2, which is a yellow oil, was obtained by drying with sodium sulfate (Na2SO4), concentrating under reduced pressure, purifying on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), and then purifying by prep-HPLC (C8, 50*40 mm, 5 μm, purified water / acetonitrile (ACN) = 35 / 65 to 5 / 95, 20 min). 1 The results of the H NMR analysis were as follows.

[0128] 1 H NMR (400 MHz, CDCl3) δ 8.16 (d, J = 6.4 Hz, 2H), 6.43 (d, J = 6.8 Hz, 2H), 3.98 (d, J = 5.6 Hz, 4H), 3.26 (t, J= 8.0 Hz, 4H), 2.31 (t, J = 7.2 Hz, 4H), 1.53 - 1.67 (m, 10H), 1.24 - 1.35 (m, 52H), 0.81 - 0.97 (m, 12H).

[0130] 1-3. Preparation of Compound 3

[0131] As shown in Fig. 3, compound 3-a (25.1 g, 100 mmol), compound 3-b (7.60 g, 45.5 mmol), K2CO3 (31.4 g, 227 mmol), and sodium iodide (NaI) (17.0 g, 114 mmol) were added to DMF (150 mL) and stirred at 80°C for 12 hours. The mixture was diluted with purified water (500 mL), the extract with EA (300 mL × 3 times) was washed with brine (500 mL × 3 times), dried with Na2SO4, filtered, concentrated under reduced pressure, and purified on silica gel (DCM / MeOH = 100 / 0 to 97 / 23) to obtain compound 3-c, a pale yellow oil. Compound 3-c (21.5 g, 42.4 mmol) was added to DCE (220 mL), and DIEA (11.0 g, 84.7 mmol) and chloroethyl chloroformate (7.27 g, 50.8 mmol) were added at 0°C, followed by stirring at 20°C for 2 hours. The mixture was concentrated under reduced pressure, dissolved in EtOH (200 mL), stirred at 60°C for 0.5 hours, and concentrated under reduced pressure. The solution was diluted with EA (100 mL), saturated sodium bicarbonate solution (sat. NaHCO3) (200 mL) was added, and the extract was extracted with EA (200 mL × 2 times). The resulting liquid was dried and filtered with Na2SO4, concentrated under reduced pressure, and purified on silica gel (EA / PE = 0 / 100 to 6 / 94) to obtain compound 3-d, a yellow solid. Compound 3-d (10.9 g, 30.4 mmol) and compound 3-e (11.4 g, 75.9 mmol hydrochloride), DavePhos (4.78 g, 12.1 mmol), Pd2(dba)3 (5.56 g, 6.07 mmol), and t-BuONa (17.5 g, 182 mmol) were dissolved in toluene (120 mL) and stirred under nitrogen gas at 100°C for 15 hours. Sat. NaHCO3 (250 mL) was added to the mixture and filtered. The extract was then extracted with EA (200 mL × 3 times), dried and filtered with Na2SO4, and concentrated under reduced pressure.After purifying twice on silica gel (MeOH / DCM = 0 / 100 to 5 / 95), the solution was dissolved in EA (100 mL), washed with 10% (2R)-2-acetamido-3-sulfanylpropanoic acid solution ((2R)-2-acetamido-3-sulfanylpropanoic acid in water) (80 mL × 2 times), filtered, washed with sat. NaHCO3 (50 mL × 2 times), dried and filtered with Na2SO4, and concentrated under reduced pressure to obtain compound 3-f, an orange oil. Compound 3-f was added to THF (15 mL), MeOH (5 mL), and purified water (5 mL), sodium hydroxide (870 mg, 21.7 mmol) was added, and the mixture was stirred at 20°C for 2 hours. The mixture was concentrated and diluted with DCM (20 mL) and purified water (10 mL), then adjusted to pH 4 using a 2 M hydrochloric acid solution. The extract was then extracted with DCM / MeOH (10 / 1, 30 mL × 3 times), dried with Na2SO4, and concentrated under reduced pressure to obtain compound 3-g, a yellow solid. Compound 3-g (350 mg, 0.925 mmol), compound 3-h (517 mg, 2.77 mmol), EDCI (532 mg, 2.77 mmol), and DMAP (339 mg, 2.77 mmol) were dissolved in DMF (10 mL) and stirred at 30°C for 12 hours. Saturated saturated Na2CO3 (40 mL) was added to the mixture, and the extract was extracted with EA (40 mL × 3 times) and washed with brine (50 mL × 2 times). After drying with Na2SO4, filtering, and vacuum concentration, the product was purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), purified by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoroacid) / ACN = 90 / 10 to 20 / 80, 22 min), and freeze-dried. The dried product was diluted with DCM (30 mL) and sat.After washing with Na2CO3 (30 mL) and brine (30 mL), the DCM layer was dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain compound 3, which is a yellow oil. 1 The results of the H NMR analysis were as follows.

[0132] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 6.8 Hz, 2H), 6.42 (d, J = 6.4 Hz, 2H), 3.98 (d, J = 6.0 Hz, 4H), 3.21 - 3.32 (m, 4H), 2.31 (t, J = 7.6 Hz, 4H), 1.54 - 1.68 (m, 10H), 1.25 - 1.37 (m, 44H), 0.85 - 0.94 (m, 12H).

[0134] 1-4. Preparation of Compound 4

[0135] As shown in Figure 4, compound 4-a (380 mg, 1.00 mmol) and compound 4-b (646 mg, 3.01 mmol) were added to DMF (10 mL), followed by the addition of EDCI (578 mg, 3.01 mmol) and DMAP (368 mg, 3.01 mmol), and the mixture was stirred at 30°C for 12 hours. After adding saturated sat. NaHCO3 (50 mL) to the mixture, the extract was extracted with EA (50 mL × 4 washes) and washed with brine (50 mL × 2 washes). The mixture was dried with Na2SO4 and concentrated under reduced pressure, then purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97) and purified by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoroacid) / ACN = 90 / 10 to 20 / 80, 22 min). After adding sat. Na2CO3 (30 mL) to the purified material, it was extracted with DCM (30 mL × 2 times), washed with brine (30 mL), dried with Na2SO4, and concentrated under reduced pressure to obtain compound 4, which is a yellow oil. 1 The results of the H NMR analysis were as follows.

[0136] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 6.4 Hz, 2H), 6.42 (d, J = 6.4 Hz, 2H), 3.98 (d, J = 5.6 Hz, 4H), 3.21 - 3.29 (m, 4H), 2.31 (t, J = 7.6 Hz, 4H), 1.54 - 1.67 (m, 10H), 1.24 - 1.36 (m, 52H), 0.89 (t, J = 6.4 Hz, 12H).

[0138] 1-5. Preparation of Compound 5

[0139] As shown in Fig. 5, compound 5-a (350 mg, 0.925 mmol) and compound 5-b (673 mg, 2.77 mmol) were added to DMF (10 mL), followed by the addition of EDCI (532 mg, 2.77 mmol) and DMAP (339 mg, 2.77 mmol), and the mixture was stirred at 30°C for 14 hours. The mixture was saturated with saturates. The liquid extracted with EA (80 mL × 2 times) after dilution with NaHCO3 (100 mL) was washed with brine (120 mL × 4 times), dried with Na2SO4, filtered, concentrated under reduced pressure, purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97), and then purified by prep-HPLC (C4, 150 × 19 mm, 5 μm, purified water (fluoroacid) / ACN = 90 / 10 to 20 / 80, 22 min). The product was diluted with DCM (30 mL), washed with sat. NaHCO3 (40 mL), extracted with DCM (30 mL), washed with brine (40 mL), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain Compound 5, a pale yellow oil, and of Compound 5 1 The results of the H NMR analysis were as follows.

[0140] 1 H NMR (400 MHz, CDCl3) δ 8.17 (d, J = 6.4 Hz, 2H), 6.35 - 6.47 (m, 2H), 3.97 (d, J = 6.0 Hz, 4H), 3.26 (d, J = 8.0 Hz, 4H), 2.31 (d, J = 7.6 Hz, 4 H), 1.49 - 1.70 (m, 10H), 1.20 - 1.38 (m, 60 H), 0.75 - 1.01 (m, 12H).

[0142] 1-6. Preparation of Compound 6

[0143] As shown in Fig. 6, compound 6-b (500 mg, 2.99 mmol), compound 6-a (3.14 g, 7.48 mmol), K2CO3 (1.24 g, 8.97 mmol), cesium carbonate (Cs2CO3) (974 mg, 2.99 mmol), and NaI (89.6 mg, 598 μmol) were added to ACN (30 mL) and stirred at 80°C for 12 hours under nitrogen gas. Purified water (100 mL) was added to the mixture, and the extract obtained with EA (100 mL × 2 times) was washed with brine (100 mL × 2 times). After drying with Na2SO4 and filtering, the solution was concentrated under reduced pressure and purified on silica gel (DCM / MeOH = 0 / 100 to 5 / 95) to obtain compound 6-c, a yellow oil. Compound 6-c (2.20 g, 2.61 mmol) was added to DCE (20 mL), and DIEA (674 mg, 5.21 mmol) and Compound 6-d (447 mg, 3.13 mmol) were added at 0°C and stirred at 25°C for 5 hours. The mixture was concentrated under reduced pressure, dissolved in MeOH (20 mL), stirred at 60°C for 0.5 hours, and concentrated; then, sat. NaHCO3 (30 mL) was added and extracted with EA (50 mL × 2 times). The extract was washed with brine (50 mL × 2 times), dried with Na2SO4, concentrated under reduced pressure, and purified on silica gel (MeOH / DCM = 0 / 100 to 3 / 97, containing 0.5% ammonia water) to obtain Compound 6-e, a white oil. Compound 6-g (10.0 g, 88.1 mmol) was added to 2-aminoethanol (100 mL), degassed by purging with nitrogen gas three times, and stirred at 110°C for 3 hours under nitrogen gas. The mixture was purified on silica gel (DCM / MeOH = 0 / 100 to 10 / 90) to obtain compound 6-h, a white solid. Compound 6-h (3.60 g, 26.1 mmol) and di-tert-butyldicarbonate (Boc2O) (11.4 g, 52.1 mmol) were added to EtOH (40 mL).1 mmol) was added and stirred at 25°C for 12 hours. After concentrating the mixture, it was purified on silica gel (MeOH / DCM = 2 / 98 to 3 / 97, containing 0.5% ammonia water) to obtain compound 6-f, a yellow oil. Compound 6-f (1.17 g, 4.90 mmol), compound 6-g (1.09 g, 5.39 mmol), and triethylamine (Et3N) (991 mg, 9.80 mmol) were added to 2-methyltetrahydrofuran (2-MeTHF) (20 mL) and stirred at 20°C for 5 hours, after which compound 6-e (1.70 g, 2.45 mmol) was added and stirred at 20°C for 12 hours. The mixture was diluted with purified water (100 mL), and the liquid extracted with EA (100 mL × 2 times) was sat. Compound 6-h, a white oil, was obtained by washing with Na2CO3 (100 mL × 2 times) and brine (200 mL), drying with Na2SO4, filtering, concentrating under reduced pressure, and purifying on silica gel (EA / PE = 0 / 100 to 25 / 75). Compound 6-h was added to a mixture of trifluoroacetic acid (TFA) (3 mL) and DCM (3 mL) and stirred at 20°C for 12 hours, then the pH was adjusted to 9 using sat. Na2CO3 (20 mL), extracted with DCM (20 mL × 2 times), dried with Na2SO4, filtered, and concentrated under reduced pressure. The oil obtained by purifying on silica gel (MeOH / DCM = 0 / 100 to 2 / 98, containing 0.5% ammonia water) and purifying by prep-HPLC (C4, 150*19 mm, 5 μm, purified water (fluoric acid) / ACN = 80 / 20 to 20 / 80, 22 min) was dissolved in DCM (20 mL), washed with sat. Na2CO3 (10 mL), dried with Na2SO4, filtered and concentrated, and freeze-dried to obtain Compound 6, a white oil. 1 The results of the H NMR analysis were as follows.

[0144] 1 H NMR (400 MHz, CDCl3) δ 8.19 (d,J = 6.0 Hz, 2H), 6.47 (d, J = 6.4 Hz, 2H), 4.95 (s, 1H), 4.33 (t, J = 5.2 Hz, 2H), 4.06 (t, J = 6.8 Hz, 4H), 3.42 (q, J = 5.2 Hz, 2H), 3.07 - 3.28 (m, 4H), 2.25 - 2.39 (m, 2H), 1.55 - 1.64 (m, 8H), 1.39 - 1.48 (m, 6H), 1.20 - 1.38 (m, 50H), 0.88 (t, J = 6.4 Hz, 12H).

[0146] <Example 2> Preparation of lipid nanoparticles

[0147] Lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNAGene, Korea) were prepared using the compound prepared in Example 1 above (ionized lipid), dioleoylphosphatidylethanolamine (DOPE) or distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and PEG 2000-DMG (polymerized lipid).

[0148] A lipid solution was prepared by composing ionized lipids, DOPE / DSPC, cholesterol, and PEG 2000-DMG in a molar ratio of 49:10:38.5:2.5 and dissolving them in ethanol at a concentration of 4.8 mg / mL. Separately, an active ingredient solution was prepared by dissolving Firefly Luciferase mRNA in a citric acid buffer at pH 4.0±1.0 or an acetic acid buffer at pH 5.0±1.0 such that the weight ratio of mRNA to ionized lipids was 1:16. Subsequently, lipid nanoparticles were prepared by passing the lipid solution to the active ingredient solution through a microfluidic mixing device (Benchtop Nanoassemblr, Precision Nanosystems) at a flow rate of approximately 15 mL / min so that the volume ratio was 1:3. The prepared lipid nanoparticles were diluted with Tris buffer containing 8.7% sucrose and dialyzed using a dialysis centrifuge tube until the ethanol content was less than 1%, and the final concentration was prepared to be 0.2 mg / mL based on mRNA.

[0149] For comparison, lipid nanoparticles encapsulating Firefly Luciferase mRNA (RNAGene, Korea) were prepared using MC3 ionized lipid, SM-102 ionized lipid, distearoylphosphatidylcholine (DSPC) (neutral lipid), cholesterol (steroid), and DMG-PEG2000 (polymerized lipid). MC3, DSPC, cholesterol, and DMG-PEG2000 or SM-102, DSPC, cholesterol, and DMG-PEG2000 were prepared in a molar% ratio of 50:10:38.5:1.5 and used as lipid nanoparticles for comparison.

[0151] <Example 3> Confirmation of lipid nanoparticle size and polydispersity

[0152] The size and surface charge of lipid nanoparticles according to the ionized lipids prepared in Example 1 above were measured. To measure the size of the lipid nanoparticles, the mRNA contained in each lipid nanoparticle was diluted with PBS to a concentration of 1 μg / mL, and the diameter and polydispersity index (PDI) of the LNPs were measured using Dynamic Light Scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0153] As a result, as shown in Table 1 below, all lipid nanoparticles exhibited a size of about 120 nm or less and a good polydispersity of within 0.2.

[0154] Compound number Particle size (nm) PDI 1 77 0.06 2 77 0.12 3 83 0.11 4 84 0.14 5 85 0.13 6 115 0.09

[0156] <Example 4> Confirmation of gene encapsulation rate

[0157] To verify the gene encapsulation rate of lipid nanoparticles, the Quant-iT™ Ribogreen™ RNA Reagent and Kit was used. 5 μL of ribosomal RNA standard (100 μg / mL in TE buffer) was taken and diluted with 245 μL of TE buffer or 0.4% Triton-TE buffer, respectively, to prepare stock solutions for the calibration curve. 2, 5, 10, 25, and 50 μL of the stock solutions were taken and diluted with TE buffer or 0.4% Triton-TE buffer to adjust the total volume to 100 μL. Then, 100 μL of Quant-iT™ Ribogreen™ RNA Reagent was added and mixed, and the fluorescence intensity (excitation 475 nm and emission 500–550 nm) was measured to construct the calibration curve.

[0158] 5 μL of the nanoparticle solution prepared in Example 2 above was taken and diluted with 245 μL of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5 in DEPC-treated water). 50 μL of either TE buffer or 2% Triton-TE buffer was added to 50 μL of the diluted nanoparticle solution, respectively, and incubated at 37°C for approximately 10 minutes. Subsequently, 40 μL of each culture medium was placed in a microplate, 60 μL of TE buffer, and 100 μL of Quant-iT™ Ribogreen™ RNA Reagent were sequentially added and mixed, after which the fluorescence intensity was measured. The measured fluorescence intensity was applied to a calibration curve to calculate the gene mass of TE buffer (TO) and the gene mass of 2% Triton-TE buffer (T2), and then the gene inclusion rate was calculated using the following Equation 1. The gene inclusion rates were confirmed and listed in Table 2. As a result, all showed good gene encapsulation rates.

[0159] [Mathematical Formula 1]

[0160]

[0161] As a result, as shown in Table 2 below, it was confirmed that all compounds exhibited a good gene encapsulation rate of 85% or higher.

[0162] Compound number Gene Encapsulation Rate (%) 1 89.9 2 93.3 3 94.4 4 94.7 5 97.1 6 97.6

[0164] <Example 5> Confirmation of lipid nanoparticle gene transfer efficiency

[0165] 5-1. Verification of In vivo Gene Delivery Efficiency 1

[0166] To confirm the in vivo gene delivery efficiency of lipid nanoparticles, a single intravenous injection of 1 mg / kg of lipid nanoparticles containing luciferase gene was administered to 7-week-old C57BL / 6 strain mice, and the mice were sacrificed and their livers were removed after 4 hours.

[0167] To determine the luciferase expression level in tissue samples, liver tissue sections were weighed and transferred to tubes containing 3 mm metal beads. 500 μl of Glo-Lysis Buffer (Promega, WI, USA) was added per 50 mg of the section, and the tissue was fragmented using a bead homogenizer. Subsequently, the supernatant from the centrifuged fragments was collected and diluted with Glo-Lysis Buffer at an appropriate ratio. 50 μl each of the diluted tissue fragments and Steady-Glo Luciferase Assay solution were then transferred to 96-well white plates at a 1:1 ratio and reacted at room temperature. After 5 minutes, luminescence values ​​were detected using a GloMax Discover microplate reader (Promega). A calibration curve was calculated using recombinant luciferase (Promega) as a standard, and the luciferase expression level (ng / g tissue) of the sample was derived from this.

[0168] As a result, as shown in Table 3, it was confirmed that the lipid nanoparticles of the ionized lipid of the present invention exhibited excellent gene delivery effects through systemic administration by showing good levels of luciferase expression in liver tissue upon intravenous injection.

[0169] Compound number Luciferase expression levels in the liver after intravenous injection (Mean±SD, ng / g liver) 1 96,246 ± 27,097 2 21,597 ± 11,480 3 8,248 ± 1,030 4 29,027 ± 853 5 46,333 ± 32,091 MC3 13,845 ± 3,787

[0171] 5-2. Verification of In vivo Gene Delivery Efficiency 2

[0172] To confirm the in vivo gene delivery efficiency following repeated administration of lipid nanoparticles, lipid nanoparticles encapsulated with EPO mRNA (TriLink BioTechnologies, CA, USA) were intravenously administered to 7-week-old C57BL / 6 lineage mice at a gene-based dose of 0.5 mg / kg. To confirm in vivo EPO expression levels, blood was collected 6 hours after administration, and the collected blood was treated with anticoagulation to separate the plasma.

[0173] To confirm the concentration of EPO protein in plasma, an EPO enzyme immunoassay (ELISA) kit (R&D Systems, MN, USA) was used to perform the analysis according to the manufacturer's instructions. More specifically, plasma samples were diluted to a ratio of 1:3000 using the specimen diluent in the kit, and 100 μL of each diluent (at a 1:1 ratio with the assay diluent) was added to each micro-well and reacted at room temperature for 2 hours. Subsequently, all wells were removed, 200 μL of EPO conjugate was added, and the mixture was reacted again at room temperature for 2 hours. After washing four times with 300 μL of washing buffer, 200 μL of substrate solution was added and reacted for 20 minutes. To terminate the reaction, 100 μL of stop solution was added, and absorbance was measured at wavelengths of 450 nm and 600 nm using a microplate (Promega). A calibration curve was calculated using standard substances, and the EPO protein concentration (ng / mL) of each sample was determined within the defined range of the standard substances.

[0174] As a result, as shown in Table 4, it was confirmed that the lipid nanoparticles produced by the ionized lipid of the present invention showed a good level of EPO expression in the blood compared to the lipid nanoparticles produced by MC3 and SM-102 ionized lipids used as a comparison when administered intravenously, thereby demonstrating an excellent gene transfer effect through systemic administration.

[0175] Compound number EPO expression levels in blood after intravenous injection (Mean±SD, ng / mL) 1 4,424 ± 588 MC3 384 ± 73 SM-102 1,154 ± 364

[0177] 5-3. Verification of In vivo Gene Delivery Efficiency 3

[0178] To confirm the gene delivery capacity of lipid nanoparticles in liver injury disease, liver injury was induced in 6-week-old C57BL / 6 strain rats by intraperitoneally administering 2 mL / kg of CCl4 diluted to 20% in olive oil three times a week for two weeks. When plasma ALT and AST levels were checked the day after the last CCl4 administration, ALT was 2,592 U / L and AST was 5,652 U / L, which was compared to plasma ALT 56 U / L and AST 34 U / L in the olive oil-administered vehicle group, confirming that liver injury had been induced. A single intravenous injection of 1 mg / kg of the luciferase gene encapsulated in each lipid nanoparticle was administered to the liver-induced rats (liver injury model) and the rats administered olive oil (normal model), and the rats were sacrificed and their livers were extracted 6 hours after administration.

[0179] To determine the luciferase expression level in tissue samples, liver tissue sections were weighed and transferred to tubes containing 3 mm metal beads. 500 μL of Glo-Lysis Buffer (Promega, WI, USA) was added per 50 mg of the section, and the tissue was fragmented using a bead homogenizer. Subsequently, the supernatant from the centrifuged fragments was collected and diluted with Glo-Lysis Buffer at an appropriate ratio. 50 μL each of the diluted tissue fragments and Steady-Glo Luciferase Assay solution were transferred to 96-well white plates at a 1:1 ratio and reacted at room temperature. After 5 minutes, luminescence values ​​were detected using a GloMax Discover microplate reader (Promega). A calibration curve was calculated using recombinant luciferase (Promega) as a standard, and the luciferase expression level (ng / g tissue) of the sample was derived from this.

[0180] As a result, as shown in Table 5, the lipid nanoparticles produced by the ionized lipids of the present invention exhibited excellent gene delivery capabilities without difference from normal animal models when intravenously injected into an animal model of liver damage. In contrast, the lipid nanoparticles produced by SM-102 used as a comparison exhibited reduced gene delivery capabilities in the animal model of liver damage.

[0181] Compound number Luciferase expression levels in the liver after intravenous injection (Mean±SD, ng / g liver) Normal model Liver injury model 1 65,704 ± 12,003 86,676 ± 8,499 SM-102 21,152 ± 197 4,040 ± 1,438

[0183] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

Claim 1 delete Claim 2 delete Claim 3 delete Claim 4 delete Claim 5 Compounds selected from the group of compounds below, pharmaceutically acceptable salts, tautomers, or stereoisomers thereof, comprising: (1) (pyridin-4-ylazanediyl)bis(hexane-6,1-dyl)bis(2-hexyldecanoate) [(pyridin-4-ylazanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (2) bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate) [bis(2-butyloctyl)10,10'-(pyridin-4-ylazanediyl)bis(decanoate)]; (3) bis(2-butyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (4) bis(2-hexyloctyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (5) bis(2-hexyldecyl)8,8'-(pyridin-4-ylazanediyl)dioctanoate; (6) (((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate) [(((2-(pyridin-4-ylamino)ethoxy)carbonyl)azanediyl)bis(hexane-6,1-diyl)bis(2-hexyldecanoate)]; (7) bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate [bis(2-hexyldecyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate]; and (8) bis(2-hexyloctyl)6,6'-(pyridin-4-ylazanediyl)dihexanoate. Claim 6 A lipid nanoparticle composition comprising a compound according to claim 5. Claim 7 A lipid nanoparticle composition according to claim 6, characterized in that the compound is an ionized lipid. Claim 8 A lipid nanoparticle composition according to claim 6, characterized in that the lipid nanoparticle composition further comprises one or more selected from the group consisting of neutral lipids, steroids, and polymeric lipids. Claim 9 A lipid nanoparticle composition according to claim 8, characterized in that the neutral lipid is a phospholipid or a glycolipid. Claim 10 A lipid nanoparticle composition according to claim 8, wherein the steroid is one or more selected from the group consisting of cholesterol, bile acid derivatives and choline acid derivatives. Claim 11 A lipid nanoparticle composition according to claim 8, wherein the polymerized lipid is a pegylated lipid and is one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Claim 12 A lipid nanoparticle composition according to claim 8, characterized in that the lipid nanoparticle composition comprises 20 to 65 mol% ionized lipid, 2.5 to 30 mol% neutral lipid, 25 to 60 mol% steroid, and 0.5 to 5 mol% polymerized lipid. Claim 13 A lipid nanoparticle composition according to claim 6, characterized in that the composition further comprises a preventive or therapeutic agent. Claim 14 A lipid nanoparticle composition according to claim 13, wherein the prophylactic or therapeutic agent is one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), and mixtures thereof. Claim 15 A lipid nanoparticle composition according to claim 6; and a drug delivery composition for treating liver damage, comprising a prophylactic or therapeutic agent. Claim 16 A drug delivery composition for treating liver damage according to claim 15, wherein the prophylactic or therapeutic agent is one or more selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), and mixtures thereof.

Images