Lipid nanoparticle formulations comprising ionized lipids having branched structures and uses thereof

Abstract

The present invention relates to: an ionized lipid comprising a lipid having a branched structure; lipid nanoparticle formulations using the ionized lipids and uses thereof. The ionized lipid provided by the invention is a biodegradable lipid material, and a branched heteroamine structure exists in the lipid structure of the ionized lipid. Moreover, the lipid nanoparticles using the ionized lipid can efficiently deliver nucleic acid drugs and the like, and thus can be effectively used in related technical fields, such as mRNA vaccines and therapeutic agents.

Description

[0001] Cross Reference to Related Applications

[0002] This application claims priority to Application No. 10-2022-0189895 filed on December 29, 2022 and Application No. 10-2023-0156562 filed on November 13, 2023, the entire contents of which are incorporated herein by reference. TECHNICAL FIELD

[0003] The present invention relates to a lipid nanoparticle formulation comprising an ionizable lipid having a branched structure and uses thereof. BACKGROUND

[0004] In the pharmaceutical industry, a drug delivery system (DDS) aimed at effectively delivering the required amount of a drug while reducing the side effects of the drug and maximizing its efficacy and effectiveness is a core technology with high value and high success potential, which can create economic benefits comparable to new drug development and aims to improve the quality of medical care through effective drug management.

[0005] Nucleic acids such as siRNA and mRNA are substances that can control the expression of specific proteins in the body and are attracting attention as important tools for treating cancer, genetic diseases, infectious diseases, and autoimmune diseases. Nucleic acids are macromolecular anionic substances, are difficult to deliver directly into cells, and are easily decomposed by enzymes in the blood, and thus a large amount of research is being conducted to overcome these problems.

[0006] Until now, a method of transporting nucleic acids into cells by mixing nucleic acids with positively charged lipids or polymers, called lipoplexes and polyplexes, respectively, has been mainly used. Lipoplexes are widely used at the cellular level because they bind to nucleic acids and well deliver nucleic acids into cells. However, when injected locally in the body, they often cause inflammation in the body, and when injected in blood vessels, they are mainly accumulated in tissues such as the lung, liver, and spleen, which are first-pass organs.

[0007] The initially developed ionizable lipid 1,2-dihydrononyl-3-dimethylaminopropane (DLinDAP) has low gene transfer efficiency, and a large amount of siRNA is required to achieve a therapeutic effect. Thereafter, by structural changes in the amine head group and linker, about 300 ionizable lipids were screened in a mouse coagulation factor FVII model, and ED 50 (Dlin-MC3-DMA) [Acc. Chem. Res. 2019] having a very low value (0.005 mg / kg) was selected. This is more effective than existing ionizable lipids, about 1000 times, and is used as a a delivery carrier of The siRNA therapy for hereditary amyloidosis (hATTR) was first approved by the U.S. Food and Drug Administration (FDA) in 2018.

[0008] Moderna and Pfizer developed the first mRNA lipid nanoparticle vaccine to overcome the COVID-19 pandemic. Currently, vaccination has been successfully carried out in several countries including the United States and Israel. The mRNA lipid nanoparticle of Moderna and Pfizer's coronavirus vaccine uses an ionizable lipid. The ionizable lipid in the Moderna vaccine is SM-102 (Arbutus){{heptadecan-9-yl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate}} and the ionizable lipid in the Pfizer vaccine is ALC-0315 (Genevant) {[(4-hydroxybutyl)azanediyl]bis(hexan-6,1-diyl)bis(2-hexyldecanoate)}.

[0009] The ionizable lipid is a structure having a tertiary amine head group, the degree of ionization of which varies depending on the pH in the body and a long hydrocarbon tail connected by a linker, and since the degree of ionization thereof varies depending on the pH, it can surround the nucleic acid drug. Then, these ionizable lipids are formulated with other components to form a solid lipid nanoparticle structure, the inside of which is encapsulated with a nucleic acid drug. The ionizable lipids are cationic, surround the nucleic acid drug during low-pH formulation, and at physiological pH, they have a neutral surface charge and form a lipid nanoparticle by hydrophobic interaction. The lipid nanoparticle introduced into the endosome by endocytosis becomes cationic again as the pH of the endosome decreases, interacts with the anionic lipids in the endosome membrane, and is able to release the enclosed nucleic acid drug into the cell by endosomal escape.

[0010] As prior art, Korean Patent Publication No. 10-2020-0040586 discloses the preparation of ionizable lipids into lipid nanoparticles for in vivo drug delivery with a 6-membered heterocyclic amine and 1,2-epoxydodecane, and Korean Patent Publication Nos. 10-2022-0103968 and 10-2022-0103968 disclose various ionizable lipids and lipid nanoparticle compositions.

[0011] Accordingly, the present inventors have made great efforts to develop a new type of particle having excellent drug encapsulation efficiency and capable of effectively delivering anionic drugs, nucleic acids, etc. to target organs or cells. Accordingly, the present invention is accomplished by preparing a lipid nanoparticle including an ionizable lipid having a branched structure, and confirming that the lipid nanoparticle can efficiently encapsulate a nucleic acid drug and induce immunity by the encapsulated nucleic acid. SUMMARY

[0012] The object of the present invention is to provide a novel ionizable lipid compound having a branched structure.

[0013] Another object of the present invention is to provide lipid nanoparticles comprising ionizable lipid compounds.

[0014] Another object of the present invention is to provide a composition for drug delivery, which comprises lipid nanoparticles and anionic drugs, nucleic acids, or a combination thereof.

[0015] Another object of the present invention is to provide use of a composition for a drug delivery system, wherein the composition for a drug delivery system comprises lipid nanoparticles and anionic drugs, nucleic acids, or a combination thereof.

[0016] Another object of the present invention is to provide a method for drug delivery, comprising the step of administering to a subject a composition for drug delivery, the composition for drug delivery comprising lipid nanoparticles and anionic drugs, nucleic acids, or a combination thereof.

[0017] In order to achieve the above object, the present invention provides an ionizable lipid compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof:

[0018] [Formula 1]

[0019]

[0020] R1 and R2 are each independently selected from -H, -C 1-10 Alkyl, -Y or -C 1-10 Alkyl-NR A R B any group,

[0021] R3 and R4 are each independently selected from -H, -C 1-10 Alkyl, -Y or -C 1-10 Alkyl-NR A R B or are connected to each other to form a 4- to 8-membered heterocycloalkyl group having two N elements,

[0022] However, at least one of R1 to R4 is -C 1-10 Alkyl-NR A R B ,

[0023] R A and R B are independently -H, -C 1-10 Alkyl or -Y,

[0024] n is an integer between 0 and 6,

[0025] There is at least one -Y substituent,

[0026] Y is represented by the following [Formula 2],

[0027] When there are a plurality of Y, they can be different,

[0028] [Formula 2]

[0029]

[0030] o, p, and q are each independently any integer from 1 to 12.

[0031] The present application also provides a use of the ionizable lipid compound represented by [Formula 1] or a pharmaceutically acceptable salt thereof in the preparation of a lipid nanoparticle for drug delivery.

[0032] The present application also provides a use of the ionizable lipid compound represented by [Formula 1] or a pharmaceutically acceptable salt thereof in the preparation of a drug delivery system comprising a lipid nanoparticle.

[0033] The present application also provides a lipid nanoparticle comprising the ionizable lipid compound or a pharmaceutically acceptable salt thereof.

[0034] The present application also provides a composition for drug delivery comprising a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof.

[0035] The present application also provides a use of the composition comprising a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof as a drug delivery system.

[0036] In addition, the present application provides a method of drug delivery comprising the step of administering to a subject a composition for drug delivery comprising a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof.

[0037] Advantages

[0038] The present application relates to an ionizable lipid comprising a lipid having a branched structure, a lipid nanoparticle formulation using the same, and use thereof. The ionizable lipid of the present application is a biodegradable lipid material in which a branched heteroamine structure is present in the lipid structure thereof. Also, a lipid nanoparticle using the ionizable lipid can efficiently deliver a nucleic acid drug or the like, and thus can be effectively used in the relevant technical field, such as an mRNA vaccine and a therapeutic agent. BRIEF DESCRIPTION OF DRAWINGS

[0039] FIG. 1 is a schematic diagram showing a synthetic method of a novel ionizable lipid having a branched structure.

[0040] FIG. 2 is a schematic diagram showing MS spectrum results to confirm the synthesis of the ionizable lipid.

[0041] FIG. 3 is a graph showing luminescent gene expression levels and cytotoxicity of ionizable lipid nanoparticles in Hela cells.

[0042] FIG. 4 is a graph showing luminescent gene expression levels and cytotoxicity of ionizable lipid nanoparticles in HEK 293 cells.

[0043] FIG. 5 is a graph showing AST levels in the blood of mice after administration of EW244-E-7 lipid nanoparticles.

[0044] FIG. 6 is a graph showing ALT levels in the blood of mice after administration of EW244-E-7 lipid nanoparticles.

[0045] FIG. 7 is a schematic showing bioluminescence of mFluc-loaded EW221-E-7 lipid nanoparticles injected intravenously into mice.

[0046] FIG. 8 is a schematic showing bioluminescence of mFluc-loaded EW244-E-7 lipid nanoparticles injected intravenously into mice.

[0047] FIG. 9 is a schematic showing bioluminescence of mFluc-loaded EW221-E-7 lipid nanoparticles injected intramuscularly into mice.

[0048] FIG. 10 is a schematic showing bioluminescence of mFluc-loaded EW244-E-7 lipid nanoparticles injected intramuscularly into mice.

[0049] FIG. 11 is a graph showing expression levels of EPO and MCP-1 after injection of hEPO mRNA-loaded EW244-E-7 lipid nanoparticles into mice.

[0050] FIG. 12 is a graph showing expression levels of MCP-1 after injection of hEPO mRNA-loaded EW244-E-7 lipid nanoparticles into mice.

[0051] FIG. 13 is a graph showing neutralizing antibody titers after injection of COVID-19 spike mRNA-loaded EW244-E-7 lipid nanoparticles into mice.

[0052] FIG. 14 is a graph showing IFN-g induction after injection of COVID-19 spike mRNA-loaded EW244-E-7 lipid nanoparticles into mice.

[0053] FIG. 15 is a graph showing neutralizing antibody titers against SARS-CoV-2 original strain (S) after injection of lipid nanoparticles loaded with in-silico spike mRNA into mice.

[0054] FIG. 16 is a graph showing neutralizing antibody titers against SARS-CoV-2 Delta variant strain (Delta) after injection of lipid nanoparticles loaded with in-silico spike mRNA into mice.

[0055] FIG. 17 is a graph showing neutralizing antibody titers against Omicron (BA.5) after injection of lipid nanoparticles loaded with in-silico spike mRNA into mice.

[0056] FIG. 18 is a graph showing IFN-γ induction after injection of lipid nanoparticles loaded with in-silico spike mRNA into mice.

[0057] FIG. 19 is a graph showing IgG antibody titers after injection of EW244-E-7 lipid nanoparticles loaded with mRNA encoding RSV into mice. DETAILED DESCRIPTION

[0058] Hereinafter, the present application is described in detail.

[0059] The present application provides an ionizable lipid compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof:

[0060] [Formula 1]

[0061]

[0062] R1and R2are each independently any group selected from -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B of any group,

[0063] R3and R4are each independently any group selected from -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B or are linked to each other to form a 4- to 8-membered heterocycloalkyl having two N elements,

[0064] However, at least one of R1to R4is -C 1-10 alkyl-NR A RB ,

[0065] R A and R B each independently is -H, -C 1-10 alkyl or -Y,

[0066] n is an integer between 0 and 6,

[0067] there are at least one -Y substituent,

[0068] Y is represented by the following [Formula 2],

[0069] when there are multiple Ys, they can be different,

[0070] [Formula 2]

[0071]

[0072] o, p, and q are each independently any integer from 1 to 12.

[0073] As another embodiment of the present application,

[0074] in the above Formula 1,

[0075] R1and R2are each independently any group selected from -H, -C 1-6 alkyl, -Y, or -C 1-6 alkyl-NR A R B ,

[0076] R3and R4are each independently any group selected from -H, -C 1-6 alkyl, -Y, or -C 1-6 alkyl-NR A R B , or are linked to each other to form a 4- to 8-membered heterocyclic alkyl group having two N elements,

[0077] However, at least one of R1to R4is -C 1-6 alkyl-NR A R B ,

[0078] R A and R B are each independently -H, -C 1-6 alkyl or -Y,

[0079] n is an integer between 0 and 4,

[0080] there are 2 to 6 -Y substituents,

[0081] Y is represented by the following [Formula 2],

[0082] each Y can be different,

[0083] [Formula 2]

[0084]

[0085] o and p are each independently an integer of 1 to 9, and

[0086] q is an integer of 1 to 5.

[0087] As another embodiment of the present application,

[0088] In the above Formula 1,

[0089] R1and R2are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B ,

[0090] R3and R4are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B , or are linked to each other to form a 5- to 6-membered heterocyclic alkyl group having two N elements,

[0091] However, at least one of R1to R4is -C 1-4 alkyl-NR A R B ,

[0092] R A and R B are each independently -H, -C 1-4 alkyl, or -Y,

[0093] n is an integer between 1 and 2,

[0094] 3 to 6 -Y substituents are present,

[0095] Y is represented by the following [Formula 2],

[0096] each Y can be different,

[0097] [Formula 2]

[0098]

[0099] o and p are each independently an integer of 1 to 9, and

[0100] q is an integer of 1 to 5.

[0101] As another embodiment of the present application,

[0102] In the above Formula 1,

[0103] R1and R2are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B ,

[0104] R3and R4are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B or linked to each other to form a 5- to 6-membered heterocyclic alkyl group having two N elements,

[0105] However, at least one of R1to R4is -C 1-4 alkyl-NR A R B ,

[0106] R A and R B are each independently -H, -C 1-4 alkyl, or -Y,

[0107] N is an integer between 1 and 2,

[0108] 3 to 6 -Y substituents are present,

[0109] Y is represented by the following [Formula 2],

[0110] Each Y can be different,

[0111] [Formula 2]

[0112]

[0113] o and p are each independently an integer of 3 to 9, and

[0114] q is an integer of 1 to 3.

[0115] As another embodiment of the present application, the compound can be selected from the following compounds:

[0116]

[0117] The above ionizable lipid refers to an amine-containing lipid that is easily protonated, for example, a lipid whose charge state changes depending on the surrounding pH.

[0118] The ionizable lipid described above can be protonated (positively charged) at a pH value lower than the pKa of the cationic lipid, and can be substantially neutral at a pH value higher than the pKa.

[0119] In the present application, the ionizable lipid is an ionizable compound having a lipid property, and is used for efficiently encapsulating a drug (e.g., an anionic drug and / or a nucleic acid) into a lipid nanoparticle by electrostatic interaction with the drug.

[0120] The ionizable lipid according to the present application can be used in the form of a pharmaceutically acceptable salt, wherein the salt is preferably an acid addition salt formed from a pharmaceutically acceptable free acid. Inorganic acids and organic acids can be used as the free acid. The inorganic acids that can be used include hydrochloric acid, bromic acid, sulfuric acid, and phosphoric acid, and the organic acids that can be used include citric acid, acetic acid, lactic acid, maleic acid, fumaric acid, gluconic acid, methanesulfonic acid, glycolic acid, succinic acid, tartaric acid, 4-toluenesulfonic acid, galacturonic acid, methylenebis-hydroxy-picolinic acid, glutamic acid, and aspartic acid.

[0121] The ionizable lipid according to the present application includes not only a pharmaceutically acceptable salt, but also all salts, isomers, hydrates, and solvates that can be prepared by a conventional method.

[0122] The ionizable lipid compound having a branched structure according to the present application can move to a cell membrane by destroying the structure of the cell membrane and facilitate endosome escape, or can slow down the rate of hydrolysis or enzymatic degradation of an ester due to the branched structure, so that the drug delivery efficiency is excellent.

[0123] The present application also provides a lipid nanoparticle including the ionizable lipid compound or a pharmaceutically acceptable salt thereof.

[0124] The lipid nanoparticle can further include a phospholipid, cholesterol, and a lipid-PEG (polyethylene glycol) conjugate.

[0125] The phospholipid plays a role in wrapping and protecting a core formed by the interaction of the ionizable lipid and the drug within the lipid nanoparticle, and binds to a phospholipid bilayer of a target cell to facilitate endosome escape and passage through a cell membrane during intracellular delivery of the drug.

[0126] According to embodiments, the use of a phospholipid can be unlimited as long as the phospholipid can facilitate fusion of the lipid nanoparticles. For example, the phospholipid can be at least one selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), egg yolk phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (DSPE), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine (POPE), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine] (DOPS), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], etc. In particular, the phospholipid is DOPE, and the lipid nanoparticle including DOPE can be effective for mRNA delivery (excellent drug delivery efficiency for mRNA).

[0127] Cholesterol imparts rigidity in the charge morphology of the lipids within the lipid nanoparticle, and is dispersed in the core and surface of the nanoparticle to enhance the stability of the nanoparticle.

[0128] Lipid-PEG (polyethylene glycol) conjugate, lipid-PEG, PEG-lipid, or lipid-PEG refers to a form in which a lipid and PEG are conjugated, and refers to a lipid having a hydrophilic polymer polyethylene glycol (PEG) polymer attached to one end. The lipid-PEG conjugate contributes to the stability of the nanoparticle in the serum within the lipid nanoparticle, and plays a role in preventing aggregation between the nanoparticles. In addition, the lipid-PEG conjugate can enhance the stability of the nucleic acid in the body by protecting the nucleic acid from degradation enzymes during in vivo delivery, and can increase the half-life of the drug encapsulated in the nanoparticle.

[0129] In the lipid-PEG conjugate, the PEG can be directly conjugated to the lipid, or can be linked to the lipid through a linker moiety. Any linker moiety suitable for binding PEG to the lipid can be used, including, for example, ester-free linker moieties and ester-containing linker moieties. The ester-free linker moieties include amido (-C(O)NH-), amino (-NR-), carbonyl (-C(O)-), carbamate (-NHC(O)O-), urea (-NHC(O)NH-), disulfide (-S-S-), ether (-O-), succinyl (- (O)CCH2CH2C(O)-), succinamide (-NHC(O)CH2CH2C(O)NH-), ether, disulfide, and combinations thereof (e.g., a linker containing both a carbamate linker moiety and an amido linker moiety). The ester-containing linker moieties include, for example, carbonate (-OC(O)O-), succinyl, phosphate (-O-(O)POH-O-), sulfonate, and combinations thereof, but are not always limited thereto.

[0130] The lipid in the lipid-PEG conjugate can be used without limitation, as long as it is a lipid that can be bound to polyethylene glycol, and phospholipid and / or cholesterol, which are other components of the lipid nanoparticle, can also be used. In particular, the lipid in the lipid-PEG conjugate can be ceramide, dimyristoylglycerol (DMG), succinyl diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), or cholesterol, more particularly, C16-PEG2000 ceramide.

[0131] The PEG in the lipid-PEG conjugate is a hydrophilic polymer having the ability to inhibit adsorption of plasma proteins, thereby increasing the circulation time of the lipid nanoparticle in the body and preventing aggregation between the nanoparticles. In addition, the lipid-PEG conjugate can exhibit a stealth function in the body, preventing degradation of the nanoparticles.

[0132] The lipid nanoparticle can include an ionizable lipid: phospholipid: cholesterol: lipid-PEG conjugate in a molar ratio of (15-35):(15-35):(40-60):(0.1-5), preferably in a molar ratio of (25-40):(10-25):(40-60):(0.5-3), more preferably in a molar ratio of (25-30):(17-22):(50-55):(1-2).

[0133] The present application also provides a composition for drug delivery, which includes a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof.

[0134] The anionic drug can be at least one selected from the group consisting of a peptide, a protein drug, a protein-nucleic acid construct, and an anionic biopolymer-drug conjugate.

[0135] The nucleic acid can be at least one selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), guide ribonucleic acid (gRNA), single-stranded guide ribonucleic acid, antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and deoxyribozyme.

[0136] The composition for drug delivery can include a physiologically active substance such as an anionic drug and / or a therapeutic nucleic acid enclosed in a lipid nanoparticle, in which the physiologically active substance such as an anionic drug or a therapeutic nucleic acid is stably and efficiently enclosed, thereby enabling the delivery composition to exhibit an excellent therapeutic effect. In addition, there is an advantage in that the type of drug enclosed in the lipid nanoparticle can be varied according to a therapeutic purpose.

[0137] The lipid nanoparticle can have an anionic drug and / or a nucleic acid enclosed therein (in the lipid nanoparticle). The description of the lipid nanoparticle in which the anionic drug and / or the nucleic acid are enclosed (in the lipid nanoparticle) is the same as the above-described lipid nanoparticle.

[0138] The weight ratio of the ionizable lipid contained in the lipid nanoparticle and the drug (anionic drug, nucleic acid, or a combination thereof) can be (1-20): 1, preferably (1-15): 1, (1-10): 1, more preferably (7.5-10): 1.

[0139] In the present application, the composition for drug delivery can be used as a pharmaceutical composition for preventing and treating a disease.

[0140] The pharmaceutical composition can be administered systemically or locally, in particular, by a delivery route selected from the group consisting of intradermal, subcutaneous, intramuscular, intraocular, intraarticular, intraventricular, intracerebroventricular, intrathecal, oral, intravenous, intratracheal, intraperitoneal, intranasal, intrauterine delivery, or any combination thereof.

[0141] The pharmaceutical composition is administered in a pharmaceutically effective dose. In the present invention, the term "pharmaceutically effective dose" refers to an amount sufficient to treat a disease, which has a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined depending on factors including the type and severity of the disease, drug activity, drug sensitivity, administration time, administration route, and excretion rate, treatment duration, concomitant drugs, and other factors well known in the medical field. The pharmaceutical composition according to one embodiment can be administered as a separate therapeutic agent or in combination with other therapeutic agents, can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered as a single dose or multiple doses. In consideration of all the above factors, it is important to administer the minimum amount that can achieve the maximum effect without side effects, which can be easily determined by those skilled in the art. Specifically, the pharmaceutically effective dose according to the present invention can vary depending on the age, sex, and weight of the patient, and can be administered daily, every other day, or divided into 1 to 3 times a day. However, since the dose can be increased or decreased depending on the administration route, obesity severity, sex, body weight, age, etc., the dose does not limit the scope of the present invention in any way.

[0142] The present invention also provides the use of a composition comprising a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof as a drug delivery system.

[0143] In addition, the present invention provides a drug delivery method comprising the step of administering to a subject a composition for drug delivery comprising a lipid nanoparticle and an anionic drug, a nucleic acid, or a combination thereof.

[0144] The subject includes all mammals, including humans, non-human primates, dogs, cats, horses, sheep, goats, cows, rabbits, pigs, rats, and mice, but is not always limited thereto. The administration can be systemic administration or local administration, and in particular, can be selected from the group consisting of intradermal, subcutaneous, intramuscular, intraocular, intraarticular, intracerebroventricular, intrathecal, oral, intravenous, intratracheal, intraperitoneal, intranasal, intrauterine delivery, or any combination thereof.

[0145] In specific examples and experimental examples, the present inventors synthesized an ionizable lipid having a biodegradable functional group with a heteroamine structure introduced (see FIG. 1 ), and confirmed that the ionizable lipid was synthesized well (see FIG. 2 ). In addition, cholesterol, phospholipid, and a lipid-PEG conjugate were dissolved in the synthesized ionizable lipid, and a lipid nanoparticle encapsulating a nucleic acid was prepared by mixing the lipid and mRNA at a volume ratio of 10:1. The prepared nucleic acid-loaded lipid nanoparticle exhibited good particle size, uniformity, and excellent drug loading rate for drug release.

[0146] Subsequently, the luminescence of the mRNA-loaded lipid nanoparticle was measured to confirm excellent intracellular nucleic acid delivery efficiency and no cytotoxicity (FIG. 3 and FIG. 4 ). In addition, the lipid nanoparticles were administered to mice and confirmed that they did not cause liver toxicity to the animals ( FIG. 5 and FIG. 6 ). In addition, it has been confirmed that the lipid nanoparticles loaded with mFluc are delivered to the liver by intravenous injection into mice ( FIG. 7 and FIG. 8 ), the lipid nanoparticles are delivered to the injection site by intramuscular injection ( FIG. 9 and FIG. 10 ), indicating that the lipid nanoparticles are suitable for in vivo drug delivery. In addition, the present inventors delivered the lipid nanoparticles loaded with hEPO mRNA to mice and confirmed that they are suitable for in vivo protein expression and have excellent primary immune generation capacity ( FIG. 11 and FIG. 12 ). In addition, the lipid nanoparticles loaded with COVID-19 mRNA or respiratory syncytial virus-encoding mRNA were delivered to mice and found to increase the levels of neutralizing antibody titers, IFN-γ secretion, and IgG titers ( FIG. 13 to FIG. 19 ), thereby confirming that the lipid nanoparticles induced a humoral or cellular immune response.

[0147] As can be seen from the above results, the lipid nanoparticles of the present application including the ionizable lipid having a branched structure can efficiently deliver nucleic acid gene therapy agents and vaccines, and thus can be effectively used in the field of lipid nanoparticle-mediated mRNA vaccines, gene therapy, and other related technologies.

[0148] Embodiment of the present application

[0149] Example 1: Synthesis of ionizable lipids

[0150] <1-1> Synthesis of ionizable lipids

[0151] A biodegradable functional group was introduced in the imine structure to synthesize an ionizable lipid having a branched structure.

[0152] Specifically, 9-bromononanoic acid, DIC (1.5 equivalents), and DMAP (0.2 equivalents) were added to 3-octanol in a DCM solvent and reacted overnight at 25°C ( FIG. 1). After that, the reaction was purified using a CombiFlash column with hexane / ethyl acetate (5:1 v / v). The solvent was evaporated, and the product was dissolved in ethanol. Then, DIPEA (1 equivalent) and an amine having the formula in Table 1 below (0.3 equivalent) were added thereto, and reacted at 25°C for 3 days. The reaction was purified using a CombiFlash column with DCM / MeOH (9:1 v / v). Thus, high-quality ionizable lipids containing various amine head groups and ester bonds were obtained. Depending on the type of amine, the obtained ionizable lipids were named EW221-E-7, EW244-E-7, and EW246-E-7.

[0153] [Table 1]

[0154]

[0155] <1-2> Confirmation of synthesis of ionizable lipids

[0156] To confirm the ionizable lipids synthesized in the above Example <1-1>, nuclear magnetic resonance analysis (NMR spectrum) was performed.

[0157] Specifically, 5 μg of the ionizable lipids (EW244-E-7) synthesized in Example <1-1> were diluted in 0.5 ml of CDC13 (Sigma, USA) to prepare a concentration of 100 millimoles. Then, 0.5 ml of the lipid solution was added to a 400 MHz NMR tube, capped and sealed with a sealing film to obtain an NMR spectrum using an Agilent 400 MHZ FT-NMR (Agilent, USA).

[0158] Thus, as shown in FIG. 2 , it was found that the signals representing each functional group of EW244-E-7 were saturated.

[0159] In addition, mass spectrometry (MS) was performed to identify the synthesized ionizable lipids (EW244-E-7).

[0160] Specifically, the ionizable lipids were diluted in ethanol to a concentration of 0.5 ppm or less, and analyzed by 6230 LC / MS (Agilent Technologies, Palo Alto, CA, USA) using a Zorbax SB-C18 separation column (Agilent Technologies, 100 mm x 2.1 mm i.d., 3.5 μm).

[0161] Thus, as shown in Table 2, it was confirmed that the measured mass-to-charge ratio (m / z) and the calculated mass-to-charge ratio of the ionizable lipids (EW244-E-7) were almost the same.

[0162] [Table 2]

[0163] Formula Calculated m / z Measured m / z [C 57 H 111 N3O6]]> 933.8476 933.8550

[0164] From the above results, it can be confirmed that the ionizable lipids are synthesized well.

[0165] Example 2: Preparation of lipid nanoparticles

[0166] <2-1> Preparation of lipid nanoparticles loaded with nucleic acids

[0167] The ionizable lipids (EW244-E-7) synthesized in Example <1-1>, phospholipid (DOPE) (Avanti, USA), cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥ 99%, Sigma, Korea), and lipid-PEG conjugate (C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 26.5:20:52:1.5 (Table 3). The mRNA was dissolved in a 10 mM sodium citrate (Sigma, Korea) buffer. The ethanol containing ionizable lipids, cholesterol, phospholipid, and lipid-PEG dissolved therein and the citric acid buffer were mixed at a volume ratio of 1:3, respectively, at a flow rate of 12 ml / min by a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) to prepare a nucleic acid-loaded lipid nanoparticle (LNP).

[0168]

Table 3

[0169] Molar ratio (%) EW244-E-7 lipid nanoparticles Ionizable lipid (EW244-E-7) 26.5 Phospholipid (DOPE) 20 Cholesterol 52 Lipid-PEG (Ceramide C16 PEG) 1.5

[0170]

Table 4

[0171] Weight ratio EW244-E-7 lipid nanoparticles Lipid / mRNA 10

[0172] <2-2> Physicochemical properties of nucleic acid-loaded lipid nanoparticles

[0173] <2-2-1> Measurement of particle size

[0174] The size of the mRNA-loaded lipid nanoparticle synthesized in the above Example <2-1> was measured.

[0175] Specifically, the firefly luciferase mRNA (mFluc, SEQ. ID. No: 1) contained in the EW244-E-7 lipid nanoparticle synthesized in the above Example <2-1> was diluted with PBS to a concentration of 1 µg / ml, and the diameter, polydispersity index (PDI), and surface charge (ζ potential) of the lipid nanoparticle were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nanoparticle Size Analyzer (Malvern Instruments, UK).

[0176] Accordingly, as shown in Table 5, the EW244-E-7 lipid nanoparticle exhibited excellent particle size for drug release, and it was found that the particles were uniform.

[0177] <2-2-2> Measurement of drug encapsulation efficiency

[0178] Ribogreen assay was performed to measure the nucleic acid drug encapsulation efficiency.

[0179] Specifically, for the Ribogreen assay (Quant-iT TM RNA, Invitrogen), the nucleic acid drug-loaded lipid nanoparticle was diluted with 50 μl of 1x TE buffer to a final concentration of 4-7 μg / ml of RNA in a 96-well plate. To the group not treated with Triton-X (Triton-X LNP(-)), 50 μl of 1x TE buffer was added, and to the group treated with Triton-X (Triton-X LNP(+)), 50 μl of 2% Triton-X buffer was added. The above mixture was incubated at 37℃ for 10 minutes to decompose the lipid nanoparticle with Triton-X and release the encapsulated nucleic acid. 100 μl of Ribogreen reagent was added to each well. After 5 minutes, the fluorescence intensity (FL) was measured at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. 200 PRO NanoQuant (Tecan) with wavelength bandwidth (excitation: 485 nm, emission: 528 nm). The drug encapsulation efficiency (%) was calculated as follows.

[0180] Drug encapsulation efficiency (%) = (fluorescence intensity of Triton LNP(+) - fluorescence intensity of Triton LNP(-)) / (fluorescence intensity of Triton LNP(+)) x 100

[0181] Accordingly, as shown in Table 5, it was confirmed that the EW244-E-7 lipid nanoparticle could efficiently encapsulate the drug.

[0182]

Table 5

[0183]

[0184]

[0185] Experimental Example 1: Intracellular nucleic acid delivery and cytotoxicity of lipid nanoparticles

[0186] Experiments were performed using the mFluc-encapsulated lipid nanoparticle prepared in Example <2-1> to confirm the intracellular nucleic acid delivery efficiency and cytotoxicity.

[0187] Specifically, luminescence was measured 24 hours after 20 ng of mFluc-encapsulated lipid nanoparticles were treated to Hela cells or HEK293 cells.

[0188] Thus, as shown in FIG. 3 , in Hela cells, EW244-E-7 lipid nanoparticles showed a higher expression effect compared to other lipid nanoparticles. In addition, as shown in FIG. 4 , the high expression effect of EW221-E-7 and EW244-E-7 lipid nanoparticles in HEK293 cells was confirmed. It was also confirmed that EW221-E-7 and EW244-E-7 lipid nanoparticles had little toxicity.

[0189] Experimental Example 2: Confirmation of hepatotoxicity of lipid nanoparticles

[0190] An experiment was conducted to confirm animal toxicity by administering mFluc-encapsulated lipid nanoparticles prepared in Example <2-1> to mice.

[0191] AST (aspartate aminotransferase) and ALT (alanine aminotransferase) are values that can measure the presence of diseases such as liver cell diseases or hepatitis. They are usually present in blood at low levels, but when liver cells are damaged, they are released and the concentration in blood can increase. Thus, they can be used as an indicator to confirm liver toxicity.

[0192] Specifically, mRNA-encapsulated lipid nanoparticles were intravenously administered to 7-week-old C57BL / 6 mice at a dose of 2 mg / kg based on mRNA. 24 hours after administration, blood samples were collected to measure the AST and ALT levels in the blood. As a control, ionizable lipids SM-102 of the Moderna vaccine and ALC-0315 of the Pfizer vaccine were used.

[0193]

Table 6

[0194] Injection (2 mg / kg) AST (mean ± standard deviation, U / L) ALT (mean ± standard deviation, U / L) PBS 51.75±18.63 21.25±1.89 SM-102 287.75±121.81 223.25±142.59 ALC-0315 144.75±82.25 109.50±100.06 244-BC 136.00±19.00 50.67±6.66

[0195] Thus, as shown in Table 6 and FIG. 5 and FIG. 6 , mice administered with EW244-E-7 lipid nanoparticles showed lower levels of liver toxicity compared to mice administered with SM-102 of Moderna or ALC-0315 of Pfizer. This indicates that EW244-E-7 lipid nanoparticles are very safe.

[0196] Experimental Example 3: Confirmation of in vivo expression of lipid nanoparticles

[0197] <3-1> Delivery of lipid nanoparticles by intravenous injection

[0198] First, mFluc-encapsulated lipid nanoparticles were prepared and their physicochemical properties were confirmed.

[0199] Therefore, as shown in Table 7, it was confirmed that the size of the mFluc-encapsulated EW221-E-7 and EW244-E-7 lipid nanoparticles was effective for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent.

[0200]

Table 7

[0201] EW221-E-7 lipid nanoparticles EW244-E-7 lipid nanoparticles Hit number 7.60 x 10 7 ]]> 5.72 x 10 7 ]]> Size 88.29 nm 99.47 nm PDI 0.227 0.177 Drug encapsulation efficiency 60.6％ 87.1％

[0202] Subsequently, the mFluc-encapsulated lipid nanoparticles were delivered to mice by intravenous injection, and bioluminescence was observed to confirm the in vivo drug delivery efficiency.

[0203] Specifically, 2 μg of mFluc-encapsulated EW221-E-7 and EW244-E-7 lipid nanoparticles were intravenously injected into 7-week-old C57BL / 6 mice, and 0.25 mg / kg of luciferin was intraperitoneally administered 3 hours later, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) device.

[0204] Therefore, as shown in Table 8, it was confirmed that the size of the mFluc-encapsulated EW221-E-7 and EW244-E-7 lipid nanoparticles was effective for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent. FIG. 7 and FIG. 8 As shown in Table 8, it was confirmed that most of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the liver.

[0205] The above results indicate that in vivo drug delivery can be performed by intravenous administration of lipid nanoparticles.

[0206] <3-2> Delivery of lipid nanoparticles by intramuscular injection

[0207] First, mFluc-encapsulated lipid nanoparticles were prepared and their physicochemical properties were confirmed.

[0208] Therefore, as shown in Table 8, it was confirmed that the size of the mFluc-encapsulated EW221-E-7 and EW244-E-7 lipid nanoparticles was effective for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent.

[0209]

Table 8

[0210] EW221-E-7 lipid nanoparticles EW244-E-7 lipid nanoparticles Hit number 7.60 x 10 7 ]]> 5.72 x 10 7 ]]> Size 88.29 nm 99.47 nm PDI 0.227 0.177 Drug encapsulation efficiency 60.6％ 87.1％

[0211] Subsequently, the mFluc-encapsulated lipid nanoparticles were delivered to mice by intramuscular injection, and bioluminescence was observed to confirm the in vivo drug delivery efficiency.

[0212] Specifically, 2 pg of mFluc-encapsulated EW221-E-7 and EW244-E-7 lipid nanoparticles were intramuscularly injected into 7-week-old C57BL / 6 mice, and 3 hours later, 0.25 mg / kg of luciferin was intraperitoneally administered, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) device.

[0213] Accordingly, as shown in FIGS. 1 and 2, it was confirmed that most of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the injection site. FIG. 9 and FIG. 10 Accordingly, as shown in FIGS. 1 and 2, it was confirmed that most of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the injection site.

[0214] The above results indicate that drug delivery in vivo can be performed by intramuscular administration of lipid nanoparticles.

[0215] Experimental Example 4: Confirmation of primary immunization generation ability of lipid nanoparticles

[0216] First, hEPO (human EPO) mRNA (SEQ. ID. NO: 2)-encapsulated lipid nanoparticles were prepared, and as a control, the same mRNA was encapsulated in the ionizable lipid SM-102 of the Moderna vaccine, and their physicochemical properties were compared (Table 9).

[0217]

Table 9

[0218] EW244-E-7 SM-102 Drug encapsulation efficiency 95.9 95.8 Size (nm) 77.88 52.18 PDI 0.072 0.088

[0219] After the hEPO mRNA-encapsulated nanolipoplex was delivered to mice, the concentrations of human EPO and MCP-1 in the blood were measured to confirm the initial protein expression and the primary immunization generation ability.

[0220] Specifically, 0.5 mg / kg of hEPO mRNA-encapsulated lipid nanoparticles were intravenously injected into 7-week-old Balb / c mice. 6 hours later, blood was collected to obtain serum, and hEPO and MCP-1 were quantitatively analyzed using an hEPO ELISA kit and an MCP-1 ELISA kit. The ionizable lipid SM-102 in the Moderna vaccine was used as a control.

[0221] Accordingly, as shown in FIGS. 1 and 2, it was confirmed that most of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the injection site. FIG. 11 and FIG. 12 Accordingly, as shown in FIGS. 1 and 2, it was confirmed that most of the EW221-E-7 and EW244-E-7 lipid nanoparticles were delivered to the injection site.

[0222] The above results indicate that the EW244-E-7 lipid nanoparticle has a high protein expression ability and excellent primary immunization generation ability.

[0223] The above results indicate that the EW244-E-7 lipid nanoparticle has a high protein expression ability and excellent primary immunization generation ability.Experimental Example 5: Confirmation of immunization generation ability of lipid nanoparticles

[0224] <5-1> Confirmation of coronavirus immunization-inducing ability

[0225] First, lipid nanoparticles encapsulating COVID-19 spike mRNA (SEQ.ID.NO: 3) were prepared, and physicochemical properties thereof were confirmed. The COVID-19 spike mRNA was provided by Seoul National University, and ALC-0315 was used as a positive control.

[0226] Therefore, as shown in Table 10, the size of the EW244-E-7 lipid nanoparticles encapsulating the COVID-19 spike mRNA was confirmed to be effective for drug delivery, the particles were uniform, and the drug encapsulation efficiency was excellent.

[0227]

Table 10

[0228]

[0229]

[0230] Subsequently, the lipid nanoparticles encapsulating the COVID-19 spike mRNA were delivered to mice, and neutralizing antibody titers and IFN-γ levels were measured.

[0231] Specifically, first, 0.25 mg / kg of mRNA was administered to 7-week-old Balb / c mice by intramuscular injection. Three weeks later, the second administration was performed in the same manner. Three weeks after the second administration, serum was collected and the spleen was extracted, and neutralizing antibody titers against SARS-CoV-2 and IFN-γ levels were measured, respectively.

[0232] Therefore, as shown in Table 11, it was confirmed that the EW244-E-7 lipid nanoparticles exhibited similar neutralizing antibody titers compared to the positive control ALC-0315. In addition, as shown in Table 12, it was confirmed that IFN-γ secretion increased as the concentration of the peptide in the EW244-E-7 lipid nanoparticles increased. FIG. 13 FIG. 14

[0233] The above results indicate that the EW244-E-7 lipid nanoparticles can induce both humoral and cellular immune responses.

[0234] <5-2> Confirmation of immunization-inducing ability of COVID 19 universal vaccine

[0235] First, lipid nanoparticles encapsulating computer-simulated spike mRNA (SEQ.ID.NO: 4) were prepared. After the lipid nanoparticles encapsulating the COVID-19 mRNA were delivered to mice, neutralizing antibody titers and IFN-γ levels were measured.

[0236] ​​Specifically, 0.25 mg / kg of mRNA was first administered to 7-week-old Balb / c mice by intramuscular injection. Three weeks later, a second administration was performed in the same manner. Three weeks after the second administration, serum was collected and the spleen was extracted, and neutralizing antibody titers against SARS-CoV-2 and IFN-γ levels were measured, respectively.

[0237] As a result, as shown in FIG. 15 to FIG. 17 The mice inoculated with the EW244-E-7 lipid nanoparticle were confirmed to have formed similar levels of neutralizing antibody titers against the SARS-CoV-2 original strain (S), the SARS-CoV-2 Delta variant strain (Delta), and Omicron (BA.5) (i.e., all COVID-19 strains). In addition, as shown in FIG. 18 The T cell response was confirmed to be active since IFN-γ secretion was confirmed to be high.

[0238] The above results indicate that the EW244-E-7 lipid nanoparticle can induce both humoral and cellular immune responses and can have universal applicability against various COVID-19 strains.

[0239] <5-3> Confirmation of immunization-inducing ability against cellular respiratory syncytial virus

[0240] First, a lipid nanoparticle encapsulating mRNA encoding respiratory syncytial virus (RSV) (SEQ. ID. NO: 5) was prepared, and its physicochemical properties were confirmed.

[0241] As a result, as shown in Table 11, it was confirmed that the EW244-E-7 lipid nanoparticle encapsulating mRNA encoding RSV was effective in drug delivery in terms of size, the particles were uniform, and the drug encapsulation effect was excellent.

[0242]

Table 11

[0243]

[0244] Subsequently, the lipid nanoparticle encapsulating mRNA encoding RSV was delivered to mice, and then IgG titers were measured.

[0245] Specifically, 0.5 mg / kg of mRNA was first administered to 7-week-old Balb / c mice by intramuscular injection. Two weeks later, a second administration was performed in the same manner. One week after the second administration, serum was collected, and neutralizing antibody titers against RSV were measured. A positive control group [(+) control] was administered an adenovirus vector-based vaccine rAd / 3xGmFcm, and a negative control group [(-) control] was not inoculated with a vaccine.

[0246] Therefore, as shown in FIG. 19As shown in the middle, the IgG titer was higher in the group administered with EW244-E-7 lipid nanoparticles than in the positive control group after the second administration.

[0247] The above results show that the EW244-E-7 lipid nanoparticles have excellent immune induction ability against respiratory syncytial virus. CLAIM (AMENDED IN ACCORDANCE WITH ARTICLE 19 OF THE TREATY) 1. An ionizable lipid compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof: [Formula 1] R1and R2are each independently any group selected from -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B , R3and R4are each independently any group selected from -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B , or are linked to each other to form a 4- to 8-membered heterocycloalkyl group having two N elements, However, at least one of R1to R4is -C 1-10 alkyl-NR A R B , R A and R B are each independently -H, -C 1-10 alkyl, or -Y, N is an integer between 0 and 6, at least one -Y substituent is present, Y is represented by the following [Formula 2], when a plurality of Ys are present, they can be different, [Formula 2] o, p, and q are each independently an integer of 1 to 12. 2. The ionizable lipid compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein: In Formula 1, R1and R2are each independently any group selected from -H, -C 1-6 alkyl, -Y, or -C 1-6 alkyl-NR A R B , R3and R4are each independently any group selected from -H, -C 1-6 alkyl, -Y, or -C1-6 alkyl-NR A R B any group of R1to R4, or connected to each other to form a 4- to 8-membered heterocyclic alkyl group having two N elements, However, at least one of R1to R4is -C 1-6 alkyl-NR A R B , R A and R B each independently -H, -C 1-6 alkyl, or -Y, n is an integer between 0 and 4, 2 to 6 -Y substituents are present, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer of 1 to 9, and q is an integer of 1 to 5. 3. The ionizable lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In Formula 1, R1and R2are each independently selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B any group of R1to R4, R3and R4are each independently selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B any group of R1to R4, or connected to each other to form a 5- to 6-membered heterocyclic alkyl group having two N elements, However, at least one of R1to R4is -C 1-4 alkyl-NR A R B , R A and R B each independently -H, -C 1-4 alkyl, or -Y, n is an integer between 1 and 2, 3 to 6 -Y substituents are present, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer of 1 to 9, and q is an integer of 1 to 5. 4. The ionizable lipid compound or pharmaceutically acceptable salt thereof according to claim 1, wherein: In Formula 1, R1and R2are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B , R3and R4are each independently any group selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B or are linked to each other to form a 5- to 6-membered heterocycloalkyl group having two N elements, However, at least one of R1to R4is -C 1-4 alkyl-NR A R B , R A and R B are each independently -H, -C 1-4 alkyl, or -Y, n is an integer between 1 and 2, 3 to 6 -Y substituents are present, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer of 3 to 9, and q is an integer of 1 to 3. 5. The ionizable lipid compound or pharmaceutically acceptable salt thereof according to claim 1, wherein the compound is selected from the following compounds: 6. A lipid nanoparticle comprising the ionizable lipid compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 5. 7. The lipid nanoparticle according to claim 6, wherein the lipid nanoparticle further comprises a phospholipid, cholesterol, or a lipid-PEG (polyethylene glycol). 8. The lipid nanoparticle according to claim 7, wherein the lipid nanoparticle comprises an ionizable lipid: phospholipid: cholesterol: lipid-PEG conjugate in a molar ratio of (15-35):(15-35):(40-60):(0.1-5). 9. The lipid nanoparticle of claim 8, wherein the phospholipid is at least one selected from the group consisting of DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE, POPC, DOPS, and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine]. 10. The lipid nanoparticle of claim 8, wherein the lipid in the lipid-PEG conjugate is at least one selected from the group consisting of ceramide, dimyristyl glycerol (DMG), succinyl dicacyl glycerol (s-DAG), distearoyl phosphatidyl choline (DSPC), distearoyl phosphatidyl ethanolamine (DSPE), and cholesterol. 11. A composition for drug delivery, comprising: (1) the lipid nanoparticle of claim 6; and (2) an anionic drug, a nucleic acid, or a combination thereof. 12. The composition for drug delivery of claim 11, wherein the anionic drug is at least one selected from the group consisting of a peptide, a protein, a protein-nucleic acid construct, and an anionic biopolymer-drug conjugate. 13. The composition for drug delivery of claim 11, wherein the nucleic acid is at least one selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), guide ribonucleic acid (gRNA), single guide ribonucleic acid (sgRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and deoxyribozyme. 14. The composition for drug delivery of claim 11, wherein the anionic drug, the nucleic acid, or the combination thereof is encapsulated within the lipid nanoparticle. 15. The composition for drug delivery of claim 11, wherein the composition is administered systemically or topically.

Claims

1. An ionizable lipid compound represented by the following [Formula 1] or a pharmaceutically acceptable salt thereof: [Formula 1] R1and R2are each independently selected from -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B of any group, R3and R4are each independently selected from the group consisting of -H, -C 1-10 alkyl, -Y, or -C 1-10 alkyl-NR A R B any group of R1, or connected to each other to form a 4- to 8-membered heterocycloalkyl having two N elements, However, at least one of R1to R4is -C 1-10 alkyl-NR A R B , R A and R B each independently -H, -C 1-10 alkyl or -Y, N is an integer between 0 and 6, there is at least one -Y substituent, Y is represented by the following [Formula 2], when there are multiple Ys, they can be different, [Formula 2] 2. The ionizable lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In Formula 1, R1and R2are each independently selected from -H, -C 1-6 alkyl, -Y, or -C 1-6 alkyl-NR A R B of any group, R3and R4are each independently selected from the group consisting of -H, -C 1-6 alkyl, -Y, or -C 1-6 alkyl-NR A R B any group of R1, or connected to each other to form a 4- to 8-membered heterocycloalkyl having two N elements, However, at least one of R1to R4is -C 1-6 alkyl-NR A R B , R A and R B each independently -H, -C 1-6 alkyl or -Y, n is an integer between 0 and 4, there are 2 to 6 -Y substituents, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer from 1 to 9, and q is an integer from 1 to 5.

3. The ionizable lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In Formula 1, R1and R2are each independently selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B of any group, R3and R4are each independently selected from the group consisting of -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B any group of R1, or connected to each other to form a 5- to 6-membered heterocyclic alkyl having two N elements, However, at least one of R1to R4is -C 1-4 alkyl-NR A R B , R A and R B each independently -H, -C 1-4 alkyl or -Y, n is an integer between 1 and 2, there are 3 to 6 -Y substituents, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer from 1 to 9, and q is an integer from 1 to 5.

4. The ionizable lipid compound or a pharmaceutically acceptable salt thereof according to claim 1, wherein: In Formula 1, R1and R2are each independently selected from -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B of any group, R3and R4are each independently selected from the group consisting of -H, -C 1-4 alkyl, -Y, or -C 1-4 alkyl-NR A R B any group of R1, or connected to each other to form a 5- to 6-membered heterocyclic alkyl having two N elements, However, at least one of R1to R4is -C 1-4 alkyl-NR A R B , R A and R B each independently -H, -C 1-4 alkyl or -Y, n is an integer between 1 and 2, there are 3 to 6 -Y substituents, Y is represented by the following [Formula 2], each Y can be different, [Formula 2] o and p are each independently an integer from 3 to 9, and q is an integer from 1 to 3.

5. The ionizable lipid compound or pharmaceutically acceptable salt thereof of claim 1, wherein, the compound is selected from the following compounds:

6. A lipid nanoparticle comprising the ionizable lipid compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5.

7. The lipid nanoparticle of claim 6, wherein, the lipid nanoparticle further comprises a phospholipid, a cholesterol, or a lipid-PEG (polyethylene glycol).

8. The lipid nanoparticle of claim 7, wherein, the lipid nanoparticle comprises a molar ratio of (15-35):(15-35):(40-60):(0.1-5) of the ionizable lipid: the phospholipid: the cholesterol: the lipid-PEG conjugate.

9. The lipid nanoparticle of claim 8, wherein, the phospholipid is at least one selected from the group consisting of DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE, POPC, DOPS, and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

10. The lipid nanoparticle of claim 8, wherein, the lipid in the lipid-PEG conjugate is at least one selected from the group consisting of ceramide, dimyristylglycerol (DMG), succinyl dicacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and cholesterol.

11. A composition for drug delivery, comprising: (1) the lipid nanoparticle according to claim 6; and (2) an anionic drug, a nucleic acid, or a combination thereof.

12. The composition for drug delivery according to claim 11, wherein, the anionic drug is at least one selected from the group consisting of a peptide, a protein, a protein-nucleic acid construct, and an anionic biopolymer-drug conjugate.

13. The composition for drug delivery according to claim 11, wherein, The nucleic acid is at least one selected from the group consisting of messenger ribonucleic acid (mRNA), small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, transfer ribonucleic acid (tRNA), guide ribonucleic acid (gRNA), single guide ribonucleic acid (sgRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA, and deoxyribozyme.

14. The composition for drug delivery according to claim 11, wherein, The anionic drug, nucleic acid, or combination thereof is encapsulated within the lipid nanoparticle.

15. The composition for drug delivery according to claim 11, wherein, The composition is administered systemically or locally.

Images