Biodegradable disulfide bond-containing ionizable lipids and lipid nanoparticles containing the same

Abstract

The present invention relates to a novel ionizable lipid containing a biodegradable disulfide bond. The ionizable lipid containing a disulfide bond of the present invention stably delivers anionic drugs when preparing lipid nanoparticles, and is particularly effective in nucleic acid delivery, and can be useful in related technical fields such as lipid nanoparticle-mediated gene therapy.

Description

[Technical field]

[0001] The present invention relates to a novel ionizable lipid containing a biodegradable disulfide bond. Specifically, the present invention relates to a novel ionizable lipid containing a biodegradable disulfide bond, lipid nanoparticles produced using the same, and uses thereof. [Background technology]

[0002] Drug delivery systems (DDS) are technologies designed to reduce side effects of drugs, maximize efficacy and effectiveness, and efficiently deliver the required amount of drugs. In particular, traditional viral carriers have been proven to be effective as drug carriers in gene therapy, but the use of viruses as gene delivery systems has been limited due to several shortcomings, such as immunogenicity, limited size of injected DNA, and difficulty in mass production.

[0003] As an alternative to the viral system, the main method for transporting nucleic acids into cells has been to transport them in a mixture with positively charged lipids or polymers (termed lipid-DNA conjugates (lipoplexes) and polymer-DNA conjugates (polyplexes), respectively) (Hirko et al., Curr. Med. Chem., 10, 1185-1193, 2003; Merdan et al., Adv. Drug. Deliv. Rev., 54, 715-758, 2002; Spagnou et al., Biochemistry, 43, 13348-13386, 2004). In particular, lipid-DNA conjugates are widely used at the cellular level because they bind to nucleic acids and effectively deliver the nucleic acids into cells. However, when injected locally in vivo, they often cause inflammation in the body (Filonand and Phillips, Biochim. Biophys / Acta, 1329, 345-356, 1997), and when injected intravascularly, they have the disadvantage of accumulating in tissues such as the lungs, liver, and spleen, which are primary passage organs (Renet et al., Gene Therapy. 7, 764-768, 2000).

[0004] Under these circumstances, the present inventors made extensive efforts to develop a novel substance that has an excellent drug encapsulation rate and can efficiently deliver anionic drugs such as nucleic acids to target organs or cells. As a result, the present inventors have completed the present invention by confirming the excellent drug delivery effect of the novel ionizable lipid containing a biodegradable disulfide bond of the present invention. Summary of the Invention [Problem to be solved by the invention]

[0005] One object of the present invention is to provide a novel structure of a biodegradable disulfide bond-containing ionizable lipid, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

[0006] Another object of the present invention is to provide lipid nanoparticles comprising said ionizable lipid, its stereoisomer or a pharma- ceutically acceptable salt thereof.

[0007] Another object of the present invention is to provide a drug delivery composition and a pharmaceutical composition for preventing or treating liver diseases, which comprise the lipid nanoparticles and an anionic drug. [Means for solving the problem]

[0008] This will be specifically described as follows. Each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of all elements disclosed in the present invention belong to the scope of the present invention. In addition, the scope of the present invention cannot be considered to be limited by the specific descriptions described below.

[0009] In order to achieve the above objective, the present inventors have conducted extensive research and have found that an ionizable lipid containing a disulfide bond, represented by the following formula 1, can stably and effectively deliver drugs during the preparation of lipid nanoparticles and has few side effects such as liver toxicity, thereby completing the present invention.

[0010] Ionizable lipids containing disulfide bonds To achieve the above object, one embodiment of the present invention is an ionizable lipid represented by the following formula 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

[0011] [ka] Here, A is [ka] and R1 and R2 are each independently -H, -C 1-6 Alkyl, -C 1-6 Alkyl-NR 3 R 4 , -C 1-3 Alkyl-SSC 6-16 Alkyl, -C 1-3Alkyl-SSC 1-3 Alkyl-(C=O)OC 4-12 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 4-12 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 4-12 alkyl, R3 and R4 are each independently -H, -C 1-6 Alkyl, -C 1-3 Alkyl-SSC 6-16 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 4-12 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 4-12 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 4-12 alkyl, R 5 -C 1-6 Alkyl-NR 3 R 4 , -C 1-3 Alkyl-SSC 6-16 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 4-12 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 4-12 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 4-12 is alkyl, X is null, -O- or -NH-; m is an integer from 0 to 5; n is an integer from 1 to 3, l is an integer from 1 to 3; p is an integer from 0 to 2; q is 0 or 1, and r is an integer from 2 to 10.

[0012] Although not limited thereto, the compound represented by the formula 1 specifically includes the following in the formula 1: A is, [ka] and R1 and R2 are each independently -H, -C 1-3 Alkyl, -C 1-4 Alkyl-NR 3 R 4 , -C 1-3 Alkyl-SSC 8-14 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 6-10 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 6-10 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 6-10 alkyl, R3 and R4 are each independently -H, -C 1-3 Alkyl, -C 1-3 Alkyl-SSC 8-14 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 6-10 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 6-10 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 6-10 alkyl, X is null, -O- or -NH-; m is an integer from 0 to 3; n is an integer from 1 to 3, l is an integer from 1 to 3; q is 0 or 1, and r may be an integer from 5 to 9.

[0013] In addition, the compound represented by the formula 1 may be, but is not limited to, the compound represented by the formula 1, A is, [ka] and R3 and R4 are each independently -H, -C 1-3 Alkyl, -C 1-3 Alkyl-SSC 8-14 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 6-10 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 6-10 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 6-10 alkyl, R 5 -C 1-4 Alkyl-NR 3 R 4 , -C 1-3 Alkyl-SSC 8-14 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)OC 6-10 Alkyl, -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)-C 6-10 Alkyl or -C 1-3 Alkyl-SSC 1-3 Alkyl-(C=O)NH-C 6-10 is alkyl, X is null, -O- or -NH-; m is an integer from 0 to 3; n is an integer from 1 to 3, l is an integer from 1 to 3; p is 1, q is 0 or 1, and r may be an integer from 5 to 9.

[0014] According to an embodiment of the present invention, the compound represented by formula 1 may be selected from the group consisting of the compounds listed in Table 1 below.

[0015] [Table 1] TIFF2025504802000007.tif154161

[0016] In the present invention, the term "alkyl" means, unless otherwise specified, a straight or branched chain non-cyclic saturated hydrocarbon. For example, "C 1-6 "Alkyl" can mean an alkyl having 1 to 6 carbon atoms. In the alkyl structure of the present invention, even if a simple substituent is added, it is included in the scope of the present invention in an equivalent range as long as it has the same effect as the ionizable lipid of the present invention.

[0017] In the present invention, the term "ionizable lipid" refers to an amine-containing lipid that can be easily protonated, and is also called a lipid analogue. The ionizable lipid can change its charge state depending on the surrounding pH, and thus plays a role in encapsulating an anionic drug in the lipid nanoparticle with high efficiency through electrostatic interaction with the drug, and contributes to the formation of the lipid nanoparticle structure. The ionizable lipid of the present invention is characterized in that an alkyl chain containing a disulfide bond is bonded to an amine head containing at least one primary amine, and has excellent efficacy such as tissue specificity and gene expression rate in vivo when delivering nucleic acid, and is easily decomposed in vivo after drug delivery, and has the advantage of not causing significant side effects such as hepatotoxicity.

[0018] In the present invention, the term "stereoisomer" refers to a compound of the present invention having the same chemical or molecular formula, but different in stereochemistry. Each such stereoisomer and mixtures thereof are included within the scope of the present invention as well. Unless otherwise indicated, a solid bond (-) connected to an asymmetric carbon atom is replaced by a solid wedge bond indicating the absolute configuration of the stereocenter. [ka] or dashed wedge bond [ka] may include.

[0019] The compound of formula 1 in the present invention can be in the form of a "pharmaceutical acceptable salt". As the salt, an acid addition salt formed with a pharmaceutical acceptable free acid can be useful, but is not limited thereto. The term "pharmaceutical acceptable salt" in the present invention refers to any and all organic or inorganic acid addition salts or base addition salts of the compound represented by formula 1, which have a relatively non-toxic and non-harmful effective concentration in patients, and the side effects caused by the salt do not reduce the beneficial efficacy of the compound represented by formula 1.

[0020] The acid addition salts can be prepared in the usual manner, for example by dissolving the compound in an excess of aqueous acid and precipitating the salt with a water-miscible organic solvent, for example methanol, ethanol, acetone or acetonitrile. Equal molar amounts of the compound and the acid or alcohol in water can be heated and then the mixture can be evaporated to dryness or the precipitated salt can be filtered off with suction.

[0021] In this case, the free acid may be an organic acid or an inorganic acid. The inorganic acid may be hydrochloric acid, phosphoric acid, sulfuric acid, or nitric acid. The organic acid may be methanesulfonic acid, p-toluenesulfonic acid, acetic acid, trifluoroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic acid, propionic acid, citric acid, lactic acid, glycolic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, or hydroiodic acid, but is not limited thereto.

[0022] Pharmaceutically acceptable metal salts can also be prepared using bases. Alkali metal salts or alkaline earth metal salts can be obtained, for example, by dissolving the compound in a solution of excess alkali metal hydroxide or alkaline earth metal hydroxide, filtering the salt of the undissolved compound, and evaporating and drying the filtrate. In this case, the metal salt can be, for example, sodium, potassium, or calcium salt, but is not limited thereto. Corresponding silver salts can also be obtained by reacting the alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

[0023] Lipid nanoparticles comprising ionizable lipids containing disulfide bonds - Patent Application 20070229633 Another aspect of the present invention is a lipid nanoparticle comprising an ionizable lipid represented by the above formula 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof. The lipid nanoparticle of the present invention may comprise one type of ionizable lipid represented by formula 1, or may comprise two or more types. In addition, it may further comprise an ionizable lipid other than that of the present invention depending on the purpose.

[0024] The lipid nanoparticles may further include at least one selected from the group consisting of phospholipids, structured lipids, and PEG-lipids, but are not limited thereto.

[0025] The phospholipids surround and protect the core formed by the interaction of the ionizable lipid and the drug in the lipid nanoparticles, and bind to the phospholipid bilayer of the target cell to facilitate cell membrane passage and endosomal escape during intracellular delivery of the drug. The phospholipids can be any phospholipid that can promote the fusion of lipid nanoparticles, and examples of such phospholipids include dioleoylphosphatidylethanolamine (DOPE), distearoylphosphatidylcholine (DSPC), palmitoyloleoylphosphatidylcholine (POPC), egg phosphatidylcholine (EGG), and phospholipids that can promote the fusion of lipid nanoparticles. phosphatidylcholine (EPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), distearoylphosphatidylethanolamine (distearoylphosphatidylethanolamine) amine (DSPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), phosphatidylethanolamine (PE), dipalmitoylphosphatidylethanolamine (dipalmitoylphosphatidylethanolamine), 1,2-dioleoyl-sn-glycero-3-phosphate (18-PA), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine (ME16:0PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine phoethanolamine, POPE), 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine], DOPS, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, DLPC, 1,2-dimyristoyl-sn-glycero-3-phosphocholine, DMPC, 1,2-diundecanoyl-sn-glycero-phosphocholine, DUPC, 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine ...2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine 3-phosphoethanolamine), 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, sphingomyelin, or a mixture thereof.

[0026] The structural lipids provide morphological rigidity to lipid packing within lipid nanoparticles and are distributed in the core and surface of nanoparticles to improve nanoparticle stability. The structural lipids may be, for example, cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid, alpha-tocopherol, or mixtures thereof, but are not limited thereto.

[0027] The PEG-lipid in the present invention refers to a form in which a lipid and PEG are conjugated, and means a lipid having a hydrophilic polymer, polyethylene glycol polymer, bound to one end. The PEG-lipid contributes to the particle stability of nanoparticles in serum in lipid nanoparticles and prevents aggregation between nanoparticles. In addition, the PEG-lipid protects nucleic acids from decomposition enzymes during in vivo delivery of nucleic acids, strengthens the stability of nucleic acids in the body, and increases the half-life of drugs encapsulated in nanoparticles. The PEG-lipid may be, for example, PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, or a mixture thereof, but is not limited thereto.

[0028] As a specific example, when the ionizable lipid of the present invention is mixed with a phospholipid, a structural lipid, and a PEG lipid to produce lipid nanoparticles, the molar ratio of the ionizable lipid:phospholipid:structural lipid:PEG-lipid may be 20-50:10-30:30-60:1-5. The molar ratio may also be 40-50:10-15:40-50:1-5 or 20-30:15-25:45-55:1-5, but is not limited thereto.

[0029] The lipid nanoparticles of the present invention have a pKa of 6.0 to 7.0, specifically 6.5 to 7.0, and are positively charged under acidic pH conditions, and can easily form a complex with a therapeutic agent such as a negatively charged nucleic acid or an anionic drug through electrostatic interaction, and can encapsulate an anionic drug with high efficiency, and can be used as a drug delivery composition in cells or in vivo. Therefore, the lipid nanoparticles of the present invention can be usefully used to deliver not only nucleic acids but also any form of drug that is anionic. That is, the lipid nanoparticles of the present invention can be produced in a form that finally contains an anionic drug (encapsulated form).

[0030] In the present invention, the term "encapsulation" refers to encapsulating a delivery substance to surround it for efficient in vivo uptake, and drug encapsulation efficiency refers to the content of drug encapsulated in lipid nanoparticles relative to the total content of drug used in the preparation.

[0031] The anionic drug may be a nucleic acid, a small molecule compound, a peptide, a protein, a protein-nucleic acid structure, or an anionic biopolymer-drug conjugate, but is not limited thereto, as long as it can form lipid nanoparticles together with the ionizable lipid of the present invention and can be delivered stably and efficiently.

[0032] The nucleic acid in the present invention may be, but is not limited to, siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, DNAzyme, or a mixture thereof.

[0033] In the lipid nanoparticles, the weight ratio of ionizable lipid / nucleic acid may be 1 to 20. For example, when siRNA is used, the weight ratio of ionizable lipid / siRNA may be 5 to 10, and when mRNA is used, the weight ratio of ionizable lipid / mRNA may be 7.5 to 12.5, but is not limited thereto.

[0034] The lipid nanoparticles in the present invention may have a diameter of, for example, 60 to 100 nm, specifically 70 to 90 nm, and more specifically 75 to 85 nm, but are not limited thereto.

[0035] Drug delivery and pharmaceutical compositions comprising lipid nanoparticles Another aspect of the present invention is a drug delivery composition comprising anionic drug-containing lipid nanoparticles according to the present invention.

[0036] Another aspect of the present invention is a pharmaceutical composition comprising the anionic drug-containing lipid nanoparticles according to the present invention as an active ingredient.

[0037] The lipid nanoparticles and the anionic drug are as described above.

[0038] The lipid nanoparticles of the present invention form a stable complex with anionic drugs such as nucleic acids, and exhibit low cytotoxicity and effective cellular absorption, making them effective for delivering anionic drugs. Therefore, the lipid nanoparticles have preventive or therapeutic effects on related diseases depending on the type of anionic drug and the type of nucleic acid used, and have unlimited potential for use as a drug delivery composition.

[0039] The term "treatment" in the present invention refers to intervention to modify the natural processes of an individual or cell with a disease, which can be performed during the progression of a pathological condition or to prevent it. The intended therapeutic effects include preventing the onset or recurrence of the disease, relieving symptoms, reducing all direct or indirect pathological consequences associated with the disease, preventing metastasis, reducing the rate of disease progression, alleviating or palliating the disease state, and improving the prognosis. In particular, the present invention includes any action that improves the course of a disease by administering lipid nanoparticles containing a disulfide bond-containing ionizable lipid, a stereoisomer thereof or a pharma- ceutically acceptable salt thereof, and an anionic drug as an active ingredient. In addition, the term "prevention" refers to any action that inhibits or delays the onset of a disease by administering the lipid nanoparticles. When the lipid nanoparticles of the present invention are used for therapeutic or prophylactic purposes, they are administered to an individual in a therapeutically effective amount.

[0040] The term "therapeutically effective amount" used in the present invention refers to an effective amount of anionic drug-containing lipid nanoparticles. Specifically, "therapeutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level can be determined according to factors including the type and severity of the individual, age, sex, type of disease, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment period, drugs used simultaneously, and other factors well known in the medical field. The pharmaceutical composition of the present invention can be administered as a single therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with commercially available therapeutic agents. It can also be administered once or multiple times. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with the minimum amount without side effects, which can be easily determined by those skilled in the art. The dosage of the pharmaceutical composition of the present invention can be determined by an expert depending on various factors such as the patient's condition, age, sex, and complications. The active ingredient of the composition of the present invention is excellent in safety, so it can be used even at or above the determined dosage.

[0041] The composition containing the lipid nanoparticles as an active ingredient can be administered by oral, intramuscular, intravenous, arterial, subcutaneous, peritoneal, pulmonary, and nasal injection, but is not limited thereto.

[0042] The composition of the present invention may further include one or more additional pharma- ceutically acceptable carriers for administration. As the pharma-ceutically acceptable carrier, saline, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other conventional additives such as antioxidants, buffers, bacteriostatic agents, etc. may be added as necessary. In addition, the composition of the present invention may be formulated in the form of an injection such as an aqueous solution, suspension, emulsion, etc., pills, capsules, granules, tablets, suppositories, etc. by additionally adding diluents, dispersants, surfactants, binders, and lubricants. Thus, the composition of the present invention may be a patch, liquid, pill, capsule, granule, tablet, suppository, etc. These preparations may be prepared by conventional methods used in the art for formulation or methods disclosed in the literature [Remington's Pharmaceutical Science, Mack Publishing Company, Easton PA], and may be formulated into various preparations according to each disease or component.

[0043] The embodiments of the present invention can be modified in various different forms, and the scope of the present invention is not limited to the embodiments described below. Moreover, the embodiments of the present invention are provided to more completely explain the present invention to those having average knowledge in the art. Furthermore, throughout the specification, "including" a certain component does not exclude other components, and means that other components can be further included, unless otherwise specified to the contrary. Effect of the Invention

[0044] The disulfide bond-containing ionizable lipid of the present invention stably delivers anionic drugs when preparing lipid nanoparticles, and is particularly effective in nucleic acid delivery, and can be useful in related technical fields such as lipid nanoparticle-mediated gene therapy. [Brief description of the drawings]

[0045] [Figure 1]This is the result of pKa measurement by TNS experiment of 221-SS lipid nanoparticles. [Diagram 2] This is a photograph of the internal structure of 221-SS lipid nanoparticles taken with a cryo-TEM electron microscope. [Diagram 3] The results of measuring luminescence intensity to examine the efficacy of siRNA-encapsulated lipid nanoparticles containing the ionizable lipid of the present invention in hepatocytes through in vitro efficacy experiments are shown below. [Figure 4] The figures show the results of measuring luminescence intensity to examine the efficacy of mRNA-encapsulating lipid nanoparticles containing the ionizable lipid of the present invention in hepatocytes through an in vitro efficacy experiment. [Diagram 5] In order to examine the hepatocyte targeting potential of siRNA-encapsulated lipid nanoparticles containing the ionizable lipid of the present invention through in vivo efficacy experiments, the results of confirming the siFVII (Factor VII) knockout effect are shown. [Figure 6] This is a photograph showing bioluminescence after intravenous injection into mice to confirm in vivo delivery of mRNA-encapsulated 221-SS 3 tail lipid nanoparticles. [Figure 7] This is a graph comparing bioluminescence after intravenous injection into mice to confirm the in vivo delivery efficacy depending on the number of chains of mRNA-encapsulated 221-SS lipid nanoparticles. [Figure 8] To confirm in vivo delivery of mRNA-encapsulated 221-SS lipid nanoparticles, a photograph was taken confirming bioluminescence after intramuscular injection into mice. [Figure 9] This is a graph comparing bioluminescence after intramuscular injection into mice to confirm the in vivo delivery efficacy depending on the number of chains of mRNA-encapsulated 221-SS lipid nanoparticles. [Figure 10] To confirm the cytotoxicity of 221-SS lipid nanoparticles, lipid nanoparticles without nucleic acid drugs were prepared and treated with the lipid nanoparticles at various concentrations in four cell lines, HepG2, MEF, Hela, and KB-GFP, and the cytotoxicity was confirmed. [Figure 11]To confirm the hepatotoxicity of 221-SS lipid nanoparticles, lipid nanoparticles encapsulating mFLuc were prepared and administered, and then AST and ALT levels were measured and compared with those of SM-102 lipid. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0046] The present invention will be described in detail below with reference to examples and experimental examples. However, the following examples and experimental examples are merely for the purpose of illustrating the present invention, and the scope of the present invention is not limited thereto.

[0047] Example 1. Preparation of disulfide bond-containing ionizable lipids Example 1-1. Synthesis of disulfide bond-containing ionizable lipids [ka] [ka] Using the synthetic methods of Schemes 1 and 2 above, various types of amine head groups were coupled with alkyl chains containing disulfide bonds to prepare the disulfide bond-containing ionizable lipids of the present invention.

[0048] Preparation of Compound 1 Compound 1 was synthesized using reaction formula 1. 1 equivalent of amine head 201 (N1,N1-dimethylethane-1,2-diamine) and 2.4 equivalents of ethylene sulfide were added to THF (tetrahydrofuran) solvent. Then, the reaction was carried out at 150 ° C for 1.5 hours using an electromagnetic reactor. 3 equivalents of 2,2'-dipyridyl sulfide were added to 1 equivalent of the product and reacted at 25 ° C for 2 hours. After concentrating the reaction product, the product was dissolved in DCM (dichloromethane), 3.6 equivalents of alkanethiol were added, and the reaction was carried out overnight. Then, purification was carried out with DCM / MeOH (5: 1 v / v ratio) using a CombiFlash column to synthesize compound 1.

[0049] Preparation of compound 2 Compound 2 was synthesized using reaction scheme 1. Compound 2 was synthesized in the same manner as compound 1 using the amine head 202 (N1,N1-diethylpropane-1,3-diamine).

[0050] Preparation of compound 3 Compound 3 was synthesized using reaction scheme 1. Compound 3 was synthesized in the same manner as in the synthesis of compound 1 using the amine head 205 (N1,N1-dimethylpropane-1,3-diamine).

[0051] Preparation of compound 4 Compound 4 was synthesized using reaction scheme 1. 1 equivalent of amine head 211 (N1-(3-aminopropyl)-N3,N3-dimethylpropane-1,3-diamine) and 3.6 equivalents of ethylene sulfide were added to THF (tetrahydrofuran) solvent. Then, the reaction was carried out at 150 °C for 1.5 hours using an electromagnetic reactor. 4.5 equivalents of 2,2'-dipyridyl sulfide were added to 1 equivalent of the product and reacted at 25 °C for 2 hours. After concentrating the reaction product, the product was dissolved in DCM (dichloromethane), 6 equivalents of alkanethiol were added, and the reaction was carried out overnight. Then, purification was carried out using a CombiFlash column with DCM / MeOH (5:1 v / v ratio) to synthesize compound 4.

[0052] Preparation of compound 5 Compound 5 was synthesized using reaction scheme 2. 1 equivalent of amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine) and 6 equivalents of ethylene sulfide were added to a toluene solvent. The mixture was then reacted at 100°C for 16 hours in a sealed tube. The reactant was filtered, concentrated, and purified using a C18 reverse phase column with MeCN / water (15:85 v / v ratio) to obtain an intermediate. 1 equivalent of the product and 3.5 equivalents of 2-(alkyldisulfanyl)pyridine were added, dissolved in methanol, and reacted at 20°C for 16 hours. Compound 5 was then synthesized by purifying the mixture using a CombiFlash column with EA / MeOH (93:7 v / v ratio).

[0053] Preparation of compound 6 Compound 6 was synthesized using reaction scheme 2. 1 equivalent of amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine) and 10 equivalents of ethylene sulfide were added to a toluene solvent. The mixture was then reacted at 100°C for 16 hours in a sealed tube. The reactant was filtered, concentrated, and purified using a C18 reverse phase column with MeCN / water (0:100 v / v ratio) to obtain an intermediate. 1 equivalent of the product and 4.3 equivalents of 2-(alkyldisulfanyl)pyridine were added, dissolved in methanol, and reacted at 20°C for 16 hours. Compound 6 was then synthesized by purifying the mixture using a CombiFlash column with EA / MeOH (93:7 v / v ratio).

[0054] Preparation of compound 7 Compound 7 was synthesized using reaction scheme 1. 1 equivalent of amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine) and 4.8 equivalents of ethylene sulfide were added to THF (tetrahydrofuran) solvent. Then, the reaction was carried out at 150 °C for 1.5 hours using an electromagnetic reactor. 6 equivalents of 2,2'-dipyridyl sulfide were added to 1 equivalent of the product and reacted at 25 °C for 2 hours. After concentrating the reaction, the product was dissolved in DCM (dichloromethane), 8 equivalents of alkanethiol were added, and the reaction was carried out overnight. Then, purification was carried out using a CombiFlash column with DCM / MeOH (5:1 v / v ratio) to synthesize compound 7.

[0055] Preparation of compound 8 Compound 8 was synthesized using reaction scheme 1. Compound 8 was synthesized in the same manner as in the synthesis of compound 7 using the amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine).

[0056] Preparation of compound 9 Compound 9 was synthesized using reaction scheme 1. Compound 9 was synthesized in the same manner as in the synthesis of compound 7 using the amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine).

[0057] Preparation of compound 10 Compound 10 was synthesized using reaction scheme 1. Compound 10 was synthesized in the same manner as in the synthesis of compound 7 using the amine head 221 (N1-(3-aminopropyl)-N1-methylpropane-1,3-diamine).

[0058] Preparation of compound 11 Compound 11 was synthesized using reaction scheme 1. Compound 11 was synthesized in the same manner as compound 4 using the amine head 244 (1-(2-aminoethyl)piperazine).

[0059] Preparation of compound 12 Compound 12 was synthesized using reaction scheme 1. 1 equivalent of amine head 246 (1,4-bis(3-aminopropyl)piperazine) and 6 equivalents of ethylene sulfide were added to THF (tetrahydrofuran) solvent. Then, the reaction was carried out at 150 °C for 1.5 hours using an electromagnetic reactor. 8 equivalents of 2,2'-dipyridyl sulfide were added to 1 equivalent of the product and reacted at 25 °C for 2 hours. After concentrating the reaction, the product was dissolved in DCM (dichloromethane), 10 equivalents of alkanethiol were added, and the reaction was carried out overnight. Then, purification was carried out using a CombiFlash column with DCM / MeOH (5:1 v / v ratio) to synthesize compound 12.

[0060] Specific examples of disulfide bond-containing ionizable lipids of the present invention are shown in Table 2 below.

[0061] [Table 2] TIFF2025504802000013.tif174161

[0062] Example 1-2. Confirmation of synthesis of disulfide bond-containing ionizable lipids In order to confirm the synthesis of the ionizable lipid prepared in Example 1-1, 1 H NMR was performed.

[0063] Specifically, 5 μg of the synthesized ionizable lipid was diluted in 0.5 ml of DMSO (Sigma, USA) to prepare a concentration of 100 mmol, and then 0.5 ml was placed in a 400 MHz NMR tube, the top was closed, and the tube was sealed with parafilm. NMR spectra were obtained using an Agilent 400 MHZ FT-NMR (Agilent, USA).

[0064] 221-SS 3 tail intermediates and final products 1 The results of H-NMR are as follows:

[0065] Intermediate 1 H-NMR (400MHz, METHANOL-d4) δ(ppm): 3.40-3.32(m,4H), 3.31-3.13(m,12H), 2.93(s,3H), 2.91-2.80(m,4H), 2.32-2.08(m,4H)

[0066] The final product 1 H-NMR (400MHz, CHLOROFORM-d) δ (ppm): 2.98-2.91 (m, 2H), 2.86-2.74 (m, 10H), 2.73-2.62 (m, 8H), 2.5 1(m,2H), 2.44-2.34(m,4H), 2.22(s,3H), 1.71-1.65(m,8H), 1.38-1.25(m,56H), 0.88(t,J=6.7Hz, 9H)

[0067] In addition, in order to confirm the synthesis of the compound produced in Example 1-1, the compound produced was diluted in ethanol to a concentration of 0.5 ppm or less and subjected to MS analysis. The equipment used for the analysis was 6230LC / MS manufactured by Agilent Technologies (Palo Alto, USA), and the separation column was Zorbax SB-C18 (100 mm x 2.1 mm id, 3.5 μm) manufactured by Agilent Technologies. The results of the MS analysis are shown in Table 3 below.

[0068] [Table 3]

[0069] Example 2. Preparation of lipid nanoparticles Example 2-1. Formulation parameters Lipid nanoparticles containing ionizable lipids of the present invention were prepared as shown in Tables 4 and 5 below.

[0070] [Table 4]

[0071] [Table 5]

[0072] Example 2-2. Preparation of lipid nanoparticles encapsulating siRNA The ionizable lipid prepared in Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≧99%, Sigma, Korea), phospholipid (DSPC) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 42.5:13:43:1.5.

[0073] Then, siRNA was dissolved in 50 mM sodium acetate (Sigma, Korea) buffer, and the ethanol and acetate buffer in which the ionizable lipids, cholesterol, phospholipids, and lipid-PEG were dissolved were mixed at a volume ratio of 1:3 at a flow rate of 12 ml / min using a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) to prepare lipid nanoparticles.

[0074] Example 2-3. Preparation of lipid nanoparticles encapsulating mRNA The ionizable lipid prepared in Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≧99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol at a molar ratio of 26.5:20:52:1.5.

[0075] Then, the mRNA was dissolved in 10 mM sodium citrate (Sigma, Korea) buffer, and the ethanol and citrate buffer in which the ionizable lipids, cholesterol, phospholipids, and lipid-PEG were dissolved were mixed at a volume ratio of 1:3 and a flow rate of 12 ml / min using a microfluidic mixer (Benchtop Nanoassembly; PNI, Canada) to produce lipid nanoparticles.

[0076] Experimental Example 1: Confirmation of physicochemical properties of lipid nanoparticles Experimental Example 1-1. Measurement of particle size, polydispersity index (PDI), zeta potential, and drug encapsulation rate The size of lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) or FVII siRNA (SEQ ID NO: 2 and SEQ ID NO: 3) prepared in Example 2 was measured. Specifically, 221-SS 3 tail lipid nanoparticles encapsulating mRNA or siRNA were prepared, and then diluted with PBS so that the concentration of mRNA or siRNA contained in each lipid nanoparticle was 1 μg / ml. Then, the diameter, polydispersity index (PDI), and zeta potential of the lipid nanoparticles were measured using dynamic light scattering (DLS) with a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0077] Next, the encapsulation efficiency (drug encapsulation rate, %) of the lipid nanoparticles encapsulating the mRNA or siRNA was measured by Ribogreen analysis (Quant-iT™ RiboGreen™ RNA, Invitrogen). Lipid nanoparticles encapsulating the mRNA or siRNA were diluted with 50 μl of 1xTE buffer so that the final concentration of mRNA or siRNA was 4 to 7 μg / ml in a 96-well plate. 50 μl of 1xTE buffer was added to the Triton-X non-treated group (Triton-xLNP(-)), and 50 μl of 2% Triton-X buffer was added to the Triton-X treated group (Triton-X LNP(+)). The lipid nanoparticles were decomposed with Triton-X by incubation at 37°C for 10 minutes, and the encapsulated nucleic acid was released. Then, 100 μl of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNP(-) and Triton LNP(+) was measured using an Infinite (registered trademark) 200 PRO NanoQuant (Tecan) with a wavelength bandwidth (excitation: 485 nm, emission: 528 nm), and the drug encapsulation rate (encapsulation efficiency, %) was calculated using the following formula 1.

[0078]

number

[0079] The respective result values ​​are as follows (Table 6).

[0080] [Table 6]

[0081] Experimental Example 1-2. pKa measurement In this experimental example, the pKa of the lipid nanoparticles encapsulating firefly luciferase mRNA (SEQ ID NO: 1) formulated in Example 2-3 was calculated by in vitro TNS (2-(p-toluidino)naphthalene-6-sulfonic acid) analysis.

[0082] Specifically, the pH of a solution containing 20 mM sodium phosphate, 25 mM citric acid, 20 mM ammonium acetate, and 150 mM sodium chloride was adjusted from pH 3.5 to pH 11 in 0.5 intervals using 0.1 N sodium hydroxide and / or 0.1 N hydrochloric acid to prepare solutions with various pH units. 100 μl of each solution having each pH was added to a black 96-well plate, and 300 μM TNS stock solution was added to each of the solutions to a final concentration of 6 μM. Formulated lipid nanoparticles were added to the mixed solution to a final concentration of 20 μM, and the fluorescence intensity was measured (excitation: 325 nm, emission: 435 nm) using a Tecan instrument. Here, the pKa for the lipid nanoparticles was calculated as the pH value at which half of the maximum fluorescence was reached.

[0083] As a result, it was confirmed that the lipid nanoparticles of the present invention have a pKa value of about 6.8 and show an s-shaped curve on the graph (Figure 1). Anionic TNS becomes lipophilic by interacting with positively charged ionizable lipids, and as the pH value approaches the pKa value of LNP, the lipophilicity of TNS decreases and more water molecules quench the TNS fluorescence, so lipid nanoparticles with a pKa of 6.0 to 7.0 have excellent in vivo drug delivery efficiency. In addition, lipid nanoparticles that show an "s-shaped curve" on the graph showing fluorescence by pH are easy to interact with endosomal membranes and can easily escape from the endosome upon acidification.

[0084] Experimental Example 1-3. Cryo-TEM Measurement The internal structure of the lipid nanoparticles was measured by cryo-TEM.

[0085] Specifically, lipid nanoparticles encapsulating mRNA were concentrated to a total lipid final concentration of 15-25mg / ml, and 2-4μl of LNP solution was placed on a copper grid and blotted.Then, the internal image of the lipid nanoparticles was measured using a cryo-TEM (FEI Tecnai F20 G2) at the Characterization Center of the Korea Advanced Institute of Science and Technology (KIST) (Figure 2).

[0086] Experimental Example 2: Confirmation of in vitro efficacy of nucleic acid-encapsulated lipid nanoparticles Experimental Example 2-1. Confirmation of efficacy of siRNA-encapsulated lipid nanoparticles To confirm the efficacy of siRNA-encapsulated lipid nanoparticles in vitro, screening was performed using lipid nanoparticles synthesized with various amine head groups.

[0087] The test was carried out by delivering siRNA that inhibits luciferase expression to Hela cells (Hela-Luc) that had been engineered to express luciferase using each lipid nanoparticle (Compounds 1 to 5, Compound 11, and Compound 12) synthesized with various amine head groups, and measuring the luminescence intensity to confirm the expression of the gene.

[0088] Specifically, lipid nanoparticles encapsulating Luc siRNA (SEQ ID NO: 4 and SEQ ID NO: 5) were added at 5 nM / well (based on siRNA concentration) at 1x10 6 Hela cell line (Nexel, Korea) was treated with cells / well in 96-well plates. After 24 hours, Bright-Glo™ luciferase assay solution (Promega, USA) was added at 100 μl / well, and the cells were left at room temperature for 10 minutes. The luminescence intensity of the lysed cells was measured using an Infinite M200 luminometer (Tecan, US) (Figure 3). As shown in Figure 3, the luminescence intensity was significantly reduced in all lipid nanoparticles tested, and it was confirmed that the lipid nanoparticles containing compound 1 (201-SS) or compound 5 (221-SS 3 tail) were able to deliver drugs to cells more efficiently than the lipid nanoparticles containing ionizable lipids prepared with other amine head groups.

[0089] Experimental Example 2-2. Confirmation of efficacy of mRNA-encapsulated lipid nanoparticles To confirm the efficacy of lipid nanoparticles encapsulating mRNA in vitro, a screening was performed using lipid nanoparticles synthesized with various amine head groups.

[0090] The test was carried out by using each lipid nanoparticle (compound 1 to compound 5, compound 11 and compound 12) synthesized with various amine head groups to deliver GFP mRNA to hepatocytes (HeLa), measuring the luminescence intensity and confirming the expression of the gene.

[0091] Specifically, 100 ng of GFP mRNA (SEQ ID NO: 6) was encapsulated in lipid nanoparticles at 0.1 μg / well (based on mRNA concentration) at 1×10 5The cells were treated with hepatocyte cell line (Nexel, Korea) dispensed into 96-well plates at 100 μl / well. After 24 hours, the cells were treated with 100 μl / well of cell lysis buffer (Thermofischer, US) and left at room temperature for 10 minutes. The fluorescence intensity of the lysed cells was measured using an Infinite M200 luminometer (Tecan, US) (Figure 4). As shown in Figure 4, the luminescence intensity was significantly increased in most of the lipid nanoparticles tested, and it was confirmed that the lipid nanoparticles containing compound 1 (201-SS), compound 3 (205-SS), or compound 5 (221-SS 3 tail) were able to deliver drugs to cells more efficiently than the lipid nanoparticles containing ionizable lipids prepared with other amine head groups.

[0092] Experimental Example 3: Confirmation of the in vivo effect of lipid nanoparticles Experimental Example 3-1. Effect of delivery of siRNA-encapsulated lipid nanoparticles Since FVII is specifically expressed in hepatocytes, we attempted to confirm the hepatocyte targeting potential of lipid nanoparticles through the FVII (Factor VII) knockout effect using siFVII.

[0093] Specifically, 221-SS 3 tail lipid nanoparticles encapsulating FVII-targeting siRNA (SEQ ID NO: 2 and SEQ ID NO: 3) were prepared by the method of Example 2-2 (Table 7).

[0094] [Table 7]

[0095] The lipid nanoparticles were intravenously injected at a dose of 0.2 mg / kg (based on siRNA) into C57BL / 6 female mice aged 7 weeks and weighing 20 g. Three days later, blood was collected through the tail vein and analyzed according to the protocol of the Coaset FVII assay kit. A standard curve was drawn using blood from mice administered PBS to measure the amount of FVII expression.

[0096] As a result, as shown in Figure 5, lipid nanoparticles produced using the ionizable lipid of the present invention effectively suppressed FVII expression in vivo, confirming that the lipid nanoparticles of the present invention can deliver nucleic acids targeting hepatocytes.

[0097] Experimental Example 3-2. Confirmation of efficacy of mRNA-encapsulated lipid nanoparticle delivery via intravenous injection To examine mRNA delivery of 221-SS lipid nanoparticles, luciferase mRNA was delivered to mice, after which bioluminescence was confirmed and gene expression was confirmed.

[0098] Specifically, 221-SS 3 tail lipid nanoparticles encapsulating Luc mRNA (SEQ ID NO: 1) were prepared by the method of Example 2-3. The prepared lipid nanoparticles were intravenously injected into 7-week-old C57BL / 6 mice at a dose of 0.1 mg / kg (based on mRNA), and 3 hours later, luciferin was intraperitoneally administered at 0.25 mg / kg, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) device. As a result, it was confirmed that most of the lipid nanoparticles were delivered to the liver (Table 8 and Figure 6).

[0099] [Table 8]

[0100] Next, to confirm the effect of the number of disulfide bond chains (tails), an in vivo assay was performed by intravenous injection of 2μg (0.1mg / kg) of Luc mRNA (SEQ ID NO: 1) containing 221-SS 3 tail (compound 5) or 221-SS 4 tail (compound 6). In addition, a similar experiment was performed using lipid nanoparticles produced by mixing 3-chain and 4-chain 221-SS at a 1:1 ratio, and SM-102, an ionizable lipid approved by the FDA, was used as a control group.

[0101] The drug encapsulation rate, size, and PDI of each lipid nanoparticle used in the experiment are shown in Table 9 below.

[0102] [Table 9]

[0103] After delivering each lipid nanoparticle containing luciferase mRNA to mice, bioluminescence was confirmed and gene expression was confirmed.

[0104] Specifically, each lipid nanoparticle was intravenously injected into 7-week-old C57BL / 6 mice at a dose of 0.1 mg / kg (based on mRNA), and 3 hours later, luciferin was intraperitoneally administered at 0.25 mg / kg, and bioluminescence was confirmed using an IVIS (PerkinElmer, USA) device.

[0105] The experimental results showed that both 221-SS 3 tail and 221-SS 4 tail alone showed superior effects to the negative control group, and in particular, when both ionizable lipids were mixed to produce lipid nanoparticles, they showed superior effects at a level equivalent to that of SM-102 (Figure 7).

[0106] Experimental Example 3-3. Effect of intramuscular injection of mRNA-encapsulated lipid nanoparticles Luc mRNA (SEQ ID NO: 1) was delivered to mice by intramuscular injection, and bioluminescence was observed to confirm gene expression.

[0107] Specifically, lipid nanoparticles containing Luc mRNA (221-SS 3 tail, 221-SS 4 tail alone and mixed) were prepared as described above, and then intramuscularly injected into C57BL / 6 mice at a concentration of 0.1 mg / kg (based on mRNA). Three hours later, luciferin was administered intraperitoneally at 0.25 mg / kg, and bioluminescence was observed using an IVIS (PerkinElmer, USA) device.

[0108] As a result, it was confirmed that when the 221-SS 3 tail lipid nanoparticles were injected intramuscularly, most of the lipid nanoparticles were delivered to the injection site (Table 10 and FIG. 8).

[0109] [Table 10]

[0110] In addition, when comparing the difference in effect depending on the number of strands, it was confirmed that both 221-SS 3 tail and 221-SS 4 tail showed superior effects compared to the negative control group when used alone, and that when mixed, they showed superior effects equivalent to those of SM-102 (Figure 9).

[0111] Experimental Example 4: Confirmation of cytotoxicity of lipid nanoparticles CCK-8 (tetrazolium salt) is reduced by mitochondrial dehydrogenase in living cells to form orange formazan, making it possible to confirm cell viability through absorbance analysis.

[0112] Various types of cells (HeLa, HepG2, MEF, KB-GFP) were seeded (0.1x10 5 Then, the components of the lipid nanoparticles excluding mRNA were mixed using a microfluidic mixer (Benchtop Nanoassemblyr; PNI, Canada) to produce 221-SS 3 tail lipid nanoparticles without encapsulating mRNA. 24 hours after cell seeding, lipid nanoparticles (based on ionizable lipids) were treated in amounts of 0.5 μg, 5 μg, 50 μg, or 100 μg per well. 24 hours after lipid nanoparticle treatment, Cell Counting Kit-8 (Sigma-Aldrich, 96992) was added at 10 μl per well. After 4 hours of incubation, the absorbance at 450 nm was measured using Infinite® 200 PRO NanoQuant (Tecan).

[0113] As a result, it was confirmed that, although some cytotoxicity was observed even at low concentrations in HepG2 and MEF cells, no cytotoxicity was observed up to 0.5 μg in cancer cell lines such as HeLa and KB-GFP (Figure 10).

[0114] Experimental Example 5: Confirmation of hepatotoxicity of lipid nanoparticles AST (Aspartate transaminase) and ALT (Alanine transaminase) are values ​​that can be used to measure the presence or absence of diseases such as hepatocellular disease and hepatitis. Normally present in the blood at low concentrations, they leak out when liver cells are damaged and their concentrations increase.

[0115] To confirm liver toxicity, 221-SS 3 tail lipid nanoparticles containing firefly luciferase mRNA (SEQ ID NO: 1) were prepared and then administered once to 7-week-old C57BL / 6 mice at a dose of 1 mg / kg or 2 mg / kg (based on mRNA). SM-102 was used as a control. 24 hours after administration, blood was collected and AST and ALT levels were confirmed.

[0116] As a result, AST was confirmed at a level similar to that of the FDA-approved SM-102 lipid, while ALT levels were significantly reduced in the lipid nanoparticles of the present invention, demonstrating little liver toxicity (Figure 11).

[0117] From the above description, a person skilled in the art to which the present invention pertains can understand that the present invention can be embodied in other specific forms without changing the technical idea or essential features thereof. In this regard, it should be understood that the above-described embodiments are illustrative in all respects and are not limiting. The scope of the present invention should be interpreted as including all modifications and variations derived from the meaning and scope of the claims below, and their equivalent concepts, rather than the above detailed description.

Claims

1. An ionizable lipid represented by the following formula 1, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof: 【Chemistry 1】 Here, A is 【Chemistry 2】 and R １ and R ２ are each independently -H, -C １－６ Alkyl, -C １－６ Alkyl-NR ３ R ４ , -C １－３ Alkyl-S-S-C ６－１６ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ４－１２ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ４－１２ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ４－１２ alkyl, R ３ and R ４ are each independently -H, -C １－６ Alkyl, -C １－３ Alkyl-S-S-C ６－１６ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ４－１２ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ４－１２ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ４－１２ alkyl, R ５ is -C １－６ Alkyl-NR ３ R ４ , -C １－３ Alkyl-S-S-C ６－１６ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ４－１２ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ４－１２ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ４－１２ is alkyl, X is null, -O- or -NH-; m is an integer from 0 to 5; n is an integer from 1 to 3, l is an integer from 1 to 3; p is an integer from 0 to 2, q is 0 or 1, and and r is an integer from 2 to 10.

2. A is, 【Chemistry 3】 and R １ and R ２ are each independently -H, -C １－３ Alkyl, -C １－４ Alkyl-NR ３ R ４ , -C １－３ Alkyl-S-S-C ８－１４ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ６－１０ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ６－１０ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ６－１０ alkyl, R ３ and R ４ are each independently -H, -C １－３ Alkyl, -C １－３ Alkyl-S-S-C ８－１４ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ６－１０ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ６－１０ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ６－１０ alkyl, X is null, -O- or -NH-; m is an integer from 0 to 3; n is an integer from 1 to 3, l is an integer from 1 to 3; q is 0 or 1, and 2. The ionizable lipid of claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof, wherein r is an integer from 5 to 9.

3. A is, 【Chemistry 4】 and R ３ and R ４ are each independently -H, -C １－３ Alkyl, -C １－３ Alkyl-S-S-C ８－１４ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ６－１０ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ６－１０ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ６－１０ alkyl, R ５ is -C １－４ Alkyl-NR ３ R ４ , -C １－３ Alkyl-S-S-C ８－１４ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)O-C ６－１０ Alkyl, -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)-C ６－１０ Alkyl or -C １－３ Alkyl-S-S-C １－３ Alkyl-(C=O)NH-C ６－１０ is alkyl, X is null, -O- or -NH-; m is an integer from 0 to 3; n is an integer from 1 to 3, l is an integer from 1 to 3; p is 1, q is 0 or 1, and 2. The ionizable lipid of claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof, wherein r is an integer from 5 to 9.

4. The ionizable lipid according to claim 1, its stereoisomer or a pharma- ceutically acceptable salt thereof, is selected from the group consisting of compounds 1 to 12 in the following table: 【Table 1】 。

5. A lipid nanoparticle comprising the ionizable lipid according to any one of claims 1 to 4, a stereoisomer thereof, or a pharma- ceutically acceptable salt thereof.

6. The lipid nanoparticle of claim 5, further comprising at least one selected from the group consisting of phospholipids, structured lipids, and PEG-lipids.

7. The phospholipids include DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, DOTAP, phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphate, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1,2-diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-diphytanoyl-sn-glycero-3-phosphoethanolamine, POPE, DOPS, DLPC, DMPC, DUPC, 1,2-di-O-octadecenyl-sn-glycero-3 1,2-diphosphocholine, 1-oleoyl-2-cholesterylhemisuccinoyl-sn-glycero-3-phosphocholine, 1-hexadecyl-sn-glycero-3-phosphocholine, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine, 1,2-didocosahexaenoyl-sn-glycero-3-phosphoethanolamine, and sphingomyelin. The lipid nanoparticle according to claim 6, which is any one or more selected from the group consisting of sphingomyelin.

8. The lipid nanoparticle according to claim 6, wherein the PEG-lipid is at least one selected from the group consisting of PEG-ceramide, PEG-DMG, PEG-c-DOMG, PEG-DLPE, PEG-DMPE, PEG-DPPC and PEG-DSPE.

9. The lipid nanoparticle of claim 6, wherein the structural lipid is any one or more selected from the group consisting of cholesterol, cholestenol, spinasterol, fecosterol, sitosterol, ergosterol, ergostenol, campesterol, stigmasterol, brassicasterol, tomatidine, ursolic acid and alpha-tocopherol.

10. The lipid nanoparticle of claim 6, wherein the lipid nanoparticle comprises a molar ratio of ionizable lipid:phospholipid:structural lipid:PEG-lipid of 20-50:10-30:30-60:1-5.

11. The lipid nanoparticle of claim 6, further comprising an anionic drug.

12. The lipid nanoparticle of claim 11, wherein the anionic drug is at least one selected from the group consisting of a nucleic acid, a low molecular weight compound, a peptide, a protein, a protein-nucleic acid structure, and an anionic biopolymer-drug conjugate.

13. The lipid nanoparticle according to claim 12, wherein the nucleic acid is at least one selected from the group consisting of siRNA, rRNA, DNA, aptamer, mRNA, tRNA, antisense oligonucleotide, shRNA, miRNA, sgRNA, tracrRNA, gRNA, ribozyme, PNA, and DNAzyme.

14. The lipid nanoparticle of claim 13, wherein the weight ratio of ionizable lipid to nucleic acid in the lipid nanoparticle is 1 to 20.

15. A drug delivery composition comprising the lipid nanoparticles described in claim 11.

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