Novel lipid based on oligo-gamma-glutamic acid derivative, lipid nanoparticles comprising same, and use thereof

Abstract

The present invention relates to a novel lipid derivative compound comprising an oligo-gamma-glutamic acid, a lipid nanoparticle composition comprising the same, and the like. According to the present invention, the compound can form lipid nanoparticles by replacing PEGylated lipids, and thus can prevent side effects such as anaphylaxis and has excellent in vivo stability.

Description

Novel lipids based on oligo-gamma-glutamic acid derivatives, lipid nanoparticles containing the same, and uses thereof

[0001] The present invention relates to a novel lipid based on oligo-γ-glutamic acid, a lipid nanoparticle containing the same, and a vaccine composition further comprising a therapeutic agent such as a nucleic acid in the lipid nanoparticle.

[0002] Nucleic acid-based medicines, which began about 40 years ago by injecting plasmid DNA into the body to help produce deficient proteins, have since been reported to include various types, including antigene, decoy, antisense, siRNA, and miRNA, which inhibit gene transcription and translation. Nucleic acid-based medicines target DNA or RNA, rather than proteins, and have garnered attention as personalized treatments through complementary binding with specific DNA or RNA sequences. Nucleic acid-based medicines are utilized not only as therapeutic agents but also as preventive agents that protect against diseases by injecting genes that can express antigens for specific diseases. Gene-based vaccines are categorized as DNA vaccines, RNA vaccines, and viral vector vaccines. Among them, RNA vaccines inject messenger RNA (mRNA) encoding antigens into the body, causing the antigens to be expressed in the body and inducing antibody formation. RNA vaccines do not carry the potential risks of viral vector-based vaccines, such as infection, or the genetic mutations of DNA vaccines. They also offer the advantage of rapid development, garnering attention as an effective response to the COVID-19 outbreak in 2019.

[0003] Current lipid nanoparticles are generally composed of a mixture of four components: ionizable lipids, phospholipids (helper lipids), cholesterol (structural maintenance lipids), and PEGylated lipids in a specific ratio. PEGylated lipids, in particular, are widely used in pharmaceutical lipid nanoparticle (LNP) formulations of anticancer drugs such as doxorubicin, irinotecan, and cisplatin, as well as in mRNA vaccines. However, hypersensitivity reactions, including anaphylaxis, and unexpected immune responses have been reported with many PEG-containing formulations. Therefore, there is an urgent need to develop alternatives to PEGylated lipids that have fewer side effects and maintain stability in the body.

[0004] The technical problem to be achieved by the present invention is to provide a novel lipid derivative compound containing oligo-gamma-glutamic acid.

[0005] Another technical task to be achieved by the present invention is to provide a lipid nanoparticle composition comprising the compound.

[0006] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

[0007] To solve the above problem, the present inventors provide a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:

[0008] [Chemical Formula 1]

[0009]

[0010] The above X is absent or And,

[0011] The above x is an integer from 1 to 6,

[0012] The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms,

[0013] The above Y is [A a B b C c D d E e F f ] n is a polymer represented by

[0014] The above A is ,

[0015] The above B is ,

[0016] The above C is ,

[0017] The above D is ,

[0018] The above E is ,

[0019] The above F is and,

[0020] The above a to f are integers from 0 to 24, and the above n is an integer from 1 to 8, The value is between 1 and 24.

[0021] According to one aspect, R1 and R2 may each independently be an alkyl group having 6 to 17 carbon atoms.

[0022] According to one side, the above X is absent, or In this case, x may be 4, and R1 and R2 may be straight-chain alkyl having 13 carbon atoms.

[0023] According to one side, the above n is 5, the above a and b are each 1 or 2, and c to f can be 0.

[0024] According to one side, the above n is 5, the above a is 2, c, e and f are 0 or 1 and at least one of them is 1, and b and d can be 0.

[0025] According to one aspect, the compound may be at least one selected from the group consisting of the following chemical formulas 1-1 to 1-12:

[0026] [Chemical Formula 1-1]

[0027]

[0028] [Chemical Formula 1-2]

[0029]

[0030] [Chemical Formula 1-3]

[0031]

[0032] [Chemical Formula 1-4]

[0033]

[0034] [Chemical Formula 1-5]

[0035]

[0036] [Chemical Formula 1-6]

[0037]

[0038] [Chemical Formula 1-7]

[0039]

[0040] [Chemical Formula 1-8]

[0041]

[0042] [Chemical Formula 1-9]

[0043]

[0044] [Chemical Formula 1-10]

[0045]

[0046] [Chemical Formula 1-11]

[0047]

[0048] [Chemical Formula 1-12]

[0049] .

[0050] According to another embodiment of the present invention, a lipid nanoparticle composition comprising the compound is provided.

[0051] According to one aspect, the compound may replace PEGylated lipids to increase colloidal stability and prevent interparticle aggregation.

[0052] According to one aspect, the composition may further comprise one or more selected from the group consisting of ionizable lipids, helper lipids and structural lipids.

[0053] According to one side, the helper lipids are 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-Oleoyl-2-cholesterylhexylsuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), It may be at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

[0054] According to one aspect, the structural lipid may be at least one selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

[0055] According to one aspect, the composition may include all of an ionizable lipid, a helper lipid and a structural lipid, and may include 1.5 to 15 mol% of the compound, 40 to 60 mol% of the ionizable lipid, 0 to 15 mol% of the helper lipid and 25 to 40 mol% of the structural lipid.

[0056] According to one aspect, the composition may comprise 10 mol% of the compound represented by Chemical Formula 1-4, 45 to 50 mol% of the ionizable lipid, 5 to 7 mol% of the helper lipid, and 35 to 40 mol% of the structural lipid.

[0057] According to another aspect, the composition may comprise 1.5 mol% of the compound of any one of Chemical Formula 1-9, Chemical Formula 1-11 or Chemical Formula 1-12, 50 mol% of the ionizable lipid, 10 mol% of the halophilic lipid and 38.5 mol% of the structural lipid.

[0058] According to one aspect, the compound may be any one selected from the group consisting of the following chemical formulas 1-1 to 1-12:

[0059] [Chemical Formula 1-1]

[0060]

[0061] [Chemical Formula 1-2]

[0062]

[0063] [Chemical Formula 1-3]

[0064]

[0065] [Chemical Formula 1-4]

[0066]

[0067] [Chemical Formula 1-5]

[0068]

[0069] [Chemical Formula 1-6]

[0070]

[0071] [Chemical Formula 1-7]

[0072]

[0073] [Chemical Formula 1-8]

[0074]

[0075] [Chemical Formula 1-9]

[0076]

[0077] [Chemical Formula 1-10]

[0078]

[0079] [Chemical Formula 1-11]

[0080]

[0081] [Chemical Formula 1-12]

[0082]

[0083] According to one aspect, the lipid nanoparticle composition described above may contain a therapeutic or preventive agent therein.

[0084] According to one aspect, the therapeutic or prophylactic agent may be selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof.

[0085] According to one aspect, the therapeutic or preventive agent is messenger RNA, and the lipid nanoparticle may preferably have a diameter of 70 to 200 nm and an internal zeta potential of -70 to -20 mV.

[0086] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided.

[0087] The present invention provides a novel oligo-gamma-glutamic acid-based lipid compound, which can be used to form lipid nanoparticles by replacing PEGylated lipids. Lipid nanoparticles comprising the compound of the present invention exhibit fewer side effects, such as allergic reactions, and superior in vivo stability compared to lipid nanoparticles comprising PEGylated lipids.

[0088] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0089] Figure 1 shows the results of a gel shift analysis using lipid nanoparticles using derivatives of chemical formulas 1-1 to 1-5 to measure the encapsulation efficiency of mRNA.

[0090] Figure 2 shows the results of a gel shift analysis using lipid nanoparticles with a derivative of chemical formula 1-4 to measure the encapsulation efficiency of mRNA.

[0091] Figure 3 shows the results of measuring the intracellular mRNA delivery and translation efficiency of lipid nanoparticles using a derivative of chemical formula 1-4 using a flow cytometer.

[0092] Figure 4 shows the results of size analysis according to the refrigerated storage period of lipid nanoparticles using a derivative of chemical formula 1-4.

[0093] Figure 5 shows the results of gel shift analysis of lipid nanoparticles manufactured with oligo-gamma-glutamic acid derivatives of chemical formulas 1-6 to 1-12, whose structures were modified to optimize the structure of chemical formula 1-4.

[0094] Figure 6 shows the results of measuring the intracellular mRNA delivery and translation efficiency of lipid nanoparticles OGA070, 073, and 076 using a flow cytometer.

[0095] Figure 7 shows the results of gel shift analysis of lipid nanoparticles using derivatives of chemical formulas 1-3 to 1-4 to measure the encapsulation efficiency of siRNA.

[0096] The present inventors have noticed that PEGylated lipid, which is one of the components of lipid nanoparticles, increases colloidal stability and prevents particle aggregation, thereby improving stability in the body, but has the disadvantage of causing allergic reactions and anaphylaxis. Therefore, the present inventors have invented and provided a novel lipid derivative compound based on glutamic acid to replace PEGylated lipid, a lipid nanoparticle composition comprising the same, a vaccine composition comprising the same, and a method for preparing the same.

[0097] The present inventors provide a compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof:

[0098] [Chemical Formula 1]

[0099]

[0100] The above X is absent or And,

[0101] The above x is an integer from 1 to 6,

[0102] The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms,

[0103] The above Y is [A a B b C c D d E e F f ] n is a polymer represented by

[0104] The above A is ,

[0105] The above B is ,

[0106] The above C is ,

[0107] The above D is ,

[0108] The above E is ,

[0109] The above F is and,

[0110] The above a to f are integers from 0 to 24, and the above n is an integer from 1 to 8, The value is between 1 and 24.

[0111] The term 'saturated hydrocarbon' used in the present invention means a hydrocarbon composed of single bonds, and 'unsaturated hydrocarbon' means a hydrocarbon having one or more double bonds or triple bonds.

[0112] The term 'polymer' used in the present invention may be a polymer formed by polymerization of a single monomer or a copolymer formed by polymerization of one or more different monomers. Preferably, it may be a polymer formed by copolymerization of the above A to F, and the above Since the value is from 1 to 24, the compound of the present invention may contain a total of 1 to 24 monomers, and most preferably may be composed of 15 monomers.

[0113] According to one aspect, R1 and R2 may each independently be an alkyl group having 6 to 17 carbon atoms, most preferably 13 to 15 carbon atoms.

[0114] The term “alkyl group” used in the present invention means an alkane with one hydrogen atom removed, preferably -(CH2) a -Can be expressed as CH3.

[0115] According to one side, the above X is absent, or In this case, x may be 4, and R1 and R2 may be straight-chain alkyl having 13 carbon atoms. The straight-chain alkyl refers to a structure in which several atoms are connected in a long line in a chemical structural formula.

[0116] According to one aspect, the n is 5, a and b are each 1 or 2, and c to f may be 0. Therefore, the Y may preferably be (A1B2)5 or (A2B1)5, and most preferably (A2B1)5. According to Example 3 or 4 of the present invention, when adopting the copolymer structure as described above, the most preferable physical properties for carrying mRNA or siRNA were exhibited.

[0117] According to one aspect, n is 5, a is 2, c, e and f are 0 or 1 and at least one of them is 1, and b and d may be 0. Therefore, Y may preferably be (A2C1)5 or (A2E1)5 or (A2F1)5. According to Example 5 of the present invention, when adopting the copolymer structure as described above, it exhibited desirable properties for carrying mRNA.

[0118] According to one aspect, the compound may be at least one selected from the group consisting of the following chemical formulas 1-1 to 1-12:

[0119] [Chemical Formula 1-1]

[0120]

[0121] [Chemical Formula 1-2]

[0122]

[0123] [Chemical Formula 1-3]

[0124]

[0125] [Chemical Formula 1-4]

[0126]

[0127] [Chemical Formula 1-5]

[0128]

[0129] [Chemical Formula 1-6]

[0130]

[0131] [Chemical Formula 1-7]

[0132]

[0133] [Chemical Formula 1-8]

[0134]

[0135] [Chemical Formula 1-9]

[0136]

[0137] [Chemical Formula 1-10]

[0138]

[0139] [Chemical Formula 1-11]

[0140]

[0141] [Chemical Formula 1-12]

[0142]

[0143] According to another embodiment of the present invention, a lipid nanoparticle composition comprising the compound is provided.

[0144] The above lipid nanoparticles refer to nano-sized spherical particles composed of lipids.

[0145] According to one aspect, the compound may replace PEGylated lipids to increase colloidal stability and prevent interparticle aggregation.

[0146] According to one aspect, the composition may further comprise at least one selected from the group consisting of ionizable lipids, helper lipids and structural lipids, and preferably may comprise all of ionizable lipids, helper lipids and structural lipids.

[0147] The ionizable lipid is an ionizable compound having properties similar to lipids, and can play a role in efficiently encapsulating a drug (e.g., anionic drug and / or nucleic acid) into lipid nanoparticles through electrostatic interaction with the drug. The ionizable lipid has a pK of the ionizable lipid. a It can be protonated (positively charged) at pH below pK a At higher pH, it can be substantially neutral. In one example, the lipid nanoparticle may comprise a protonated ionizable lipid and / or an ionizable lipid that exhibits neutrality. The ionizable lipid may be a cationic lipid, and a non-limiting example is SM-102.

[0148] The above helper lipid may promote the fusion of lipid nanoparticles, and may be 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-Oleoyl-2-cholesterylhexylsuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), It may be at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE), but most preferably DSPC.

[0149] The above structural lipid refers to a lipid that provides morphological rigidity to lipid charging within lipid nanoparticles and plays a role in improving the stability of nanoparticles by being dispersed in the core and surface of the nanoparticles, and may be at least one selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene, but most preferably cholesterol.

[0150] According to one aspect, the composition may include all of an ionizable lipid, a helper lipid and a structural lipid, and may include 1.5 to 15 mol% of the compound, 40 to 60 mol% of the ionizable lipid, 0 to 15 mol% of the helper lipid and 25 to 40 mol% of the structural lipid.

[0151] According to one aspect, the composition may comprise 10 mol% of the compound represented by Chemical Formula 1-4, 45 to 50 mol% of the ionizable lipid, 5 to 7 mol% of the helper lipid, and 35 to 40 mol% of the structural lipid.

[0152] According to another aspect, the composition may comprise 1.5 mol% of the compound of any one of Chemical Formula 1-9, Chemical Formula 1-11 or Chemical Formula 1-12, 50 mol% of the ionizable lipid, 10 mol% of the halophilic lipid and 38.5 mol% of the structural lipid.

[0153] As shown in Examples 4 and 7 below, optimal RNA loading efficiency and intracellular mRNA translation efficiency and stability were demonstrated within the composition range described above.

[0154] According to one aspect, the compound may be any one selected from the group consisting of the following chemical formulas 1-1 to 1-12:

[0155] [Chemical Formula 1-1]

[0156]

[0157] [Chemical Formula 1-2]

[0158]

[0159] [Chemical Formula 1-3]

[0160]

[0161] [Chemical Formula 1-4]

[0162]

[0163] [Chemical Formula 1-5]

[0164]

[0165] [Chemical Formula 1-6]

[0166]

[0167] [Chemical Formula 1-7]

[0168]

[0169] [Chemical Formula 1-8]

[0170]

[0171] [Chemical Formula 1-9]

[0172]

[0173] [Chemical Formula 1-10]

[0174]

[0175] [Chemical Formula 1-11]

[0176]

[0177] [Chemical Formula 1-12]

[0178]

[0179] According to one aspect, the lipid nanoparticle composition described above may contain a therapeutic or preventive agent therein, and the therapeutic or preventive agent may be preferably selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA) and mixtures thereof, and most preferably messenger RNA. When the therapeutic or preventive agent is messenger RNA, the lipid nanoparticle may preferably have a diameter of 70 to 200 nm and an internal zeta potential of -70 to -20 mV.

[0180] According to another embodiment of the present invention, a vaccine composition comprising the lipid nanoparticle composition is provided. The vaccine composition may preferably be an mRNA vaccine and may further comprise a pharmaceutically acceptable salt or adjuvant.

[0181] The vaccine composition of the present invention may be administered via, for example, the oral or parenteral route. Here, parenteral refers to a broad route of administration, and includes, for example, intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intranasal, sublingual, intrathecal, inhalation, ocular, rectal, vaginal, and intracerebroventricular administration.

[0182] When formulating the above composition, it is manufactured using diluents or excipients such as commonly used fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants.

[0183] Solid preparations for oral administration include tablets, tablets, powders, granules, capsules, troches, etc., and these solid preparations are prepared by mixing one or more compounds according to the present invention with at least one excipient, such as starch, calcium carbonate, sucrose, lactose, or gelatin. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, and syrups, and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, flavoring agents, and preservatives may be included.

[0184] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0185] Non-aqueous solvents and suspending agents include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cocoa butter, laurin, glycerol, and gelatin.

[0186] The composition according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment. The effective dosage level may be determined based on the type and severity of the patient's disease, the activity and sensitivity of the drug to the drug, the time of administration, the route of administration and excretion rate, the duration of treatment, concomitant drugs, and other factors well known in the medical field. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or in multiple doses. It is important to administer an amount that achieves the maximum effect with the minimum amount without causing side effects by taking all of the above factors into consideration, and this can be easily determined by those skilled in the art.

[0187] The terms used in the examples are for illustrative purposes only and should not be construed as limiting. Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to specify the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0188] Unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0189] The present invention is susceptible to various modifications and embodiments. Specific embodiments are illustrated in the drawings and described in detail in the following detailed description. However, this is not intended to limit the present invention to specific embodiments, but rather to encompass all modifications, equivalents, and alternatives falling within the spirit and technical scope of the present invention. In describing the present invention, detailed descriptions of related known technologies will be omitted if they are deemed to obscure the gist of the present invention.

[0190] Example 1. Synthesis of oligo-gamma-glutamic acid derivatives

[0191] Oligo-gamma-glutamic acid derivatives (12 types below) were designed as follows. Gamma-glutamic acid monomer A and its derivative monomers B to F were designed first, and then C, E, and F, which are not commercially available, were synthesized as follows. Oligo-gamma-glutamic acid derivatives were basically composed of lipids composed of alkyl chains and 15 gamma-glutamic acid monomers A to F having a repeating pattern. All monomers, including the synthesized monomers, of the designed oligo-gamma-glutamic acid derivatives were provided to Dandicure Co., Ltd., and the synthesis was requested in compliance with the general solid-phase peptide synthesis method.

[0192] Example 1-1: Synthesis of chemical formula C

[0193] Synthesis of 5-(tert-butyl) 1-ethyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate

[0194]

[0195] (S)-2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(tert-butoxy)-5-oxopentanoic acid (1.09 g) was dissolved in 20 mL of dimethylformaldehyde, and potassium carbonate (0.23 g) was added under strong stirring. Bromoethane (91 μl) was added to the reaction mixture, and the mixture was stirred at room temperature for 8 hours. Distilled water was added to the reaction mixture, and the solvent was removed under high vacuum. The reaction mixture was extracted into the organic layer by adding distilled water and ethyl acetate, and the organic layer was washed with a saturated aqueous solution of sodium chloride. The organic layers were combined, and the solvent was removed under high vacuum. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 9:1) to obtain the compound 5-(tert-butyl) 1-ethyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (1.06 g, 92%).

[0196] 1H NMR (400 MHz, Chloroform-d) δ 7.78 (dd,J= 7.6, 1.0 Hz, 2H), 7.61 (dd,J= 7.7, 7.6 Hz, 2H), 7.41 (dd,J= 7.5, 7.5 Hz, 2H), 7.33 (dd,J= 7.4, 7.4 Hz, 2H), 5.50 (d,J= 8.3 Hz, 1H), 4.59 - 4.31 (m, 2H+1H), 4.23 (q,J= 7.3 Hz, 2H+1H), 2.50 - 2.24 (m, 2H), 2.23 - 2.13 (m, 1H), 2.02 - 1.93 (m, 1H), 1.46 (s, 9H), 1.30 (t,J= 7.1 Hz, 6H).

[0197] 13 C NMR (101 MHz, Chloroform-d) δ 172.04, 155.95, 143.88, 143.69, 141.26, 127.67, 127.03, 125.09, 119.94, 80.80, 67.01, 61.60, 53.50, 47.11, 31.42, 28.03, 27.59, 14.13.

[0198]

[0199] (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-ethoxy-5-oxopentanoic acid (C)의 합성

[0200]

[0201] The above 5-(tert-butyl) 1-ethyl (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (807.7 mg) was dissolved in 9 mL of dichloromethane, and then trifluoroacetic acid (9 mL) was added. The mixture was stirred in an ice bath for 3 hours, and 20 mL of a saturated aqueous solution of sodium bicarbonate was added, followed by stirring for an additional 10 minutes. The organic layer was washed with distilled water and a saturated aqueous solution of sodium chloride, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 94:6) to obtain a compound of formula C (424 mg, 59.9%).

[0202] 1 H NMR (400 MHz, DMSO-d6) δ 7.89 (d,J= 7.5 Hz, 2H), 7.81 (d,J= 7.9 Hz, 1H), 7.72 (dd,J= 7.1, 2.2 Hz, 2H), 7.49 - 7.37 (m, 2H), 7.39 - 7.27 (m, 2H), 4.48 - 4.27 (m, 2H), 4.26 - 4.21 (m, 1H), 4.11 - 4.04 (m, 2H+1H), 2.46 - 2.25 (m, 2H), 2.07 - 1.91 (m, 1H), 1.88 - 1.70 (m, 1H), 1.17 (t,J= 7.1 Hz, 3H).

[0203] 13 C NMR (101 MHz, DMSO-d6) δ 173.74, 172.15, 156.18, 143.85, 140.78, 127.71, 127.12, 125.26, 120.18, 65.71, 60.61, 53.17, 46.68, 30.00, 26.01, 14.09.

[0204]

[0205] Example 1-2: Synthesis of chemical formula E

[0206] Synthesis of 5-(tert-butyl) 1-(2-ethoxyethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate

[0207]

[0208] N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Hydrochloride (451.9 mg) and 4-Dimethylamino pyridine (29.1 mg) were dissolved in 85 mL of dichloromethane and stirred in an ice bath under argon for 10 minutes. 2-Ethoxyethanol (230 μL) was mixed with 35 mL of dichloromethane and slowly added dropwise to the reaction mixture in an ice bath under argon for 30 minutes. The reaction mixture was warmed to room temperature and stirred for 23.5 hours. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 93:7) to obtain compound 5-(tert-butyl) 1-(2-ethoxyethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (510.9 mg, 87.0%).

[0209] 1H NMR (400 MHz, Chloroform-d) δ 7.77 (d,J= 7.5 Hz, 2H), 7.61 (dd,J= 7.7, 7.5 Hz, 2H), 7.41 (dd,J= 7.4, 7.4 Hz, 2H), 7.36 - 7.25 (m, 2H), 5.57 (d,J= 8.2 Hz, 1H), 4.47 - 4.34 (m, 2H+1H+1H), 4. 31 - 4.46 (m, 1H), 4.23 (t,J= 7.1 Hz, 1H), 3.64 (t,J= 4.8 Hz, 2H), 3.52 (q,J= 6.9 Hz, 2H), 2.42 - 2.26 (m, 2H), 2.26 - 2.12 (m, 1H), 2.08 - 1.94 (m, 1H), 1.46 (s, 9H), 1.21 (t,J= 7.0 Hz, 3H).

[0210] 13 C NMR (101 MHz, Chloroform-d) δ 172.02, 155.87, 143.85, 143.65, 141.21, 127.64, 127.01, 125.07, 119.91, 80.72, 67.98, 66.98, 66.55, 64.51, 53.47, 47.08, 31.33, 28.00, 27.54, 15.03.

[0211]

[0212] (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(2-ethoxyethoxy)-5-oxopentanoic acid (E)의 합성

[0213]

[0214] The above 5-(tert-butyl) 1-(2-ethoxyethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (749 mg) was dissolved in 7.5 mL of dichloromethane, and then trifluoroacetic acid (7.5 mL) was added. The mixture was stirred in an ice bath for 3 hours, and 15 mL of a saturated aqueous sodium bicarbonate solution was added, followed by stirring for an additional 10 minutes. The organic layer was washed with distilled water and a saturated aqueous sodium chloride solution, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 94:6) to obtain a compound of chemical formula E (570 mg, 86.0%).

[0215] 1 H NMR (400 MHz, Chloroform-d) δ 7.76 (d,J= 7.5 Hz, 2H), 7.60 (d,J= 7.5 Hz, 2H), 7.40 (dd,J= 7.5 Hz, 7.5 Hz, 2H), 7.32 (ddd,J= 7.5, 7.5 Hz, 1.4 Hz, 2H), 5.58 (d,J= 8.2 Hz, 1H), 4.52 - 4.38 (m, 2H+1H), 4.38 - 4.24 (m, 2H), 4.22 (t,J= 6.9 Hz, 1H), 3.64 (t,J= 4.8 Hz, 2H), 3.52 (q,J= 7.0 Hz, 2H), 2.54 - 2.39 (m, 2H), 2.28 - 2.20 (m, 1H), 2.06 - 1.97 (m, 1H), 1.20 (t,J= 7.0 Hz, 3H).

[0216] 13C NMR (101 MHz, Chloroform-d) δ 177.72, 171.91, 156.04, 143.84, 141.32, 127.76, 127.12, 125.13, 120.03, 67.98, 67.12, 66.68, 64.68, 53.26, 47.15, 29.87, 27.54, 15.03.

[0217]

[0218] Example 1-3: Synthesis of chemical formula F

[0219] Synthesis of 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate

[0220]

[0221] N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide Hydrochloride (465.0 mg) and 4-Dimethylamino pyridine (31.5 mg) were dissolved in 85 mL of dichloromethane and stirred in an ice bath under argon gas for 10 minutes. Diethyleneglycol monomethyl ether (280 μL) was mixed with 35 mL of dichloromethane and slowly added dropwise to the reaction mixture in an ice bath under argon gas for 30 minutes. The reaction mixture was warmed to room temperature and stirred for 17 hours. The organic layer was washed with a saturated aqueous solution of sodium bicarbonate and a saturated aqueous solution of sodium chloride, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 93:7) to obtain the compound 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (621 mg, quantitative yield).

[0222] 1H NMR (400 MHz, Chloroform-d) δ 7.77 (dd,J= 7.5, 1.1 Hz, 2H), 7.61 (dd,J= 7.7, 7.6 Hz, 2H7.47 - 7.37 (m, 2H), 7.32 (ddd,J= 7.4, 7.4, 0.9 Hz, 2H), 5.57 (d,J= 8.2 Hz, 1H), 4.52 - 4.36 (m, 2H+1H), 4.35 - 4.30 (m, 2H), 4.23 (t,J= 7.0 Hz, 1H), 3.72 (t,J= 4.8 Hz, 2H), 3.63 (dd,J= 5.7, 3.5 Hz, 2H), 3.53 (dd,J= 5.8, 3.4 Hz, 2H), 3.37 (s, 3H), 2.45 - 2.26 (m, 2H), 2.25 - 2.11 (m, 1H), 2.04 - 1.94 (m, 1H), 1.46 (s, 9H).

[0223] 13 C NMR (101 MHz, Chloroform-d) δ 172.04, 155.91, 143.87, 143.68, 141.25, 127.66, 127.03, 125.08, 119.93, 80.76, 71.82, 70.46, 68.82, 66.98, 64.43, 59.00, 53.49, 47.11, 31.38, 28.03, 27.53.

[0224]

[0225] (S)-4-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-5-(2-(2-methoxyethoxy)ethoxy)-5-oxopentanoic acid (F)의 합성

[0226]

[0227] 5-(tert-butyl) 1-(2-(2-methoxyethoxy)ethyl) (((9H-fluoren-9-yl)methoxy)carbonyl)-L-glutamate (824 mg) was dissolved in 7.5 mL of dichloromethane, and then trifluoroacetic acid (7.5 mL) was added. The mixture was stirred in an ice bath for 2 hours, and 15 mL of a saturated aqueous solution of sodium bicarbonate was added, followed by stirring for an additional 10 minutes. The organic layer was washed with distilled water and a saturated aqueous solution of sodium chloride, and the remaining water was removed with sodium sulfate. The reaction mixture was purified by column chromatography (SiO2, dichloromethane → dichloromethane:methanol 94:6) to obtain a compound of formula F (714 mg, 97.0%).

[0228] 1 H NMR (400 MHz, Chloroform-d) δ 7.76 (d,J= 7.5 Hz, 2H), 7.60 (dd,J= 7.7, 7.6 Hz, 2H), 7.40 (ddd,J= 7.6, 7.6, 1.4 Hz, 2H), 7.32 (ddd,J= 7.4, 7.4, 1.4 Hz, 2H), 5.64 (d,J= 8.1 Hz, 1H), 4.56 - 4.33 (m, 2H+1H), 4.32 - 4.17 (m, 2H+1H), 3.70 (t,J= 4.7 Hz, 2H), 3.66 - 3.58 (m, 2H), 3.58 - 3.54 (m, 2H), 3.38 (s, 3H), 2.53 - 2.40 (m, 2H), 2.27 - 2.18 (m, 1H), 2.11 - 2.02 (m, 1H).

[0229] 13C NMR (101 MHz, Chloroform-d) δ 176.56, 171.81, 155.90, 143.65, 141.24, 127.68, 127.04, 125.06, 119.94, 71.80, 70.13, 68.75, 67.00, 64.61, 58.84, 53.24, 47.08, 29.77, 27.34.

[0230]

[0231] [Chemical Formula 1-1]

[0232]

[0233] [Chemical Formula 1-2]

[0234]

[0235] [Chemical Formula 1-3]

[0236]

[0237] [Chemical Formula 1-4]

[0238]

[0239] [Chemical Formula 1-5]

[0240]

[0241] [Chemical Formula 1-6]

[0242]

[0243] [Chemical Formula 1-7]

[0244]

[0245] [Chemical Formula 1-8]

[0246]

[0247] [Chemical Formula 1-9]

[0248]

[0249] [Chemical Formula 1-10]

[0250]

[0251] [Chemical Formula 1-11]

[0252]

[0253] [Chemical Formula 1-12]

[0254]

[0255]

[0256] Example 2. Preparation of lipid nanoparticles for mRNA delivery

[0257] Using the 12 types of oligo-gamma-glutamic acids described above, lipid nanoparticle carriers were manufactured with the compositions shown in Table 1. Lipid nanoparticles can be manufactured using a rotary heating stirrer (TS-100, Biosan), a low-volume laboratory mixer and emulsifier (NanoAssemblr Spark, Precision Nanosystems, Inc.), and a high-volume laboratory mixer and emulsifier (NanoAssemblr Ignite, Precision Nanosystems, Inc.). The size of the lipid nanoparticles is manufactured in a smaller and more uniform manner as the order of manufacturing proceeds. Therefore, the first screening experiment is conducted using a rotary heating stirrer, the intracellular mRNA delivery and translation efficiency experiment is conducted using a low-volume laboratory mixer and emulsifier, and the stability experiment of the finally selected lipid nanoparticles is conducted using a high-volume laboratory mixer and emulsifier.

[0258] Lipid nanoparticles were prepared by rapidly mixing RNA solution (50 mM sodium citrate buffer, 110 mM sodium chloride, pH=4.0) and lipid mixture solution (ethanol, dimetyl sulfoxide) using a laboratory rotary stirrer (TS-100, Biosan), a low-volume laboratory mixer and emulsifier (NanoAssemblr Spark, Precision Nanosystems, Inc.), or a high-volume laboratory mixer and emulsifier (NanoAssemblr Ignite, Precision Nanosystems, Inc.), and then solvent-switching to saline or phosphate buffered saline was performed using a centrifugal filter tube (UFC5010, Amicon).

[0259] In the primary lipid nanoparticle screening study, lipid nanoparticles were specifically prepared using a laboratory rotary heating stirrer (TS-100, Biosan) using a lipid mixture solution containing oligo-gamma-glutamic acid derivatives (chemical formulae 1-1 to 1-5) in the contents shown in Table 1. The sizes below represent diameters.

[0260] LNP No. Ionized lipid (mol%) Helper lipid (phospholipid) (mol%) Cholesterol (mol%) γ-OGA derivative lipid (mol%) N / P Size (nm) PDI Zeta potential (mV) PEG-mSM-102 (50) DSPC (10) Cholesterol (38.5) DMG-PEG2000 (1.5) 6158.3 ± 39.4 0.19 12.4 ± 0.5 OGA001 SM-102 (50) DSPC (10) Cholesterol (38.5) Chemical formula 1-1 (1.5) 61711 ± 3 15.5 0.9 3 -10.0 ± 5.3 OGA002 SM-102 (50) DSPC (10) Cholesterol (35) Chemical formula 1-1 (5) 61410 ± 202.90.95-38.9 ± 1.9OGA003SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-1(10)6129.3 ± 0.80.14-45.0 ± 1.0OGA004SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-2(1.5)63600 ± 306.91.00-6.4 ± 2.0OGA005SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-2(5)62750 ± 38.21.00-5.0 ± 4.6OGA006SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-2(10)61922 ± 5700.93-12.2 ± 1.4OGA007SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-3(1.5)62961 ± 662.51.00-3.4 ± 1.0OGA008SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-3(5)62362 ± 553.71.00-11.1 ± 4.0OGA009SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-3(10)63356 ± 684.91.00-23.2 ± 2.5OGA010SM-102(50)DSPC(10)Cholesterol(38.5)Chemical formula 1-4(1.5)62624 ± 263.21.00-11.0 ± 0.8OGA011SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-4(5)62801 ± 683.91.00-8.7 ± 1.9OGA012SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-4(10)6207.2 ± 4.10.23-47.1 ± 0.3OGA013SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-5(1.5)62402 ± 164.01.00-17.2 ± 3.3OGA014SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-5(5)6798.3 ± 96.70.70-45.0 ± 2.2OGA015SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-5(10)6116.7 ± 2.30.28-54.5 ± 0.9OGA016SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-6(1.5)61179 ± 104.90.91-12.3 ± 3.9OGA017SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-6(5)62549 ± 166.61.00-6.2 ± 2.6OGA018SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-6(10)63150 ± 88.51.00-6.6 ± 4.3.

[0261]

[0262] Example 3. Analysis of structural physicochemical properties of lipid nanoparticles and mRNA encapsulation efficiency.

[0263] In the composition of lipid nanoparticles PEG-m containing DMG-PEG2000, the mol% of DMG-PEG2000 and cholesterol were replaced with oligo-gamma-glutamic acid derivatives (chemical formulae 1-1 to 1-6) at 1.5, 5, and 10 mol%, respectively. The sizes, PDI, and zeta potentials of 18 lipid nanoparticles were measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.) and are shown in Table 1. Since oligo-gamma-glutamic acid derivatives have a negative charge at neutral pH, all 18 lipid nanoparticles have negative zeta potential values. Since lipid nanoparticles were manufactured using a rotary heating stirrer for rapid screening, considering that the size and PDI values ​​would decrease when manufactured using a laboratory mixer and emulsifier, lipid nanoparticles with a size of 300 nm or less and a PDI value of 0.3 or less were selected as suitable mRNA delivery vehicles. Lipid nanoparticles (OGA003, 012, 015) containing 10 mol% of each of chemical formulae 1-1, 1-4, and 1-5 each exhibited suitable sizes and PDI values.

[0264] To measure the encapsulation efficiency of mRNA in mRNA-encapsulated lipid nanoparticles, a gel retardation assay was performed and is shown in Fig. 1. The gel retardation assay was performed as follows. The mRNA standard solutions at various concentrations and the mRNA-encapsulated lipid nanoparticle samples were mixed 5:1 with loading buffer (36% glycerol) and loaded into each well of a 1% agarose gel (containing Midori green dye). The agarose gel with the loaded sample was carefully placed into an electrophoresis device (mupid-2plus, ADVANCE) containing 1X MOPS buffer solution so that it was sufficiently submerged. After operating the electrophoresis device for approximately 5 minutes, the agarose gel was taken out and fluorescence imaging was measured using a gel imaging system (Gel doc, Bio-Rad). Since Midori Green dye fluoresces when bound to mRNA, the higher the fluorescence intensity of the band appearing on the same line as the mRNA standard solution, the lower the encapsulation efficiency of the lipid nanoparticle. When examining three types of lipid nanoparticles with a size and PDI suitable for mRNA delivery, among the lipid nanoparticles (OGA003, 012, 015) containing 10 mol% each of Chemical Formula 1-1, 1-4, and 1-5, OGA012 containing Chemical Formula 1-4 and OGA015 containing Chemical Formula 1-5 showed high mRNA encapsulation efficiencies of over 90%, and in particular, the encapsulation efficiency of OGA012 was measured to be the highest at close to 100%. Lipid nanoparticle OGA003 containing Chemical Formula 1-1 showed an mRNA encapsulation efficiency of approximately 50%.

[0265] As shown in the above results, it was confirmed that the lipid nanoparticle containing 10 mol% of the oligo-gamma-glutamic acid derivative represented by [Chemical Formula 1-4] in the present invention instead of OGA012, i.e. PEG lipid, which has an appropriate size and PDI value and a high mRNA encapsulation efficiency close to 100%, is the most excellent. In the examples described below, the composition of the lipid nanoparticles manufactured using 10 mol% of the oligo-glutamic acid derivative of [Chemical Formula 1-4] was optimized.

[0266]

[0267] Example 4. Optimization of the composition of lipid nanoparticles for mRNA delivery.

[0268] As described above, lipid nanoparticles were manufactured using a laboratory low-volume mixer and emulsifier (NanoAssemblr Spark, Precision Nanosystems, Inc.) with various compositions containing 10 mol% of the oligo-gamma-glutamic acid derivative represented by the above-described [Chemical Formula 1-4], and these are shown in Table 2 below.

[0269] No. Ionized lipid (mol%) Helper lipid (mol%) Cholesterol (mol%) γ-OGA derivative lipid (mol%) N / P Size (nm) PDI Zeta potential (mV) PEG-mSM-102(50) DSPC(10) Cholesterol (38.5) DMG-PEG2000 (1.5) 610 6.6 ± 1.4 0.0 63.9 ± 1.1 OGA01 2 SM-102(50) DSPC(10) Cholesterol (30) Chemical formula 1-4(10) 613 8.6 ± 3.3 0.10-36.0 ± 3.6 OGA04 6 SM-102(40) DSPC(10) Cholesterol (40) Chemical formula 1-4(10) 614 9.3 ± 1.2 0.11-31.7 ± 0.7OGA047SM-102(45)DSPC(10)Cholesterol(35)Chemical Formula 1-4(10)6127.7 ± 1.80.10-36.4 ± 0.7OGA048SM-102(45)DSPC(5)Cholesterol(40)Chemical Formula 1-4(10)6145.6 ± 0.20.16-38.6 ± 0.4OGA049SM-102(50)DSPC(5)Cholesterol(35)Chemical Formula 1-4(10)6117.7 ± 0.50.07-36.9 ± 1.2OGA050SM-102(47)DSPC(7)Cholesterol(36)Chemical Formula 1-4(10)6127.4 ± 0.80.09-32.1 ± 1.4OGA054SM-102(50)DSPC(0)Cholesterol(40)Chemical formula 1-4(10)6127.8 ± 1.60.08-45.4 ± 1.9

[0270] The size, PDI, and zeta potential of the lipid nanoparticles manufactured above were measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.) and are shown in Table 2. When the lipid nanoparticles were manufactured using a low-capacity laboratory mixer and emulsifier, they were generally manufactured to be 20 to 70 nm smaller and more uniform than those manufactured using a rotary heating stirrer.

[0271] All lipid nanoparticles (PEG-m and OGA012, 046, 047, 048, 049, 050, 054) with different composition ratios of ionizable lipid, helper lipid, and cholesterol lipid, including the control PEG-m and superior OGA012, exhibited sizes (106.6 to 149.3 nm) and PDI (0.06 to 0.16) suitable as mRNA delivery vehicles.

[0272] The results of gel shift analysis of the manufactured LNPs are shown in Fig. 2. All lipid nanoparticles (128-m and OGA012, 046, 047, 048, 049, 050, 054) with changed composition ratios of ionizable lipids, helper lipids, and cholesterol lipids, including the control PEG-m and the superior OGA012, exhibited high mRNA encapsulation efficiencies close to 100%.

[0273] Similarly, the intracellular mRNA delivery and translation efficiency of the manufactured LNPs were measured using a flow cytometer (CytoFLEX, Beckman Coulter Life Sciences), and the results are shown in Fig. 3. After Huh7 cells were cultured in a 24-well cell culture plate for one day, lipid nanoparticles (PEG-m and OGA012, 046, 047, 048, 049, 050, 054) encapsulating GFP mRNA encoding green fluorescent protein (GFP) and DPBS as a negative control were treated to each well in which the cells were cultured. After 24 hours of culture, the cells were removed from the cell culture plate, and the quantification of GFP-expressing cells was confirmed using a flow cytometer. In Fig. 3, the more the graph is shifted to the right, the more cells express a high level of GFP. All lipid nanoparticles (OGA012, 046, 047, 048, 049, 050, 054) containing 10 mol% of chemical formula 1-4 exhibited high intracellular delivery and translation efficiencies of GFP mRNA, similar to those of PEG-m lipid nanoparticles containing PEG. In particular, lipid nanoparticles (OGA048 to 050) prepared with 45 to 50 mol% of ionizable lipid, 5 to 7 mol% of helper lipid, 35 to 40% of cholesterol, and 10 mol% of chemical formula 1-4 exhibited the highest intracellular delivery and translation efficiencies.

[0274] Finally, the stability of the control group, PEG-m (LNP manufactured using PEG lipid) and lipid nanoparticle OGA050 according to long-term storage was tested as follows, and the results are shown in Fig. 4. Lipid nanoparticles were manufactured using a high-capacity laboratory mixer and emulsifier (NanoAssemblr Ignite, Precision Nanosystems, Inc.) and stored in a refrigerator at 4°C. The size of the lipid nanoparticles was measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.) at intervals of 2 to 3 days. As a result, the lipid nanoparticles manufactured using OGA instead of PEG of the present invention maintained their size for more than 35 days, confirming excellent stability.

[0275]

[0276] Example 5. Optimization study of the structure of chemical formula 1-4.

[0277] In order to optimize the structure of the oligo-gamma-glutamic acid derivative selected as a substitute for PEG, the following 7 types of oligo-gamma-glutamic acid derivatives were designed by modifying the structure as follows. Since the oligo-gamma-glutamic acid derivative of Chemical Formula 1-4 has a saturated hydrocarbon structure with 13 carbon atoms in R1 in Chemical Formula 1, oligo-gamma-glutamic acid derivatives of Chemical Formula 1-7 and Chemical Formula 1-8 were designed in which R1 was changed to a saturated hydrocarbon structure with 15 and 17 carbon atoms, respectively. In addition, since the oligo-gamma-glutamic acid derivative of Chemical Formula 1-4 has an oligomeric structure in which gamma-glutamic acid (A) and the gamma-glutamic acid derivative (B) are repeated 5 times in the form of AAB, oligo-gamma-glutamic acid derivatives of Chemical Formulas 1-9 to 1-12 were designed in which the gamma-glutamic acid derivative (B) was changed to a gamma-glutamic acid derivative of C to F, respectively. As described above, lipid nanoparticles containing oligo-gamma-glutamic acid derivatives represented by the chemical formulas 1-7 to 1-12 were prepared using a laboratory rotary heating stirrer (TS-100, Biosan), and the results are shown in Table 3 below.

[0278] No. Ionized lipid (mol%) Helper lipid (mol%) Cholesterol (mol%) γ-OGA derivative lipid (mol%) N / P Size (nm) PDI Zeta potential (mV) OGA064SM-102(50)DSPC(10)Cholesterol(38.5) Chemical formula 1-7(1.5) 6628.8 ± 341.4 0.62-30.7 ± 2.7 OGA065SM-102(50)DSPC(10)Cholesterol(35) Chemical formula 1-7(5) 6348.4 ± 9.2 0.39-53.4 ± 2.0 OGA066SM-102(50)DSPC(10)Cholesterol(30) Chemical formula 1-7(10) 677.6 ± 2.0 0.29-58.4 ± 0.8OGA067SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-8(1.5)6448.6 ± 194.50.51-17.1 ± 1.2OGA068SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-8(5)6259.4 ± 7.40.21-51.7 ± 3.2OGA069SM-102(50)DSPC(10)Cholesterol(30)Chemical Formula 1-8(10)672.9 ± 1.00.16-48.9 ± 3.2OGA070SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-9(1.5)6122.8 ± 0.50.10-39.8 ± 0.7OGA071SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-9(5)689.5 ± 3.10.21-39.2 ± 2.0OGA072SM-102(50)DSPC(10)Cholesterol(30Chemical Formula 1-9(10)683.9 ± 1.30.22-40.4 ± 3.9OGA079SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-10(1.5)63361 ± 24800.94-45.4 ± 1.2OGA080SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-10(5)6397.4 ± 99.70.37-22.2 ± 3.9OGA081SM-102(50)DSPC(10)Cholesterol(30Chemical formula 1-10(10)6172 ± 1.70.08-42.3 ± 2.1OGA073SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-11(1.5)6169.6 ± 2.00.14-40.3 ± 1.2OGA074SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-11(5)6104.2 ± 1.50.24-40 ± 0.8OGA075SM-102(50)DSPC(10)Cholesterol(30Chemical Formula 1-11(10)674.3 ± 1.40.17-38.3 ± 0.5OGA076SM-102(50)DSPC(10)Cholesterol(38.5)Chemical Formula 1-12(1.5)6113.3 ± 0.70.08-38.1 ± 2.3OGA077SM-102(50)DSPC(10)Cholesterol(35)Chemical Formula 1-12(5)6117.9 ± 0.80.17-45.9 ± 2.5OGA078SM-102(50)DSPC(10)Cholesterol(30Chemical Formula 1-12(10)695.3 ± 2.40.17-40.5 ± 1.5.

[0279]

[0280] The size, PDI, and zeta potential of the manufactured lipid nanoparticles were measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.) and are shown in Table 3. It was confirmed that the lipid nanoparticles (OGA066, 068, 069, 070, 071, 072, 081, 073, 074, 075, 076, 077, 078) were manufactured appropriately as mRNA delivery vehicles. In the case of Chemical Formulas 1-7 and 1-10, only the lipid nanoparticles (OGA066, 081) containing 10 mol% showed the appropriate size and PDI value. In the case of Chemical Formula 1-8, the lipid nanoparticles (OGA068, 069) containing 5 and 10 mol% showed the appropriate size and PDI value. For chemical formulas 1-9, 1-11, and 1-12, all lipid nanoparticles (OGA070, 071, 072, 071, 072, 073, 074, 075, and 076) containing 1.5, 5, and 10 mol% showed suitable sizes and PDI values.

[0281] To measure the encapsulation efficiency of mRNA in mRNA-encapsulated lipid nanoparticles, a gel retardation assay was performed, and the results are shown in Fig. 5. When 13 types of lipid nanoparticles having a size and PDI suitable as mRNA delivery vehicles were examined, lipid nanoparticle OGA068 containing 5 mol% of chemical formula 1-8 showed an mRNA encapsulation efficiency of over 90%, and lipid nanoparticle OGA069 containing 10 mol% of chemical formula 1-8 showed an encapsulation efficiency of about 70%. Lipid nanoparticles (OGA070, 071, 072, 073, 074, 075, 076, 077, 078) containing 1.5, 5, and 10 mol% of chemical formulas 1-9, 1-11, and 1-12, respectively, showed a decrease in mRNA encapsulation efficiency as the mol% increased. Lipid nanoparticles OGA070 containing 1.5 mol% of chemical formula 1-9, lipid nanoparticles OGA073 containing 1.5 mol% of chemical formula 1-11, and lipid nanoparticles OGA076 containing 1.5 mol% of chemical formula 1-12 showed high mRNA encapsulation efficiencies of over 90%.

[0282] As shown in the above results, OGA070, OGA073, OGA076, which have suitable sizes and PDI values ​​and mRNA encapsulation efficiency of over 90%, were confirmed to be the most excellent lipid nanoparticles containing 1.5 mol% of oligo-gamma-glutamic acid derivatives represented by [Chemical Formula 1-9], [Chemical Formula 1-11], and [Chemical Formula 1-12] in place of PEG lipids.

[0283] The intracellular mRNA delivery and translation efficiency of lipid nanoparticles (OGA070, 073, 076) selected as excellent mRNA delivery vehicles were measured using a flow cytometer (CytoFLEX, Beckman Coulter Life Sciences), and the results are shown in Fig. 6. Lipid nanoparticle OGA070 containing 1.5 mol% of Chemical Formula 1-9 showed high intracellular mRNA delivery and translation efficiency similar to OGA050 containing 10 mol% of Chemical Formula 1-4. Lipid nanoparticle OGA 073 containing 1.5 mol% of Chemical Formula 1-11 showed slightly lower intracellular mRNA delivery and translation efficiency than OGA050 containing 10 mol% of Chemical Formula 1-4. Lipid nanoparticle OGA 076 containing 1.5 mol% of chemical formula 1-12 showed lower intracellular mRNA delivery and translation efficiency than lipid nanoparticle OGA 073 containing 1.5 mol% of chemical formula 1-11, but showed significantly higher intracellular mRNA delivery and translation efficiency than the negative control.

[0284] In the case of lipid nanoparticles containing oligo-gamma-glutamic acid derivatives of chemical formulae 1-9, 1-11, and 1-12, lipid nanoparticles (OGA070, 073, and 076) prepared with 50 mol% of ionizable lipid, 10 mol% of helper lipid, 38.5% of cholesterol, and 1.5 mol% of oligo-gamma-glutamic acid derivatives exhibited sizes and PDI suitable for mRNA delivery and high intracellular mRNA delivery and translation efficiency.

[0285]

[0286] Example 6. Preparation of lipid nanoparticles for siRNA delivery

[0287] As described above in Example 2, lipid nanoparticles containing oligo-gamma-glutamic acid derivatives represented by the chemical formulas 1-3 and 1-4 were prepared using a laboratory rotary heating stirrer (TS-100, Biosan), and the results are shown in Table 4 below.

[0288] No. Ionized lipid (mol%) Helper lipid (mol%) Cholesterol (mol%) γ-OGA derivative lipid (mol%) N / P Size (nm) PDI Zeta potential (mV) PEG-siSM-102(50) DSPC(10) Cholesterol(38.5) DMG-PEG2000(1.5) 6137.9 ± 1.3 0.2 114.6 ± 2.3 OGA027 SM-102(50) DSPC(10) Cholesterol(30) Chemical formula 1-3(10) 6811.8 ± 162.5 0.63-28.1 ± 2.4 OGA030 SM-102(50) DSPC(10) Cholesterol(30) Chemical formula 1-4(10) 6169.7 ± 1.7 0.06-46.8 ± 1.1OGA037SM-102(50)DSPC(10)Cholesterol(30)Formula 1-3 / Formula 1-4(9 / 1)6604.5 ± 33.90.61-18.0 ± 2.5OGA038SM-102(50)DSPC(10)Cholesterol(30)Formula 1-3 / Formula 1-4(5 / 5)6134.2 ± 1.60.09-38.6 ± 2.1OGA039SM-102(50)DSPC(10)Cholesterol(30)Formula 1-3 / Formula 1-4(1 / 9)6125.6 ± 2.30.06-39.9 ± 1.3

[0289]

[0290] Example 7. Encapsulation and characterization of siRNA in lipid nanoparticles

[0291] The size, PDI, and zeta potential of the manufactured lipid nanoparticles were measured using a dynamic light scattering particle size analyzer (Zetasizer, Malvern Panalytical Ltd.) and are shown in Table 4. Since oligo-gamma-glutamic acid derivatives have a negative charge at neutral pH, all six lipid nanoparticles have negative zeta potential values. Since lipid nanoparticles were manufactured using a rotary heating stirrer for rapid screening, considering that the size and PDI values ​​would decrease when manufactured using a laboratory mixer and emulsifier, lipid nanoparticles with a size of 300 nm or less and a PDI value of 0.3 or less were selected as suitable siRNA delivery vehicles. Lipid nanoparticles OGA027, 037, 038, 039, and 030 containing the mol% ratios of Chemical Formula 1-3 and Chemical Formula 1-4 as 10:0, 9:1, 5:5, 1:9, and 0:10, respectively, showed that the size and PDI value of lipid nanoparticles decreased as the mol% of Chemical Formula 1-4 increased. Lipid nanoparticles (OGA038, 039, and 030) containing 5:5, 1:9, and 0:10 mol% of Chemical Formula 1-3 and Chemical Formula 1-4 showed suitable sizes and PDI values.

[0292] To measure the encapsulation efficiency of siRNA in FITC-siRNA-encapsulated lipid nanoparticles, a gel retardation assay was performed and is shown in Fig. 7. The gel retardation assay was performed as follows. FITC-siRNA standard solutions at various concentrations and FITC-siRNA-encapsulated lipid nanoparticle samples were mixed 5:1 with loading buffer (36% glycerol) and loaded into each well of a 1% agarose gel. The agarose gel loaded with the samples was carefully placed into an electrophoresis apparatus (mupid-2plus, ADVANCE) containing 1X MOPS buffer solution so that it was sufficiently submerged. After operating the electrophoresis apparatus for approximately 5 minutes, the agarose gel was taken out and fluorescence imaging was measured using a gel imaging system (Gel doc, Bio-Rad). Since fluorescence is generated from the FITC fluorophore bound to siRNA, the higher the fluorescence intensity of the band appearing on the same line as the siRNA standard solution, the lower the encapsulation efficiency of the lipid nanoparticle. When examining three types of lipid nanoparticles with suitable sizes and PDIs as siRNA delivery vehicles, among lipid nanoparticles (OGA038, 039, 030) containing 5:5, 1:9, and 0:10 mol% of Chemical Formula 1-3 and Chemical Formula 1-4, lipid nanoparticle OGA038 containing 5 mol% each of Chemical Formula 1-3 and Chemical Formula 1-4 showed a high siRNA encapsulation efficiency of over 90%. Lipid nanoparticle OGA039 containing 1 and 9 mol% of chemical formula 1-3 and chemical formula 1-4, respectively, hardly encapsulated siRNA, while lipid nanoparticle OGA030 containing 10 mol% of chemical formula 1-4 showed an encapsulation efficiency of more than 50%.

[0293]

[0294] As shown in the above results, OGA038, which has an appropriate size and PDI value and an siRNA encapsulation efficiency of 90% or more, was confirmed to be the most excellent siRNA delivery vehicle, i.e., lipid nanoparticles containing 5 mol% each of oligo-gamma-glutamic acid derivatives represented by [Chemical Formula 1-3] and [Chemical Formula 1-4] in place of PEGlyated lipids.

[0295]

[0296] Although the embodiments described above have been described with limited drawings, those skilled in the art will appreciate that various technical modifications and variations can be applied based on the above. For example, appropriate results can still be achieved even if the described techniques are performed in a different order than described, and / or components of the described systems, structures, devices, circuits, etc. are combined or combined in a different manner than described, or are replaced or substituted with other components or equivalents.

[0297] Therefore, other implementations, other manufacturing examples and equivalents to the patent claims also fall within the scope of the claims described below.

Claims

1. A compound represented by the following chemical formula 1, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1] The above X is absent or And, The above x is an integer from 1 to 6, The above R1 and R2 are each independently a saturated or unsaturated hydrocarbon having 6 to 22 carbon atoms, The above Y is [A a B b C c D d E e F f ] n is a polymer represented by The above A is , The above B is , The above C is , The above D is , The above E is , The above F is and, The above a to f are integers from 0 to 24, and the above n is an integer from 1 to 8, The value is between 1 and 24.

2. In paragraph 1, A compound wherein R1 and R2 are each independently an alkyl group having 6 to 17 carbon atoms, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.

3. In paragraph 1, The above X is absent, or If x is 4, The compound wherein R1 and R2 are straight-chain alkyl having 13 carbon atoms, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.

4. In paragraph 1, A compound wherein n is 5, a and b are each 1 or 2, and c to f are 0, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof.

5. In paragraph 1, The compound is characterized by being at least one selected from the group consisting of the following chemical formulas 1-1 to 1-12, a stereoisomer thereof, a racemate thereof, or a pharmaceutically acceptable salt thereof: [Chemical Formula 1-1] [Chemical Formula 1-2] [Chemical Formula 1-3] [Chemical Formula 1-4] [Chemical Formula 1-5] [Chemical Formula 1-6] [Chemical Formula 1-7] [Chemical Formula 1-8] [Chemical Formula 1-9] [Chemical Formula 1-10] [Chemical Formula 1-11] [Chemical Formula 1-12] 6. A lipid nanoparticle composition comprising the compound of paragraph 1.

7. A lipid nanoparticle composition according to claim 6, wherein the compound replaces PEGylated lipid to increase colloidal stability and prevent inter-particle aggregation.

8. A lipid nanoparticle composition according to claim 7, characterized in that the composition further comprises at least one selected from the group consisting of ionizable lipids, helper lipids, and structurally maintaining lipids.

9. In the 8th paragraph, the helper lipid is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-diundecanoyl-sn-glycero-phosphocholine (DUPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2-di-O-octadecenyl-sn-glycero-3-phosphocholine (18:0 diether PC), 1-Oleoyl-2-cholesterylhexylsuccinoyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-Dilinoyl-sn-glycero-3-phosphocholine, 1,2-Diarachidonoyl-sn-glycero-3-phosphocholine, 1,2-Didocosahexaenoyl-sn-glycero-3-phosphocholine, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Diphytanoyl-sn-glycero-3-phosphoethanolamine (ME 16.0 PE), 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, 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), A lipid nanoparticle composition characterized by comprising at least one selected from the group consisting of dipalmitoyl-phosphatidyl-ethanolamine (DPPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, and lysophosphatidylethanolamine (LPE).

10. A lipid nanoparticle composition according to claim 8, characterized in that the structural lipid is at least one selected from the group consisting of cholesterol, bile acid derivatives including butyl lithocholate, cholanic acid derivatives, lithocholic acid derivatives, flavonoids, vitamin A and its derivatives, vitamin E, vitamin K, coenzyme Q10, and beta-carotene.

11. In paragraph 8, A lipid nanoparticle composition, characterized in that the composition comprises all of an ionizable lipid, a helper lipid, and a structural lipid, and comprises 1.5 to 15 mol% of the compound, 40 to 60 mol% of the ionizable lipid, 0 to 15 mol% of the helper lipid, and 25 to 40 mol% of the structural lipid.

12. In paragraph 11, A lipid nanoparticle composition characterized in that the composition comprises 10 mol% of the compound, 47 mol% of the ionizable lipid, 7 mol% of the helper lipid, and 36 mol% of the structural lipid, or 1.5 mol% of the compound, 50 mol% of the ionizable lipid, 10 mol% of the helper lipid, and 38.5 mol% of the structural lipid.

13. In the 12th paragraph, the compound is a lipid nanoparticle composition selected from the group consisting of the following chemical formulas 1-1 to 1-12: [Chemical Formula 1-1] [Chemical Formula 1-2] [Chemical Formula 1-3] [Chemical Formula 1-4] [Chemical Formula 1-5] [Chemical Formula 1-6] [Chemical Formula 1-7] [Chemical Formula 1-8] [Chemical Formula 1-9] [Chemical Formula 1-10] [Chemical Formula 1-11] [Chemical Formula 1-12] 14. A lipid nanoparticle composition according to any one of claims 8 to 13, wherein the lipid nanoparticle composition contains a therapeutic or preventive agent therein.

15. A lipid nanoparticle composition according to claim 14, characterized in that the therapeutic or preventive agent is selected from the group consisting of interfering RNA (siRNA), asymmetric interfering RNA (aiRNA), microRNA (miRNA), dicer-substrate RNA (dsRNA), small hairpin RNA (shRNA), messenger RNA (mRNA), and mixtures thereof.

16. In paragraph 15, the therapeutic or preventive agent is messenger RNA, A lipid nanoparticle composition, characterized in that the lipid nanoparticle has a diameter of 70 to 200 nm and an internal zeta potential of -70 to -20 mV.

17. A vaccine composition comprising the lipid nanoparticle composition of Article 14.

Images