Lipid nanoparticles for localized delivery of nucleic acid delivery using cationic cholesterol

Abstract

The present invention relates to lipid nanoparticles for topical delivery of nucleic acid delivery using cationic cholesterol, and when topical administration, side effects caused by systemic drug delivery can be minimized, and protein expression can be limited at the site of administration. In addition, the duration of protein expression at the administration site can be increased, and thus the lipid nanoparticles can be used in the technical field related to nucleic acid therapy.

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles for nucleic acid delivery, and more particularly to lipid nanoparticles for local delivery comprising cationic cholesterol, lipid nanoparticle compositions comprising the nanoparticles and nucleic acids, and methods of preventing or treating diseases using the same. Background Art

[0002] With the recent emergence of COVID-19 vaccines, the expectations and importance of mRNA-based vaccines are increasing. Two licensed vaccines (Spikevax TM , Comirnaty TM ) all use lipid nanoparticles (LNPs) as delivery vehicles, and efforts are underway to continuously improve efficacy by optimizing LNP composition and developing additional ionizable lipids.

[0003] However, in general, intramuscularly injected LNPs diffuse throughout the body via lymphatic vessels and blood, as well as the injected muscle site, which increases the risk of systemic reactions. In particular, for COVID-19 vaccines, there is an increased likelihood of myocarditis, an inflammation of the heart muscle, due to some cardiac-delivered vaccines. Therefore, delivery vehicles with improved safety are needed for the development of preventive vaccines for the general healthy population after the pandemic ends.

[0004] The goal is to address these limitations, and in the present invention, attempts have been made to develop delivery vectors that reduce systemic reactions by expressing mRNA only at the injection site and minimizing systemic distribution. In the future, such delivery vectors are expected to be suitable for local administration, such as intratumoral injection, intradermal injection, intracerebral injection, etc., in addition to intramuscular injection. It is expected that by limiting mRNA expression to the administration site and reducing the amount of loss outside the administration site, the administration dose can be fully utilized.

[0005] In addition, it has been confirmed that the present invention can increase the duration of protein expression at the muscle site. Typically, the reduction of protein expression at the muscle site after intramuscular injection shows zero-order kinetics, and it seems that the present invention can significantly increase the duration of protein expression. Therefore, it is expected that the therapeutic dose can be reduced.

[0006] In summary, the present invention relates to lipid nanoparticles comprising cationic cholesterol and compositions comprising the lipid nanoparticles, and in particular to delivery vehicles that can 1) increase safety by limiting protein expression to a local site and 2) increase the duration of protein expression.

[0007] The above information described in the Background section is only for enhancement of understanding of the background of the invention and it may not include information that forms the prior art that is already known to one of ordinary skill in the art to which the invention belongs. Summary of the invention

[0008] An object of the present invention is to provide lipid nanoparticles and lipid nanoparticle compositions comprising the lipid nanoparticles and nucleic acids, which, when topically administered, limit the delivery of nucleic acids to only the administration site, thereby reducing systemic side effects, thereby increasing safety, and increasing the duration of effective expression at the administration site.

[0009] Another object of the present invention is to provide a vaccine comprising the lipid nanoparticle composition.

[0010] Still another object of the present invention is to provide a method for preventing or treating a disease, comprising administering a lipid nanoparticle composition to a subject.

[0011] Yet another object of the present invention is to provide use of the lipid nanoparticle composition for preventing or treating diseases.

[0012] Still another object of the present invention is to provide use of the lipid nanoparticle composition for the manufacture of a medicament for preventing or treating a disease.

[0013] In order to achieve the above-mentioned object, the present invention provides a lipid nanoparticle (LNP), comprising (A) ionizable lipid; (B) cationic cholesterol; (C) cholesterol; (D) auxiliary lipid; and (E) PEG lipid (polyethylene glycol lipid), wherein the molar ratio of (B) cationic cholesterol to (C) cholesterol is 1:0.1 to 1:10.

[0014] In addition, the present invention provides a lipid nanoparticle composition comprising a lipid nanoparticle and a nucleic acid.

[0015] In addition, the present invention provides a vaccine comprising the lipid nanoparticle composition.

[0016] In addition, the present invention provides a method for preventing or treating a disease, comprising administering the lipid nanoparticle composition to a subject.

[0017] In addition, the present invention provides use of the lipid nanoparticle composition for preventing or treating diseases.

[0018] In addition, the present invention provides use of the lipid nanoparticle composition for manufacturing a medicament for preventing or treating a disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Shown are graphs showing protein expression profiles after intramuscular injection of a lipid nanoparticle composition including cationic cholesterol according to an embodiment of the present invention in mice, together with results of measurements of luminescence in liver and muscle tissues.

[0020] Figure 2An exemplary structure of cationic cholesterol of the present invention is shown.

[0021] Figure 3 Exemplary structures of ionizable lipids evaluated in the present invention are shown.

[0022] Figure 4 Shown is the protein expression profile after intramuscular injection into mice of lipid nanoparticles using 9 different ionizable lipids without / with cationic cholesterol.

[0023] Figure 5 Shown are protein expression levels in liver and muscle tissues 6 hours and 7 days after injection following intramuscular administration to mice.

[0024] Figure 6 Shown are the results of measuring the particle size and zeta potential of lipid nanoparticles used in an embodiment of the present invention.

[0025] Figure 7 Shown is the distribution of protein expression in mice after intramuscular injection, depending on changes in the ratio of cationic cholesterol in lipid nanoparticles.

[0026] Figure 8 It is shown from Figure 7 Figure 3. Results of luminescence measurements of muscle tissue.

[0027] Fig. 9 It is shown from Figure 7 Figure 3 is a graph of the results of luminescence measurement of liver tissue.

[0028] Fig.10 It is shown from Figure 7 Graph of the luminescence ratio of muscle tissue to liver tissue.

[0029] Fig.11 Shown is the distribution of protein expression in mice after intramuscular injection, depending on the type of cationic cholesterol used in the lipid nanoparticles.

[0030] Fig.12 It is shown from Fig.11 Figure 3. Results of luminescence measurements of muscle tissue.

[0031] Fig.13 It is shown from Fig.11 Figure 3 is a graph of the results of luminescence measurement of liver tissue.

[0032] Fig.14 It is shown from Fig.11 Graph of the luminescence ratio of muscle tissue to liver tissue.

[0033] Fig.15Shown are protein expression levels in liver and muscle tissues 6 hours and 7 days after injection following intramuscular administration to mice, depending on the type of cationic cholesterol.

[0034] Fig.16 Shown are antigen-specific antibody titers induced after two intramuscular injections of lipid nanoparticles without / with cationic cholesterol into mice, measured 4, 6, 8 and 11 weeks after the first immunization.

[0035] Fig.17 Shown are the protein expression kinetics after subcutaneous injection of lipid nanoparticles without / with cationic cholesterol into mice.

[0036] Fig.18 Shown are the plasma concentration kinetics of erythropoietin in the blood after intramuscular injection of lipid nanoparticles without / with cationic cholesterol into mice.

[0037] Fig.19 Shown are the protein expression kinetics following intramuscular injection of lipid nanoparticles with cationic cholesterol or the cationic lipid DOTAP into mice.

[0038] Fig. 20 Shown are the protein expression kinetics in mice after intramuscular injection of lipid nanoparticles with cationic cholesterol and self-amplified mRNA.

[0039] Detailed description and preferred embodiments of the invention

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. Generally, the nomenclature used herein is well known and typical in the art.

[0041] In an embodiment of the present invention, it is expected that mRNA expression is limited only to the administration site, and the dosage is maintained by reducing the amount lost outside the administration site. In addition, it was confirmed that the administration of the lipid nanoparticle composition of the present invention increases the duration of protein expression at the intramuscular or subcutaneous administration site.

[0042] Therefore, in one aspect, the present invention relates to lipid nanoparticles (LNPs) comprising (A) ionizable lipids; (B) cationic cholesterol; (C) cholesterol; (D) helper lipids; and (E) PEG lipids (polyethylene glycol lipids), wherein the molar ratio of (B) cationic cholesterol to (C) cholesterol is 1:0.1 to 1:10.

[0043] In the lipid nanoparticles according to the present invention, the molar ratio of (B) cationic cholesterol to (C) cholesterol may be 1: 0.1 to 1: 10, preferably 1: 0.2 to 1: 5, more preferably 1: 0.33 to 1: 3, and most preferably 1: 1. Here, it was confirmed that protein expression in liver tissue decreased when the ratio of cationic cholesterol increased, and it was also confirmed that protein expression in muscle tissue decreased when the ratio of cationic cholesterol exceeded a specific ratio.

[0044] In the lipid nanoparticles according to the present invention, the molar ratio of (B) cationic cholesterol to (A) ionizable lipid may be 1:0.5 to 1:20, preferably 1:1 to 1:10, more preferably 1:2 to 1:5, and most preferably 1:2.59.

[0045] In the lipid nanoparticles according to the present invention, the molar ratio of (B) cationic cholesterol to (D) helper lipid can be 1:0.2 to 1:10, preferably 1:0.33 to 1:5, more preferably 1:0.5 to 1:2, and most preferably 1:0.518 (1.93:1).

[0046] In the lipid nanoparticles according to the present invention, the molar ratio of (B) cationic cholesterol to (E) PEG lipid may be 1:0.01 to 1:1, preferably 1:0.02 to 1:0.2, more preferably 1:0.05 to 1:0.1, and most preferably 1:0.078 (12.87:1).

[0047] In addition, the lipid nanoparticles according to the present invention preferably include 30 to 80 mol% of ionizable lipids; 0.01 to 50 mol% of cationic cholesterol; and 0.01 to 50 mol% of cholesterol, more preferably 40 to 60 mol% of ionizable lipids; 5 to 25 mol% of cationic cholesterol; and 5 to 25 mol% of cholesterol, most preferably 45 to 55 mol% of ionizable lipids; 15 to 25 mol% of cationic cholesterol; and 15 to 25 mol% of cholesterol.

[0048] Furthermore, the lipid nanoparticles preferably further comprise 0.01 to 20 mol %, more preferably 5 to 15 mol %, most preferably 8 to 12 mol % of a helper lipid (phospholipid).

[0049] Furthermore, the lipid nanoparticles preferably further comprise 0.01 to 10 mol %, more preferably 0.01 to 5 mol %, most preferably 1 to 2 mol % of PEG lipid.

[0050] The lipid nanoparticles according to the present invention can have a zeta potential of 5mV to 15mV and a particle size (Z mean value) of 50nm to 250nm. The particle size (Z mean value) and zeta potential of lipid nanoparticles are measured using Zetasizer Pro (Malvern Instruments, United Kingdom). Particle size is measured after dilution with 1X DPBS, and zeta potential measurement is performed using 10mM NaCl. Based on the measurement results, the particle size is similar, but the composition including cationic cholesterol shows a higher zeta potential.

[0051] In the lipid nanoparticles according to the present invention, the cationic cholesterol may be, but is not limited to, at least one selected from the group consisting of: AC-cholesterol (3β-[N-(aminoethane)carbamoyl]-cholesterol), MC-cholesterol (3β-[N-(N'-methylaminoethane)carbamoyl]-cholesterol), DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), DMHAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)dimethylammonio]propyl]carbamate]), DMP-cholesterol (3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), AC-cholesterol (3-[[3-(dimethylamino)propyl]carbamate]), MHAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)methylamino]propyl]carbamate]), HAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)amino]propyl]carbamate]), OH-cholesterol (N-[2-[(2-hydroxyethyl)amino]ethyl]-(3β)-cholester-5-ene-3-carboxamide) and OH-C-cholesterol (3-[N-[2-[(2-hydroxyethyl)amino]ethyl]carbamate]).

[0052] In the lipid nanoparticles according to the present invention, the ionizable lipid can be, but is not limited to, at least one selected from the group consisting of: DLin-DMA (1,2-dilinoleyloxy-N,N-dimethylaminopropane), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-triacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate), DODAP (1,2-dioleoyl-3-dimethylammoniumpropane), DODMA (N,N-dimethyl-(2,3-dioleyloxy)propylamine), cKK-E12 shown in the following chemical formula 1, C12-200 shown in the following chemical formula 2, ATX-002 shown in the following chemical formula 3, and SM-102 shown in the following chemical formula 4.

[0053] [Chemical formula 1]

[0054]

[0055] [Chemical formula 2]

[0056]

[0057] [Chemical formula 3]

[0058]

[0059] [Chemical formula 4]

[0060]

[0061] In addition, in the lipid nanoparticles according to the present invention, the ionizable lipid may be, but is not limited to, at least one selected from the group consisting of: 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dioctadecylamidoglycylcarboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67), biguanidinium-spermidine-cholesterol (BGTC), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)bis(dodecanol) Alkane-2-ol (C12-200), N-tert-butyl-N'-tetradecyl-amino-propionamidine (bis-C14-amidine), dimethyl dioctadecyl ammonium bromide (DDAB), N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), dioleyloxypropyl-3-dimethylhydroxyethylammonium bromide (DORIE ), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyltrimethylpropaneammonium chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA) and aminopropyl-dimethyl-bis(dodecyloxy)-propylammonium bromide (GAP-DLRIE).

[0062] Furthermore, in the lipid nanoparticles according to the present invention, the ionizable lipid is more preferably a lipid containing a tertiary amine.

[0063] In the lipid nanoparticles according to the present invention, the auxiliary lipid (phospholipid) can be, but is not limited to, at least one selected from the group consisting of: DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPI (1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPI (1,2-distearoyl-sn-glycero-3-phosphoinositol) and DLPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine).

[0064] In addition, in the lipid nanoparticles according to the present invention, the auxiliary lipid (phospholipid) can be but is not limited to at least one selected from the group consisting of: 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 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-cholesteryl hemisuccinyl-sn-glycero-3-phosphocholine (OChemsPC), 1-hexadecyl-sn-glycero-3-phosphocholine (C16 Lyso PC), 1,2-dilinolenoyl-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 (4ME 16:0PE), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinolenoyl-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), palmitoyl phosphatidylethanolamine (POPE), distearoyl-phosphatidyl-ethanolamine (DSPE), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), 1-stearoyl-2-oleoyl-phosphatidylcholine (SOPC), sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyloleoylphosphatidylcholine, lysophosphatidylcholine and lysophosphatidylethanolamine (LPE).

[0065] In the lipid nanoparticles according to the present invention, the PEG lipid may be, but is not limited to, at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

[0066] In addition, the PEG lipid preferably includes a PEG moiety having a size of 100 Da to 20 kDa, and is more preferably, but not limited to, at least one selected from the group consisting of DMG-PEG2000 (1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000), DSPE-PEG2000 (1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)-2000], and ceramide-PEG2000 (N-palmitoyl-sphingosine-1-{succinyl[methoxy(polyethylene glycol))2000]}).

[0067] In another aspect, the present invention relates to a lipid nanoparticle composition comprising a lipid nanoparticle and a nucleic acid.

[0068] In the lipid nanoparticle composition according to the present invention, the nucleic acid may be at least one selected from the group consisting of mRNA, siRNA, aiRNA, miRNA, dsRNA, shRNA, lncRNA, saRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, tRNA, piRNA, circRNA, antisense oligonucleotide, ribozyme, PNA and DNAzyme, and most preferably mRNA, but not limited thereto.

[0069] The N / P ratio of the lipid nanoparticle composition according to the present invention is preferably 2 to 12, more preferably 4 to 8. The N / P ratio is determined by dividing the molar number N of protonatable amine groups included in the lipid nanoparticle composition by the molar number P of phosphate groups in the mRNA.

[0070] In still another aspect, the present invention relates to a vaccine comprising the lipid nanoparticle composition.

[0071] In the present invention, the term "vaccine" is understood as a preventive or therapeutic substance that provides at least one antigen, preferably an immunogen. The antigen or immunogen can be derived from any substance suitable for vaccination. For example, the antigen or immunogen can be derived from a pathogen, such as a bacterial or viral particle, or a tumor or cancerous tissue. The antigen or immunogen stimulates the body's adaptive immune system, which provides an adaptive immune response.

[0072] In yet another aspect, the present invention relates to a method for preventing or treating a disease, the method comprising administering a lipid nanoparticle composition to a subject.

[0073] In another aspect, the present invention relates to the use of the lipid nanoparticle composition for preventing or treating a disease.

[0074] In still another aspect, the present invention relates to use of the lipid nanoparticle composition for the manufacture of a medicament for preventing or treating a disease.

[0075] As used herein, the term "prevention" refers to any action of preventing the onset of a disease or delaying the progression of a disease by administering a composition. In addition, the term "treatment" refers to any action of improving disease symptoms or alleviating or eliminating symptoms by administering a composition.

[0076] As used herein, the term "subject" refers to a mammal, preferably a human, suffering from or at risk of a condition or disease that can be alleviated, inhibited or treated by administering a composition according to the present invention.

[0077] As used herein, the term "administration" refers to the act of introducing the composition of the present invention into a subject by any appropriate method, and the route of administration may include various oral or parenteral routes, as long as the drug can reach the target tissue. Parenteral administration may be intramuscular (IM), intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intratumor (IT), intradermal (ID) or intracerebral injection, and the dosage may vary according to the patient's state and weight, the severity of the disease, the type of drug, and the route and time of administration, but may be appropriately selected by those skilled in the art.

[0078] The dosage of the composition of the present invention for human administration may vary according to the patient's age, weight, sex, dosage form, health condition and severity of the disease.

[0079] In the present invention, when formulating the composition, a diluent or excipient such as a filler, a bulking agent, a binder, a wetting agent, a disintegrant, a surfactant, etc. can generally be used. Preparations for parenteral administration may include sterile aqueous solutions, non-aqueous solvents, suspending agents, emulsions, lyophilized preparations, suppositories, etc. Examples of non-aqueous solvents or suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate, etc. Examples of the base ingredients of suppositories may include Witepsol, Macrogol, Tween 61, cocoa butter, laurin butter, glycerin, gelatin, etc.

[0080] The present invention can be better understood by the following examples. These examples are set forth only to illustrate the present invention and should not be construed as limiting the scope of the present invention, as will be apparent to those skilled in the art.

[0081] Example 1: Preparation of lipid nanoparticles containing cationic cholesterol and confirmation of their properties

[0082] Ionizable lipids, cholesterol, cationic cholesterol, phospholipids and PEG lipids were dissolved in ethanol at a molar ratio of 50:19.25:19.25:10:1.5, and then mixed with mRNA dissolved in citrate buffer (pH 4.0, 50 mM) at a volume ratio of 1:3. For the control, ionizable lipids, cholesterol, phospholipids and PEG lipids were used at a molar ratio of 50:38.5:10:1.5, excluding cationic cholesterol ( Figure 1 The cationic cholesterol used in the examples is DC-cholesterol (AvantiPolar Lipids), HAPC-cholesterol (GLPBIO, USA), DMPAC-cholesterol (GLPBIO, USA) or DMHAPC-cholesterol (GLPBIO, USA) ( Figure 2 ), the ionizable lipids used were D-Lin-MC3-DMA (MedChemExpress, USA), D-Lin-DMA (MedChemExpress, USA), D-Lin-KC2-DMA (MedChemExpress USA), cKK-E12 (Organix, USA), C12-200 (Organix, USA), ATX-002 (Organix, USA), DODAP (Avanti Polar Lipids, USA), DOMDA (Avanti Polar Lipids, USA) or SM-102 (Xiamen Sinopeg Biotech, China) ( Figure 3 ). In addition, the phospholipid used was 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids, USA), and the PEG lipid used was 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (Avanti Polar Lipids, USA). As mRNA, Firefly luciferase mRNA (TriLink, USA), Erythropoietin mRNA (TriLink, USA), HA mRNA (produced in-house) or self-amplifying mRNA (produced in-house).

[0083] To produce lipid nanoparticles, use Ignite TM (Precision Nanosystems, Inc. Canada), and the total flow rate was set to 12 mL / min. Ultracentrifugal filters MWCO 10 kDa (Millipore, USA) were used for ethanol removal, buffer exchange and concentration of the prepared lipid nanoparticles. 1X DPBS (Thermo Scientific, USA) was used for dilution and buffer exchange.

[0084] In order to analyze the characteristics of the lipid nanoparticles formed, the particle size (Z average value) and zeta potential were measured using Zetasizer Pro (Malvern Instruments, UK). The particle size was measured after dilution with 1X DPBS, and zeta potential was measured using 10mM NaCl. The measurement results are shown in Table 1 below. The amount of mRNA was measured using Ribogreen RNA assay kit (Invitrogen, USA).

[0085] [Table 1]

[0086]

[0087] For lipid nanoparticles formed by the addition of cationic cholesterol, the particle size was similar, and the cationic zeta potential was increased.

[0088] Example 2: Evaluation of drug delivery profile and duration following intramuscular injection of lipid nanoparticles comprising cationic cholesterol

[0089] In order to evaluate the drug delivery profile after local administration of the substance prepared in Example 1, lipid nanoparticles corresponding to 0.25 mg / kg mRNA were injected intramuscularly into the thigh of Balb / c mice (male, 5 weeks old) (injection volume: 50 μL). 6 hours or 7 days after injection, 150 mg / kg of D-luciferin (Perkin Elmer, USA) was intraperitoneally administered thereto, and 15 minutes later, bioluminescence was measured using IVIS Lumina XR (Perkin Elmer, USA).

[0090] The luminescence levels of the administered muscle and liver tissues were measured using whole body images, and the results of drug delivery distribution after intramuscular injection of lipid nanoparticles without / with cationic cholesterol using 9 ionizable lipids into mice are shown in Figure 4 Specifically, mice were injected intramuscularly with lipid nanoparticles using 9 ionizable lipids (D-Lin-MC3-DMA, D-Lin-DMA, D-Lin-KC2-DMA, cKK-E12, C12-200, ATX-002, DODAP, DODMA, SM-102) with encapsulated luciferase mRNA, and after 6 hours or 7 days, luminescence images were measured to evaluate the distribution and kinetics of protein expression. The luminescence signals of muscle and liver tissues at 6 hours and 7 days ( Figure 5 ) and the physical properties of lipid nanoparticles are presented in the figure ( Figure 6 ).like Figure 5 As shown, for conventional lipid nanoparticles without cationic cholesterol, a large amount of protein expression was observed in the liver in addition to the administration site. However, when cationic cholesterol was included, protein expression was restricted to muscle tissue. In addition, at day 7 after injection, protein expression by conventional lipid nanoparticles was greatly reduced, while lipid nanoparticles with cationic cholesterol continued to maintain a significant level of protein expression.

[0091] The above results were obtained regardless of the type of ionizable lipids tested, indicating that the composition ratios can be generally applied to lipid nanoparticles using ionizable lipids.

[0092] Example 3: Evaluation of drug delivery profile after intramuscular injection of lipid nanoparticles according to the ratio of cationic cholesterol

[0093] To confirm the optimal composition of cationic cholesterol, protein expression profiles were evaluated with different ratios of cationic cholesterol, as shown in Table 2. The ionizable lipid used was D-Lin-MC3-DMA.

[0094] [Table 2]

[0095]

[0096] Six hours after intramuscular injection (0.25 mg / kg mRNA) into the thigh of Balb / c mice (male, 5 weeks old), bioluminescence was measured using IVIS Lumina XR, and the results are shown in Figure 7 In particular, lipid nanoparticles containing luciferase mRNA using D-Lin-MC3-DMA were encapsulated by including cationic cholesterol at different ratios, and luminescence images were measured 6 hours after intramuscular injection into mice. The measurement results of luminescence in the muscle tissue of the administration are shown in Figure 8 The results of the luminescence measurements of liver tissue are shown in Fig. 9 , and the luminescence ratio of muscle tissue to liver tissue is shown in Fig.10 It was confirmed that protein expression in liver tissue decreased as the ratio of cationic cholesterol increased, and it was also confirmed that protein expression in muscle tissue decreased when the ratio of cationic cholesterol exceeded a specific ratio (D). Therefore, a composition ratio (D; cholesterol: cationic cholesterol = 1:1) that can maintain the protein expression level in muscle tissue and minimize systemic delivery was determined.

[0097] Example 4: Evaluation of drug delivery profile after intramuscular injection of lipid nanoparticles according to the type of cationic cholesterol

[0098] In order to confirm the drug delivery distribution after intramuscular injection according to the type of cationic cholesterol, lipid nanoparticles were prepared and evaluated using the composition ratio selected in Example 3 (ionizable lipid: cationic cholesterol: cholesterol: phospholipid: PEG lipid = 50: 19.3: 19.3: 10: 1.5 molar ratio). By including different types of cationic cholesterol, lipid nanoparticles containing luciferase mRNA using D-Lin-MC3-DMA were prepared.

[0099] Bioluminescence was measured using IVIS Lumina XR 6 hours or 7 days after intramuscular injection (0.25 mg / kg mRNA) into the thigh of Balb / c mice (male, 5 weeks old), and the results are shown in Fig.11 In particular, the results of the measurement of luminescence in the muscle tissue of the drug administration are shown in Fig.12 The results of the luminescence measurements of liver tissue are shown in Fig.13 The luminescence ratio of muscle tissue to liver tissue is shown in Fig.14 , and the results of the measurement of protein expression levels in muscle tissue and liver tissue 6 hours and 7 days after injection are shown in Fig.15 It was demonstrated that protein expression in liver tissue was reduced by the inclusion of cationic cholesterol, regardless of the type of cationic cholesterol evaluated (HAPC-cholesterol, DMPAC-cholesterol, DMHAPC-cholesterol).

[0100] Example 5: Use of influenza vaccine in intramuscular injection (IM) route

[0101] In order to confirm the suitability of lipid nanoparticles including cationic cholesterol for vaccines, lipid nanoparticles including influenza HAmRNA and lipid nanoparticles including cationic cholesterol were prepared. Here, the ionizable lipid used is MC3. LNPs including 2 μg or 10 μg of HA mRNA were injected intramuscularly into the right thigh of Balb / c mice (female, 6 weeks old) using an insulin syringe, twice at an interval of 2 weeks. Serum was obtained by retroorbital bleeding 2, 4, 6 and 9 weeks after the second immunization, and HA antigen-specific IgG levels were measured by ELISA to confirm antigen-specific immunogenicity. As a result, at both 2 μg and 10 μg, it was confirmed that the response induced by lipid nanoparticles including cationic cholesterol was similar to that induced by lipid nanoparticles ( Fig.16 ).

[0102] Example 6: Confirmation of suitability for subcutaneous (SC) route

[0103] To evaluate the drug delivery profile after subcutaneous injection of SM-102LNP in the material prepared in Example 1, lipid nanoparticles corresponding to 0.25 mg / kg mRNA were subcutaneously (sc) injected into the nape of the neck of Balb / c mice (male, 6 weeks old) (injection volume: 50 μL). 6 hours, 5 days, 10 days or 21 days after injection, 150 mg / kg of D-luciferin (Perkin Elmer, USA) was intraperitoneally administered, and 15 minutes later, bioluminescence was measured using IVIS Lumina XR (Perkin Elmer, USA). Images of the administration site and the measurement results of the luminescence value are shown in Fig.17 It was confirmed that when lipid nanoparticles including cationic cholesterol were used, the duration of protein expression was continuously maintained up to 10 days even when injected subcutaneously, as was the result after intramuscular injection in Example 2, compared to when conventional lipid nanoparticles were used.

[0104] Example 7: Confirmation of Minimized Systemic Delivery of Lipid Nanoparticles Including Cationic Cholesterol

[0105] To evaluate systemic exposure protein levels after intramuscular injection of SM-102LNP in the material prepared in Example 1, lipid nanoparticles corresponding to 2 mg / kg mRNA (erythropoietin mRNA) were injected intramuscularly (im) into Balb / c mice (male, 6 weeks old) (injection volume: 50 μL). Blood was collected at 3 hours, 1 day, 4 days, 7 days and 14 days after injection, and serum erythropoietin concentrations were measured. The results of blood EPO concentration measurements over time are shown in Fig.18 The amount of protein in the blood was observed to be approximately 10 times smaller when lipid nanoparticles including cationic cholesterol were used than when conventional lipid nanoparticles were used, indicating that the amount of systemically exposed protein can be reduced when cationic cholesterol is included.

[0106] Example 8: Comparison of the effects of using cationic substances other than cholesterol

[0107] In order to compare the effect of using cationic lipids other than cationic cholesterol, lipid nanoparticles were prepared and evaluated using the composition ratio selected in Example 3 (ionizable lipid: cationic cholesterol: cholesterol: phospholipid: PEG lipid = 50: 19.3: 19.3: 10: 1.5 molar ratio). Here, the cationic lipid used was DOTAP. In order to evaluate the distribution of drug delivery after intramuscular injection, lipid nanoparticles corresponding to 0.25 mg / kg mRNA were injected intramuscularly (0.25 mg / kg mRNA) into the thighs of Balb / c mice (male, 6 weeks old), and after 6 hours, 7 days and 14 days, bioluminescence was measured using IVIS Lumina XR, and the results are shown in Fig.19 Even when DOTAP was used instead of cationic cholesterol, the distribution of protein expression was similarly restricted to the administration site, but the duration of protein expression at the administration site was relatively reduced (e.g., about 7 days for DOTAP vs. about 14 days for cationic cholesterol). Therefore, cholesterol-based cationic substances were shown to be necessary for lipid nanoparticles according to the present invention.

[0108] Example 9: Confirmation of suitability for self-amplifying RNA

[0109] To evaluate the suitability of SM-102LNP in the material prepared in Example 1 for self-amplifying RNA (saRNA), lipid nanoparticles corresponding to 0.1 mg / kg mRNA were injected intramuscularly (im) into Balb / c mice (male, 6 weeks old) (injection volume: 50 μL). Bioluminescence was measured using IVIS Lumina XR (Perkin Elmer, USA) 6 hours, 7 days, 14 days and 21 days after injection. Images of the administration site and the measurement results of the luminescence value are shown in Fig. 20 It was confirmed that when lipid nanoparticles including cationic cholesterol were used, the duration of protein expression was increased compared to when conventional lipid nanoparticles were used, even when applied to saRNA, as was the result when applied to conventional mRNA (Example 1).

[0110] Industrial Applicability

[0111] According to the present invention, the lipid nanoparticles for nucleic acid delivery effectively minimize the systemic delivery of the drug during local administration and only deliver the drug to the administration site. This reduces the amount of drug lost outside the administration site and maintains the dosage of the target site. In addition, after local administration according to the lipid nanoparticles of the present invention, the duration of protein expression at the muscle site can be increased, which potentially reduces the therapeutic dose.

[0112] Although the specific embodiments of the present invention have been disclosed in detail above, it will be apparent to those skilled in the art that this description is only a preferred exemplary embodiment and should not be interpreted as limiting the scope of the present invention. Therefore, the essential scope of the present invention will be limited by the appended claims and their equivalents.

Claims

1. A lipid nanoparticle (LNP), comprising: (A) Ionizable lipids; (B) cationic cholesterol; (C) Cholesterol; (D) helper lipids; as well as (E) PEG lipids (polyethylene glycol lipids), Wherein, the molar ratio of (B) cationic cholesterol to (C) cholesterol is 1:0.1 to 1:

10.

2. The lipid nanoparticle according to claim 1, wherein The molar ratio of (B) cationic cholesterol to (A) ionizable lipid ranged from 1:0.5 to 1:

20.

3. The lipid nanoparticle according to claim 1, wherein The molar ratio of (B) cationic cholesterol to (D) helper lipid ranged from 1:0.2 to 1:

10.

4. The lipid nanoparticle according to claim 1, wherein The molar ratio of (B) cationic cholesterol to (E) PEG lipid ranged from 1:0.01 to 1:

1.

5. The lipid nanoparticle according to claim 1, wherein The lipid nanoparticles include 30 to 80 mol% of the ionizable lipid; 0.01 to 50 mol% of the cationic cholesterol; and 0.01 to 50 mol% of the cholesterol.

6. The lipid nanoparticle according to claim 5, wherein The lipid nanoparticles further include 0.01 to 20 mol % of the helper lipid.

7. The lipid nanoparticle according to claim 5, wherein The lipid nanoparticle further comprises 0.01 to 10 mol % of the PEG lipid.

8. The lipid nanoparticle according to claim 1, wherein The lipid nanoparticles have a zeta potential of 5 mV to 15 mV.

9. The lipid nanoparticle according to claim 1, wherein The lipid nanoparticles have a particle size (Z-average) of 50 nm to 250 nm.

10. The lipid nanoparticle according to claim 1, wherein The cationic cholesterol is at least one selected from the group consisting of AC-cholesterol (3β-[N-(aminoethane)carbamoyl]-cholesterol), MC-cholesterol (3β-[N-(N'-methylaminoethane)carbamoyl]-cholesterol), DC-cholesterol (3β-[N-(N',N'-dimethylaminoethane)carbamoyl]-cholesterol), DMHAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)dimethylammonio]propyl]carbamate]), DMPAC-cholesterol (3- [[3-(dimethylamino)propyl]carbamate]), MHAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)methylamino]propyl]carbamate]), HAPC-cholesterol (3-[N-[3-[(2-hydroxyethyl)amino]propyl]carbamate]), OH-cholesterol (N-[2-[(2-hydroxyethyl)amino]ethyl]-(3β)-cholester-5-ene-3-carboxamide) and OH-C-cholesterol (3-[N-[2-[(2-hydroxyethyl)amino]ethyl]carbamate]).

11. The lipid nanoparticle according to claim 1, wherein The ionizable lipid is at least one selected from the group consisting of DLin-DMA (1,2-dilinoleyloxy-N,N-dimethylaminopropane), DLin-KC2-DMA (2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane), DLin-MC3-DMA ((6Z,9Z,28Z,31Z)-heptatriacontane-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butyrate), DODAP (1,2-dioleoyl-3-dimethylammoniumpropane), DODMA (N,N-dimethyl-(2,3-dioleyloxy)propylamine), cKK-E12 represented by the following chemical formula 1, C12-200 represented by the following chemical formula 2, ATX-002 represented by the following chemical formula 3, and SM-102 represented by the following chemical formula 4: [Chemical formula 1] [Chemical formula 2] [Chemical formula 3] [Chemical formula 4] 12. The lipid nanoparticle according to claim 1, wherein The helper lipid is at least one selected from the group consisting of: DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), DOPI (1,2-dioleoyl-sn-glycero-3-phospho-(1'-myo-inositol)), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), DSPC (1,2-distearoyl-sn-glycero-3-phosphocholine), DSPI (1,2-distearoyl-sn-glycero-3-phosphoinositol) and DLPC (1,2-dilinoleoyl-sn-glycero-3-phosphocholine).

13. The lipid nanoparticle according to claim 1, wherein The PEG lipid is at least one selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol.

14. A lipid nanoparticle composition comprising the lipid nanoparticle according to any one of claims 1 to 13, and a nucleic acid.

15. The lipid nanoparticle composition according to claim 14, wherein The nucleic acid is at least one selected from the group consisting of mRNA, siRNA, aiRNA, miRNA, dsRNA, shRNA, lncRNA, saRNA, rRNA, RNA, DNA, cDNA, plasmid, aptamer, tRNA, piRNA, circRNA, antisense oligonucleotide, ribozyme, PNA and DNAzyme.

16. The lipid nanoparticle composition according to claim 14, wherein The composition has an N / P ratio of 2 to 12.

17. A vaccine comprising the lipid nanoparticle composition according to claim 14.

18. A method for preventing or treating a disease, comprising administering the lipid nanoparticle composition according to claim 14 to a subject.

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