Lipid nanoparticles using cationic cholesterol for topical delivery for nucleic acid delivery

Abstract

The present invention relates to lipid nanoparticles using cationic cholesterol for local delivery for nucleic acid delivery, which can minimize side effects due to systemic delivery of a drug upon local administration and obtain an effect of limiting protein expression at the administration site. Further, there is an advantage that the duration of protein expression at the administration site can be increased, and it can be usefully used in the technical field related to nucleic acid therapeutic agents.

Description

Technical Field

[0001] The present invention relates to lipid nanoparticles for nucleic acid delivery, and more particularly to lipid nanoparticles for topical delivery containing cationic cholesterol, a lipid nanoparticle composition containing the nanoparticles and nucleic acids, and a method for preventing or treating diseases using the same.

Background Art

[0002] Recently, with the emergence of COVID-19 vaccines, the expectations and importance for mRNA-based vaccines have been increasing. Two approved vaccines (Spikevax TM , Comirnaty TM ) both use lipid nanoparticles (LNP) as carriers and are working to continuously improve efficacy through optimization of the LNP composition and development of further ionizable lipids.

[0003] However, in the case of generally intramuscularly injected LNP, it has the characteristic of spreading not only to the injected muscle site through lymphatic vessels and blood but also throughout the body, which raises the risk of systemic reactions. In particular, in the case of COVID-19 vaccines, there is a possibility that the vaccine transmitted to some hearts causes myocarditis that inflames the myocardium. Therefore, for the development of preventive vaccines for general healthy people after the end of the pandemic, carriers with an improved safety profile are required.

[0004] To improve such limitations, the present invention attempted to develop a carrier that expresses mRNA only at the injection site, minimizes systemic distribution, and reduces systemic reactions. In the future, it is expected to be applicable to local administrations such as intratumoral injection, intradermal injection, and intracerebral injection in addition to intramuscular injection. By limiting mRNA expression only to the administration site, it is expected to enhance stability and at the same time maintain the administration dose by reducing the amount that disappears outside the administration site.

[0005] Furthermore, according to the present invention, it was confirmed that the duration of protein expression in the muscle site was increased. Generally, after intramuscular injection, the decrease in protein expression at the muscle site shows zero-order kinetics, but according to the present invention, it was shown that the duration of protein expression can be significantly increased. Thereby, it is expected that the therapeutic dose can be reduced.

[0006] In summary, the present invention relates to lipid nanoparticles containing cationic cholesterol, and the lipid nanoparticles and compositions containing the same relate to a carrier that 1) limits protein expression to a local site to enhance safety and 2) increases the duration of protein expression.

[0007] The information described in this background art section is only for deepening the understanding of the background of the present invention, and may not include information forming prior art that is already known to those with ordinary knowledge in the technical field to which the present invention pertains.

Summary of the Invention

Problems to be Solved by the Invention

[0008] An object of the present invention is to provide lipid nanoparticles and a lipid nanoparticle composition containing the lipid nanoparticles and nucleic acids, wherein upon local administration, the delivery of nucleic acids is limited only to the administration site, thereby increasing safety through a reduction in systemic side effects and increasing the duration of the onset of effect at the administration site.

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

[0010] Another object of the present invention is to provide a method for preventing or treating a disease, which includes the step of administering the lipid nanoparticle composition to an individual.

[0011] Another object of the present invention is to provide the use of the lipid nanoparticle composition for preventing or treating a disease.

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

Means for Solving the Problems

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

[0014] The present invention also provides a lipid nanoparticle composition comprising the lipid nanoparticle and a nucleic acid.

[0015] The present invention also provides a vaccine comprising the lipid nanoparticle composition.

[0016] The present invention also provides a method for preventing or treating a disease, comprising the step of administering the lipid nanoparticle composition to an individual.

[0017] The present invention also provides the use of the lipid nanoparticle composition for preventing or treating a disease.

[0018] The present invention also provides the use of the lipid nanoparticle composition for the manufacture of a medicament for preventing or treating a disease.

Brief Description of the Drawings

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Modes for Carrying Out the Invention

[0039] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the technical field to which this invention belongs. Generally, the nomenclature used herein is well known and commonly used in the relevant technical field.

[0040] In one embodiment of the present invention, it was expected that the administration dose could be maintained by limiting mRNA expression only to the administration site and reducing the amount that disappears outside the administration site. Also, it was confirmed that the duration of protein expression at the intramuscular or subcutaneous administration site increases by administering the lipid nanoparticle composition of the present invention.

[0041] Therefore, in one aspect, the present invention relates to a lipid nanoparticle (LNP) comprising (A) an ionizable lipid, (B) a cationic cholesterol, (C) a cholesterol, (D) a helper lipid, and (E) a polyethylene glycol lipid, wherein the molar ratio of the (B) cationic cholesterol to the (C) cholesterol is from 1:0.1 to 1:10.

[0042] In the lipid nanoparticles according to the present invention, the molar ratio of the (B) cationic cholesterol:(C) cholesterol is 1:0.1 to 1:10, preferably 1:0.2 to 1:5, more preferably 1:0.33 to 1:3, and most preferably may be 1:1. At this time, when the composition of the cationic cholesterol increases, the protein expression in the liver tissue decreases, and at the same time, when the composition of the cationic cholesterol exceeds a specific ratio, it has been confirmed that the protein expression in the muscle tissue also decreases.

[0043] In the lipid nanoparticles according to the present invention, the molar ratio of the (B) cationic cholesterol:(A) ionizable lipid is 1:0.5 to 1:20, preferably 1:1 to 1:10, more preferably 1:2 to 1:5, and most preferably may be 1:2.59.

[0044] In the lipid nanoparticles according to the present invention, the molar ratio of the (B) cationic cholesterol:(D) helper lipid is 1:0.2 to 1:10, preferably 1:0.33 to 1:5, more preferably 1:0.5 to 1:2, and most preferably may be 1:0.518 (1.93:1).

[0045] In the lipid nanoparticles according to the present invention, the molar ratio of the (B) cationic cholesterol:(E) PEG lipid is 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 may be 1:0.078 (12.87:1).

[0046] In addition, the lipid nanoparticles according to the present invention preferably contain 30 to 80 mol% of ionizable lipid, 0.01 to 50 mol% of cationic cholesterol, and 0.01 to 50 mol% of cholesterol, more preferably 40 to 60 mol% of ionizable lipid, 5 to 25 mol% of cationic cholesterol, and 5 to 25 mol% of cholesterol, and most preferably may contain 45 to 55 mol% of ionizable lipid, 15 to 25 mol% of cationic cholesterol, and 15 to 25 mol% of cholesterol.

[0047] In addition, the lipid nanoparticles preferably further contain 0.01 to 20 mol% of helper lipid (phospholipid), more preferably 5 to 15 mol%, and most preferably may be 8 to 12 mol%.

[0048] In addition, the lipid nanoparticles preferably further contain 0.01 to 10 mol% of PEG lipid, more preferably 0.01 to 5 mol%, and most preferably may be 1 to 2 mol%.

[0049] The lipid nanoparticles according to the present invention may have a zeta potential of 5 mV to 15 mV and a particle size (Z-average) of 50 nm to 250 nm. The particle size (Z-average) and zeta potential of the lipid nanoparticles were measured using a Zetasizer Pro (Malvern Instruments, United Kingdom). After dilution using 1X DPBS, the particle size was measured, and 10 mM NaCl was used for the zeta potential measurement. As a result of the measurement, the particle sizes were similar, but in the case of the composition containing cationic cholesterol, a more increased zeta potential value was obtained.

[0050] In the lipid nanoparticles according to the present invention, the cationic cholesterol may be one or more 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)-cholest-5-ene-3-carboxamide), and OH-C-cholesterol (3-[N-[2-[(2-hydroxyethyl)amino]ethyl]carbamate]), but is not limited thereto.

[0051] In the lipid nanoparticles according to the present invention, the ionizable lipid may be one or more 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)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DODMA (N,N-dimethyl-(2,3-dioleoyloxy)propylamine), cKK-E12 represented by Chemical Formula 1, C12-200 represented by Chemical Formula 2, ATX-002 represented by Chemical Formula 3, and SM-102 represented by Chemical Formula 4, but is not limited thereto.

Chem.

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[0052] In addition, in the lipid nanoparticles according to the present invention, the ionizable lipid may be one or more selected from the group consisting of 1-linoleoyl-2-linoleoyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dilinoleoylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 2,2-dilinoleoyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), dioctadecylamidoglycyl carboxyspermine (DOGS), spermine cholesteryl carbamate (GL-67), bis-guanidinium-spermidine-cholesterol (BGTC), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydecyl)amino)ethyl)(2-hydroxydecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol (C12-200), N-t-butyl-N'-tetradecyl-amino-propionamidine (diC14-amidine), dimethyldioctadecylammonium 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-dioleyloxylpropyl)-N-2-(sperminecarboxamide)ethyl)-N,N-dimethylammonium trifluoroacetate (DOSPA), 1,2-dioleoyltrimethylammonium propane chloride (DOTAP), N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), and aminopropyl-dimethyl-bis(dodecyloxy)-propanaminium bromide (GAP-DLRIE), but is not limited thereto.

[0053] In addition, in the lipid nanoparticles according to the present invention, the ionizable lipid is more preferably a lipid containing a tertiary amine.

[0054] In the lipid nanoparticles according to the present invention, the helper lipid (phospholipid) may be one or more 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), but is not limited thereto.

[0055] In addition, in the lipid nanoparticles according to the present invention, the helper lipid (phospholipid) is 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLPC), 1,2-didecanoyl-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 hemisuccinoyl-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 16:0 PE), 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,It may be one or more selected from the group consisting of 2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG), dipalmitoylphosphatidylglycerol (DPPG), palmitoyloleoylphosphatidylethanolamine (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), but is not limited thereto.,

[0056] In the lipid nanoparticles according to the present invention, the PEG lipid may be one or more selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol, but is not limited thereto.,

[0057] Also, the PEG lipid preferably contains a PEG moiety having a size of 100 Da to 20 kDa, more preferably, it may be 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]}), but is not limited thereto. From another aspect, the present invention also relates to a lipid nanoparticle composition containing the lipid nanoparticles and nucleic acid.

[0058] In the lipid nanoparticle composition according to the present invention, the nucleic acid can be characterized as being 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, it may be mRNA, but is not limited thereto.

[0059] The N / P ratio of the lipid nanoparticle composition according to the present invention is preferably 2 to 12, and more preferably may be 4 to 8. The N / P ratio is calculated by dividing the number of moles of protonatable amine groups contained in the lipid nanoparticle composition by N, and dividing this by P, which is the number of moles of phosphate groups in the mRNA, to calculate the ratio.

[0060] From another aspect, the present invention also relates to a vaccine containing the lipid nanoparticle composition.

[0061] In the present invention, the term "vaccine" is understood as a prophylactic 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 bacteria or virus particles, etc., or from pathogens such as tumors or cancer tissues. The antigen or immunogen stimulates the adaptive immune system of the body to provide an adaptive immune response.

[0062] From another aspect, the present invention relates to a method for preventing or treating a disease, comprising the step of administering the lipid nanoparticle composition to an individual.

[0063] From another aspect, the present invention relates to the use of the lipid nanoparticle composition for preventing or treating a disease.

[0064] From another aspect, the present invention relates to the use of the lipid nanoparticle composition for the manufacture of a medicament for preventing or treating a disease.

[0065] The term "prevention" in the present invention means any act of preventing the onset of a disease or delaying its progression by administering the composition. Also, the term "treatment" used in the present invention means any act of improving the symptoms of a disease, or reducing or curing the symptoms by administering the composition.

[0066] In the present invention, the term "individual" means a mammal that may be alleviated, suppressed, or treated by administering the composition according to the present invention, or that has or is at risk of having a condition or disease, preferably a human.

[0067] As used herein, the term "administering" of the present invention means the act of introducing the composition of the present invention into an individual by any suitable method, and the administration route may be through various oral or parenteral routes as long as the target tissue can be reached. Parenteral administration may be intramuscular (IM), intravenous (IV), subcutaneous (SC), intraperitoneal (IP), intratumoral (IT), intradermal (ID) or intracerebral injection, and the dosage varies depending on the patient's condition and weight, the degree of the disease, the drug form, the administration route and time, but may be appropriately selected by a person of ordinary skill in the art.

[0068] The dosage of the composition of the present invention for administration to humans varies depending on the patient's age, weight, sex, dosage form, health condition and the degree of the disease.

[0069] In the present invention, when formulating the composition, it is usually prepared using diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrants, surfactants and the like. Preparations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories and the like. As the non-aqueous solvent and suspension, propylene glycol, polyethylene glycol, vegetable oils such as olive oil, injectable esters such as ethyl oleate and the like may be used. As the base of the suppository, witepsol, macrogol, tween 61, cacao butter, laurin fat, glycerol, gelatin and the like may be used.

[0070] Hereinafter, the present invention will be described in more detail by way of examples. It will be apparent to those having ordinary knowledge in the art that these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0071] Example 1: Production of Cationic Cholesterol-Containing Lipid Nanoparticles and Confirmation of Their Physical Properties 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 the dissolved mRNA at a volume ratio of 1:3 on citrate buffer (pH 4.0, 50 mM). In the case of the control group, ionizable lipids, cholesterol, phospholipids, and PEG-lipids without cationic cholesterol were used at a molar ratio of 50:38.5:10:1.5 (Figure 1). The cationic cholesterol used in the examples was DC-cholesterol (Avanti Polar Lipids), HAPC-cholesterol (GLPBIO, USA), DMPAC-cholesterol (GLPBIO, USA), or DMHAPC-cholesterol (GLPBIO, USA) (Figure 2), and the ionizable lipids 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). 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC) (Avanti Polar Lipids, USA) was used for the phospholipids, and 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG2000) (Avanti Polar Lipids, USA) was used for the PEG-lipids.mRNA was used, including CleanCap® Firefly Luciferase mRNA (TriLink, USA), CleanCap® Erythropoietin mRNA (TriLink, USA), HA mRNA (in-house production), or self-amplifying mRNA (in-house production).

[0072] NanoAssemblr® Ignite TM (Precision Nanosystems, Inc. Canada) was used for the generation of lipid nanoparticles, and the total flow rate was 12 mL / min. The prepared lipid nanoparticles were subjected to ethanol removal, buffer exchange, and concentration using Amicon® Ultra Centrifugal Filter, MWCO 10 kDa (Millipore, USA). 1X DPBS (Thermo Scientific, USA) was used for dilution and concentration exchange.

[0073] For the physical property analysis of the formed lipid nanoparticles, the particle size (Z-average) and zeta potential were measured using a Zetasizer Pro (Malvern Instruments, United Kingdom). After dilution with 1X DPBS, the particle size was measured, and 10 mM NaCl was used for the zeta potential measurement. The measurement results are shown in Table 1 below. The mRNA content was measured using a Ribogreen RNA assay kit (Invitrogen, USA).

[0074]

Table 1

[0075] In the case of lipid nanoparticles formed by the addition of cationic cholesterol, the particle sizes were similar, showing a more increased cationic zeta potential.

[0076] Example 2: Evaluation of Drug Delivery Distribution and Duration after Intramuscular Injection of Cationic Cholesterol-Containing Lipid Nanoparticles To evaluate the drug delivery distribution after local administration of the substance prepared in Example 1, lipid nanoparticles corresponding to 0.25 mg / kg mRNA were intramuscularly injected into the thigh of Balb / c mice (male, 5-week-old) (dose 50 μL). At 6 hours or 7 days after injection, 150 mg / kg of D-Luciferin (Perkin Elmer, USA) was intraperitoneally administered, and 15 minutes later, bioluminescence was measured using an IVIS Lumina XR (Perkin Elmer, USA) device.

[0077] Using whole-body images, the luminescence values of the administered muscle tissue and liver tissue were measured, and the results of the drug delivery distribution after intramuscular injection of lipid nanoparticles using nine ionizable lipids before and after applying cationic cholesterol are shown in Fig. 4. Specifically, lipid nanoparticles (D-Lin-MC3-DMA, D-Lin-DMA, D-Lin-KC2-DMA, cKK-E12, C12-200, ATX-002, DODAP, DODMA, SM-102) using nine ionizable lipids encapsulating Luciferase mRNA were intramuscularly injected into mice, and luminescence images were measured at 6 hours or 7 days later to evaluate the protein expression distribution and kinetics. The luminescence signals of muscle tissue and liver tissue at 6 hours or 7 days later (Fig. 5) and the physical properties of the lipid nanoparticles (Fig. 6) are shown graphically. As shown in Fig. 5, in the case of lipid nanoparticles without conventional cationic cholesterol, a significant amount of protein expression was observed in the liver in addition to the administration site. However, when cationic cholesterol was included, it was confirmed that protein expression was limited to muscle tissue. Also, on the 7th day after injection, while protein expression significantly decreased in conventional lipid nanoparticles, lipid nanoparticles containing cationic cholesterol sustained a considerable level of protein expression.

[0078] The above results suggest that it is a composition ratio that can be generally applied to lipid nanoparticles using ionizable lipids, regardless of the type of ionizable lipid tested.

[0079] Example 3: Evaluation of Drug Delivery Distribution after Intramuscular Injection of Lipid Nanoparticles with Different Cationic Cholesterol Composition Ratios To confirm the optimal composition of cationic cholesterol, as shown in Table 2, protein expression profiles were evaluated with various ratios of cationic cholesterol. For ionizable lipids, D-Lin-MC3-DMA was used.

[0080] [Table 2]

[0081] 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 an IVIS Lumina XR device, and the results are shown in Figure 7. Specifically, lipid nanoparticles containing Luciferase mRNA were encapsulated using different cationic cholesterol composition ratios, and luminescence images were measured 6 hours after intramuscular injection into mice. The luminescence measurement values in the muscle tissue after liver administration are shown in Figure 8, the luminescence measurement values in the liver tissue are shown in Figure 9, and the luminescence ratio of the liver tissue to the muscle tissue is shown in Figure 10. As the cationic cholesterol composition increased, a tendency for a decrease in protein expression in the liver tissue was confirmed. At the same time, it was confirmed that when the cationic cholesterol composition exceeded a specific ratio (D), protein expression in the muscle tissue also decreased. Therefore, a composition ratio (D; cholesterol:cationic cholesterol = 1:1) that can minimize systemic delivery while maintaining the protein expression level in the muscle tissue was confirmed.

[0082] Example 4: Evaluation of Drug Delivery Distribution after Intramuscular Injection of Lipid Nanoparticles with Different Types of Cationic Cholesterol To confirm the drug delivery distribution after intramuscular injection due to changes in 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). Lipid nanoparticles containing D-Lin-MC3-DMA and Luciferase mRNA were prepared including different types of cationic cholesterol.

[0083] Bioluminescence was measured using an IVIS Lumina XR device 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. Specifically, the luminescence measurement values in the liver-administered muscle tissue are shown in Fig. 12, the luminescence measurement values in the liver tissue are shown in Fig. 13, the luminescence ratio of liver tissue to muscle tissue is shown in Fig. 14, and the measurement results of the protein expression levels in muscle tissue and liver tissue 6 hours and 7 days after injection are shown in Fig. 15. Regardless of the type of cationic cholesterol evaluated (HAPC-cholesterol, DMPAC-cholesterol, DMHAPC-cholesterol), it was confirmed that the inclusion of cationic cholesterol tended to decrease protein expression in liver tissue.

[0084] Example 5: Application of the Intramuscular Injection (IM) Route for Influenza Vaccine To confirm the vaccine applicability of lipid nanoparticles containing cationic cholesterol, lipid nanoparticles containing influenza HA mRNA and lipid nanoparticles containing cationic cholesterol were prepared. MC3 was used as the ionizable lipid. LNP containing 2 μg or 10 μg of HA mRNA was intramuscularly injected into the right thigh of Balb / c mice (female, 6 weeks old) twice at 2-week intervals using an insulin syringe. After the secondary immunization, blood was collected from the orbital sinus at 2, 4, 6, and 9 weeks to obtain serum, and the HA antigen-specific IgG level was measured by ELISA to confirm the antigen-specific immunogenicity. As a result, it was confirmed that the responses induced by the cationic cholesterol-containing lipid nanoparticles were at similar levels to those induced by the lipid nanoparticles in both the 2 μg and 10 μg administrations (Fig. 16).

[0085] Example 6: Confirmation of the Applicability of the Subcutaneous Injection (SC) Route Among the substances prepared in Example 1, to evaluate the drug delivery distribution after subcutaneous injection of SM-102 LNP, lipid nanoparticles corresponding to 0.25 mg / kg mRNA were subcutaneously (s.c.) injected into the larynx of Balb / c mice (male, 6 weeks old) (dose 50 μL). At 6 hours, 5 days, 10 days, or 21 days after injection, 15 minutes after intraperitoneal administration of 150 mg / kg D-Luciferin (Perkin Elmer, USA), bioluminescence was measured using an IVIS Lumina XR (Perkin Elmer, USA) device. The measurement images and luminescence measurement values at the administration site are shown in Fig. 17. Similar to the results after intramuscular injection in Example 2, it was confirmed that when cationic cholesterol was included during subcutaneous injection, the duration of protein expression was continuously maintained up to 10 days compared to conventional lipid nanoparticles.

[0086] Example 7: Confirmation of Minimizing Systemic Delivery of Cationic Cholesterol-Containing Lipid Nanoparticles Among the substances prepared in Example 1, to evaluate the systemic exposure protein level after intramuscular injection of SM-102 LNP, lipid nanoparticles corresponding to 2 mg / kg mRNA (erythropoietin mRNA) were intramuscularly (i.m.) injected into Balb / c mice (male, 6 weeks old) (dose 50 μL). Blood was collected at 3 hours, 1 day, 4 days, 7 days, and 14 days after injection, and the serum erythropoietin concentration was measured. The measured values of blood EPO concentration over time are shown in Fig. 18. It was shown that when cationic cholesterol was included, the amount of protein in the blood was observed at a level less than about 10-fold, indicating that the protein exposed systemically could be reduced when cationic cholesterol was included compared to conventional lipid nanoparticles.

[0087] Example 8: Comparison of Effects when Applying Cationic Substances Other than Cholesterol-Based Ones To compare with 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). DOTAP was used as the cationic lipid. To evaluate the drug delivery distribution after intramuscular injection, bioluminescence was measured using an IVIS Lumina XR device 6 hours, 7 days, and 14 days after intramuscular injection (0.25 mg / kg mRNA) of lipid nanoparticles corresponding to 0.25 mg / kg mRNA into the thigh of Balb / c mice (male, 6 weeks old), and the results are shown in Fig. 19. Even when DOTAP was used instead of cationic cholesterol, the protein expression distribution was limited to the administration site in the same way, but the protein expression period at the administration site decreased relatively (for example, DOTAP about 7 days vs. cationic cholesterol about 14 days). Therefore, it was proved that a cholesterol-based cationic substance is essential for the lipid nanoparticles according to the present invention.

[0088] Example 9: Confirmation of the Applicability of Self-amplifying RNA To evaluate the applicability of the substance produced in Example 1 to self-amplifying RNA (saRNA) of SM-102 LNP, lipid nanoparticles corresponding to 0.1 mg / kg mRNA were intramuscularly (i.m.) injected into Balb / c mice (male, 6 weeks old) (dose 50 μL). Bioluminescence was measured using an IVIS Lumina XR (Perkin Elmer, USA) device 6 hours, 7 days, 14 days, and 21 days after injection. The measurement images and luminescence measurement values at the administration site are shown in Fig. 20. Similar to the results of conventional mRNA application (Example 1), it was shown that when applying saRNA, the protein expression period increased when cationic cholesterol was included compared to conventional lipid nanoparticles.

Industrial Applicability

[0089] The lipid nanoparticles for nucleic acid delivery according to the present invention are effective in minimizing systemic delivery of a drug upon local administration and delivering the drug only to the administration site. This can maintain the administered dose at the target site by reducing the amount of the drug that disappears outside the administration site. Further, upon local administration of the lipid nanoparticles according to the present invention, the duration of protein expression in the muscle site may increase, which can potentially reduce the therapeutic dose.

[0090] As described above, specific parts of the content of the present invention have been described in detail. However, it will be apparent to those with ordinary knowledge in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the present invention. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (A) Ionizable lipid, (B) Cationic cholesterol, (C) Cholesterol, (D) Helper lipid, and (E) Polyethylene glycol lipid A lipid nanoparticle (LNP) comprising the above, 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 is 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 is 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 is 1:0.01 to 1:

1.

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

6. The lipid nanoparticle according to claim 5, further comprising 0.01 to 20 mol% of helper lipid.

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

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

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

10. The cationic cholesterol is one or more 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)-cholest-5-ene-3-carboxamide), and OH-C-cholesterol (3-[N-[2-[(2-hydroxyethyl)amino]ethyl]carbamate]), and the lipid nanoparticle according to claim 1 is characterized by this.

11. The ionizable lipid is one or more 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)-heptatriaconta-6,9,28,31-tetraene-19-yl 4-(dimethylamino)butanoate), DODAP (1,2-dioleoyl-3-dimethylammonium propane), DODMA (N,N-dimethyl-(2,3-dioleoyloxy)propylamine), cKK-E12 represented by Chemical Formula 1, C12-200 represented by Chemical Formula 2, ATX-002 represented by Chemical Formula 3, and SM-102 represented by Chemical Formula 4, the lipid nanoparticles according to claim 1: 【Chemical 1】 [Chemical Formula 2] [Chemical 3] 【Chemical Formula 4】

12. The helper lipid is one or more 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), the lipid nanoparticles according to claim 1.

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

14. A lipid nanoparticle composition comprising the lipid nanoparticles 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 any one or more 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 N / P ratio in the composition is 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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