LIPID NANOPARTICLES FOR IN-VIVO DRUG DELIVERY, AND USES THEREOF

Abstract

The present invention relates to lipid nanoparticles for in vivo drug delivery and uses thereof, and the lipid nanoparticle are liver tissue-specific, have excellent biocompatibility and can deliver a gene therapeutic agent with high efficiency, and thus it can be usefully used in related technical fields such as lipid nanoparticle mediated gene therapy.

Claims

1. A lipid nanoparticle comprising an ionizable lipid in which a 6-membered heterocyclic amine and an alkyl-epoxide are bonded; a phospholipid; cholesterol; and a lipid-PEG (polyethyleneglycol) conjugate.

2. The lipid nanoparticle according to claim 1, wherein the 6-membered heterocyclic amine is a chain or non-chain amine comprising a tertiary amine.

3. The lipid nanoparticle according to claim 1, wherein the 6-membered heterocyclic amine is selected from the group consisting of ##STR00010##

4. The lipid nanoparticle according to claim 1, wherein the alkyl-epoxide is 1,2-epoxydodecane.

5. The lipid nanoparticle according to claim 1, wherein the phospholipid is one or more kinds selected from the group consisting of DOPE, DSPC, POPC, EPC, DOPC, DPPC, DOPG, DPPG, DSPE, Phosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine, POPE, POPC, DOPS, and 1,2-dioleoyl-sn-glycero-3-[phospho-L-serine].

6. The lipid nanoparticle according to claim 1, wherein the lipid in the lipid-PEG conjugate is one or more kinds selected from the group consisting of ceramide, dimyristoylglycerol (DMG), succinoyl-diacylglycerol (s-DAG), distearoylphosphatidylcholine (DSPC), distearoylphosphatidylethanolamine (DSPE), and cholesterol.

7. The lipid nanoparticle according to claim 1, wherein the lipid-PEG conjugate is comprised in 0.25 to 10 mol %.

8. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle comprises the ionizable lipid:phospholipid:cholesterol:lipid-PEG conjugate at a molar ratio of 20 to 50:10 to 30:30 to 60:0.25 to 10.

9. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle has a pKa of 6.0 to 7.0.

10. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle specifically targets liver tissue.

11. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle targets a hepatocyte.

12. The lipid nanoparticle according to claim 1, wherein the lipid nanoparticle targets an LSEC (liver sinusoidal endothelial cell).

13. A method of delivering a drug, comprising administering a composition comprising (1) the lipid nanoparticle according to of claim 1; and (2) the drug, wherein the drug is an anionic drug, a nucleic acid or a combination thereof, to a subject in need of delivering the drug.

14. The method according to claim 13, wherein the anionic drug, nucleic acid or combination thereof is encapsulated inside of the lipid nanoparticle.

15. The method according to claim 13, wherein the lipid nanoparticle has an average diameter of 30 nm to 150 nm.

16. The method according to claim 13, wherein the anionic drug is one or more kinds selected from the group consisting of a peptide, a drug protein, a protein-nucleic acid structure, and an anionic biopolymer-drug conjugate.

17. The method according to claim 13, wherein the nucleic acid is one or more kinds selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosome ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, shRNA, miRNA, ribozyme, PNA and DNAzyme.

18. A method for preventing or treating liver disease comprising administering a composition comprising (1) the lipid nanoparticle according to claim 1; and (2) an anionic drug, a nucleic acid or a combination thereof, to a subject in need of preventing or treating the liver disease.

19. The method according to claim 18, wherein the liver disease is one or more kinds selected from the group consisting of ATTR amyloidosis, hypercholesterolemia, hepatitis B virus infection, acute liver failure, cirrhosis, and liver fibrosis.

20. The method according to claim 18, wherein the lipid nanoparticle has an average diameter of 30 nm to 150 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0131] FIG. 1a shows an exemplary structure of the lipid nanoparticle according to one example, and FIG. 1b shows an image of observing the nanoparticle according to one example by Cryo-TEM.

[0132] FIG. 2 shows the result of .sup.1H NMR (room temperature, 400 MHz) of 246-C10 in CDCl.sub.3.

[0133] FIG. 3a (241-C10 LNP to 243-C10 LNP) and FIG. 3b (244-C10 LNP to 246-C10 LNP) show the result of measuring the fluorescence intensity shown by each lipid nanoparticle in a solution having a range of pH 4.1 to pH 9.6,

[0134] FIG. 4a and FIG. 4b are results showing the intracellular gene delivery efficiency of each nanoparticle. Specifically, FIG. 4a shows the luminescence intensity measured by transforming LNP encapsulating mRNA (luc mRNA) encoding luciferase into HeLa cell and then dissolving the cell, and FIG. 4b shows the luminescence intensity measured by transforming LNP encapsulating luc mRNA to a hepatocyte and then dissolving the cell. In FIG. 4b, +ApoE refers to a group treated by ApoE3, and -ApoE refers to a group untreated by ApoE3.

[0135] FIG. 5a shows in vivo drug delivery distribution in a mouse to which 244-C10 LNP to 246-C10 LNP with Luc mRNA encapsulated is administered, and FIG. 5b shows drug delivery distribution to each organ of the mouse removed from the mouse in which 246-C10 LNP with Luc mRNA encapsulated is administered.

[0136] FIG. 6 shows the drug delivery efficiency of the lipid nanoparticle and the size of the nanoparticle, depending on the content of lipid-PEG comprised in the lipid nanoparticle in the mouse in which 246-C10 LNP comprising lipid-PEG in an amount of 1.0 to 2.5 mol % is administered.

[0137] FIG. 7 shows the result of confirming the hepatocyte targeting possibility according to the concentration of siFVII administered as encapsulated in the lipid nanoparticle through the expression of FVII.

[0138] FIG. 8 shows the size of the lipid nanoparticle and PDI value of the lipid nanoparticle according to the content of the lipid-PEG comprised in the lipid nanoparticle (left table), and shows the result of confirming the in vivo drug delivery efficiency to the hepatocyte through the expression of FVII (right graph).

[0139] FIG. 9 shows the size of the lipid nanoparticle and PDI value of the lipid nanoparticle according to the content of the lipid-PEG comprised in the lipid nanoparticle (left table), and shows the result of confirming the in vivo drug delivery efficiency to the LSEC through the expression of FVIII (right graph).

[0140] FIG. 10 shows the result of measuring the intracellular siRNA delivery efficiency of the lipid nanoparticle comprising ceramide-PEG or DSPE-PEG.

MODE FOR INVENTION

[0141] The present invention will be described in more detail by the following examples, but the scope is not intended to be limited by the following examples.

[0142] Hereinafter, the present invention will be described in more detail by examples. These examples are only for describing the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example 1. Preparation of Ionizable Lipids

Example 1-1. Preparation of Ionizable Lipids

[0143] Ionizable lipids were synthesized by reacting the amine-based compounds of Table 1 below comprising a 6-membered heterocyclic tertiary amine and 1,2-epoxidodecane (hereinafter, C10) (Sigma-Aldrich, USA) at a molar ratio of 1:n (n=primary amine×2+secondary amine×1).

TABLE-US-00001 TABLE 1 Name 241 [00004] 242 [00005] 243 [00006] 244 [00007] 245 [00008] 246 [00009]

[0144] Specifically, each of 241 to 246 amines of the Table 1 and epoxide (C10) were added at a molar ratio of 1:n (n=primary amine×2+secondary amine×1) in a 5 ml vial with a magnetic bar and reacted in a stirrer at 750 rpm, 90° C. for 3 days. Then, after purifying with WELUX fine silica column (Intertec, Korea), the molecular weight of each ionizable lipid produced by the reaction was calculated and they were stored at a concentration of 100 mg/ml using ethanol. The ionizable lipid produced by using 241 amine and C10 was named ‘241-C10’, and other ionizable lipids produced by using other kinds of amines were named ‘used amine name (241 to 246)-C10’ in the same way.

Example 1-2. Confirmation of Produced Ionizable Lipids

[0145] In order to confirm the ionizable lipids produced in the Example 1-1, 1H NMR was performed. Specifically, the ionizable lipid (246-C10) synthesized in Example 1-1 of 5 ug was prepared by diluting in CDCl.sub.3 (sigma, USA) 0.5 ml to 100 mmole concentration. Then, 0.5 ml each was put into a tube for 400 MHz NMR and the top was sealed, and then sealed with parafilm to obtain NMR spectra using Agilent 400 MHZ FT-NMR (Agilent, USA), and the result was shown in FIG. 2. As shown in FIG. 2, it could be seen that the signal representing each functional group of 246-C10 was saturated.

[0146] In addition, in order to confirm the ionizable lipids (241-C10 to 246-C10) prepared in Example 1-1, MS analysis was performed. Specifically, the ionizable lipids were diluted in ethanol at a concentration of 0.5 ppm or less and MS analysis was performed. The equipment used for the analysis was 6230 LC/MS of Agilent Technologies (Palo Alto, USA) and the Zorbax SB-C18 (100 mm×2.1 mm i.d., 3.5 μm) of Agilent Technologies was used for the separation tube, and two solvents of distilled water (A) containing 0.1% formic acid and acetonitrile (B) were gradient eluted. The solvent gradient of the mobile phase was maintained for 4 minutes until the ratio of the organic solvent acetonitrile (B) was initially increased from 30% to 80% for 2 minutes and then the ratio of the organic solvent was lowered to 30% again and stabilized. The flow rate of the mobile phase was 300 μl/min, and then, the injection volume of the analyzer was 2 μl. The result of performing the MS analysis was shown in Table 2 below. As shown in Table 2, it could be confirmed that the measured m/z ratio and calculated m/z ratio of the ionizable lipids were almost identical.

TABLE-US-00002 TABLE 2 Chemical Calculated Observed formula m/z ratio m/z ratio 241-C10 C.sub.32H.sub.66N.sub.2O.sub.2 510.87864 511.5201 242-C10 C.sub.31H.sub.64N.sub.2O.sub.2 496.85206 497.5043 243-C10 C.sub.31H.sub.65N.sub.3O.sub.2 511.8667 513.5186 244-C10 C.sub.42H.sub.87N.sub.3O.sub.3 682.15848 682.6821 245-C10 C.sub.43H.sub.89N.sub.3O.sub.3 696.18506 696.7045 246-C10 C.sub.48H.sub.120N.sub.4O.sub.4 937.5978 937.9383

[0147] From the result, it could be confirmed that the ionizable lipids were well made in Example 1.1.

Example 2. Preparation of Lipid Nanoparticles

Example 2-1. Preparation of Lipid Nanoparticles

[0148] The ionizable lipids (241-C10 to 246-C10) prepared in the Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥99%, sigma, Korea), phospholipid (DSPC) (Avanti, US), and a lipid-PEG conjugate (ceramide-PEG conjugate; C16 PEG2000 Ceramide, Avanti, US) were dissolved in ethanol at a molar ratio of 42.5:13:43:1.5.

[0149] The ethanol in which the ionizable lipids, cholesterol, phospholipid and lipid-PEG were dissolved and acetate buffer were mixed with a microfluid mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min in a volume ratio of 1:3, thereby preparing lipid nanoparticles (LNPs).

Example 2-2. Preparation of Nucleic Acid-Encapsulated Lipid Nanoparticles

[0150] The ionizable lipids (241-C10 to 246-C10) prepared in the Example 1-1, cholesterol (Cholesterol powder, BioReagent, suitable for cell culture, ≥99%, sigma, Korea), phospholipid (DSPC or DOPE) (18:0 PC (DSPC), 18:1 (Δ9-Cis) PE (DOPE), Avanti, US), and a lipid-PEG conjugate (ceramide-PEG conjugate; C16 PEG2000 Ceramid, Avanti, US) were dissolved in ethanol. An RNA therapeutic agent, mRNA (luciferase mRNA; SEQ ID NO: 1) 30 ug was diluted in sodium citrate 0.75 ml, or siRNA (siFVII; SEQ ID NOs: 2 and 3 were mixed at the same molar ratio, or siFVIII; SEQ ID NOs: 4 to 11 were mixed at the same molar ratio, or siLuc: SEQ ID NOs: 12 and 13 were mixed at the same molar ratio) 30 ug was diluted in sodium acetate (50 mM) 0.75 ml to prepare an aqueous phase.

[0151] The used siRNA sequences are as follows: SEQ ID NO: 2 (FVII target siRNA_sense; 5′-GGAUCAUCUCAAGUCUUACdtdt-3′), SEQ ID NO: 3 (FVII target siRNA_antisense; 5′-GUAAGACUUGAGAUGAUCCdtdt-3′), SEQ ID NO: 4 (FVIII target siRNA_sense_1; 5′CUUAUAUCGUGGAGAAUUAdtdt-3′) SEQ ID NO: 5 (FVIII target siRNA_antisense_1; 5′-UAAUUCUCCACGAUAUAAGdtdt-3′), SEQ ID NO: 6 (FVIII target siRNA_sense_2; 5′-UCAAAGGAUUCGAUGGUAUdtdt-3′), SEQ ID NO: 7 (FVIII target siRNA_antisense_2; 5′-AUACCAUCGAAUCCUUUGAdtdt-3′), SEQ ID NO: 8 (FVIII target siRNA_sense_3; 5′-CAAGAGCACUAGUGAUUAUdtdt-3′), SEQ ID NO: 9 (FVIII target siRNA_antisense_3; 5′-AUAAUCACUAGUGCUCUUGdtdt-3′), SEQ ID NO: 10 (FVIII target siRNA_sense_4; 5′-GGGCACCACUCCUGAAAUAdtdt-3′), SEQ ID NO: 11 (FVIII target siRNA_antisense_4; 5′-UAUUUCAGGAGUGGUGCCCdtdt-3′), SEQ ID NO: 12 (siLuc_sense; 5′-AACGCUGGGCGUUAAUCAAdtdt-3′), SEQ ID NO: 13 (siLuc_antisense; 5′-UUGAUUAACGCCCAGCGUUdtdt-3′).

[0152] The aqueous phase (sodium acetate or sodium citrate) in which the organic phase (ethanol) in which the ionizable lipids, cholesterol, phospholipid and lipid-PEG conjugate (hereinafter, lipid-PEG) were dissolved and an RNA therapeutic agent (nucleic acid) were dissolved were mixed through a microfluid mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min, to prepare lipid nanoparticles (LNPs) in which the nucleic acid was encapsulated. (i) In order to prepare a lipid nanoparticle in which mRNA is encapsulated, the ionizable lipid:phospholipid (DOPE):cholesterol:lipid-PEG (C16-PEG2000 ceramide) were dissolved in ethanol at a molar ratio of 26.5:20:52.5 to 51:1.0 to 2.5 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100), and the organic phase and the aqueous phase were mixed so that the mRNA (luciferase mRNA; SEQ ID NO:1):ionizable lipid was at the weight ratio of 1:10, and thereby a lipid nanoparticle was prepared. (ii) In order to prepare a lipid nanoparticle in which siRNA is encapsulated, the ionizable lipid:phospholipid (DSPC):cholesterol:lipid-PEG (C16-PEG2000 ceramide) were dissolved in ethanol at a molar ratio of 42.5:13:44 to 39.5:0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100), and the organic phase and the aqueous phase were mixed so that the siRNA (siFVII; SEQ ID NOs: 2 and 3 were mixed at the same molar ratio, or siFVIII; SEQ ID NOs: 4 to 11 were mixed at the same molar ratio, or siLuc: SEQ ID NOs: 12 and 13 were mixed at the same ratio):ionizable lipid was at the weight ratio of 1:7.5 and thereby a lipid nanoparticle (LNP) was prepared.

[0153] The prepared LNPs were dialyzed against PBS for 16 hours using a 3500 MWCO dialysis cassette to remove ethanol and adjust the body pH and the pH of the nanoparticles.

[0154] The lipid nanoparticles comprising the ionizable lipid ‘241-C10’ were named ‘241-C10 LNP’, and the lipid nanoparticles prepared by using the ionizable lipid comprising amine (including lipid nanoparticles in which a nucleic acid was encapsulated) were named ‘comprised amine name (214 to 246)-C10 LNP’.

Example 2-3. Observation of Nucleic Acid-Encapsulated Lipid Nanoparticles

[0155] The Lipid nanoparticles in which siLuc (SEQ ID NOs: 12 and 13) were encapsulated was prepared by using a ceramide-PEG conjugate (C16-PEG2000 ceramide) as Example 2-2. The prepared lipid nanoparticles (comprising 1.5 mol % of ceramide-PEG conjugate) were loaded on 200 mesh carbon lacey film Cu-grid in an amount of 60 ug based on siRNA concentration and were immersed in ethane liquefied with vitrobot (about −170 degrees or less) and were plunge frozen to be prepared, and then were observed with Cryo-TEM (Tecnai F20, FEI), and the result was shown in FIG. 1b. As shown in FIG. 1b, spherical particles with a solid shape were observed.

Example 3. pKa of Lipid Nanoparticles

[0156] In the present example, pKa of each lipid nanoparticle (LNP) formulated in the Example 2-1 was calculated through In vitro TNS assay. Anionic TNS becomes lipophilic by interacting with a positively charged ionizable lipid, and as the pH value becomes close to the pKa value of each LNP, the lipophilic property of TNS becomes lower and more water molecules quench the TNS fluorescence, and therefore, lipid nanoparticles having a pKa of 6.0 to 7.0 have excellent in vivo drug delivery efficiency, and lipid nanoparticles showing a “s-type curve” in the graph representing fluorescence according to pH mean that they are easy to interact with the endosome membrane and can easily escape the endosome during acidification.

[0157] Specifically, the pH of the solution comprising 20 mM sodium phosphate, 25 mM citrate, 20 mM ammonium acetate, and 150 mM NaCl with 0.1N NaOH and/or 0.1N HCl at an interval of 0.5 from pH 4.1 to pH 9.6 to prepare solutions of various pH units. 100 μl of each solution having each pH (pH with an interval of 0.5 from pH 4.1 to pH 9.6) was added to a black 96 well plate and each was added to a solution having the pH in the range so as to be the final concentration of 6 uM using a TNS stock solution of 300 uM. 241-C10 LNP to 246-C10 LNP were added to the mixed solution so that the final concentration is 20 uM. The fluorescence intensity was measured by excitation at 325 nm and emission at 435 nm through a Tecan equipment, and the fluorescence intensity for each lipid nanoparticle was shown in FIG. 3a and FIG. 3b, and the pKa for each lipid nanoparticle was calculated as a pH value reaching half of the maximum fluorescence and shown in Table 3 below. As shown in FIG. 3b, it could be seen that 244-C10 LNP to 246-C10 LNP exhibit a fluorescence titration s-shaped curve through nonlinear regression.

TABLE-US-00003 TABLE 3 Lipid nanoparticle pKa 241-C10 LNP 7.7 242-C10 LNP 8.7 243-C10 LNP 8.2 244-C10 LNP 6.8 245-C10 LNP 6.9 246-C10 LNP 7

[0158] As confirmed in the Table 3, it was confirmed that the lipid nanoparticles according to one example showed pKa 6.0 to 7.0 range in which in vivo safety and drug release are excellent.

[0159] The LNPs in which a nucleic acid was encapsulated, prepared by the method as Example 2-2, also showed the same pattern according to the type of ionizable lipids contained (type of amine contained in the ionizable lipids).

Example 4. Confirmation of Characteristics of Lipid Nanoparticles

Example 4-1. Particle Size Measurement

[0160] In the present example, the size of the lipid nanoparticles (LNP; comprising 1.5 mol % of lipid-PEG) in which mRNA was encapsulated measured in Example 2-2 was to be measured. It was diluted using PBS so that the concentration of RNA (luciferase mRNA; SEQ ID NO: 1) comprised in each lipid nanoparticle prepared in Example 2-2 was 1 ug/ml, and the diameter and polydispersity index (PDI) of the LNPs were measured using dynamic light scattering (DLS) in Malvern Zetasizer Nano (Malvern Instruments, UK), and the result was described in Table 4 below.

TABLE-US-00004 TABLE 4 Lipid nanoparticle Diameter (nm) PDI 241-C10 LNP 128 0.259 242-C10 LNP 77 0.210 243-C10 LNP 56 0.225 244-C10 LNP 66 0.149 245-C10 LNP 70 0.210 246-C10 LNP 68 0.143

[0161] As confirmed in the Table 4, the lipid nanoparticles according to one example showed the particle size that is easy to be introduced into hepatocytes and has excellent drug release, and it could be found that the PDI values were small and the particles were uniform in order of 241-C10 LNP>243-C10 LNP>242-C10 LNP=245-C10 LNP>244-C10 LNP>246-C10 LNP.

Example 4-2. Measurement of Encapsulation Efficiency

[0162] The encapsulation efficiency (drug encapsulation efficiency, %) of each LNP (comprising 1.5 mol % of lipid-PEG) in which siRNA (siFVII siRNA) was encapsulated as a nucleic acid drug was measured through Ribogreen analysis (Quant-iT™ RiboGreen® RNA, Invitrogen). The LNPs in which a nucleic acid drug was encapsulated prepared in the Example 2-2 were diluted with 1×TE buffer solution 500 in a 96 well plate so that the final concentration of siRNA was 4˜7 ug/ml. To the group untreated with Triton-X (Triton-x LNP(−)), 1×TE buffer 50 μl was added, and to the group treated with Triton-X (Triton-x LNP(+)), 2% Triton-X buffer 50 μl was added. By incubating at 37° C. for 10 minutes, the nucleic acid encapsulated by degrading LNPs with Triton-X was released. Then, Ribogreen reagent 100 μl was added to each well. The fluorescence intensity (FL) of Triton LNP(−) and Triton LNP(+) was measured by the wavelength bandwidth (excitation: 485 nm, emission: 528 nm) in Infinite® 200 PRO NanoQuant (Tecan), and the drug encapsulation efficiency (encapsulation efficiency, %) was calculated as the following Equation 3. The drug encapsulation efficiency (%) for each LNP was shown in Table 5 below as the average value of the results measured repeatedly twice.

Drug encapsulation efficiency (%)=(Fluorescence intensity of Triton LNP(+)−Fluorescence intensity of Triton LNP(−))/(Fluorescence intensity of Triton LNP(+))×100  (Equation 3)

TABLE-US-00005 TABLE 5 Lipid nanoparticle Encapsulation efficiency (%) 241-C10 LNP 84 242-C10 LNP 83 243-C10 LNP 91 244-C10 LNP 87 245-C10 LNP 91 246-C10 LNP 94

[0163] As confirmed in the Table 5, it was confirmed that the lipid nanoparticles according to one example could encapsulate a drug with high efficiency.

Example 5. Confirmation of Intracellular Nucleic Acid Delivery Using Lipid Nanoparticles

Example 5-1. Nucleic Acid Delivery Effect According to Types of Ionizable Lipids Comprised in LNP

[0164] One day prior to transfection of LNP according to one example into cells, HeLa cells (Korea Cell Line Bank) were aliquoted at 0.01×10.sup.6 cells/well in a white plate (96 well) and were cultured under the condition of 37° C., 0.5˜3% CO.sub.2 in DMEM media (SH30022, Hyclone, USA). After stirring LNPs (241-C10 LNP to 246-C10 LNP comprising 1.5 mol % of lipid-PEG) in which mRNA (luc mRNA; SEQ ID NO: 1) encoding a luciferase gene with ApoE30.1 ug/ml by pipetting and then incubating at a room temperature for 10 minutes, they were treated (100 ng/well based on the mRNA comprised in the lipid nanoparticles) in HeLa cells. ApoE3 binds to the LNP surface and plays a role in allowing LNP to enter the cell through endocytosis through an LDL receptor expressed on the cell surface.

[0165] In 24 hours, after treating 1000/well of Bright-Glo™ Luciferase Assay solution (promega, USA) each and leaving them at a room temperature for 10 minutes, the luminescence intensity was measured for the dissolved cells using Infinite M200 luminescence measuring device (Tecan, USA), and the result was shown in FIG. 4a. As shown in FIG. 4a, 244-C10 LNP, 245-C10 LNP, and 246-C10 LNP having a pKa range of 6.0 to 7.0 showed strong luminescence intensity, and among them, 246-C10 LNP had the highest luminescence intensity, and therefore, it could be seen that 246-C10 LNP had the highest intracellular drug delivery efficiency.

Example 5-2. Confirmation of Nucleic Acid Delivery in Hepatocytes

[0166] The luminescence intensity was measured by delivering luc mRNA into hepatocytes using 246-C10 lipid nanoparticle prepared in Example 2-2, thereby confirming expression of the gene.

[0167] Specifically, after combining 246-C10 LNP (comprising 1.5 mol % of lipid-PEG) in which luc mRNA (SEQ ID NO: 1) was encapsulated with ApoE35 ug/ml, the LNP was treated into a hepatocyte cell line (Nexel, Korea) aliquoted at 1×105 cells/well at 0.2 ug/well, 0.5 ug/well, or 1 ug/well based on the mRNA concentration comprised in the nanoparticle. In 6 hours, Bright-Glo™ Luciferase Assay solution (promega, USA) of 100 μl/well was treated and left at a room temperature for 10 minutes, and then the luminescence intensity was measured for the dissolved cells using Infinite M200 luminescence measuring device (Tecan, US) and the result was shown in FIG. 4b.

[0168] As confirmed in FIG. 4b, it was confirmed that the lipid nanoparticle according to one example was easy to introduce into cells through binding to ApoE3, increased the amount of drug (nucleic acid) delivery in a concentration-dependent manner, and could deliver the drug to hepatocytes with high efficiency.

Example 6. Confirmation of In Vivo Expression Using Lipid Nanoparticles

[0169] As confirmed in the Example 5-1, in vivo drug delivery efficiency and biodistribution of 244-C10 LNP to 246-C10 LNP showing an excellent gene expression effect (gene delivery effect) in vitro were to be confirmed in the present example.

[0170] 244-C10 to 246-C10 LNP (comprising 1.5 mol % of lipid-PEG) in which luc mRNA (SEQ ID NO: 1) was encapsulated by the method of Example 2-2 were prepared, and each nanoparticle was dialyzed in PBS for 16 hours to remove ethanol. In 3 hours after intravenously (i.v) injecting the lipid nanoparticle in which mRNA was encapsulated into C57BL/6 Female 7-week-old mice (Orient Bio) in an amount of 0.1 mg/kg based on the mRNA comprised in the lipid nanoparticle, luciferin 0.25 mg/kg was intraperitoneally administered and the bioluminescence was confirmed through IVIS (PerkinElmer, USA) equipment, and the result was shown in FIG. 5a.

[0171] Mice in which luc mRNA-encapsulated 246-C10 LNP was administered were sacrificed and organs were removed, and the biodistribution of the lipid nanoparticle was confirmed in each organ through IVIS equipment and the result was shown in FIG. 5b.

[0172] As shown in FIG. 5a, mice in which luc mRNA-encapsulated 244-C10 LNP to 246-C10 LNP were administered showed high luminescence intensity, and this corresponds to the result of the Example 5-1. In particular, as shown in FIG. 5a and FIG. 5b, through systemic imaging and ex vivo organ imaging, it was confirmed that luc mRNA-encapsulated 246-C10 LNP showed high luminescence intensity specifically to liver, and thereby it could be confirmed that the lipid nanoparticle according to one example showed high biodistribution to the liver.

Example 7. Confirmation of Composition Ratio of Lipid Nanoparticles Optimal for Nucleic Acid Delivery

[0173] In the present example, the composition ratio of the lipid nanoparticle with the most excellent drug delivery efficiency specifically to liver in vivo was to be confirmed.

[0174] In the preparation of the lipid nanoparticle, the lipid nanoparticle (246-C10 LNP) in which luc mRNA (SEQ ID NO: 1) was encapsulated was prepared by the method of Example 2-2 by mixing lipid-PEG (C16-PEG2000 ceramide) at 1.0 to 2.5 mol %. The weight ratio of the ionizable lipid:mRNA comprised in the lipid nanoparticle was 10:1, and the molar ratio of the ionizable lipid (246-C10):phospholipid (DOPE):cholesterol:lipid-PEG (C16-PEG2000 ceramide) comprised in the LNP was 26.5:20:52.5 to 51:1.0 to 2.5 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

[0175] For the 246-C10 LNP in which lipid-PEG was contained at 1.0 mol %, 1.5 mol %, or 2.5 mol % and luc mRNA was encapsulated, similarly to the method of Example 6, in 3 hours after mRNA-encapsulated lipid nanoparticle was intravenously (i.v) injected to C57BL/6 Female 7-week-old mice (Orient Bio) at a dose of 0.1 mg/kg based on the luc mRNA contained in the lipid nanoparticle, luciferin 0.25 mg/kg was intraperitoneally administered through IVIS (PerkinElmer, USA) equipment to confirm bioluminescence, and the result was shown in FIG. 6, and the size of the lipid nanoparticles according to the lipid-PEG content was measured as same as the method of Example 4-1 and was described in Table 6 and FIG. 6 below.

TABLE-US-00006 TABLE 6 Lipid-PEG content comprised in LNP Diameter (nm) 1.0 mol% 90 1.5 mol% 67 2.5 mol% 55

[0176] As shown in FIG. 6, it could be confirmed that the group in which the lipid nanoparticle according to one example was administered had excellent drug delivery efficiency to the liver, and the LNP size comprising lipid-PEG of 1.5 mol % was about 70 nm.

Example 8. Confirmation of Hepatocyte-Specific Drug Delivery Effect

Example 8-1. Confirmation of Knockout Effect of FVII Using Lipid Nanoparticles

[0177] FVII is expressed specifically in hepatocytes and therefore, in the present example, the hepatocyte targetability of the lipid nanoparticles according to one example was to be confirmed through an FVII (Factor VII) knockout effect using siFVII.

[0178] So that a concentration based on the concentration of siRNA comprised in the lipid nanoparticle was 0.03 mg/kg, 0.1 mg/kg, or 0.3 mg/kg, in 3 days after the 246-C10 lipid nanoparticle (comprising lipid-PEG of 1.5 mol %) in which FVII target siRNA (SEQ ID NOs: 2 and 3) was encapsulated, prepared in Example 2-2 was intravenously injected to C57BL/6 female 7-week-old 20 g mice, blood was collected through tail veins, and blood analysis was performed according to the protocol of the coaset FVII assay kit, and a standard curve was drawn with blood of mice administered with PBS and the FVII expression was measured and the result was shown in FIG. 7. As shown in FIG. 7, as FVII expression was inhibited in vivo dependently on the siRNA concentration encapsulated in the 246-C10 lipid nanoparticle, it was confirmed that the lipid nanoparticle according to one example could deliver a nucleic acid to hepatocytes as a target.

Example 8-2. Drug Delivery Effect to Hepatocytes According to Lipid-PEG Content

[0179] The lipid nanoparticle (246-C10 LNP) in which siFVII (SEQ ID Nos: 2 and 3) was encapsulated by the method of Example 2-2, by modifying the content of lipid-PEG comprised in the lipid nanoparticle to 0.5 to 5.0 mol %, was prepared. The weight ratio of the ionizable lipid:siRNA comprised in the lipid nanoparticle was 7.5:1, and the molar ratio of the ionizable lipid (246-C10):phospholipid(DSPC):cholesterol:lipid-PEG (C16-PEG2000 ceramide) comprised in the LNP was 42.5:13:44 to 39.5:0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

[0180] The diameter and polydispersity index of the lipid nanoparticles prepared above were measured as same as the method of Example 4-1, and were shown in Table 7 and FIG. 8 (left table) below.

TABLE-US-00007 TABLE 7 Lipid-PEG (%) Average diameter (nm) PDI 0.5 120 0.018 1 78 0.106 1.5 52 0.159 3 42 0.152 5 37 0.226

[0181] So that a concentration based on the concentration of siRNA comprised in the lipid nanoparticle was 0.2 mg/kg, in 3 days after the lipid nanoparticle (comprising lipid-PEG of 0.5 to 5 mol %) in which siFVII was encapsulated was intravenously injected to C57BL/6 female 7-week-old 20 g mice, blood was collected through tail veins, and similarly to the method of Example 8-1, using coaset FVII assay kit, the FVII expression was measured and the result was shown in FIG. 8 (right graph). As shown in FIG. 8, it was confirmed that when the lipid nanoparticle according to one example was administered, the FVII expression in vivo was reduced, and when the lipid nanoparticle having a lipid-PEG content of 0.5 to 5.0 mol % was administered, the FVII expression was excellently inhibited.

Example 9. LSEC-Specific Drug Delivery Effect

[0182] As FVIII is specifically expressed in LSEC (liver sinusoidal endothelial cells), in the present example, the LSEC targetability of the lipid nanoparticle according to one example was to be confirmed through a knockout effect of FVIII (Factor VIII) using siFVIII, and the drug delivery effect according to the lipid-PEG content was examined.

[0183] By modifying the content of lipid-PEG comprised in the lipid nanoparticle to 0.5 to 5.0 mol %, lipid nanoparticles (246-C10 LNP) in which siFVIII (SEQ ID Nos: 4 to 11) was encapsulated were prepared by the method of Example 2. The weight ratio of the ionizable lipid:siRNA comprised in the lipid nanoparticle was 7.5:1, and the ionizable lipid (246-C10):phospholipid(DSPC):cholesterol:lipid-PEG (C16-PEG2000 ceramide) comprised in the LNP=42.5:13:44 to 39.5:0.5 to 5.0 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100).

[0184] The diameter and PDI of the lipid nanoparticles prepared above were measured by the same method of Example 4-1, and were shown in Table 8 and FIG. 9 (left table) below.

TABLE-US-00008 TABLE 8 Average diameter Lipid-PEG (%) (nm) PDI 0.5 166 0.018 1 87 0.106 1.5 78 0.159 3 42 0.152 5 35.6 0.226

[0185] So that a concentration based on the concentration of siRNA comprised in the lipid nanoparticle was 0.5 mg/kg, in 2 days after the lipid nanoparticle (comprising lipid-PEG of 0.5 to 5 mol %) in which siFVIII was encapsulated was intravenously injected to C57BL/6 female 7-week-old 20 g mice, blood was collected through tail veins, and similarly to the method of Example 8-1, using coaset FVII assay kit, the FVIII expression was measured and the result was shown in FIG. 9 (right graph). As shown in FIG. 9, it was confirmed that when the lipid nanoparticle according to one example was administered, the FVIII expression in vivo was reduced, and the lipid nanoparticle according to one example could target the LSEC, and when the lipid nanoparticle having a lipid-PEG content of 0.5 to 5.0 mol % was administered, the FVIII expression was excellently inhibited.

Example 10. Drug Delivery Effect According to Types of Lipid-PEG Conjugates

[0186] Lipid nanoparticles (comprising a lipid-PEG conjugate of 0.25 to 10.0 mol %) comprising a ceramide-PEG conjugate (C16-PEG 2000 ceramide; Avanti, US) or PEG-DSPE (Avanti, US) as a lipid-PEG conjugate were prepared similarly to the method of Example 2-2.

[0187] The weight ratio of the ionizable lipid:siRNA (siLuc) comprised in the lipid nanoparticle was 7.5:1, and the molar ratio of the ionizable lipid (2464-C10):phospholipid (DSPC):cholesterol:lipid-PEG (ceramide-PEG or PEG-DSPE) comprised in the LNP was 42.5:13:44.25 to 34.5:0.25 to 10 (adjusting the content of cholesterol and lipid-PEG so that the total sum of the molar ratio is 100). The sequence of the used siLuc (siRNA targeting a luciferase gene; SEQ ID Nos: 12 and 13) was described in the Example 2-2.

[0188] One day prior to transfection of LNP according to one example into cells, HeLa cells (Korea Cell Line Bank) were aliquoted at 0.01×10.sup.6 cells/well in a white plate (96 well) and were cultured under the condition of 37° C., 0.5˜3% CO.sub.2 in DMEM media (SH30022, Hyclone, USA). In 24 hours after treating the lipid nanoparticle in which siLuc was encapsulated to the HeLa-Luc cell line at 10 nM based on the siRNA concentration, Bright-Glo™ Luciferase Assay solution (promega, USA) was treated by 100 μl/well each and was left at a room temperature for 10 minutes, and then for the dissolved cells, the luminescence intensity was measured using Infinite M200 luminescence measuring device (Tecan, USA), and the result was shown in FIG. 10. The measured result was represented by mean±SD. The result value was statistically verified by the T-test method, and a case of p<0.05 or more was defined as statistically significant.

[0189] As shown in FIG. 10, the lipid nanoparticle according to one example had an excellent nucleic acid delivery effect to cells, and in particular, in case of comprising the ceramide-PEG conjugate as a lipid-PEG conjugate, the nucleic acid delivery effect was excellent.