IONIZABLE LIPID CONTAINING BIODEGRADABLE ESTER BOND AND LIPID NANOPARTICLES COMPRISING SAME

Abstract

The present disclosure relates to a novel ionizable lipid containing a biodegradable ester bond. The ionizable lipid containing an ester bond, according to the present disclosure, stably delivers an anionic drug when prepared into lipid nanoparticles, and exhibits an excellent effect, in particular, in delivering nucleic acids, and thus can be effectively used in related technical fields such as lipid nanoparticle-mediated gene therapy.

Claims

1. An ionizable lipid represented by the following Formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: ##STR00033## wherein A is ##STR00034## R.sub.1 and R.sub.2 are each independently any one selected from H, C.sub.1-6alkyl, C.sub.1-6alkyl-NR.sub.3R.sub.4, or C.sub.2-12alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-12alkyl, R.sub.3 and R.sub.4 are each independently any one selected from H, C.sub.1-6alkyl, or C.sub.2-12alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-12alkyl, R.sub.5 is C.sub.1-6alkyl-NR.sub.3R.sub.4 or C.sub.2-12alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-12alkyl, Y is O or NR.sub.6, R.sub.6 is H or C.sub.1-3alkyl, m is an integer from 0 to 5, n is an integer from 1 to 11, x is an integer from 1 to 5, l is an integer from 2 to 10, and p is an integer from 0 to 2.

2. The ionizable lipid, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof of claim 1, wherein A is ##STR00035## R.sub.1 and R.sub.2 are each independently any one selected from H, C.sub.1-3alkyl, C.sub.1-4alkyl-NR.sub.3R.sub.4, or C.sub.4-8 alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-8alkyl, R.sub.3 and R.sub.4 are each independently any one selected from H, C.sub.1-3alkyl, or C.sub.4-8 alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-8 alkyl, Y is O, m is an integer from 0 to 3, n is an integer from 3 to 7, x is an integer from 1 to 3, l is an integer from 2 to 6, and p is 1.

3. The ionizable lipid, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof of claim 1, wherein A is ##STR00036## R.sub.3 and R.sub.4 are each independently any one selected from H, C.sub.1-3alkyl, or C.sub.4-8 alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-Balkyl, R.sub.5 is C.sub.1-4alkyl-NR.sub.3R.sub.4 or C.sub.4-8 alkyl-(CO)Y (CH.sub.2).sub.xCHCHC.sub.4-8alkyl, Y is O, m is an integer from 0 to 3, n is an integer from 3 to 7, x is an integer from 1 to 3, l is an integer from 2 to 6, and p is 1.

4. The ionizable lipid, the stereoisomer thereof, or the pharmaceutically acceptable salt thereof of claim 1, wherein the ionizable lipid is selected from the group consisting of Compounds 1 to 11 listed in Table below: TABLE-US-00011 Compound Structure 1 2 3 4 5 6 7 8 9 10 11

5. A lipid nanoparticle comprising the ionizable lipid, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof according to claim 1.

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

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

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

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

10. The lipid nanoparticle of claim 6, wherein the lipid nanoparticle comprising ionizable lipid: phospholipid: cholesterol: lipid-PEG conjugate in a molar ratio of 10 to 40:10 to 30:40 to 70:1 to 5.

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

12. The lipid nanoparticle of claim 11, wherein the anionic drug is at least any one selected from the group consisting of nucleic acids, small molecule compounds, peptides, proteins, protein-nucleic acid constructs, and anionic biopolymer-drug conjugates.

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

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

15. A method for drug delivery, comprising administering the lipid nanoparticle according to claim 11 to a patient in need thereof.

Description

DESCRIPTION OF DRAWINGS

[0071] FIG. 1 shows the pKa measurement of 244-9-cis lipid nanoparticles by the TNS experiment.

[0072] FIG. 2 shows the results of siFVII (Factor VII) knockout effect in an in vivo efficacy experiment, obtained by testing the hepatocyte targeting potential of siRNA-encapsulated lipid nanoparticles containing ionizable lipid of the present disclosure.

[0073] FIG. 3 shows hEPO levels analyzed in blood collected from mice after intravenous injection to confirm in vivo delivery of hEPO mRNA-encapsulated 244-9-cis lipid nanoparticles.

[0074] FIG. 4 is an image showing bioluminescence after intramuscular injection into mice to confirm in vivo delivery of mFLuc encapsulated 244-9-cis lipid nanoparticles.

[0075] FIG. 5 shows the results of cytotoxicity of 244-9-cis lipid nanoparticles obtained after manufacturing lipid nanoparticles without encapsulating nucleic acid drugs and then treating four cell lines, HepG2, MEF, Hela, and KB-GFP, with the lipid nanoparticles at different concentrations to confirm cytotoxicity thereof.

[0076] FIG. 6 shows the results of AST and ALT levels after preparation and administration mRNA-encapsulated lipid nanoparticles to mice in order to confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, as compared to ALC-0315 lipid.

[0077] FIG. 7 shows the results of ALP and ALB levels after preparation and administration of mRNA-encapsulated lipid nanoparticles in order to confirm the hepatotoxicity of 244-9-cis lipid nanoparticles, as compared to ALC-0315 lipid.

[0078] FIG. 8 shows a liver histogram confirming the liver toxicity of 244-9-cis lipid nanoparticles 24 hours after mFLuc-encapsulated lipid nanoparticles were manufactured and administered to mice, as compared to ALC-0315.

BEST MODE

[0079] Herein after, the present disclosure will be described in more detail through Preparation Examples, Examples and Experimental Examples. However, the following Preparation Examples, Examples and Experimental Examples are provided only for illustrating the present disclosure, and the scope of the present disclosure is not limited thereto.

Example 1. Preparation of an Ionizable Lipid Containing Ester Bond

Example 1-1. Synthesis of an Ionizable Lipid Containing Ester Bond

[0080] An ionizable lipid containing an ester bond of the present disclosure was prepared by combining various kinds of amine headgroups with alkyl chains containing an ester bond and a double bond.

[0081] As to Compound 1 to Compound 4, specifically, 9-bromo nonanoic acid and N,N-diisopropylcarbodiimide (DIC) (each 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.) were added per cis-2-nonen-1-ol in dichloromethane (DCM) solvent and reacted overnight at 25 C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) was added, followed by the addition of a substance containing primary amine (amine head) at 0.3 eq. (for Compound 1), 0.2 eq. (for Compounds 2 and 4), and 0.15 eq. (for Compound 3), and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

[0082] As to Compound 5 to Compound 9, specifically, 9-bromo nonanoic acid for Compound 5, 5-bromo pentanoic acid for Compound 6, 6-bromo hexanoic acid for Compound 7, 7-bromo heptanoic acid for Compound 8, or 8-bromo octanoic acid for Compound 9, and N,N-diisopropylcarbodiimide (DIC) (each at 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.) were added per cis-2-nonen-1-ol in dichloromethane (DCM) solvent and reacted overnight at 25 C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) and a substance containing primary amine (amine head) (0.3 eq.) were added, and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

[0083] For example, the specific Reaction Scheme for 244-9-cis is shown in Reaction Scheme 1 below.

##STR00016##

[0084] As to Compound 10 to Compound 11, specifically, 9-bromo nonanoic acid and N,N-diisopropylcarbodiimide (DIC) (each at 1.5 eq.), and 4-dimethylaminopyridine (DMAP) (0.2 eq.), were added per cis-3-nonen-1-ol for Compound 10, or cis-4-decen-1-ol for Compound 11 in dichloromethane (DCM) solvent and reacted overnight at 25 C., followed by purification in hexane/ethyl acetate (5:1 v/v ratio) using a CombiFlash column. After the solvent was blown off, each product was dissolved in ethanol. Then, N,N-diisopropylethylamine (DIPEA) (1 eq.) and a substance containing primary amine (amine head) (0.3 eq.) was added, and reacted for 3 days. Each reaction product was purified in DCM/MeOH (9:1 v/v ratio) using a CombiFlash column to synthesize the ionizable lipid.

[0085] Specific exemplary compounds of the ionizable lipids containing ester bonds of the present disclosure are shown in Table 2 below.

TABLE-US-00002 TABLE 2 Final product No. Amine head (Ionizable lipid) Compound 1 (211-cis) [00017] [00018] Compound 2 (221- cis) [00019] [00020] Compound 3 (222- cis) [00021] [00022] Compound 4 (246-cis) [00023] [00024] Compound 5 (244-9-cis) [00025] [00026] Compound 6 (244-5-cis) [00027] Compound 7 (244-6-cis) [00028] Compound 8 (244-7-cis) [00029] Compound 9 (244-8-cis) [00030] Compound 10 (244-cis- 3-nonen) [00031] Compound 11 (244-cis- 4-decan) [00032]

Example 1-2. Confirmation of Synthesis of the Ionizable Lipid Containing Ester Bonds

[0086] To confirm the synthesis of the ionizable lipids prepared in Example 1-1 above, MS analysis was performed. Specifically, the ionizable lipids were diluted in ethanol to a concentration of 0.5 ppm or less for MS analysis, wherein the instrument used for the analysis was a 6230 LC/MS from Agilent Technologies (Palo Alto, USA), and the separation column was Zorbax SB-C18 (100 mm2.1 mm i.d., 3.5 m) from Agilent Technologies. The MS analysis results are shown in Table 3 below.

TABLE-US-00003 TABLE 3 Chemical Calculated m/z Observed m/z Formula ratio ratio 244-5-cis C.sub.48H.sub.87N.sub.3O.sub.6 802.2209 802.6668 244-6-cis C.sub.51H.sub.93N.sub.3O.sub.6 844.3006 844.7140 244-7-cis C.sub.54H.sub.99N.sub.3O.sub.6 886.3804 886.7616 244-8-cis C.sub.57H.sub.105N.sub.3O.sub.6 928.4601 928.8082 244-9-cis C.sub.60H.sub.111N.sub.3O.sub.6 970.5398 970.8539 221-cis C.sub.79H.sub.147N.sub.3O.sub.8 1267.0278 1267.1263 246-cis C.sub.82H.sub.152N.sub.4O.sub.8 1322.1063 1322.1683

[0087] It could be seen from the above result that the ionizable lipids containing ester bonds in Example 1-1 were successfully synthesized.

Example 2. Manufacture of Lipid Nanoparticle

Example 2-1. Preparation Parameter

[0088] Lipid nanoparticles containing the ionizable lipids of the present disclosure were manufactured at the weight ratios in Table 4 and Table 5 below.

TABLE-US-00004 TABLE 4 Weight ratio Ionizable lipid/mRNA 10 Ionizable lipid/siRNA 10

TABLE-US-00005 TABLE 5 siRNA-encapsulated mRNA-encapsulated LNP (molar LNP (molar ratio, %) ratio, %) Ionizable lipid 26.5 26.5 Helper Lipid 20 (DOPE) 20 (DOPE) Cholesterol 52 52 PEG-lipid 1.5 (ceramide C16 1.5 (ceramide C16 PEG) PEG)

Example 2-2. Manufacture of siRNA-Encapsulated Lipid Nanoparticles

[0089] Each ionizable lipid prepared in Example 1-1 above, cholesterol powder (BioReagent, suitable for cell culture, 99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol in a molar ratio of 26.5:20:52:1.5.

[0090] Subsequently, siRNA was dissolved in 50 mM sodium acetate buffer (Sigma, Korea). The above prepared ethanol containing each ionizable lipid, cholesterol, phospholipid, and lipid-PEG dissolved therein was mixed with the acetate buffer at a volume ratio of 1:3 through a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min, thereby manufacturing lipid nanoparticles.

Example 2-3. Manufacture of mRNA-Encapsulated Lipid Nanoparticles

[0091] The ionizable lipids prepared in Example 1-1 above, cholesterol powder (BioReagent, suitable for cell culture, 99%, Sigma, Korea), phospholipid (DOPE) (Avanti, USA), and C16-PEG2000 ceramide (Avanti, USA) were dissolved in ethanol in a molar ratio of 26.5:20:52:1.5.

[0092] Subsequently, mRNA was dissolved in 10 mM sodium citrate buffer (Sigma, Korea). The above prepared ethanol containing each ionizable lipid, cholesterol, phospholipid, and lipid-PEG dissolved therein were mixed with the citrate buffer at a volume ratio of 1:3 through a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) at a flow rate of 12 ml/min, thereby manufacturing lipid nanoparticles.

Experimental Example 1: Confirmation of Physicochemical Characterization of Lipid Nanoparticles

Experimental Example 1-1. Measurement of Particle Size, Polydispersity Index (PDI), Surface Charge (Zeta Potential) and Drug Encapsulation Efficiency

[0093] The physicochemical properties of lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID No: 1) or FVII siRNA (SEQ ID Nos: 2 and 3) manufactured in Example 2-3 above were to be measured in the present Experimental Example. Specifically, 244-9-cis lipid nanoparticles encapsulated with mRNA or siRNA were each manufactured and subsequently diluted with PBS to achieve a concentration of 1 g/ml for the RNA within each lipid nanoparticle. Then, the diameter, polydispersity index (PDI), and surface charge (zeta potential) of the lipid nanoparticles were measured using dynamic light scattering (DLS) on a Malvern Zetasizer Nano (Malvern Instruments, UK).

[0094] Next, the encapsulation efficiency (drug encapsulation efficiency, %) of lipid nanoparticles encapsulated with mRNA or siRNA above was determined by Ribogreen assay (Quant-iT RiboGreen RNA, Invitrogen). The lipid nanoparticles encapsulated with mRNA were diluted with 50 l of 1TE buffer to a final concentration of 4 to 7 g/ml of mRNA in 96-well plate. 50 l of 1TE buffer was added to the group without Triton-X treatment (Triton-x LNPs()), while 50 l of 2% Triton-X buffer was added to the group treated with Triton-X (Triton-X LNPs(+)). After incubation at 370 C. for 10 minutes, the lipid nanoparticles were decomposed with Triton-X to release the encapsulated nucleic acids. Then, 100 l of Ribogreen reagent was added to each well. The fluorescence intensity (FL) of Triton LNPs () and Triton LNPs (+) was measured by wavelength bandwidth (excitation: 485 nm, emission: 528 nm) on an Infinite 200 PRO NanoQuant (Tecan), and the drug encapsulation efficiency (%) was calculated as shown in Equation 1 below.

[00001]$\begin{array}{cc}\mathrm{Drug}\mathrm{Encapsulation}\mathrm{Efficiency}\left(\%\right)=\left(\mathrm{Fluorescence}\mathrm{of}\mathrm{Triton}\mathrm{LNP}(+)-\mathrm{Fluorescence}\mathrm{of}\mathrm{Triton}\mathrm{LNP}(-)\right)/\left(\mathrm{Fluorescence}\mathrm{of}\mathrm{Triton}\mathrm{LNP}(+)\right)100& \left[\mathrm{Equation}1\right]\end{array}$

[0095] The results for each are as follows (Table 6).

TABLE-US-00006 TABLE 6 244-9-cis mRNA 244-9-cis siRNA LNP LNP Size (nm) 70.94 74.87 Polydispersity index 0.170 0.051 (PDI) Zeta potential (mV) 3.05 3.87 Drug encapsulation 90.0 94.7 efficiency (%)

Experimental Example 1-2. Measurement of pKa

[0096] In the present Experimental Example, the pKa of the firefly luciferase mRNA (SEQ ID NO: 1)-encapsulated lipid nanoparticles formulated in Example 2-3 above was calculated by in vitro TNS (2-(p-toluidino)naphthalene-6-sulfonic acid) assay.

[0097] Specifically, solutions containing 20 mM sodium phosphate, 25 mM citric acid, 20 mM ammonium acetate, and 150 mM sodium chloride were prepared at various pH levels by adjusting the pH of the solution using 0.1 N sodium hydroxide and/or 0.1 N hydrochloric acid in increments of 0.5, starting from pH 3.5 and ending at pH 11. Each (100 l) of the solutions with varying pH was added to a black 96-well plate, and 300 M of TNS stock solution was added to each of the above solutions to a final concentration of 6 M. The formulated lipid nanoparticles were added to the mixed solution to a final concentration of 20 M, and the fluorescence intensity was measured (excitation: 325 nm, emission: 435 nm) using a Tecan instrument. Here, the pKa for the lipid nanoparticles was calculated as the pH value at which half of the maximum fluorescence was reached.

[0098] As a result, it was found that the lipid nanoparticles of the present disclosure have a pKa value of about 6.2 and exhibit the shape of an s-shaped curve on the graph (FIG. 1). Anionic TNS interacts with positively charged ionizable lipids to become lipophilic, and as the pH value approaches the pKa value of each LNP, the lipophilicity of TNS decreases, leading to increased quenching of TNS fluorescence by more water molecules, and thus lipid nanoparticles with a pKa of 6.0 to 7.0 have good in vivo drug delivery efficiency. In addition, the lipid nanoparticles that exhibit an s-shaped curve in the graph of pH-dependent fluorescence indicate easy interaction with endosomal membrane and the ability to facilitate endosomal escape upon acidification.

Experimental Example 1-3. Cryo-TEM Measurement

[0099] Cryo-TEM was used to image the internal structure of the lipid nanoparticles.

[0100] Specifically, lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID NO: 1) were concentrated to a final concentration of 15 to 25 mg/ml of total lipid, and 2 to 4 l of the LNP solution was loaded onto a copper grid and blotted. An internal image of the lipid nanoparticles was then measured using a cryo-TEM (FEI Tecnai F20 G2) instrument at the KIST Advanced Analysis Center.

Experimental Example 2: Confirmation of In Vitro Efficacy of mRNA-Encapsulated Lipid Nanoparticles

[0101] To determine the efficacy of mRNA-encapsulated lipid nanoparticles in vitro, screening was performed using lipid nanoparticles synthesized with various amine head groups.

[0102] The test was conducted by treating 20 ng of mFLuc (SEQ ID NO: 1)-encapsulated lipid nanoparticles into HeLa cells using each lipid nanoparticle synthesized with various amine headgroups (Compounds 1, 2, 4, and 5). The luminescence intensity was measured at 24 hours.

[0103] As a result, as shown in Table 7 below, a significant increase in luminescence intensity was observed for most of the lipid nanoparticles tested, and in particular, it could be seen that Compound 5 (2-(4-9-cis) lipid nanoparticles showed the highest expression effect.

TABLE-US-00007 TABLE 7 Relative luminescence mean Sample value p value Negative control 2823.25 221-cis 229976.5 <0.0001 244-9-cis 1022327.75 <0.0001 221-cis 129255 <0.0001 246-cis 39970.75 0.2634 Lipofectamine 1953.5 >0.9999

Experimental Example 3: Confirmation of In Vivo Effects of Lipid Nanoparticles

Experimental Example 3-1. Delivery Effect of siRNA-Encapsulated Lipid Nanoparticles

[0104] Since FVII is specifically expressed in hepatocytes, the hepatocyte targeting potential of lipid nanoparticles was confirmed by Factor VII (FVII) knockout effect using siFVII.

[0105] Specifically, 244-9-cis lipid nanoparticles encapsulated with FVII-targeting siRNAs (SEQ ID NOs: 2 and 3) were manufactured by the method of Example 2-2 above. SiRNA-encapsulated 244-9-cis lipid nanoparticles manufactured at a concentration of 0.03, 0.1, or 0.3 mg/kg, based on the concentration of siRNA contained in the lipid nanoparticles, were injected intravenously into C.sub.57BL/6 female 7-week-old mice. After 3 days, blood was collected and analyzed according to the protocol of the coaset FVII assay kit. The expression level of FVII was measured by plotting a standard curve with the blood from mice administered with PBS.

[0106] As a result, as shown in FIG. 2, the lipid nanoparticles manufactured using the ionizable lipids of the present disclosure effectively inhibited FVII expression in vivo in a concentration-dependent manner of the encapsulated siRNA, confirming that the lipid nanoparticles of the present disclosure could effectively deliver nucleic acids to target hepatocytes. In particular, lipid nanoparticles containing the ionizable lipids of the present disclosure showed superior FVII expression inhibition compared to lipid nanoparticles manufactured with FDA-approved ALC-0315 at all doses.

Experimental Example 3-2. Confirmation of Delivery Efficacy of mRNA-Encapsulated Lipid Nanoparticles Via Intravenous Injection

[0107] To investigate the mRNA delivery of the 244-9-cis lipid nanoparticles, luciferase mRNA was delivered into mice, and gene expression was confirmed by bioluminescence.

[0108] Specifically, 244-9-cis lipid nanoparticles encapsulated with mFluc (SEQ ID NO: 1) were manufactured by the method of Example 2-3 above. Then, 7-week-old C.sub.57BL/6 mice were injected intravenously with the manufactured lipid nanoparticles (2 g based on mRNA), and 3 hours later, administered intraperitoneally with luciferin 0.25 mg/kg, and bioluminescence was detected by IVIS (PerkinElmer, USA). The results showed that most of the lipid nanoparticles were delivered to the liver (Table 8).

TABLE-US-00008 TABLE 8 Sample 244-9-cis LNP SM-102 Hit 1.75 10.sup.7 2.3 10.sup.7 Body 2.52 10.sup.4 2.91 10.sup.5 Hit/Body 4160 79 Size (nm) 53.04 76.42 Polydispersity index 0.147 0.163 (PDI) Drug encapsulation 89.6 91.9 efficiency (%)

[0109] Next, lipid nanoparticles encapsulated with hEPO mRNA (SEQ ID NO: 4) were manufactured in the same manner as above. The manufactured lipid nanoparticles at a dose of 0.1 mg/kg based on mRNA were injected intravenously into 7-week-old C.sub.57BL/6 mice, and blood was collected after 3, 6, 9, 24, and 48 hours, respectively, for quantitative analysis of hEPO using the hEPO ELISA kit. As a result, the lipid nanoparticles containing the ionizable lipid of the present disclosure effectively delivered nucleic acids in vivo, resulting in significant levels of hEPO detection in the blood, which was confirmed by an approximately 1.7-fold higher the area under the curve compared to lipid nanoparticles manufactured with ALC-0315, the control (Table 9 and FIG. 3).

TABLE-US-00009 TABLE 9 AUC Fold increase 244-9-cis LNP 8022.5 1.72 ALC-0315 LNP 4660.0

Experimental Example 3-3. Delivery Effect of mRNA-Encapsulated Lipid Nanoparticles Via Intramuscular Injection

[0110] Luciferase mRNA (SEQ ID NO: 1) was delivered to mice by intramuscular injection, and gene expression was confirmed by bioluminescence.

[0111] Specifically, mFluc-encapsulated lipid nanoparticles (2-(4-9-cis) were manufactured as described above, and 2 g (based on mRNA) of the lipid nanoparticles were intramuscularly injected into 7-week-old C.sub.57BL/6 mice. After 3 hours, luciferin 0.25 mg/kg was administered intraperitoneally and bioluminescence was confirmed by IVIS (PerkinElmer, USA).

[0112] The results showed that most of the lipid nanoparticles were well delivered to the injection site (Table 10 and FIG. 4).

TABLE-US-00010 TABLE 10 Sample 244-cis LNP Hit 1.9 10.sup.6 Body 5.7 10.sup.4 Hit/Body 333 Size (nm) 53.04 Polydispersity index 0.147 (PDI) Drug encapsulation 89.6 efficiency (%)

Experimental Example 4: Determination of Cytotoxicity of Lipid Nanoparticles

[0113] The substance CCK-8 (tetrazolium salt) forms orange formazan through reduction by dehydrogenase in the mitochondria of living cells, so cell viability can be confirmed by absorbance analysis.

[0114] Specifically, different kinds of cells (HeLa, HepG2, MEF, KB-GFP) were seeded (0.410.sup.5) in transparent 96-well plates (SPL, 30096). Subsequently, the lipid nanoparticle components, except mRNA, were mixed via a microfluidic mixing device (Benchtop Nanoassemblr; PNI, Canada) to manufacture 244-9-cis lipid nanoparticles without mRNA encapsulation. 24 hours after cell seeding, the cells were treated with lipid nanoparticles in an amount of 0.5 g, 5 g, 50 g, or 100 g (based on ionizable lipids) per well. 24 hours following lipid nanoparticle treatment, Cell counting Kit8 (Sigma-Aldrich, 96992) was added at 10 l per well. After incubation for 4 hours, the absorbance at 450 nm was measured using an Infinite200 PRO NanoQuant (Tecan).

[0115] The results showed no cytotoxicity in each cancer cell line up to 5 g (FIG. 5).

Experimental Example 5: Confirmation of Hepatotoxicity of Lipid Nanoparticles

[0116] Aspartate transaminase (AST) and Alanine transaminase (ALT), which can detect the presence of diseases such as hepatocellular disease or hepatitis, are normally present in the blood at low concentrations. However, when liver cells are damaged, these transaminases are released, leading to an increase in their concentration. Alkaline phosphatase (ALP) is an enzyme in the cells lining the bile ducts of the liver that is elevated with diseases such as obstruction of the bile ducts and the cessation of bile secretion in the liver. Albumin (ALB) is produced by the liver, and albumin levels are reduced in acute hepatitis, such as cirrhosis.

[0117] To confirm hepatotoxicity, 244-9-cis lipid nanoparticles encapsulated with firefly luciferase mRNA (SEQ ID NO: 1) were manufactured and administered once to 7-week-old C57BL/6 mice at a single dose of 2 mg/kg based on mRNA. The control group was ALC-0315. 24 hours after administration, blood was collected to determine the levels of AST, ALT, ALP, and ALB (FIGS. 6 and 7). In addition, liver tissue analysis images of each experimental group are shown in FIG. 8.

[0118] As a result, there was no hepatotoxicity compared to the FDA-approved ALC-0315 lipid.

[0119] From the above description, those skilled in the art to which the present disclosure pertains will understand that the present disclosure may be embodied in other specific forms without changing the technical ideas or essential characteristics thereof. In this regard, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. The scope of the present disclosure is indicated by the following claims rather than the detailed description, and should be construed as including all changes or modifications derived from the meaning and scope of the claims and equivalent concepts within the scope of the present disclosure.