Novel ionizable sterol derivative and lipid nanoparticle composition comprising same

Abstract

The present invention relates to a novel ionizable sterol derivative and a lipid nanoparticle composition comprising same, in which a novel ionizable lipid synthesized on the basis of a sterol structure may simultaneously exhibit the roles of cholesterol components of ionizable lipids and structural lipids of existing lipid nanoparticles, and thus lipid nanoparticles composed thereof may be utilized as a more stable and effective drug delivery platform.

Description

Novel ionized sterol derivative and lipid nanoparticle composition containing the same

[0001] The present invention relates to novel lipid compounds, and more specifically, to novel lipid compounds based on a sterol structure and lipid nanoparticle compositions containing the same.

[0002] Gene therapy, which uses nucleic acids as therapeutic agents or immune stimulants outside the body, has been researched for decades, but delivering genes into the body without a carrier is highly inefficient. In particular, while mRNA offers various advantages over plasmid DNA, such as direct protein expression in the cytoplasm, it is easily degraded by in vivo RNA degrading enzymes and has limitations in entering the cell.

[0003] Although therapies using adenoviruses or lentiviruses as vectors, such as Zolgensma, Luxturna, and Zynteglo, which have recently received FDA approval, are attracting attention, there are still limitations in clinical development due to issues with safety, immune responsiveness, insufficient study periods, and high costs.

[0004] Accordingly, various types of non-viral delivery vehicles, ranging from liposomes, cationic polymers, and dendrimers to lipid nanoparticles, are being continuously studied as solutions for safety, and the pace of such research has accelerated further since the COVID-19 pandemic.

[0005] Generally, lipid nanoparticles (LNPs) are composed of four types of lipids, such as cationic ionized lipids, phospholipids, cholesterol, and PEG lipids; however, gene expression efficiency can vary significantly depending on various factors, such as the type and composition ratio of the lipids. In mRNA-based COVID-19 vaccines, lipid nanoparticles used to protect the mRNA have been shown to successfully induce nucleic acid expression in muscle cells, demonstrating a mechanism in which antigen proteins trigger an immune response. Furthermore, some studies report that lipid nanoparticles themselves act as adjuvants that boost immunity.

[0006] Cholesterol is known to influence the release of internal transporters by regulating the structural stability of lipid nanoparticles, and it possesses numerous derivatives. Free cholesterol, increased by intracellular and extracellular factors, affects the efficiency of gene transfer. Recent studies indicate that the use of beta-sitosterol, a cholesterol derivative, alters particle morphology to enhance mRNA delivery; furthermore, it has been reported that the addition of cationic cholesterol reduces the rate of delivery to the liver and can provide targeted effects toward the heart or lungs.

[0007] Based on these factors, there is a continuous need for research and development of safer and more effective lipid nanoparticles by synthesizing various ionized lipids, which overcome the limitations of conventional gene therapy delivery vehicles.

[0008] The object of the present invention is to provide a novel ionized lipid compound.

[0009] Another objective of the present invention is to provide lipid nanoparticles comprising the above-mentioned lipid compound.

[0010] Another objective of the present invention is to provide the use of the above lipid nanoparticles as a drug delivery system.

[0011] To achieve the above objective, the present invention provides a compound selected from a compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0012] <Chemical Formula 1>

[0013]

[0014] In the above chemical formula 1, L 1 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 12 carbon atoms, X is O or S, and L 2 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 20 carbon atoms, and n is 0 or 1, and

[0015] R is H, OH, a linear or branched saturated hydrocarbon having 1 to 5 carbon atoms, or is selected from the following chemical formula 1-1, and

[0016] <Chemical Formula 1-1>

[0017]

[0018] In the above chemical formula 1-1, L 3 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, and R 1 and R 2 Each may be the same or different and may be selected from linear or branched saturated or unsaturated hydrocarbons having 1 to 5 carbon atoms; or from -OH.

[0019] The present invention provides a lipid nanoparticle composition comprising the above-mentioned compound.

[0020] In addition, the present invention provides a drug delivery composition comprising the above lipid nanoparticle composition; and a preventive or therapeutic agent.

[0021] The novel compound according to the present invention is an ionized lipid and can form lipid nanoparticles in which gene therapy drugs, etc., can be stably encapsulated, delivered, and expressed.

[0022] The novel compound according to the present invention is an ionized lipid synthesized based on a sterol structure, which can simultaneously perform the role of the ionized lipid and the cholesterol component of the structural lipid of conventional lipid nanoparticles, and thus lipid nanoparticles composed thereof can be utilized as a more economical and effective drug delivery platform.

[0023] Figure 1 shows the structure of a novel lipid compound synthesized according to a synthesis example of the present invention.

[0024] Figure 2 evaluates the cell delivery ability of lipid nanoparticles prepared using a synthesized novel sterol derivative (BKC-1).

[0025] Figure 3 evaluates the eGFP expression ability of lipid nanoparticles prepared using a novel synthesized sterol derivative.

[0026] Figure 4 is an electron microscope image of lipid nanoparticle BKLL-9 prepared using BKL-9, with a scale bar of 200 nm.

[0027] The present invention will be described in detail below.

[0028]

[0029] The inventors synthesized various forms of novel derivatives based on a sterol structure and confirmed that lipid nanoparticles can be formed using the synthesized novel derivatives, and furthermore, that the formed lipid nanoparticles possess excellent physicochemical properties and stability regarding cell efficacy and toxicity, thereby completing the present invention.

[0030]

[0031] The present invention provides a compound selected from a compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof:

[0032] <Chemical Formula 1>

[0033]

[0034] In the above chemical formula 1, L 1 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 12 carbon atoms, X is O or S, and L 2 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 20 carbon atoms, and n is 0 or 1, and

[0035] R is H, OH, a linear or branched saturated hydrocarbon having 1 to 5 carbon atoms, or is selected from the following chemical formula 1-1, and

[0036] <Chemical Formula 1-1>

[0037]

[0038] In the above chemical formula 1-1, L 3 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, and R 1 and R 2 Each may be the same or different and may be selected from linear or branched saturated or unsaturated hydrocarbons having 1 to 5 carbon atoms; or from -OH.

[0039] Preferably, L in Formula 1 above 1 is a linear or branched saturated hydrocarbon having 5 to 12 carbon atoms, and L 2 is a linear or branched saturated or unsaturated hydrocarbon having 10 to 20 carbon atoms, and

[0040] R is OH, or selected from the above formula 1-1, and L in the above formula 1-1 3 It can be selected from linear saturated hydrocarbons having 2 to 5 carbon atoms.

[0041] More preferably, the compound may be selected from the group consisting of compounds 1 to 12 of Table 1 below:

[0042] Compound Structure Formula 1 2 3 4 5 6 7 8 9 10 11 12

[0043]

[0044] In this specification, the term "stereoisomer" refers to a compound having the same chemical formula or molecular formula but different stereochemical properties, and each stereoisomer and mixtures thereof may also be included within the scope of the invention. Unless otherwise noted, solid bonds connected to asymmetric carbon atoms may include wedge-shaped solid bonds or wedge-shaped dotted bonds representing an absolute arrangement of stereocenters.

[0045]

[0046] In this specification, "pharmaceutically acceptable salt" means any organic acid or inorganic acid addition salt, or base addition salt of said compound, at a concentration that has a relatively non-toxic and harmless active effect on cells or humans exposed to said compound, and at which side effects caused by said salt do not reduce the beneficial efficacy of said compound represented by Formula 1.

[0047] In addition, the above compound may include not only the above salt, but also all salts, hydrates, solvates, derivatives, etc. that can be prepared by conventional methods.

[0048]

[0049] The compound according to the present invention may be an ionizable lipid, and preferably may simultaneously possess the characteristics of an ionizable lipid and cholesterol, thereby contributing to drug encapsulation within the nanoparticles and the formation of the nanoparticle structure in lipid nanoparticles formed from said compound.

[0050]

[0051] The present invention provides a lipid nanoparticle composition comprising the above-mentioned compound.

[0052] The above composition can simultaneously perform the roles of ionized lipids and cholesterol, and can be utilized to form lipid nanoparticles. The cholesterol can function as a structural lipid that provides structural rigidity to the lipid packing within the lipid nanoparticles and improves the stability of the nanoparticles.

[0053]

[0054] The above lipid nanoparticle composition may further include helper lipids or PEG-modified lipids.

[0055] The above helper lipid performs the role of protecting the core formed by the interaction of ionized lipids and drugs within the lipid nanoparticles by surrounding it, and can be selected from phospholipids or glycolipids capable of promoting the fusion of lipid nanoparticles.

[0056] Preferably, the phospholipid may be selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylglycerol (DPPG), and mixtures thereof. More preferably, it may be 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), but is not limited thereto.

[0057] Preferably, the glycolipid may be selected from the group consisting of glucosylceramide, galactosylceramide, glucosylsphingosine, galactosylsphingosine, phosphoglycoceramide, and mixtures thereof, but is not limited thereto.

[0058] The above PEG-modified lipid is a pegylated lipid that contributes to the particle stability of the nanoparticles within the lipid nanoparticles. Preferably, the above PEG-modified lipid may be selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof, and more preferably may be PEG-modified dimyristoylglycerol (DMG) (DMG-PEG), but is not limited thereto.

[0059] In addition, the above lipid nanoparticle composition may further include, but is not limited to, bile acid derivatives, cholanic acid derivatives, litocholic acid derivatives, flavonoids, etc. as structural lipids.

[0060]

[0061] The above lipid nanoparticle composition may include all of the compound, helper lipid, and PEG-modified lipid, wherein the compound, helper lipid, and PEG-modified lipid may each be included in a molar ratio of 1 : (0.5 to 1.5) : (0.01 to 0.1), and preferably in a molar ratio of 1 : 1 : 0.03, but is not limited thereto.

[0062]

[0063] The above lipid nanoparticle composition may further include therapeutic or prophylactic agents.

[0064] The above therapeutic or prophylactic agent may be one or more selected from the group consisting of nucleic acids, peptides, proteins, and protein-nucleic acid constructs, but is not limited thereto.

[0065] The above nucleic acid may be a gene including RNA, DNA, or a mixture thereof, and specifically may be selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), self-replicating ribonucleic acid (srRNA), circular ribonucleic acid (circRNA), and mixtures thereof, but is not limited thereto.

[0066]

[0067] In addition, the present invention provides a drug delivery composition comprising the above lipid nanoparticle composition; and a therapeutic or prophylactic agent.

[0068] Corresponding features can be substituted in the aforementioned section.

[0069]

[0070] The above drug delivery composition may be administered to mammals, including humans, via various routes including parenteral administration, which may be applied intravenously, subcutaneously, intraperitoneally, or topically, and the dosage may be appropriately selected by a person skilled in the art, although it depends on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0071] When formulating the above drug delivery composition according to one example, it is manufactured using diluents or excipients such as commonly used fillers, extenders, lyophilizers, binders, wetting agents, disintegrants, and surfactants.

[0072] Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc.

[0073] Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspension solvents. Witepsol, Macrogol, Tween 61, cocoa paste, laurin paste, glycerol, gelatin, etc. may be used as bases for suppositories.

[0074] The above drug delivery composition may be administered while containing a pharmaceutically effective amount of a therapeutic or prophylactic agent. The effective dose level of the therapeutic or prophylactic agent may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concomitant drugs, and other factors well known in the medical field. According to one example, the composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art. For example, the composition may be administered at 0.01 to 100 mg / kg, 0.1 to 50 mg / kg, or 1 to 10 mg / kg.

[0075]

[0076] In addition, the present invention provides a method for delivering a therapeutic or prophylactic agent to mammalian cells through the above-mentioned drug delivery composition.

[0077] Corresponding features can be substituted in the aforementioned parts.

[0078] A method for delivering the above therapeutic or prophylactic agent to mammalian cells comprises administering the above lipid nanoparticle composition to a subject, wherein the administration involves bringing the cell into contact with the above nanoparticle composition so that the therapeutic or prophylactic agent can be delivered to the cell.

[0079] The above mammalian cell is of a mammal, and the mammal may include humans.

[0080] Additionally, the above drug delivery composition may be administered intravenously, intramuscularly, dermally, subcutaneously, nasally, or by inhalation. A therapeutic or prophylactic dose of about 0.01 mg / kg to about 10 mg / kg may be administered to mammals.

[0081] Hereinafter, the present invention will be described in detail with reference to examples to aid in understanding. However, the following examples are merely illustrative of the content of the present invention and the scope of the present invention is not limited to the following examples. The examples of the present invention are provided to more completely explain the present invention to those with average knowledge in the art.

[0082]

[0083] <Synthesization Example 1> Synthesis of a Novel Lipid Compound

[0084] The reagents used for the synthesis of the novel lipid compounds were commercially available and used in the experiment without additional purification, while the separation and purification of the novel lipid compounds were performed using a Combi-Flash RF200. Structural analysis of the novel lipid compounds was performed using a Bruker Ascend TM Using 400 1 H-NMR spectra were analyzed, and for the NMR measurements, an appropriate mixture of Eroisotop’s CD3OD (Methanol D4) and Sigma-Aldrich’s CDCl3 (Chloroform-d) was used as the solvent, taking into account the solubility of lipids.

[0085]

[0086] As shown in Figure 1, two types of novel sterol derivatives using cholesterol and ten types of derivatives using lithocholic acid were synthesized.

[0087] Lipid derivatives in the form of esters and thioesters were synthesized via amide coupling reactions using EDC coupling reagents. The coupling reaction between the COOH present in the ribocolic acid structure and the ROH and RSH used in the reaction proceeded predominantly with the primary alcohols used, rather than competing with the secondary OH contained in the ribocolic acid structure, thereby enabling the yield of the desired lipid compounds. Since the obtained compounds are UV inactive due to the properties of the materials, 1 The structure of the compound was finally confirmed by verifying the purity through H NMR and the mass value of the compound through LC / MS measurement.

[0088]

[0089] 1-1. Synthesis of BKC-01 (Compound 1)

[0090] [Reaction Equation 1]

[0091]

[0092] Cholesterol (1.0 equiv.), 4-(dimethylamino)butanoic acid hydrochloride (1.5 equiv.), dimethylaminopyridine (DMAP) (1.0 equiv.), and 1-ethyl-(3-3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) (1.2 equiv.) were dissolved in dichloromethane (DCM) (0.1 M) and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by vacuum distillation, and the concentrated mixture was dissolved in dichloromethane. The organic layer was then washed with brine and dried with magnesium sulfate (MgSO4). After removing the solvent by vacuum distillation, a pale yellow hygroscopic solid compound was obtained by purifying it using medium-pressure liquid chromatography with dichloromethane and methanol.

[0093]

[0094] [Compound 1] BKC-01 (DN301618) :(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptane-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(dimethylamino)butanoate [(3S,8S,9S,10R,13R,14S,17R)-10,13-dimethyl-17-((R)-6-methylheptan-2-yl)-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate]

[0095] 1H NMR (400 MHz, CDCl3)δ5.37 (d,J= 4.3 Hz, 1H), 4.61 (m, 1H), 2.29 (m, 6H), 2.22 (s, z6H), 2.05 - 1.92 (m, 2H), 1.90 - 1.74 (m, 5H), 1.69 (s, 1H), 1.64 - 1.41 (m, 6H), 1.41 - 1.23 (m, 4H), 1.22 - 1.06 (m, 6H), 1.06 - 0.93 (m, 7H), 0.91 (d,J= 6.5 Hz, 3H), 0.86 (dd,J= 6.6, 1.8 Hz, 6H), 0.67 (s, 3H). MS(m / z): uv inactive (Yield: 95%).

[0096]

[0097] 1-2. Synthesis of BKC-02 (Compound 2)

[0098] [Reaction Equation 2]

[0099]

[0100] Beta-sitosterol (β-sitosterol) (1.0 equiv.), 4-(dimethylamino)butanoic acid hydrochloride (1.5 equiv.), DMAP (1.0 equiv.), and EDC (1.2 equiv.) were dissolved in dichloromethane (0.1 M) and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by vacuum distillation, the concentrated mixture was dissolved in dichloromethane, the organic layer was washed with brine, and dried with MgSO4. After removing the solvent by vacuum distillation, the mixture was purified by medium-pressure liquid chromatography using dichloromethane and methanol to obtain a pale yellow hygroscopic solid compound.

[0101]

[0102] [Compound 2] BKC-02 (DN301619) : (3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptane-2-yl)-10,13-dimethyl-2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthrene-3-yl 4-(dimethylamino)butanoate [(3S,8S,9S,10R,13R,14S,17R)-17-((2R,5R)-5-ethyl-6-methylheptan-2-yl)-10,13-dimethyl -2,3,4,7,8,9,10,11,12,13,14,15,16,17-tetradecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate]

[0103] 1 H NMR (400 MHz, CDCl3)δ5.37 (d,J= 4.6 Hz, 1H), 4.61 (m, 1H), 2.29 (m, 6H), 2.22 (s, 6H), 2.03 - 1.93 (m, 2H), 1.88 - 1.75 (m, 4H), 1.67 (m, 4H), 1.61 - 1.52 (m, 3H), 1.52 - 1.38 (m, 4H), 1.37 - 1.30 (m, 2H), 1.29 - 1.21 (m, 3H), 1.21 - 1.05 (m, 6H), 1.02 (s, 4H), 0.96 - 0.88 (m, 4H), 0.86 (s, 1H), 0.84 (s, 3H), 0.82 (d,J= 2.1 Hz, 3H), 0.80 (s, 1H), 0.68 (s, 3H) (Yield: 72%).

[0104]

[0105] 1-3. Synthesis of BKL-01 and BKL-02 (Compounds 3 and 4)

[0106] [Reaction Equation 3]

[0107]

[0108] Litocholic acid (1.0 equiv.), alcohol (1.2 equiv.), DMAP (0.1 equiv.), and EDC (1.2 equiv.) were dissolved in dichloromethane (0.1 M) and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by vacuum distillation, the concentrated mixture was dissolved in dichloromethane, the organic layer was washed with brine, and dried with MgSO4. After removing the solvent by vacuum distillation, the mixture was purified by medium-pressure liquid chromatography using dichloromethane and methanol to obtain a white solid compound.

[0109]

[0110] [Compound 3] BKL-01 (DN301936) : Decyl(4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0111]

[0112] 1 H NMR (400 MHz, MeOD)δ4.08 (t,J=6.4 Hz, 2H), 3.61 - 3.48 (m, 1H), 2.38 (m, 1H), 2.32 - 2.19 (m, 1H), 2.04 (s, 1H), 1.85 (m, 5H), 1.64 (m, 4H), 1.45 (m, 6H), 1.32 (s, 19H), 1.28 - 1.23 (m, 2H), 1.15 (m, 5H), 1.02 (m, 1H), 0.97 (s, 6H), 0.93 (t,J= 6.6 Hz, 3H), 0.71 (s, 3H) (Yield: 72%).

[0113]

[0114] [Compound 4] BKL-02 (DN301935): dodecyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0115]

[0116] 1 H NMR (400 MHz, CDCl3)δ4.07 (t,J= 6.7 Hz, 2H), 3.73 - 3.58 (m, 1H), 2.41 - 2.29 (m, 1H), 2.28 - 2.16 (m, 1H), 1.98 (d,J= 12.1 Hz, 1H), 1.84 (m, 5H), 1.73 - 1.61 (m, 3H), 1.58 (s, 3H), 1.52 (m, 1H), 1.47 - 1.32 (m, 13H), 1.28 (s, 12H), 1.21 - 0.98 (m, 7H), 0.96 - 0.87 (m, 10H), 0.66 (s, 3H) (Yield: 70%).

[0117]

[0118] 1-4. Synthesis of BKL-03 ~ 10 (Compounds 5 to 12)

[0119] [Reaction Equation 4]

[0120]

[0121] Litocholic acid (1.0 equiv.), alcohol or thiol (1.2 equiv.), DMAP (1.0 equiv.), and EDC (1.2 equiv.) were dissolved in dichloromethane (0.1 M) and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by vacuum distillation, the concentrated mixture was dissolved in dichloromethane, the organic layer was washed with brine, and dried with MgSO4. After removing the solvent by vacuum distillation, the mixture was purified by medium-pressure liquid chromatography using dichloromethane and methanol to obtain a white solid compound.

[0122] Lithocolic ester (1.0 equiv.), 4-(dimethylamino)butanoic acid hydrochloride (1.5 equiv.), DMAP (1 equiv.), and EDC (1.2 equiv.) were dissolved in dichloromethane (0.1 M) and stirred at room temperature for 24 hours. After the reaction was complete, the solvent was removed by vacuum distillation, the concentrated mixture was dissolved in dichloromethane, the organic layer was washed with brine, and dried with MgSO4. After removing the solvent by vacuum distillation, the mixture was purified by medium-pressure liquid chromatography using dichloromethane and methanol to obtain a pale yellow hygroscopic solid compound.

[0123]

[0124] [Compound 5] BKL-03 (DN301938) : Decyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0125]

[0126] 1 H NMR (400 MHz, MeOD) δ4.78 - 4.69 (m, 1H), 4.08 (t, J= 6.5 Hz, 2H), 2.82 - 2.69 (m, 2H), 2.59 (s, 6H), 2.45 - 2.32 (m, 3H), 2.28 (m, 1H), 2.04 (d,J= 12.3 Hz, 1H), 1.97 - 1.78 (m, 7H), 1.60 (m, 4H), 1.46 (s, 6H), 1.32 (s, 18H), 1.25 - 1.02 (m, 7H), 1.01 - 0.89 (m, 10H), 0.71 (s, 3H) (Yield: 45%).

[0127]

[0128] [Compound 6] BKL-04 (DN301937): dodecyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0129]

[0130] 1H NMR (400 MHz, MeOD)δ4.78 - 4.68 (m, 1H), 4.09 (t,J= 6.4 Hz, 2H), 2.74 - 2.64 (m, 2H), 2.53 (s, 6H), 2.37 (m, 3H), 2.32 - 2.22 (m, 1H), 2.04 (d,J= 12.0 Hz, 1H), 2.00 - 1.78 (m, 7H), 1.65 (m, 4H), 1.56 (m, 1H), 1.47 (s, 7H), 1.30 (s, 22H), 1.24 - 1.03 (m, 6H), 1.00 - 0.95 (m, 6H), 0.92 (t,J= 6.8 Hz, 3H), 0.71 (s, 3H) (Yield: 40%).

[0131]

[0132] [Compound 7] BKL-05 (DN302448) : tridecyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0133]

[0134] 1H NMR (400 MHz, CDCl3)δ4.79 - 4.65 (m, 1H), 4.17 - 3.90 (m, 2H), 2.57 - 2.45 (m, 2H), 2.39 (s, 6H), 2.34 (q,J= 7.5 Hz, 3H), 2.29 - 2.14 (m, 1H), 1.96 (dd,J= 19.6, 8.0 Hz, 1H), 1.93 - 1.74 (m, 7H), 1.72 - 1.48 (m, 5H), 1.49 - 1.17 (m, 24H), 1.17 - 0.98 (m, 8H), 0.97 - 0.79 (m, 14H), 0.64 (s, 3H) (Yield: 43%).

[0135]

[0136] [Compound 8] BKL-06 (DN302449): Pentadecyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0137]

[0138] 1H NMR (400 MHz, CDCl3)δ4.82 - 4.66 (m, 1H), 4.06 (t,J= 6.4 Hz, 2H), 2.71 (s, 2H), 2.40 - 2.26 (m, 9H), 2.26 - 2.16 (m, 1H), 1.97 (d,J= 11.5 Hz, 1H), 1.82 (m,J= 8.4, 6.0 Hz, 7H), 1.71 - 1.49 (m, 6H), 1.39 (m, 8H), 1.27 (s, 26H), 1.06 (m, 6H), 0.97 - 0.85 (m, 9H), 0.65 (s, 3H) (Yield: 42%).

[0139]

[0140] [Compound 9] BKL-07 (DN302450): Octadecyl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0141]

[0142] 1 H NMR (400 MHz, CDCl3)δ4.73 (m, 1H), 4.06 (t,J= 6.7 Hz, 2H), 2.70 - 2.62 (m, 2H), 2.52 (s, 6H), 2.44 - 2.30 (m, 3H), 2.22 (m, 1H), 2.02 - 1.94 (m, 1H), 1.85 (m, 6H), 1.70 - 1.51 (m, 5H), 1.42 (m, 8H), 1.34 - 1.22 (m, 34H), 1.07 (m, 6H), 0.95 - 0.86 (m, 9H), 0.65 (s, 3H) (Yield: 43%).

[0143]

[0144] [Compound 10] BKL-08 (DN303238) : (Z)-dodec-9-en-1-yl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0145]

[0146] 1 H NMR (400 MHz, CDCl3)δ5.48 - 5.23 (m, 2H), 4.84 - 4.66 (m, 1H), 4.07 (t,J= 6.7 Hz, Z2H), 2.42 (m, 2H), 2.37 - 2.30 (m, 8H), 2.29 - 2.18 (m, 1H), 2.10 - 1.95 (m, 6H), 1.93 - 1.76 (m, 7H), 1.62 (m, 7H), 1.43 (m, 8H), 1.38 - 1.22 (m, 12H), 1.18 - 1.02 (m, 6H), 0.99 (m, 2H), 0.95 (t,J= 6.2 Hz, 6H), 0.67 (s, 3H) (Yield: 43%).

[0147]

[0148] [Compound 11] BKL-09 (DN302451) : (Z)-octadec-9-en-1-yl (4R)-4-((3R,8R,9S,10S,13R,14S,17R)-3-((4-(dimethylamino)butanoyl)oxy)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)pentanoate

[0149]

[0150] 1 H NMR (400 MHz, CDCl3)δ5.47 - 5.28 (m, 2H), 4.81 - 4.68 (m, 1H), 4.07 (t,J= 6.7 Hz, 2H), 3.31 - 3.13 (m, 3H), 2.99 (s, 6H), 2.54 (t,J= 6.5 Hz, 2H), 2.36 (m, 1H), 2.22 (m, 1H), 2.09 - 1.95 (m, 6H), 1.92 - 1.76 (m, 5H), 1.74 - 1.49 (m, 6H), 1.50 - 1.19 (m, 32H), 1.18 - 0.98 (m, 6H), 0.97 - 0.85 (m, 9H), 0.66 (s, 3H) (Yield: 35%).

[0151]

[0152] [Compound 12] BKL-10 (DN302983) : (3R,8R,9S,10S,13R,14S,17R)-17-((R)-5-(dodecylthio)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate [(3R,8R,9S,10S,13R,14S,17R)-17-((R)-5-(dodecylthio)-5-oxopentan-2-yl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 4-(dimethylamino)butanoate]

[0153]

[0154] 1 H NMR (400 MHz, MeOD)δ2.87 (m, 4H), 2.67 (s, 6H), 2.64 - 2.46 (m, 2H), 2.42 (t,J= 7.2 Hz, 2H), 2.06 - 1.97 (m, 1H), 1.97 - 1.79 (m, 7H), 1.69 (m, 1H), 1.57 (m, 4H), 1.44 (m, 7H), 1.38 - 1.24 (m, 22H), 1.21 - 1.00 (m, 7H), 0.96 (m, 6H), 0.89 (t,J= 6.0 Hz, 3H), 0.69 (d,J= 9.5 Hz, 3H) (Yield: 41%).

[0155]

[0156] <Experimental Method>

[0157] 1. Cells and Chemicals

[0158] The cell lines HEK 293T, CHO, and HeLa used were obtained from ATCC and cultured in the recommended medium containing 10% FBS at 37°C with 5% CO2. FBS and cell culture medium were purchased from Welgene (Korea), and GFP mRNA (Luciferase mRNA) was purchased in the 5 moU (5-methoxyuridine) form sold by Trilink. All chemicals used for lipid nanoparticle formation and evaluation, including 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and dimyristoylglycerol (DMG)-PEG 2000, were purchased from Sigma-Aldrich.

[0159]

[0160] 2. pK of novel sterol derivatives a Value measurement

[0161] To evaluate the potential of each derivative for ionizing lipids, pK was determined using the fluorescence intensity of 6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt [TNS] as a function of pH. a The values ​​were calculated. Citrate buffer was used for pH 2.5–3.5, acetate buffer for pH 4.0–6.0, phosphate buffer for pH 6.5–8.0, and carbonate buffer for pH 8.5–10.0, respectively, and each solution was prepared at a concentration of 20 mM. Under each pH buffer, 6 μM of TNS solution and 25 μM of lipid were mixed, and then λ at 37°C ex = 321 nm, λ em= Measured at a wavelength of 447 nm using a fluorescence reader (Germini EM, Molecular Devices, USA). The point at which the maximum fluorescence intensity drops to 50% was calculated using a graph pad prism to determine pK a I obtained.

[0162]

[0163] 3. Formation of lipid nanoparticles using novel sterol derivatives

[0164] Lipid solutions were prepared by dissolving novel sterol derivatives, helper lipids, and DMG-PEG 2000 in ethanol at a molar ratio of 1:1:0.03, respectively, and mRNA was dissolved in Rnase-free water at a concentration of 10 mg / mL. Using a microfluidic instrument (Precigenome, Nanogenerator), the ethanol solution and mRNA solution were prepared with a volume ratio of 1:3. The prepared lipid nanoparticles were dialyzed for 2 hours in PBS at 4°C using a Pur-A-Lyzer (10kD, Sigma-Aldrich, USA) to remove ethanol, and stored at -80°C. All mRNAs used had uridine-substituted sequences, and eGFP mRNA was used for the evaluation of intracellular protein expression.

[0165]

[0166] 4. Evaluation of the physicochemical properties of lipid nanoparticles

[0167] A particle scattering spectrometer Nano-ZS zetasizer (Malvern, UK) was used to measure the size and zeta potential of lipid nanoparticles. The prepared lipid nanoparticles were diluted in distilled water to prepare analytical samples, which were measured according to the manufacturer's recommended protocol. Particle size and zeta potential were measured in three repetitions and presented as the mean and standard error values.

[0168]

[0169] 5. Evaluation of mRNA encapsulation rate

[0170] To evaluate the mRNA encapsulation ability of lipid nanoparticles, the Quant-it Ribogreen assay (Thermo Fisher, USA) was performed in 96-well black plates. The use of Triton x-100 allows for the measurement of mRNA concentrations inside the lipid nanoparticles, as it enables the measurement of both external and total RNA concentrations. The protocol followed the one provided by the manufacturer and, in brief, is as follows. Since the Triton / TE solution can affect the background, standard curves were plotted separately with and without Triton. The lipid nanoparticle solution was prepared by diluting it with the provided TE solution, and the Quant-iT ribogreen RNA reagent dissolved in DMSO was prepared by diluting it 20-fold with TE. Each solution was mixed at a 1:1 volume ratio and reacted at room temperature for 5 minutes, after which λ was measured using a plate reader (Biotek, Agilent, USA). ex = 485 nm, λ em = Fluorescence values ​​were measured at 528 nm. The encapsulation rate was calculated using Equation 1 below:

[0171] <Equation 1>

[0172] Encapsulation efficiency (EE) (%) = (Total mRNA - Internal mRNA) / Total mRNA × 100

[0173]

[0174] 6. Electron Microscope Observation

[0175] Lipid nanoparticles were adsorbed onto a 200-mesh grid (Ted Pella, USA) coated with Formva-carbon. The fabricated grid was stained with 1% uranyl acetate for 5 minutes, dried sufficiently at room temperature, and then observed. The samples were photographed using an electron microscope accelerated to 75 kV (HT-7800B; Hitachi, Tokyo, Japan), and the scale bar is indicated in the photograph.

[0176]

[0177] 7. Evaluation of cell efficacy

[0178] HEK 293T (12,000 cells / well), CHO (9,000 cells / well), and HeLa (7,000 cells / well) cells were seeded into 96-well plates and cultured for 24 hours. On the day of the experiment, the medium was replaced with fresh medium, lipid nanoparticles containing 500 ng of eGFP mRNA were added, and the cells were cultured for an additional 48 hours. The expressed fluorescence was captured using a fluorescence microscope, and the intensity of the fluorescence was quantified using a plate reader after cell lysis.

[0179] Cytotoxicity tests were performed using the MTT test. After 48 hours of additional incubation, 20 μL of a 5 mg / mL MTT solution was added to the plate and incubated for an additional 3 hours. The formed purple crystals were dissolved in 100 μL of DMSO, and the wavelength was measured at 550 nm and expressed as a relative value compared to the negative control.

[0180]

[0181] 8. Statistical Processing

[0182] All experiments, excluding animal experiments, were performed in triplicate and expressed as mean and standard error values. Additionally, a T-test was performed using Graphpad Prism, and data with statistical significance were marked with an asterisk (*).

[0183]

[0184] <Experimental Example 1> Characterization of Novel Lipid Compounds

[0185] Generally, the charge of ionized sterols depends on the external pH pK a The characteristic of changing to a positive charge when lower than the pK value affects various properties of lipid nanoparticles, such as surface charge, stability, and toxicity. aLipid nanoparticles formulated with ionized sterols characterized by values ​​between 5.5 and 7.4 acquire a positive charge in the acidic environment of endosomes during the process of endocytosis, and can easily release internal genes due to structural modifications of the lipid nanoparticles. Ionized sterols can be divided into a head, linker, and tail, and the pK associated with changes in each part a Novel ionized sterol derivatives were synthesized as in Synthesis Example 1 of the present invention by varying the tail length, changing the head group, or changing the linker type of the tail to predict the value.

[0186] pK of the novel sterol derivative synthesized according to Synthesis Example 1 above a A TNS assay was performed to measure the value, and the pH value that inhibits 50% of the fluorescence was calculated.

[0187] Table 2 below shows the pK of the synthesized novel sterol derivatives measured by the TNS assay. a It represents the value.

[0188] Compound pK a Compound pK a BKC-15.72BKL-57.07BKC-25.8BKL-67.38BKL-17.43BKL-77.56BKL-27.45BKL-86.29BKL-37.00BKL-97.29BKL-47.01BKL-107.43

[0189]

[0190] If we compare BKL-1 and 2, which have unsubstituted head groups, with BKL-3 and 4, which have tertiary amine heads connected by ester bonds, pK a It can be confirmed that the value decreases by approximately 0.4 points and that it possesses a head portion capable of changing to a positive charge, making it suitable for gene transfer purposes. Additionally, the pK of compounds having a primary amine as a head aThe value was found to be 9.48, which is a permanently cationic substance at approximately pH 7.4, the in vivo condition. In other words, the head group of ionized lipids is pK a The type of head plays an important role in determining the value and has a significant impact on the properties of lipid nanoparticles. Permanent cationic nanoparticles have high gene expression rates and endosome escape capabilities, but they are highly cytotoxic and have limitations in bioavailability.

[0191] When comparing compounds having the same tail, where the connecting linker is an ester (BKL-4) or a thioester (BKL-10), pK a The value was found to increase gradually, and as the electronegativity of the linker constituent atoms increased, pK a It was thought that the value decreased. Comparing the compounds BKL-3 to 7, as the length of the tail alkyl chain increased, pK a It was observed that the value increased, and when the tail consists of unsaturated lipids, as in BKL-8 and 9, pK a The values ​​showed a decreasing trend. This phenomenon is a result of the influence of increased hydrophobicity depending on the length of the alkyl chain, and pK when designing new compounds a It can be used to adjust the value.

[0192] pK a The value is an intrinsic characteristic of the compound; the electron distribution can vary depending on the type of functional group and the compound structure, and there are limitations to predicting and calculating it using computers. Furthermore, factors such as the chain lengths constituting the heads and tails, the presence or absence of unsaturated tails, and the type of linker are closely related to the ionized lipid properties.

[0193]

[0194] <Experimental Example 2> Selection of Helper Lipids for Lipid Nanoparticle Formation

[0195] To select helper lipids, lipid nanoparticles were formulated with eGFP mRNA using 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), respectively.

[0196] The molar ratios between the novel ionized sterol derivative synthesized according to Synthesis Example 1 and the helper lipid were 5:1, 1:1, and 1:5; however, conditions other than the 1:1 molar ratio were excluded because some lipids precipitated or the size distribution was poor. Lipid nanoparticles BKCl-1 formed using BKC-1 and DOPE in a 1:1 ratio showed PDI values ​​of 97.8 ± 0.4 nm and 0.22 ± 0.01, while lipid nanoparticles formed using DSPC showed PDI values ​​of 139.1 ± 1.0 nm and 0.17 ± 0.01. To evaluate the delivery capacity of internal nucleic acids of each formed lipid nanoparticle, transfection experiments were performed in HEK 293T, HeLa, and CHO cells.

[0197] As a result, as shown in Figure 2, no significant cytotoxicity was observed in either the control group or the experimental group, and interestingly, GFP expression was observed only in lipid nanoparticles using DOPE, while fluorescence was hardly observed in particles using DSPC.

[0198]

[0199] The most commonly used helper lipids when forming nanoparticles are phospholipids, which affect particle stability and delivery efficiency, and are broadly divided into phosphatidyl-ethanolamine (PE) series and phosphatidylcholine (PC) series.

[0200] A representative PC-type lipid is DSPC, which is used in Doxil, the first FDA-approved nanoparticle drug, and COVID-19 mRNA vaccines. As such, since DSPC is a lipid already clinically verified, it was expected to exhibit excellent protein expression capabilities; however, the combination with the novel ionized sterol synthesized according to the present invention was not good. DSPC possesses saturated lipid chains, resulting in a high transition temperature (approx. 54°C) and a cylindrical structure, which causes it to take on a lamellar structure when formulated as a lipid nanoparticle. It is believed that this structural form limits the delivery and expression of internal mRNA into the cell.

[0201] DOPE is a representative PE-based lipid, characterized by having two unsaturated lipid chains and forming cone-shaped nanoparticles. Additionally, due to its cone shape, it has a low transition temperature (approx. 30°C) and an inverse hexagonal structure (H₀), which is more advantageous for endosome escape. II It can easily change to the state. It is thought that lipid nanoparticles using DOPE interacted with ionized sterols to easily release internal nucleic acids inside the endosome, thereby successfully expressing proteins. Therefore, all remaining ionized sterol compounds were subsequently formulated using DOPE and evaluated, and the lipid nanoparticles were named by adding L after each compound name, such as BKLL.

[0202]

[0203] <Experimental Example 3> Analysis of the Physicochemical Properties of Lipid Nanoparticles

[0204] Lipid nanoparticles (LNPs) composed of DMG-PEG 2000 for PEGylation with synthesized novel ionized sterols and DOPE were formulated using a microfluidic instrument, and then dynamic light scattering (DLS) analysis was performed.

[0205] Table 3 below shows the physicochemical properties of lipid nanoparticles prepared using novel sterol derivatives and DOPE.

[0206] Lipid Nanoparticles Particle Size (nm) Zeta Potential (mV) Encapsulation Rate (%) BKCL-197.8±0.4 13.1±2.2 93.5 BKCL-2133.6±2.0 9.7±2.6 79.6 BKLL-1132.5±19.9 -20.6±1.7 37.8 BKLL-2196.0±5.5 6.3±0.2 37.0 BKLL-3134.4±1.2 14.0±1.0 70.7 BKLL-4125.4±1.6 11.0±1.1 84. 9BKLL-5115.0±0.712.2±1.462.7BKLL-6111.0±3.511.0±0.445.0BKLL-7387.7±9.412.1±0.230.5 BKLL-8108.4±0.310.8±0.788.0BKLL-9144.2±1.911.9±1.685.5BKLL-10186.8±0.712.9±0.951.0

[0207]

[0208] Referring to Table 3 above, it was confirmed that, with the exception of BKLL-7, all particles had sizes between 10 and 200 nm. Additionally, most exhibited zeta potential values ​​around 10–15 mV, whereas BKLL-1 demonstrated a negative charge. Since dialysis with PBS was performed during the final stage of the lipid nanoparticle formulation process, the pH of the solution containing the suspended lipid nanoparticles can be considered to be approximately 7.4; this value corresponds mostly to the pK of ionized sterols. a It is a higher value than the value.

[0209] As previously reported, zeta potential values ​​of -10 mV to 10 mV indicate a nearly neutral charge, while zeta potentials of ±30 mV are referred to as lipid nanoparticles with strong positive or negative charges. Lipid nanoparticles with strong positive charges have the advantages of favorable cell membrane penetration and easy binding with nucleic acids to form nano-sized particles, but they have limitations in bioavailability due to strong cytotoxicity, unnecessary immune stimulation, and tissue damage.

[0210] From this, it can be seen that the lipid nanoparticles formed according to the present experimental example are lipid nanoparticles that are mostly neutral or have a weak positive charge in a bioavailable pH environment. In addition, biocompatibility was significantly improved by utilizing DMG-PEG 2000. When used in vivo, PEG molecules impart stealth properties that offer various advantages, such as preventing protein aggregation, evading the immune system, and increasing circulation time in the body.

[0211]

[0212] Lipid nanoparticles offer the advantages of protecting internal nucleic acids during in vivo circulation and maximizing the efficiency of endosome escape from cells. To maximize these benefits, excellent nucleic acid encapsulation capability is required, and knowing the precise mass of nucleic acids within the lipid nanoparticles is also an essential condition for evaluating efficacy in cells and animals.

[0213] Encapsulation efficiency (EE) was expressed as a percentage by evaluating the internal volume using the fluorescence values ​​and standardization curves of RNA before and after degradation of lipid nanoparticles using the surfactant Tween 100. Referring to Table 3 above, most nanoparticles showed an encapsulation efficiency of over 80%; however, the ionized sterols constituting BKLL-01 and 02 are derivatives composed of alcohol group heads. Unlike ionized lipids with ethanolamine heads such as SM-102 and ALC-0315, they lack an ionizable head portion and thus lack a site capable of binding to negatively charged nucleic acids, which is thought to explain their encapsulation efficiency in the 30% range. In structures connected by the same linker, the encapsulation efficiency decreased as the tail chain length increased; in particular, when the carbon chain length exceeded 15, the encapsulation efficiency was found to be less than 50%.

[0214]

[0215] <Experimental Example 4> Analysis of the Gene Transfer Ability of Lipid Nanoparticles

[0216] To evaluate the gene delivery capabilities of lipid nanoparticles, eGFP expression experiments were performed on three cell types (CHO, HEK 293T, HeLa). For comparison of superiority, Pfizer lipid nanoparticles (P. LNP) were prepared as a positive control using lipids and ratios as described in the literature, and only fresh samples produced within 2 to 3 days were used.

[0217] Referring to Figure 3, BKLL-4 and 9 showed superior effects in common compared to P.LNP in all three types of cell lines.

[0218]

[0219] It has been reported that in cationic lipids, cell expression ability is superior when the tail length is shortened or when they have an unsaturated double tail. When the number of cis double bonds in the same tail increases from zero to two to become an unsaturated tail, the inverse hexagonal structure transition temperature is lowered and the fusogenic effect is increased, allowing the endosome membrane to easily collapse and release internal nucleic acids.

[0220] Similar results apply to the structures of ionized sterols synthesized according to the synthesis examples of the present invention; lipid nanoparticles having a longer chain tail than BKLL-4 showed a tendency for reduced eGFP mRNA expression ability. Furthermore, BKLL-9, having an unsaturated tail, demonstrated excellent expression ability in all cells, which is thought to be due to the structural specificity of ionized sterols. Appropriate pK a Ionized sterols with a value ionize into cations in endosomes where the internal pH is lowered, changing into an inverse hexagonal structure that is advantageous for escape, and since the unsaturated fat chain structure is relatively closer to a circular horn-shaped structure than the single saturated chain structure, mRNA can be expressed more effectively.

[0221] Electron microscopy was performed to observe the structural peculiarities of BKLL-9, which exhibited excellent delivery capabilities. As a result, as shown in Figure 4, spherical lipid nanoparticles were observed, and no peculiarities were found internally. The particle size measured in the electron microscope image was 127.8 nm, which was similar to the size obtained using the DLS method.

[0222]

[0223] Foregoing, specific parts of the present invention have been described in detail. It is evident to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. That is, the actual scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A compound selected from a compound represented by the following chemical formula 1, a stereoisomer thereof, or a pharmaceutically acceptable salt thereof: <Chemical Formula 1> In the above chemical formula 1, L 1 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 12 carbon atoms, and X is O or S, and L 2 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 20 carbon atoms, and n is 0 or 1, and R is H, OH, a linear or branched saturated hydrocarbon having 1 to 5 carbon atoms, or is selected from the following chemical formula 1-1, and <Chemical Formula 1-1> In the above chemical formula 1-1, L 3 is a linear or branched saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms, and R 1 and R 2 Each may be the same or different, and is a linear or branched saturated or unsaturated hydrocarbon having 1 to 5 carbon atoms; or selected from -OH.

2. In Paragraph 1, In the above chemical formula 1, L 1 is a linear or branched saturated hydrocarbon having 5 to 12 carbon atoms, and L 2 is a linear or branched saturated or unsaturated hydrocarbon having 10 to 20 carbon atoms, and R is OH, or selected from the above formula 1-1, and In the above chemical formula 1-1, L 3 A compound characterized by being a linear saturated hydrocarbon having 2 to 5 carbon atoms.

3. In Paragraph 1, The above compound is, Compound characterized by being selected from the group consisting of the following compounds: (Compound 1); (Compound 2); (Compound 3); (Compound 4); (Compound 5); (Compound 6); (Compound 7); (Compound 8); (Compound 9); (Compound 10); (Compound 11); and (Compound 12).

4. A lipid nanoparticle composition comprising a compound according to claim 1.

5. In Paragraph 4, The above composition is, A lipid nanoparticle composition characterized by simultaneously performing the roles of ionized lipids and cholesterol.

6. In Paragraph 4, The above composition is, A lipid nanoparticle composition characterized by further comprising a helper lipid or a PEG-modified lipid.

7. In Paragraph 6, The above helper lipid is. Lipid nanoparticles characterized by being selected from the group consisting of 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-phosphocholine (DMPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), palmitoyloleoylphosphatidylethanolamine (POPE), dipalmitoylphosphatidylglycerol (DPPG), and mixtures thereof. Composition.

8. In Paragraph 6, The above PEG-modified lipid is, A lipid nanoparticle composition characterized by being selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, and mixtures thereof.

9. In Paragraph 6, The above composition is, A lipid nanoparticle composition characterized by comprising the above compound, helper lipid, and PEG-modified lipid in a molar ratio of 1 : (0.5 to 1.5) : (0.01 to 0.1).

10. In Paragraph 4, The above composition is, A lipid nanoparticle composition characterized by further comprising a therapeutic or prophylactic agent.

11. A lipid nanoparticle composition according to claim 4; and a drug delivery composition comprising a therapeutic or prophylactic agent.

12. In Paragraph 11, The above-mentioned therapeutic or prophylactic agents are, A drug delivery composition characterized by being one or more selected from the group consisting of nucleic acids, peptides, proteins, and protein-nucleic acid structures.

13. In Paragraph 12, The above nucleic acid is, A drug delivery composition characterized by being selected from the group consisting of small interfering ribonucleic acid (siRNA), ribosomal ribonucleic acid (rRNA), ribonucleic acid (RNA), deoxyribonucleic acid (DNA), complementary deoxyribonucleic acid (cDNA), aptamer, messenger ribonucleic acid (mRNA), transfer ribonucleic acid (tRNA), antisense oligonucleotide, small hairpin ribonucleic acid (shRNA), microribonucleic acid (miRNA), asymmetric interfering ribonucleic acid (aiRNA), Dicer-substrate ribonucleic acid (dsRNA), ribozyme, peptide nucleic acid (PNA), deoxyribonucleic acid (DNAzyme), guide ribonucleic acid for gene editing (sgRNA), self-replicating ribonucleic acid (srRNA), circular ribonucleic acid (circRNA), and mixtures thereof.

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