Cationic lipids and their use

Abstract

To provide a novel, safe cationic lipid for encapsulating active pharmaceutical ingredients (API) (e.g., nucleic acids) and effectively delivering the active pharmaceutical ingredients into cells.SOLUTION: The cationic lipid has the structure of formula (I), wherein, at least one of X and Y is -(C=O)CH2NH2, and the other X, Y, and Z are independently selected from the group consisting of -(C=O)CH2NH2, -[(C=O)CH2NH](C=O)R1, -(C=O)R1, and H, wherein R1 is C9-25 alkyl, or C13-21 alkenyl.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the priority and benefit of U.S. Provisional Patent Application No. 63 / 423,506, filed on November 8, 2022, the content of which is incorporated herein by reference.

[0002] This disclosure relates to novel cationic lipids for enhancing the delivery of therapeutic agents and their use. More specifically, this disclosure relates to cationic lipids for encapsulating and delivering nucleic acids into cells and their use.

Background Art

[0003] Gene therapy is a process of introducing foreign genomic material into host cells to elicit a therapeutic effect. Various types of nucleic acids, including DNA, plasmid - type interfering nucleic acids, short - interfering nucleic acids for use in RNA interference (RNAi), antisense molecules, and aptamers, are currently being developed as therapeutic agents in gene therapy. On the other hand, two major delivery systems, viral and non - viral systems, have also been developed to encapsulate and deliver the aforementioned therapeutic agents in a stable and long - half - life form.

[0004] For decades, numerous viral and non - viral gene delivery systems have been available, but each of them is limited in some way. For example, viral delivery systems are advantageous in terms of their high transfection efficiency, but there are some concerns about their biological safety. Non - viral delivery systems (e.g., polymers, lipids, liposomes, micelles, dendrimers, and nanomaterials) are highly versatile in their ability to encapsulate nucleic acids of various sizes without affecting the recipient's immune system. On the other hand, their transfection efficiency has not yet been improved.

[0005] Lipoplexes are defined as complexes between nucleic acids and cationic lipids and have become one of the most studied non-viral delivery systems to date. Lipofectamine is a conventionally available cationic lipid, but DOSPA (2,3-dioleoyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propaniminium trifluoroacetate), one of its components, has been reported to have high cytotoxicity during gene transfection.

[0006] In view of the foregoing, in the related art, there is a need for new and safe cationic lipids for effectively delivering therapeutic agents into cells. SUMMARY OF THE INVENTION

[0007] A simplified summary of the present disclosure is presented below to provide the reader with a basic understanding. This summary is neither an exhaustive overview of the disclosure nor is it intended to identify key / important elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some of the concepts disclosed herein in a simplified form as a prelude to the more detailed description that follows.

[0008] The present disclosure provides novel cationic lipids and their compositions for encapsulating and delivering nucleic acids into cells. The treatment of diseases through the use of such cationic lipids and / or compositions is also disclosed herein.

[0009] In one aspect, the present disclosure is directed to a cationic lipid of formula (I) shown below.

Chemical formula

[0010] In this formula, at least one of X and Y is -(C=O)CH 2 NH2 wherein the other X, Y, and Z are independently selected from the group consisting of -(C=O)CH 2 NH 2 , -[(C=O)CH 2 NH](C=O)R 1 , -(C=O)R 1 and H, and R 1 is C 9-25 alkyl or C 13-21 alkenyl.

[0011] According to some embodiments of the present disclosure, in formula (I), X and Z are independently -(C=O)CH 2 NH 2 wherein Y is -[(C=O)CH 2 NH](C=O)R 1 and R 1 is C 13-21 alkenyl.

[0012] According to other embodiments of the present disclosure, in formula (I), X is -(C=O)CH 2 NH 2 wherein Y and Z are independently -[(C=O)CH 2 NH](C=O)R 1 and R 1 is C 13-21 alkenyl.

[0013] According to further embodiments of the present disclosure, in formula (I), Y is -(C=O)CH 2 NH 2 wherein X and Z are independently -(C=O)R 1 and R 1 is C 13-21 alkenyl.

[0014] According to still other embodiments of the present disclosure, in formula (I), X and Z are independently -(C=O)CH 2 NH 2 wherein Y is -(C=O)R 1 and R 1 is C 13-21 alkenyl.

[0015] According to a further embodiment of the present disclosure, in formula (I), X is -(C=O)CH 2 NH 2 and Y is H, Z is -(C=O)R 1 and R 1 is C 13-21 alkenyl.

[0016] Preferably, a subset of the cationic lipids of formula (I) is

Chemical formula

[0017] Another aspect of the present disclosure is directed to lipid nanoparticles (LNPs) comprising the cationic lipid, non-cationic lipid and pharmaceutically active ingredient (API).

[0018] According to some embodiments of the present disclosure, the API disposed in the LNP can be nucleic acid, peptide, polypeptide, protein, carbohydrate, proteoglycan, glycoprotein or a combination thereof.

[0019] Examples of nucleic acids suitable for use in the LNP include, but are not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA) and aptamers. Preferably, the miRNA is precursor miRNA or mature miRNA.

[0020] In some preferred embodiments of the present disclosure, in the LNP, the cationic lipid is

Chemical formula

[0021] In other preferred embodiments of the present disclosure, in this LNP, the cationic lipid is [Chemical formula] and the API is siRNA having the sequence of SEQ ID NO: 6 or 7.

[0022] According to some embodiments of the present disclosure, the non-cationic lipid contained in this LNP can be a helper lipid, a phospholipid, a PEGylated lipid, a PEGylated phospholipid, or a combination thereof. Examples of helper lipids suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC). According to an embodiment of the present disclosure, the non-cationic lipid contained in this LNP is a helper lipid of distearoylphosphatidylcholine (DSPC).

[0023] According to some embodiments of the present disclosure, the cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of 1:5 to 5:1, respectively.

[0024] In an alternative or optional embodiment, the LNP further comprises a steroid. Examples of steroids suitable for use in the LNP include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol. In one effective example, the LNP further comprises cholesterol.

[0025] Another aspect of the disclosure is directed to a method of treating a disease in a subject in need thereof, comprising the step of administering to the subject an effective amount of the above-described LNP.

[0026] According to an embodiment of the disclosure, the disease can be cancer, an infectious disease, or an autoimmune disease.

[0027] Examples of cancers treatable by the method include, but are not limited to, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, bile duct cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, hypopharyngeal cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, and uterine cancer.

[0028] In some preferred embodiments of the disclosure, when the disease is lung cancer, the LNP

Chemical formula

[0029] In other preferred embodiments, when the disease is lung cancer, the LNP

Chemical formula

[0030] According to some embodiments of the disclosure, the subject is human.

[0031] Still other aspects of the present disclosure are methods of delivering an API into cultured cells, comprising contacting the cultured cells with an effective amount of the above-described LNP, wherein the API is a nucleic acid, polypeptide, protein, carbohydrate, proteoglycan, glycoprotein, or a combination thereof.

[0032] Examples of nucleic acids suitable for use in the present method include, but are not limited to, mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmids, messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), and aptamers. Preferably, the miRNA is a precursor miRNA or a mature miRNA.

[0033] Examples of cultured cells suitable for use in the present method include, but are not limited to, pluripotent stem cells and immune cells.

[0034] Many of the attendant features and advantageous effects of the present disclosure will be more deeply understood by reference to the following detailed description considered in connection with the accompanying drawings.

[0035] This description will be more deeply understood from the following detailed description read in light of the accompanying drawings.

Brief Description of the Drawings

[0036]

Figure 1

Figure 2A

Figure 2B

Mode for Carrying Out the Invention

[0037] The detailed description given below in connection with the accompanying drawings is for the purpose of explaining the present examples and does not represent the only form in which the present examples can be configured or utilized. The description explains the functions of the examples and the sequence of steps for configuring and operating the examples. However, the same or equivalent functions and sequences can also be realized by different examples.

[0038] 1. Definitions For convenience, the specific terms employed in the specification, examples and appended claims are summarized herein. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the technical field to which the present invention pertains.

[0039] The singular forms "a", "an" and "the" are used herein to include plural references unless the context clearly dictates otherwise.

[0040] As used herein, the term "alkyl" refers to a straight-chain or branched hydrocarbon group containing 9 to 25 carbon atoms. C suitable for use in the present invention 9-25 Examples of alkyl include nonyl, decyl, undecyl (similarly hendecyl), dodecyl, tridecyl, tetradecyl, pentadecyl, hexadecyl, heptadecyl, octadecyl, nonadecyl, icosyl, heneicosyl (similarly heneicosyl), docosyl, tricosyl, tetracosyl and pentacosyl.

[0041] The term "alkenyl" used refers to a long straight-chain hydrocarbon group containing 13 to 21 carbon atoms and one or more double bonds. C suitable for use in the present invention 13-21Examples of alkenyl include tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, icosenyl, and heneicosenyl (similarly heneicosanyl). Generally, the total number of carbon atoms (C) and double bonds (D) in an alkene is represented by a numerical symbol of "C:D", such as 14:1, 16:1, 18:1, 18:2, 18:3, 20:4, or 20:5, respectively.

[0042] As used herein, the term "active pharmaceutical ingredient (API)" refers to a component that is biologically active in the form of or as part of a medicine, drug, composition, vaccine, and / or pharmaceutical product, by which a disease and / or condition in a subject can be delayed, improved, inhibited, or prevented. Examples of APIs suitable for use in the present disclosure include, but are not limited to, nucleic acids, polypeptides, proteins, carbohydrates, proteoglycans, glycoproteins, and combinations thereof.

[0043] As used herein, the term "cationic lipid" refers to a lipid having one or more fatty acid or fatty alkyl chains and acquiring a positive charge through one or more amines present in the polar head group. Cationic lipids are usually protonated (i.e., positively charged) at a pH below their pKa.

[0044] As used herein, the term "non-cationic lipid" refers to lipids that do not possess a positive charge, such as neutral lipids or anionic lipids. According to embodiments of the present disclosure, the term "non-cationic lipid" is also intended to include "helper lipids" that are known to stabilize the structure of LNPs and thereby enhance the delivery of APIs carried by the LNPs. Helper lipids also serve to optimize the encapsulation of APIs (e.g., nucleic acids), protect them from degradation, and facilitate their transport into target cells. Examples of helper lipids suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC).

[0045] As used herein, the term "steroid" refers to a class of chemical substances derived from the core structure of gonane or cyclopentanoperhydrophenanthrene, which are tetracyclic hydrocarbons without double bonds (i.e., the steroid nucleus). Examples of steroids that play a role in the formation of the present LNPs include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol.

[0046] As used herein, the term "microRNA" or "miRNA" refers to a class of non-coding RNAs that play a role in regulating gene expression. Most miRNAs are transcribed from DNA sequences into primary miRNAs (pri-miRNAs) and processed into precursor miRNAs (pre-miRNAs) and ultimately mature miRNAs. Accordingly, the term "miRNA" as used herein refers to all non-coding RNAs involved in the processing and maturation of miRNAs, preferably including precursor miRNAs and mature miRNAs.

[0047] As used herein, the term "infectious disease" generally refers to a disorder caused by a pathogen such as a bacterium, virus, fungus, or parasite that causes acute symptoms such as fever, inflammation, upper respiratory symptoms, diarrhea, and the like.

[0048] The terms "subject" or "patient" are used interchangeably herein and are intended to mean a mammal including the human species that can be treated with the API within this cationic lipid. The term "mammal" refers to all members of the mammalian network including humans, primates (e.g., monkeys and chimpanzees), rabbits, pigs, goats, sheep, and cows, etc., domesticated animals and livestock, as well as other animals in zoos, for sports or pets (e.g., horses, dogs, cats, etc.) and rodents such as mice, rats, guinea pigs, and hamsters. In effective examples, the subject is a human. Further, the terms "subject" or "patient" are intended to refer to both male and female genders unless a specific gender is indicated.

[0049] 2. Detailed Description of Preferred Embodiments The present disclosure is based at least in part on the discovery that some novel cationic lipids can act as vehicles for the delivery of polynucleotides into cells. Accordingly, the present disclosure provides novel cationic lipids and compositions thereof that act as vehicles for the delivery of therapeutic agents. The treatment of diseases by the use of the present cationic lipid and / or composition is also disclosed herein.

[0050] 2.1 Cationic Lipids of the Present Disclosure The present disclosure aims to provide novel cationic lipids and compositions containing the same for delivering therapeutic agents. In one aspect, the present invention relates to a cationic lipid of formula (I), [Chemical formula] At least one of X and Y is -(C=O)CH 2 NH 2 and the other X, Y, and Z are -(C=O)CH 2 NH 2 , -[(C=O)CH 2NH](C=O)R 1 、 -(C=O)R 1 and H, independently selected from the group consisting of, R 1 is C 9-25 alkyl or C 13-21 is alkenyl.

[0051] In some embodiments, R 1 is C 9-25 alkyl, and in a preferred embodiment, R 1 is C 11-23 alkyl, and in another preferred embodiment, R 1 is C 13-21 alkyl, and in yet another preferred embodiment, R 1 is C 15-19 alkyl. In some effective examples, R 1 is C 15 alkyl.

[0052] In other embodiments, R 1 is C 13-21 alkenyl, and in a preferred embodiment, R 1 is C 15-19 alkenyl. In some effective examples, R 1 is C 15 alkenyl, and in other effective examples, R 1 is C 17 alkenyl.

[0053] Exemplary compounds of formula (I) are

Chemical formula

Chemical formula

Chemical formula

Chem.

Chem.

Chem.

Chem.

[0054] The cationic lipids of the present disclosure can be prepared by the procedures described in the effective examples. Briefly, first, the amino group of glycine (NH 2) is protected by a protecting group such as a phenacyl (PAc) group or a tert-butyloxycarbonyl (Boc) group. Then, at least one of the PAc- or Boc-protected glycine is linked to glycerol via a condensation reaction (i.e., dehydration) that occurs between at least one hydroxyl group of glycerol and the carboxyl group of glycine. Finally, one or two fatty acids are linked to the remaining hydroxyl groups of glycerol to form the present cationic lipid. According to an alternative embodiment, the condensation reaction that occurs between the carboxyl group of the fatty acid and the amino group of glycine results in an amino bond therebetween, thereby producing an intermediate compound having a free carboxyl group for subsequent reaction with the hydroxyl group of glycerol, and thus producing the final cationic lipid product of the present invention. The detailed reaction conditions and chemicals for producing the present cationic lipid compound can be changed by those skilled in the art without departing from the spirit of the present disclosure.

[0055] Examples of fatty acids suitable for use in the production of the present compound of formula (I) include, but are not limited to, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid, linoleic acid, linolenic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid, erucic acid, and docosahexaenoic acid. In an effective example, the fatty acid used in the production of the present compound of formula (I) is palmitoleic acid or linoleic acid.

[0056] All stereoisomers of the present compound, such as those that may exist due to the asymmetric carbons in each of the X, Y, and Z substituents of the compound of formula (I) including enantiomeric and diastereomeric forms, are considered to be within the scope of the present invention.

[0057] 2.2 Lipid Nanoparticles Also disclosed herein are novel lipid nanoparticles (LNPs) that facilitate the intracellular delivery of biologically active therapeutic molecules into the cells of a patient.

[0058] According to an embodiment of the present disclosure, the lipid nanoparticle (LNP) contains the cationic lipid, non-cationic lipid, and pharmaceutically active ingredient (API) of the above formula (I). Examples of non-cationic lipids suitable for constructing the present LNP include, but are not limited to, helper lipids, phospholipids, PEGylated lipids, PEGylated phospholipids, and combinations thereof. Examples of helper lipids suitable for use in the present disclosure include, but are not limited to, dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE), and distearoylphosphatidylcholine (or 1,2-distearoyl-sn-glycero-3-phosphocholine, DSPC). According to an embodiment of the present disclosure, the non-cationic lipid contained in the present LNP is a helper lipid of distearoylphosphatidylcholine (DSPC).Examples of PEGylated lipids suitable for use in the present disclosure include PEG conjugated to diethylene glycol (DEG-PEG) (e.g., 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (DEG-PEG2000)), PEG conjugated to dialkyloxypropyl (PEG-DAA) (e.g., dioleoylphosphatidylethanolamine (DOPE)), PEG conjugated to diacylglycerol (PEG-DAG) (e.g., 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (DOPE-PEG2000)), PEG conjugated to phosphatidylethanolamine (PEG-PE) (e.g., 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000], DSPE-PEG2000) and combinations thereof, but are not limited thereto. In an effective embodiment, the PEGylated lipid is DEG-PEG2000.

[0059] According to embodiments of the present disclosure, the cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of 1:5 to 5:1, such as 1:5, 1:4.5, 1:4, 1:3.5, 1:3, 1:2.5, 1:2, 1:1.5, 1:1, 1.5:1, 2:1, 2.5:1, 3:1, 3.5:1, 4:1, 4.5:1 or 5:1. In some effective embodiments, the cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of 1:1. In alternative effective embodiments, the cationic lipid and the non-cationic lipid are present in the LNP at a molar ratio of about 4.3:1.

[0060] Alternatively or additionally, the present LNP may further comprise a steroid that helps to stabilize the structure of the LNP. Examples of steroids suitable for constructing the present LNP include, but are not limited to, cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol. Preferably, the present LNP further comprises cholesterol.

[0061] According to some embodiments of the present disclosure, each LNP has a hydrophilic core encapsulated by a lipid bilayer composed of both cationic (e.g., at least one compound of formula (I)) and non-cationic lipids, as well as optionally a steroid and / or helper lipid embedded within the lipid bilayer. Thus, an API that is inherently hydrophobic is inserted between the hydrophobic regions between the lipid bilayers, while an API that is inherently hydrophilic can be located within the hydrophilic core. In other embodiments, instead of having a hydrophilic core encapsulated by a lipid bilayer as shown above, each LNP has a micelle structure in which a plurality of smaller inverse micelles are encapsulated therein. The micelle has a hydrophobic core encapsulated by a monolayer of lipids (e.g., the cationic and non-cationic lipids of the present disclosure) in which the hydrophilic head groups of the lipids face outward, but the hydrophobic tails of the lipids extend towards the center. Further, a plurality of inverse micelles smaller in size than the micelle may be encapsulated within the hydrophobic core of the micelle, where the term "inverse micelle" refers to a micelle in which the hydrophobic tails of the lipids extend outward but the head groups of the lipids are at the center. Thus, a hydrophilic API (e.g., a nucleic acid) can be located inside the hydrophilic core of the inverse micelle.

[0062] This LNP may be generated via procedures and / or tools well known in the art, including thin layer hydration, ethanol injection, microfluidic devices, etc. The cationic lipid (i.e., the compound of formula (I)), non-cationic lipid and optionally steroid self-assemble to form a lipid bilayer. The API may be directly encapsulated within the LNP or may be bound to the cationic lipid prior to encapsulation within the LNP, thereby generating a complex in the form of a nanoparticle, liposome or micelle. The API delivered by this LNP in either liquid or solid form can be a nucleic acid, polypeptide, protein, antibody, carbohydrate, proteoglycan, glycoprotein, small molecule or a combination thereof.

[0063] In some embodiments of the present disclosure, the API is a nucleic acid including but not limited to mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA) or aptamer. In preferred embodiments, the API is miRNA or siRNA. Examples of miRNA include but are not limited to precursor miRNA or mature miRNA. In some effective examples, the miRNA encapsulated and delivered via this cationic lipid has the sequence of SEQ ID NO: 1, and more preferably, the miRNA has the sequence of SEQ ID NO: 2, 3, 4 or 5. In other effective examples, the siRNA used to be delivered via this cationic lipid has the sequence of SEQ ID NO: 6 or 7. In some alternative embodiments, the API is a plasmid.

[0064] 2.3 Treatment via the use of this cationic lipid The cationic lipids of formula (I) and their lipid nanoparticles are useful for the delivery of biologically active agents and, thus, can be used in the treatment of diseases. Accordingly, the present disclosure encompasses methods of treating a disease in a subject in need thereof. The method mainly includes the step of administering to the subject an effective amount of a composition (i.e., a lipid nanoparticle (LNP) composed of a compound of formula (I)).

[0065] The lipid nanoparticles containing the cationic lipid of formula (I) may be formulated into powders, granules, solutions or suspensions, suppositories or patches, and such formulations can be administered orally or parenterally to effectively transport the active ingredient to the appropriate or desired site of action and lesion site. According to the present disclosure, the lipid nanoparticles can be administered parenterally to a lesion (e.g., a tumor) of a subject. Exemplary suitable parenteral routes include, but are not limited to, transdermal, percutaneous, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intraventricular, intradermal, subcutaneous, rectal, intravaginal and intraperitoneal routes. The appropriate route will vary depending on the specific condition being treated, the severity of the condition, age, physical condition, build, gender and weight of the individual patient, including individual patient parameters, the duration of treatment, (where applicable) the nature of the combination therapy, the dose and nature of the active ingredient, genetic factors and similar factors within the knowledge and expertise of the medical practitioner, as will be recognized by those skilled in the art. These factors are well known to those skilled in the art and can be addressed by routine experimentation. Generally, the most appropriate route of administration depends on various factors including the stability of the drug in the environment of the circulatory system and / or the condition of the subject (e.g., the severity of the subject's lung cancer or whether the subject can tolerate conventional treatments). According to some embodiments of the present disclosure, the lipid nanoparticles are formulated into an injectable preparation for parenteral administration. According to other embodiments of the present disclosure, the lipid nanoparticles are formulated into a patch for transdermal administration.

[0066] The present lipid nanoparticles can be administered at a frequency that effectively prevents, inhibits, suppresses, or treats a disease, condition, or trait in a subject. In some embodiments, the lipid nanoparticles can be administered at a frequency of 4 times a day to once every 3 months, for example, 4 times a day, 3 times a day, 2 times a day, once a day, once every other day, once every 3 days, once a week, once every other week, once a month, 2 times a month, 3 times a month, once every other month, or once every 3 months. Preferably, the lipid nanoparticles are administered to the subject at a frequency of 2 times a week (once every 3 or 4 days). Optionally, the lipid nanoparticles are administered to the subject at a frequency of once a week. Further optionally, the lipid nanoparticles are administered to the subject at a frequency of once every other week.

[0067] According to embodiments of the present disclosure, diseases treatable by the present method can include cancer, infectious diseases (including inflammatory diseases), transplantation and / or tissue rejection, autoimmune diseases, and the like.

[0068] Examples of cancers treatable by the present method include, but are not limited to, bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, bile duct cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gingival cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, hypopharyngeal cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, and uterine cancer.

[0069] In a preferred embodiment, a lung cancer subject is

Chemical formula

[0070] In other preferred embodiments, a lung cancer subject has a cationic lipid of

Chemical formula

[0071] 2.4 API Delivery Platform via Use of the Present Cationic Lipids The present lipid nanoparticles (LNPs) are useful for the delivery of APIs into cultured cells and, thus, they can be used as a platform for the delivery of therapeutic materials in the preparation of cell therapies. Accordingly, the present disclosure encompasses a method of delivering an API into cultured cells. The method mainly includes the step of contacting the cultured cells with an effective amount of the present LNPs, and the API is a nucleic acid, polypeptide, protein, carbohydrate, proteoglycan, glycoprotein, or a combination thereof.

[0072] The cultured cells provided in this embodiment are isolated from or derived from a subject, and the cell grafts are useful for cell therapies applicable to selected fields including regenerative medicine, immune system disorders, and cancer, and have the efficacy to differentiate or develop into cell grafts having a therapeutic effect by receiving a gene or gene product delivered via this LNP so as to be beneficial. Generally, cultured cells include stem cells and immune cells. Examples of stem cells suitable for use in this method include pluripotent stem cells (PSCs, e.g., embryonic stem cells (ESCs), epiblast stem cells (EpiSCs), embryonic germ cells (EGCs), and induced pluripotent stem cells (iPSCs)), adult stem cells (ASCs, e.g., hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs)), and cancer stem cells (CSCs), but are not limited thereto. Examples of immune cells suitable for use in this method include, but are not limited to, T cells, dendritic cells (DCs), natural killer (NK) cells, and macrophages. Immune cells generally express genetically engineered antigen receptors, including genetically engineered T cell receptors (TCRs) and functional non-TCR antigen receptors, such as chimeric antigen receptors (CARs), including activating, stimulating, and costimulating CARs and combinations thereof.

[0073] Due to the above characteristics, the present disclosure provides a novel cationic lipid capable of encapsulating the polynucleotide filled therein and delivering it to cells, thereby enabling effective and efficient treatment of diseases including cancer.

Examples

[0074] Materials and Methods Cell Culture NCI-H460 cells (a large cell lung cancer cell line) were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with heat-inactivated 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (PS) solution. A549 cells (established from human lung cancer tissue) were cultured in Ham’s F-12K (Kaighn’s) medium supplemented with heat-inactivated 10% FBS and 1% PS solution. All cells were cultured at 37 °C in an atmosphere containing 5% CO 2 .

[0075] Plasmid Stabilization via This Cationic Lipid This cationic lipid (i.e., compound 6, 9, 11, 14, 18, 22, or 24), lecithin, and cholesterol were thoroughly mixed at a molar ratio of 2:2:1 and completely dissolved in ethanol. The solution was diluted to 30 - 50 μL with DPBS buffer. Then, the diluted solution was co-cultured with 50 μL of an aqueous sucrose solution (5% w / v) containing 1.5 μg of EGFP-plasmid (pUC19, 4564 bp, Addgene) at room temperature for 1 hour to generate a cationic lipid-DNA complex. The resulting solution was divided into two vials. To one of them, 1 μL of DNase and 10 μL of 10×DNase buffer were added, and the mixture was further incubated at 37 °C for 15 minutes. Then, 100 μL of phenol / chloroform (5 / 1) was added, and the mixture was centrifuged at 13000×g for 5 minutes to extract DNA. All samples were subjected to gel electrophoresis (1% agarose gel, 110 V).

[0076] Preparation of Cationic Lipid-RNA Complex To generate the composition, the cationic lipid (Compound 6 or 22), cholesterol, DEG-PEG 2000, and distearoyl phosphatidylcholine (DSPC) were thoroughly mixed in ethanol at a molar ratio of 50:38.5:1.5:10, with the total volume set to 500 μL, thereby generating a liquid formulation of lipid nanoparticles (LNP). As a control group, D-lin-MC3-DMA (MC30), an ionizable amino lipid conventionally used in LNP preparation, was mixed with cholesterol, DEG-PEG 2000, and DSPC at the same molar ratio as this LNP. 0.268 mM, 265 μL of miRNA or siRNA (e.g., the polynucleotides of SEQ ID NOs: 2 - 7) were added to 695 μL of an acidic buffer (composed of 100 mM citrate buffer (pH 4.0) and nuclease-free water) to generate an RNA working solution, with the total volume set to 960 μL. The RNA working solution and LNP (with a molar ratio of 1:6) were each fed into two inlets of a microfluidic device (NanoAssemblr platform) with a flow rate ratio (FRR) set to 3 and a total flow rate (TFR) set to 12 mL / min, mixed within the flow path, and the combined liquid reaching the outlet of the microfluidic device was collected at a total volume of 1.2 mL. Then, the collected mixture was filtered and centrifuged three times at 2,000 g for 20 minutes using a centrifugal filter (a membrane filter with a 10 kDa cut-off molecular weight), and the filtrate containing lipid nanoparticles encapsulating miRNA or siRNA therein (i.e., the cationic lipid-RNA complex) was stored at 4°C until use.

[0077] Characterization of the Cationic Lipid-RNA Complex The particle size and zeta potential of the cationic lipid-RNA complex were determined using a particle size and zeta potential analyzer (DelsaNano C, Beckman Coulter), respectively.

[0078] The encapsulation ratio of liposomes for encapsulating miRNA or siRNA was determined by a fluorescence reader (Infinite F200 Pro) using an RNA quantification kit containing a fluorescent dye (RiboGreen). To construct a standard curve, the absorption spectra of miRNA or siRNA with a known concentration (i.e., 100 μg / mL) were first obtained. Then, the cationic lipid-RNA complex (usually having a total volume of 50 μL for each sample well) was mixed with the reagents in the RNA quantification kit in each well. Optionally, the cationic lipid-RNA complex was exposed to a lysis buffer, thereby returning the RNA to a suspension. The amount of miRNA or siRNA was quantified by interpolating their absorption intensities at 535 nm into those of the standard curve. Assuming that Wt is the total amount of miRNA or siRNA released into the LNP lysis suspension and Wi is the total amount of miRNA or siRNA initially added during the preparation, the encapsulation efficiency (EE) was calculated by the following formula, Encapsulation efficiency (EE%) = (Wt / Wi) × 100% as follows.

[0079] Flow cytometry analysis Cell sorting was performed via flow cytometry using miRNA or siRNA conjugated with a fluorochrome. Specifically, a far-red fluorescent label Cy5 dye was conjugated to the miRNA. After the final wash before sorting, the cells were filtered through a 35-μm nylon cell strainer to remove cell clumps and then subjected to sorting using a flow cytometer (BD LSRFortessa X-20) and analysis software (FlowJo, BD Biosciences).

[0080] Example 1 Chemical synthesis of the cationic lipid The cationic lipid was synthesized by the steps described in Schemes I-IV, respectively. Generally, the amino group (NH of glycine 2)The base was first protected with a phenacyl (PAc) group or a tert-butyloxycarbonyl (Boc) group. After the condensation reaction (i.e., dehydration) between glycerol and glycine and / or between glycerol and fatty acid, the protecting group was removed and reduced to the NH 2 group.

[0081] Scheme I

Chem.

[0082] Scheme II

Chem.

[0083] Scheme III

Chem.

[0084] Scheme IV

Chem.

[0085] The method for preparing exemplary compounds and the analytical data for the compounds prepared as a result are described below.

[0086] 1.1 TGG-linoleic acid (Compound 6)

Chem.

[0087] Preparation of tert-butyl 2-((9Z,12Z)-octadeca-9,12-dienamido)acetate (1) A solution of N,N’-dicyclohexylcarbodiimide (DCC, 0.40 g, 1.96 mmol) in 10 mL of dichloromethane (DCM) was added to linoleic acid (0.5 g, 1.78 mmol), t-butyl 2-aminoacetate (0.27 mL, 1.96 mmol) and DMAP (0.13, 1.07 mmol). The solution was stirred overnight at room temperature. After thorough mixing, 1,3-dicyclohexyl urea (DCU) was removed by filtration and the solution was removed by rotary evaporator. The residual solution was purified by column chromatography eluted with ethyl acetate (hexane:EA, 3:1) to give 480 mg of a solid (Compound 1) (yield 69%). 1 H NMR (600 MHz, CDCl 3 ) δ 5.93 (s, 1H), 5.44 - 5.26 (m, 4H), 3.95 (d, J = 4.9 Hz, 2H), 2.78 (t, J = 6.9 Hz, 2H), 2.29 - 2.17 (m, 2H), 2.10 - 2.02 (m, 4H), 1.66 (dd, J = 15.7, 8.8 Hz, 3H), 1.49 (s, 9H), 1.39 - 1.28 (m, 14H), 0.91 (t, J = 7.0 Hz, 3H).

[0088] Preparation of 2-((9Z,12Z)-octadeca-9,12-dienamido)acetic acid (2) Trifluoroacetic acid (TFA, 2.80 mL, 36.59 mmol) was slowly added to a solution of Compound 1 (480 mg, 1.22 mmol dissolved in 8 mL of DCM). The solution was stirred and monitored by TLC. After complete reaction, DCM was removed under reduced pressure. The residue was washed several times with hexane and EA. The remaining solvent was removed by rotary evaporator to give Compound 2 for further use.

[0089] Preparation of 2-((tert-butoxycarbonyl)amino)acetic acid (3) To a bottle having a solution of 2N NaOH (8.66 mL, 17.32 mmol), glycine (1 g, 13.32 mmol) was added and the mixture was stirred until all of the glycine was dissolved and then Boc 2 O (3.34 g, 15.32 mmol) dissolved in THF (20 mL) was added to the bottle and stirred overnight at room temperature. The THF was removed by evaporation. To make the basic solution, 2N NaOH solution was added to the residue and washed with EA. The pH value was adjusted to 3 - 4 by adding 2M HCl. The aqueous phase was extracted with EA. The organic phase was dried over MgSO 4 and filtered, and the solvent was removed by rotary evaporator to obtain 1.38 g of a white solid product (Compound 3, yield 59%). 1H NMR (600 MHz, CDCl3) δ 5.03 (s, 1H), 3.97 (t, J = 20.4 Hz, 2H), 1.48 (s, 9H).

[0090] Preparation of 2-hydroxypropane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (4) To a solution of DCC (0.99 g, 4.78 mmol) dissolved in DCM, glycerol (0.2 g, 2.17 mmol), Compound 3 (0.80 g, 4.56 mmol) and DMAP (0.27 g, 2.17 mmol) were added and stirred overnight at room temperature. The mixture was filtered to remove DCU and evaporated to obtain the crude product. The crude product was purified by column chromatography (hexane:EA, 50:50→30:70) to obtain 130 mg of a sticky transparent liquid product (Compound 4, yield 15%). LC-MS (ESI); m / z: [M+H] + Calcd. for C17 H 30 N 2 O 9 ,406.20. Found 407.41.

[0091] Preparation of 2-(2-((9Z,12Z)-octadeca-9,12-dienamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (5) To a solution of DCC (0.073 g, 0.35 mmol) dissolved in DCM, compound 4 (0.13 g, 0.32 mmol), compound 2 (0.12 g, 0.35 mmol) and DMAP (0.039 g, 0.32 mmol) were added. The solution was stirred at room temperature overnight. After complete mixing, the solution was filtered to remove DCU and then evaporated under reduced pressure to remove DCM. The residual solution was purified by column chromatography (hexane:EA, 2:3) to give 200 mg of a white solid product (compound 5, 86% yield). 11H NMR (600 MHz, CDCl3) δ 5.36 (ddd, J = 27.2, 14.1, 6.3 Hz, 4H), 5.21 (s, 1H), 4.40 (dt, J = 18.0, 9.0 Hz, 2H), 4.34 - 4.23 (m, 2H), 4.13 - 3.97 (m, 2H), 3.97 - 3.82 (m, 4H), 2.79 (t, J = 6.8 Hz, 1H), 2.27 (dd, J = 14.3, 6.9 Hz, 2H), 2.11 (d, J = 5.8 Hz, 1H), 2.06 (dd, J = 13.8, 6.8 Hz, 3H), 1.95 (s, 2H), 1.86 (dd, J = 9.8, 4.7 Hz, 2H), 1.78 (d, J = 13.3 Hz, 2H), 1.70 - 1.61 (m, 3H), 1.47 (s, 18H), 1.41 - 1.26 (m, 16H), 0.91 (t, J = 6.6 Hz, 3H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 37 H 63 N 3 O 11 , 725.45. Found 726.7.

[0092] Preparation of 2-(2-((9Z,12Z)-octadeca-9,12-dienamido)acetoxy)propane-1,3-diyl bis(2-aminoacetate) (6) To a solution of compound 5 (0.2 g, 0.28 mmol) dissolved in 6 mL of DCM, TFA (0.42 mL, 5.51 mmol) was slowly added. The solution was stirred until the reactants were consumed. Then, the resulting solution was evaporated under reduced pressure to remove the solvent and washed with ether and DCM. The residual solvent was removed by evaporation to afford 48 mg of a sticky transparent product (compound 6, 33% yield). 11H NMR (600 MHz, MeOD) δ 5.53 - 5.44 (m, 1H), 5.41 - 5.28 (m, 4H), 4.60 - 4.37 (m, 4H), 3.94 - 3.84 (m, 6H), 2.79 (t, J = 6.7 Hz, 2H), 2.28 (dd, J = 9.8, 5.0 Hz, 2H), 2.07 (dd, J = 12.2, 6.2 Hz, 4H), 1.67 - 1.55 (m, 2H), 1.41 - 1.27 (m, 15H), 0.92 (t, J = 6.8 Hz, 3H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 27 H 47 N 3 O 7 , 525.34. Found 527.38.

[0093] 1.2 TGG - Di(linoleic acid) (TGG - di(linoleic acid)) Compound 9

[0094] Procedure 1

Chem.

[0095] Preparation of 2,3 - Dihydroxypropyl 2 - ((tert - butoxycarbonyl)amino)acetate (2,3 - dihydroxypropyl 2 - ((tert - butoxycarbonyl)amino)acetate) (7) Glycerol (0.42 g, 4.57 mmol), Compound 3 (0.2 g, 1.14 mmol), EDCI (0.35 g, 2.28 mmol) and DMAP (0.14 g, 1.14 mmol) were mixed and dissolved in DCM. The solution was stirred overnight at room temperature. The resulting solution was evaporated to remove DCM to obtain a crude product. The crude product was purified by column chromatography (EA, 100%) to obtain 120 mg of a sticky liquid product (Compound 7, yield 42%). 11H NMR (600 MHz, MeOD) δ 4.59 (s, 1H), 4.22 (dd, J = 11.3, 4.3 Hz, 1H), 4.16 - 4.10 (m, 1H), 3.88 - 3.81 (m, 2H), 3.57 (dd, J = 5.6, 2.1 Hz, 2H), 1.46 (s, 9H).

[0096] Preparation of 3-(2-((tert-butoxycarbonyl)amino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (8) Compound 7 (0.1 g, 0.40 mmol), compound 2 (0.30 g, 0.88 mmol), EDCI (0.16 g, 1.00 mmol) and DMAP (0.059 g, 0.48 mmol) were mixed and dissolved in DCM. After stirring overnight at room temperature, the mixture was evaporated to remove DCM. The residual solution was extracted with EA / H 2 2O. The organic phase was collected, dried over MgSO 4 4 and filtered. The resulting filtrate was evaporated to remove EA. The crude product was purified by column chromatography (hexane:EA, 3:7) to give 190 mg of a sticky brown liquid product (compound 9, yield 53%). 11H NMR (600 MHz, CDCl3) δ 5.46 - 5.31 (m, 8H), 4.46 (dd, J = 12.0, 3.5 Hz, 1H), 4.41 - 4.33 (m, 2H), 4.28 (ddd, J = 17.9, 12.1, 6.2 Hz, 1H), 4.14 (q, J = 7.1 Hz, 1H), 4.07 (ddd, J = 19.8, 9.8, 5.1 Hz, 3H), 4.00 (d, J = 3.6 Hz, 2H), 3.96 - 3.83 (m, 2H), 2.79 (t, J = 6.8 Hz, 3H), 2.27 (ddd, J = 9.9, 7.8, 5.2 Hz, 4H), 2.13 - 2.00 (m, 8H), 1.76 - 1.61 (m, 8H), 1.47 (s, 9H), 1.40 - 1.26 (m, 28H), 0.91 (t, J = 7.0 Hz, 6H).

[0097] Procedure 2

Chem.

[0098] Preparation of 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (25) Glycerol (0.29 g, 3.15 mmol) and trt-glycine (0.2 g, 0.63 mmol) were mixed and dissolved in a DCM / dioxane solvent to provide a mixture. DCC (0.17 g, 0.82 mmol) and DMAP (0.038 g, 0.32 mmol) were gradually added to the mixture, and the mixture was stirred overnight at room temperature. The solution was filtered to remove DCU and evaporated under reduced pressure to remove the solvent. The crude product was purified by column chromatography (Hex:EA = 1:3) to obtain 195 mg of a white solid product (Compound 25, yield 79%). 11H NMR (600 MHz, MeOD) δ 7.62 - 7.38 (m, 6H), 7.30 (dd, J = 10.6, 5.0 Hz, 6H), 7.24 - 7.16 (m, 3H), 4.11 (dd, J = 11.3, 4.2 Hz, 1H), 4.02 (dd, J = 11.3, 6.4 Hz, 1H), 3.76 (td, J = 9.9, 5.7 Hz, 1H), 3.50 (d, J = 5.7 Hz, 2H), 3.13 (s, 2H).

[0099] Preparation of 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (26) Compound 25 (0.2 g, 0.51 mmol), compound 2 (0.40 g, 1.18 mmol), EDCI (0.18 g, 1.18 mmol) and DMAP (0.062 g, 0.51 mmol) were mixed and dissolved in DCM. The solution was stirred overnight at room temperature and then evaporated to dryness under reduced pressure to remove DCM. The crude product was purified by column chromatography (hexane:EA, 3:2) to give 266 mg of a sticky white product (compound 26, 51% yield). 1 1H NMR (600 MHz, CDCl 3) δ 7.49 (d, J = 7.7 Hz, 6H), 7.36 (d, J = 6.4 Hz, 5H), 7.31 (dd, J = 6.7, 2.3 Hz, 2H), 5.46 - 5.29 (m, 6H), 5.27 (d, J = 6.0 Hz, 1H), 4.26 (ddd, J = 19.0, 11.9, 5.4 Hz, 3H), 4.14 (dd, J = 11.8, 5.8 Hz, 1H), 4.09 - 3.91 (m, 4H), 2.79 (t, J = 6.8 Hz, 3H), 2.27 (dd, J = 14.0, 6.6 Hz, 4H), 2.07 (dd, J = 12.3, 5.3 Hz, 6H), 1.62 (dd, J = 27.0, 19.6 Hz, 11H), 1.52 - 1.20 (m, 28H), 0.97 - 0.81 (m, 6H).

[0100] Preparation of 3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (9) To a solution of compound 26 (0.27 g, 0.26 mmol) dissolved in DCM, TFA (0.3 mL, 3.93 mmol) was slowly added and stirred until all the reactants were consumed. The solution was evaporated and the solvent was removed by washing several times with ether. The crude product was purified by column chromatography (Hex:EA = 1:1 → DCM:MeOH, 95:5) to obtain 131 mg of a pasty brown product (compound 9, yield 64%). 11H NMR (600 MHz, MeOD) δ 5.48 - 5.25 (m, 8H), 4.53 - 4.44 (m, 2H), 4.37 (ddd, J = 18.1, 12.1, 5.0 Hz, 2H), 4.06 - 3.81 (m, 6H), 2.79 (t, J = 6.8 Hz, 3H), 2.27 (td, J = 7.5, 4.1 Hz, 4H), 2.08 (dt, J = 10.9, 5.1 Hz, 7H), 1.72 - 1.56 (m, 4H), 1.50 - 1.14 (m, 28H), 0.92 (t, J = 7.0 Hz, 6H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 45 H 77 N 3 O 8 ,787.57. Found 789.48.

[0101] 1.3 DGG-linoleic acid (DGG-linoleic acid) (Compound 11)

Chem.

[0102] Preparation of 2-((9Z,12Z)-octadeca-9,12-dienoyloxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (10) A solution of DCC (76 mg, 0.37 mmol) dissolved in 8 mL of DCM was mixed with and added to compound 4 (0.15 g, 0.37 mmol), linoleic acid (93 mg, 0.33 mmol), and DMAP (32 mg, 0.26 mmol). The mixture was stirred overnight at room temperature and then filtered to remove DCU. Then, the solution was evaporated to obtain the crude product. The crude product was purified by column chromatography (hexane:EA, 7:3) to obtain 140 mg of a clear liquid product (compound 10, yield 63%). 1 H NMR (600 MHz, CDCl3) δ 5.33 (ddt, J = 14.9, 10.0, 7.8 Hz, 2H), 5.14 - 5.01 (m, 1H), 4.47 - 4.34 (m, 2H), 4.25 (dd, J = 11.9, 5.7 Hz, 1H), 4.22 - 4.11 (m, 1H), 4.11 - 4.00 (m, 1H), 3.93 (s,4H), 2.79 (t, J = 6.8 Hz, 1H), 2.41 - 2.28 (m, 2H), 2.16 - 2.01 (m, 2H), 1.92 - 1.83 (m, 1H), 1.69 - 1.51 (m, 9H), 1.47 (s, 18H), 1.42 - 1.20 (m, 14H), 0.91 (dd, J = 8.6, 5.3 Hz, 3H).

[0103] Preparation of 2-((9Z,12Z)-octadeca-9,12-dienoyloxy)propane-1,3-diyl bis(2-aminoacetate) (11) TFA (0.40 mL, 5.23 mmol) was slowly added to a solution containing compound 10 (0.14 g, 0.21 mmol) dissolved in 6 mL of DCM. The solution was stirred until the reactants were consumed and then evaporated to remove the solvent, and washed with ether and DCM. The resulting product was evaporated to obtain compound 11 (60 mg of a pasty clear product, yield 61%). 11H NMR (600 MHz, MeOD) δ 5.51 (s, 1H), 5.45 (dd, J = 9.3, 4.9 Hz, 1H), 5.42 - 5.29 (m, 4H), 4.57 (dt, J = 12.0, 4.1 Hz, 1H), 4.50 - 4.43 (m, 1H), 4.39 (dd, J = 12.0, 6.4 Hz, 1H), 3.96 - 3.82 (m, 3H), 2.79 (t, J = 6.7 Hz, 2), 2.38 (td, J = 7.5, 4.9 Hz, 2H), 2.16 - 1.99 (m, 3H), 1.64 (d, J = 5.9 Hz, 2H), 1.44 - 1.26 (m, 13H), 0.92 (dd, J = 9.4, 4.5 Hz, 3H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 25 H 44 N 2 O 6 , 468.32. Found 470.22.

[0104] 1.4 MGG - di(linoleic acid) (Compound 14)

Chem.

[0105] (9Z,9’Z,12Z,12’Z)-2-hydroxypropane-1,3-diyl bis(octadeca-9,12-dienoate) (12) Preparation In a round-bottomed bottle, glycerol (0.2 g, 2.17 mmol), linoleic acid (1.28 g, 4.56 mmol), EDCI (0.78 g, 5.00 mmol), and DMAP (0.27 g, 2.17 mmol) were dissolved and mixed in 12 mL of DCM. The solution was stirred overnight at room temperature and then evaporated to remove DCM. The crude product was purified by column chromatography (hexane:EA, 3:2) to obtain 310 mg of the product (Compound 12, yield 23%). 1 H NMR (600 MHz, CDCl3) δ 5.51 - 5.24 (m, 7H), 4.33 - 3.94 (m, 5H), 2.79 (t, J = 6.8 Hz, 4H), 2.37 (t, J = 7.6 Hz, 4H), 2.07 (q, J = 6.9 Hz, 8H), 1.64 (dd, J = 14.5, 7.2 Hz, 4H), 1.48 - 1.19 (m, 28H), 0.91 (t, J = 7.0 Hz, 6H).

[0106] Preparation of (9Z,9’Z,12Z,12’Z)-2-(2-((tert-butoxycarbonyl)amino)acetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate) ((9Z,9’Z,12Z,12’Z)-2-(2-((tert-butoxycarbonyl)amino)acetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate)) (13) To a solution of DCC (0.12 g, 0.60 mmol) dissolved in 10 mL of DCM, Compound 12 (0.31 g, 0.50 mmol), Compound 3 (0.11 g, 0.60 mmol), and DMAP (4.3 mg, 0.35 mmol) were added. The solution was stirred overnight at room temperature and then filtered to remove DCU. The solution was evaporated to remove the solvent and purified by column chromatography (hexane:EA, 7:3) to obtain 270 mg of the product (Compound 13, yield 70%). 1 H NMR (600 MHz, CDCl 3) δ 5.46 - 5.21 (m, 8H), 4.99 (s,1H), 4.35 (dd, J = 12.1, 4.1 Hz, 2H), 4.17 (dd, J = 12.1, 5.9 Hz, 2H), 3.95 (d, J = 5.2 Hz, 2H), 2.79 (t, J = 6.8 Hz, 4H), 2.33 (t, J = 7.6 Hz, 4H), 2.07 (q, J = 7.0 Hz, 7H), 1.62 (dd, J = 15.9, 8.6 Hz, 5H), 1.47 (s, 9H), 1.43 - 1.17 (m, 28H), 0.91 (t, J = 6.9 Hz, 6H).

[0107] (9Z,9’Z,12Z,12’Z)-2-(2-Aminoacetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate) ((9Z,9’Z,12Z,12’Z)-2-(2-aminoacetoxy)propane-1,3-diyl bis(octadeca-9,12-dienoate))(14) Preparation To a solution of compound 13 (0.27 g, 0.35 mmol) dissolved in 5 mL of DCM, TFA (0.53 mL, 6.98 mmol) was slowly added and stirred until the reactants were consumed. The solution was evaporated to remove the solvent. The residue was washed with ether and hexane, and the residual solvent was removed by evaporation under reduced pressure, thereby obtaining about 200 mg of a pasty orange product (compound 14, 85% yield). 1 H NMR (600 MHz, MeOD) δ 5.43 - 5.28 (m, 8H), 4.43 (dd, J = 12.2, 4.2 Hz, 2H), 4.26 (dd, J = 12.2, 5.7 Hz, 2H), 3.88 (s, 2H), 2.79 (t, J = 6.6 Hz, 4H), 2.37 (t, J = 7.4 Hz, 4H), 2.15 - 2.03 (m, 8H), 1.68 - 1.58 (m, 4H), 1.44 - 1.25 (m, 28H), 0.92 (t, J = 7.0 Hz, 6H). LC-MS (ESI); m / z: [M+H] +Calcd. for C 41 H 71 NO 6 , 673.53. Found 675.91.

[0108] 1.5 MGG-linoleic acid (Compound 18)

Chem.

[0109] Preparation of (9Z,12Z)-3-(2-((tert-butoxycarbonyl)amino)acetoxy)-2-hydroxypropyl octadeca-9,12-dienoate (17) In a round-bottom flask, Compound 7 (0.19 g, 0.75 mmol), linoleic acid (0.1 g, 0.36 mmol), 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI, 0.11 g, 0.73 mmol) and DMAP (44 mg, 0.36 mmol) were mixed and dissolved in 10 mL of DCM. The solution was stirred overnight at room temperature and then evaporated to remove DCM. The crude product was purified by column chromatography (Hex:EA, 2:3) to give 75 mg of a sticky liquid product (Compound 17, yield 41%). 11H NMR (600 MHz, CDCl3) δ 5.47 - 5.30 (m, 2H), 4.50 - 4.01 (m, 5H), 3.96 (d, J = 5.4 Hz, 2H), 2.79 (t, J = 6.9 Hz, 1H), 2.37 (t, J = 7.5 Hz, 2H), 2.07 (dd, J = 13.4, 7.2 Hz, 2H), 1.63 (dd, J = 23.5, 18.2 Hz, 8H), 1.53 - 1.38 (m, 9H), 1.45 - 1.19 (m, 14H), 0.99 - 0.85 (m, 3H).

[0110] Preparation of ((9Z,12Z)-3-(2-aminoacetoxy)-2-hydroxypropyl octadeca-9,12-dienoate) (18) To a solution of compound 17 (75 mg, 0.15 mmol) dissolved in 4 mL of DCM, TFA (0.22 mL, 2.93 mmol) was slowly added, stirred until the reactants were consumed, and the solution was evaporated to remove DCM. The residue was washed with ether and DCM. Then the remaining solvent was evaporated again under reduced pressure, thereby obtaining about 75 mg of a pasty light brown product (compound 18, yield 100%). 11H NMR (600 MHz, MeOD) δ 5.43 - 5.29 (m, 4H), 4.33 (dd, J = 11.4, 4.3 Hz, 1H), 4.28 (dd, J = 11.4, 5.8 Hz, 1H), 4.15 (dd, J = 5.4, 3.8 Hz, 1H), 4.11 - 4.03 (m, 1H), 3.89 (s, 2H), 2.79 (t, J = 6.6 Hz, 2H), 2.38 (t, J = 7.5 Hz, 2H), 2.11 - 2.05 (m, 4H), 1.68 - 1.59 (m, 2H), 1.41 - 1.28 (m, 14H), 0.92 (t, J = 7.0 Hz, 3H). LC-MS (ESI); m / z: [M+H]+ Calcd. for C 23 H 41 NO 5 ,411.30. Found 412.98.

[0111] 1.6 TGG-di(palmitoleic acid) (Compound 22)

[0112] Procedure 1

Chemical Structure

[0113] Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) (19) In a round-bottomed bottle, to a solution of DCC (0.97 g, 4.72 mmol) dissolved in 12 mL of DCM, palmitoleic acid (1 g, 3.93 mmol), t-butyl 2-aminoacetate (0.62 g, 4.72 mmol) and DMAP (0.34 g, 2.75 mmol) were added. The solution was stirred overnight at room temperature and then filtered to remove DCU, and evaporated to remove the solvent. The crude product was purified by column chromatography (hexane:EA, 3:1) to give an adhesive transparent liquid product of about 1.04 g (Compound 19, yield 72%). 1 H NMR (600 MHz, CDCl 3 ) δ 5.92 (s, 1H), 5.36 (ddd, J = 7.5, 3.9, 1.9 Hz, 2H), 3.96 (d, J = 4.9 Hz, 2H), 2.24 (dd, J = 10.1, 5.3 Hz, 2H), 2.03 (q, J = 6.5 Hz, 3H), 1.66 (dd, J = 14.5, 7.7 Hz, 2H), 1.49 (s, 9H), 1.31 (ddd, J = 21.0, 13.6, 7.1 Hz, 16H), 0.90 (dd, J = 9.7, 4.4 Hz, 3H).

[0114] Preparation of (Z)-2-(hexadec-9-enamido)acetic acid (20) To a solution of Compound 19 (1.04 g, 2.83 mmol) dissolved in DCM, TFA (4.33 mL, 56.59 mmol) was slowly added and stirred until the reactants were consumed. The solution was evaporated to remove DCM. The residue was washed with ether and hexane, and the remaining solvent was removed again by evaporation under reduced pressure, thereby providing a white powder product (about 870 mg, Compound 20) (yield 99%) for further use. 11H NMR (600 MHz, MeOD) δ 5.36 (t, J = 4.7 Hz, 2H), 3.90 (s, 2H), 2.26 (t, J = 7.5 Hz, 2H), 2.04 (dd, J = 11.9, 6.2 Hz, 4H), 1.73 - 1.60 (m, 2H), 1.33 (dd, J = 14.6, 8.6 Hz, 16H), 0.92 (t, J = 7.0 Hz, 3H).

[0115] (Z)-3-(2-((tert-Butoxycarbonyl)amino)acetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (21) Preparation In a round-bottom flask, compound 7 (50 mg, 0.20 mmol), compound 20 (0.14 g, 0.44 mmol), EDCI (70 mg, 0.44 mmol) and DMAP (25 mg, 0.20 mmol) were mixed and added to 10 ml of DCM. The solution was stirred overnight at room temperature and evaporated under reduced pressure to remove DCM. The crude product was purified by column chromatography (Hex:EA, 1:4) to obtain 140 mg of a sticky orange liquid product (compound 21, 84% yield). 1 1H NMR (600 MHz, CDCl 3 ) δ 5.43 - 5.33 (m, 2H), 4.41 - 4.32 (m, 2H), 4.11 - 4.03 (m, 4H), 3.95 - 3.86 (m, 2H), 2.28 (tt, J = 13.8, 6.9 Hz, 4H), 2.07 - 1.99 (m, 4H), 1.92 (tdd, J = 20.5, 12.6, 7.8 Hz, 2H), 1.86 - 1.76 (m, 2H), 1.75 - 1.51 (m, 12H), 1.48 (s, 9H), 1.44 - 1.21 (m, 32H), 0.91 (q, J = 6.9 Hz, 6H).

[0116] Preparation of (Z)-3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) (22) To a solution of compound 21 (0.14 g, 0.17 mmol) dissolved in 4 mL of DCM, TFA (0.26 mL, 3.35 mmol) was slowly added and the reaction mixture was stirred until the reactants were completely consumed. Then the solution was evaporated to remove DCM, and the residue was washed with hexane and DCM. The remaining solvent was further removed by evaporation under reduced pressure. As a result, about 80 mg of a pasty orange product was obtained (compound 22, 64% yield). 1 H NMR (600 MHz, MeOD) δ 5.37 (dd, J = 19.9, 15.0 Hz, 5H), 4.61 - 4.24 (m, 4H), 4.04 - 3.79 (m, 6H), 2.27 (td, J = 7.5, 4.0 Hz, 4H), 2.17 - 1.92 (m, 8H), 1.70 - 1.55 (m, 4H), 1.49 - 1.23 (m, 32H), 0.92 (t, J = 7.0 Hz, 6H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 41 H 73 N 3 O 8 , 735.54. Found 737.33.

[0117] Procedure 2

Chemical Structure

[0118] (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) ((Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate)) (19) Preparation In a round-bottom flask, to a solution of DCC (1.16 g, 5.66 mmol) dissolved in 10 ml of DCM, palmitoleic acid (1.2 g, 4.72 mmol), tert-butyl 2-aminoacetate (0.74 g, 5.66 mmol) and DMAP (0.4 g, 3.3 mmol) were gradually added and stirred overnight at room temperature. The mixture was filtered to remove DCU and evaporated to remove the solvent. The crude product was purified by column chromatography (hexane:EA, 4:1) to obtain about 1.37 g of a sticky transparent liquid product (Compound 19, yield 72%).

[0119] (Z)-2-(hexadec-9-enamido)acetic acid ((Z)-2-(hexadec-9-enamido)acetic acid) (20) Preparation To a solution of Compound 19 (1.37 g, 3.73 mmol) dissolved in DCM, TFA (4.28 mL, 55.91 mmol) was slowly added and stirred until the reactants were consumed. The solution was evaporated to remove DCM. The residue was washed with ether and hexane, and the remaining solvent was removed again by evaporation under reduced pressure, thereby providing a white powder product (about 1150 mg, Compound 20) (yield 99%) for further use.

[0120] 3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate) (3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((9Z,12Z)-octadeca-9,12-dienamido)acetate)) (25) Preparation Glycerol (0.29 g, 3.15 mmol) and trt-glycine (0.2 g, 0.63 mmol) were injected into a round-bottom flask, and a mixture of DCM / dioxane was added as a solvent. DCC (0.17 g, 0.82 mmol) and DMAP (38 mg, 0.32 mmol) were gradually added into the round-bottom flask. After the solution was stirred overnight at room temperature, DCU was removed. The solvent was removed under reduced pressure. The crude product was purified by column chromatography (Hex:EA = 1:3) to obtain 195 mg of a white solid product (yield 79%).

[0121] (Z)-3-(2-(Tritylamino)acetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) ((Z)-3-(2-(tritylamino)acetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate)) (27) Preparation Compound 25 (0.2 g, 0.51 mmol), compound 20 (0.37 g, 1.18 mmol), EDCI (0.18 g, 1.18 mmol) and DMAP (60 mg, 0.51 mmol) were mixed, dissolved in 10 ml of DCM, and the mixture was stirred overnight at room temperature. Then, the mixture was evaporated to remove DCM to obtain a crude product. The crude product was purified by column chromatography (Hex:EA, 3:2) to obtain 280 mg of a viscous white product (compound 27, yield 56%). 1 H NMR (600 MHz, CDCl 3) δ 7.49 (d, J = 7.6 Hz, 6H), 7.36 (s, 5H), 5.37 (dd, J = 9.6, 4.3 Hz, 2H), 5.32 - 5.20 (m, 1H), 4.31 - 4.20 (m, 3H), 4.15 (s, 1H), 4.09 - 4.02 (m, 4H), 3.96 (dd, J = 18.1, 5.3 Hz, 1H), 2.27 (td, J = 7.7, 3.9 Hz, 4H), 2.08 - 1.97 (m, 4H), 1.97 - 1.87 (m, 2H), 1.86 - 1.75 (m, 2H), 1.63 (dd, J = 23.3, 15.7 Hz, 14H), 1.49 - 1.20 (m, 30H), 0.91 (q, J = 6.9 Hz, 6H).

[0122] (Z)-3-(2-Aminoacetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate) ((Z)-3-(2-aminoacetoxy)propane-1,2-diyl bis(2-((Z)-hexadec-9-enamido)acetate)) (22) Preparation To a solution of compound 27 (0.28 g, 0.29 mmol) dissolved in 8 mL of DCM, TFA (0.33 mL, 4.29 mmol) was slowly added and stirred until the reactant was completely consumed. The solution was evaporated to remove the solvent, and the residue was purified by column chromatography (DCM:MeOH, 95:5~90:10) to obtain about 200 mg of a viscous white to pale yellow solid product (compound 22, yield 94%). 11H NMR (600 MHz, MeOD) δ 5.40 (tt, J = 6.3, 4.1 Hz, 1H), 5.37 - 5.29 (m, 3H), 4.51 - 4.43 (m, 2H), 4.37 (tt, J = 18.1, 6.2 Hz, 2H), 4.05 - 3.76 (m, 6H), 2.26 (td, J = 7.5, 4.1 Hz, 4H), 2.02 (dd, J = 20.5, 13.2 Hz, 7H), 1.71 - 1.55 (m, 5H), 1.46 - 1.17 (m, 32H), 0.90 (t, J = 7.0 Hz, 6H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 41 H 73 N 3 O 8 , 735.54. Found 737.18.

[0123] 1.7 TGG-Palmitoleic acid (TGG-palmitoleic acid) (Compound 24)

Chem.

[0124] (Z)-2-(2-(Hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate) ((Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-((tert-butoxycarbonyl)amino)acetate)) (23) Preparation In a round-bottom flask, compound 4 (0.22 g, 0.54 mmol), compound 20 (0.20 g, 0.65 mmol), EDCI (0.10 g, 0.65 mmol) and DMAP (0.07 g, 0.54 mmol) were mixed and dissolved in 10 ml of DCM. The mixture was stirred overnight at room temperature and then evaporated to remove DCM. The residue was extracted with EA / H 2 2O, the organic phase was taken, MgSO4 It was dried and filtered. The filtrate was evaporated under reduced pressure to remove the solvent. The crude product was purified by column chromatography (hexane:EA, 2:3) to obtain 290 mg of a sticky transparent liquid product (Compound 23, yield 77%). 1 H NMR (600 MHz, CDCl3) δ 5.43 - 5.31 (m, 2H), 5.21 (s, 1H), 4.50 - 4.35 (m, 2H), 4.30 (dd, J = 15.4, 8.4 Hz, 2H), 4.14 - 3.96 (m, 3H), 3.98 - 3.81 (m, 3H), 2.27 (ddd, J = 15.2, 7.3, 3.2 Hz, 2H), 2.03 (dd, J = 13.1, 6.7 Hz, 2H), 1.92 (ddd, J = 19.1, 9.5, 4.8 Hz, 1H), 1.87 - 1.75 (m, 1H), 1.75 - 1.51 (m, 8H), 1.47 (s, 15H), 1.44 - 1.23 (m, 16H), 0.91 (q, J = 6.9 Hz, 3H).

[0125] Preparation of (Z)-2-(2-(hexadec-9-enamido)acetoxy)propane-1,3-diyl bis(2-aminoacetate) (24) To a solution of Compound 23 (0.29 g, 0.41 mmol) dissolved in 5 mL of DCM, TFA (0.95 mL, 12.43 mmol) was slowly added and stirred until all the reactants were consumed. After removing the DCM in the solution by a rotary evaporator, the residue was washed with hexane and ether. The remaining solvent was removed by evaporation under reduced pressure, thereby obtaining about 170 mg of a sticky transparent product (Compound 24, yield 83%). 11H NMR (600 MHz, MeOD) δ 5.51 - 5.45 (m, 1H), 5.36 (dd, J = 7.5, 3.4 Hz, 2H), 4.57 - 4.44 (m, 3H), 3.94 - 3.88 (m, 5H), 2.33 - 2.22 (m, 2H), 2.05 (dt, J = 12.1, 4.2 Hz, 4H), 1.63 (dd, J = 14.4, 7.2 Hz, 2H), 1.45 - 1.22 (m, 16H), 0.91 (t, J = 7.0 Hz, 3H). LC-MS (ESI); m / z: [M+H] + Calcd. for C 25 H 45 N 3 O 7 499.33. Found 501.12.

[0126] Example 2 Encapsulation and Delivery Support of Nucleic Acids by This Cationic Lipid 2.1 Encapsulation of Nucleic Acids In this example, the ability of this cationic lipid to protect nucleic acids was investigated. For this purpose, the cationic lipids of Example 1 (i.e., Compounds 6, 9, 11, 14, 18, 22, or 24) were co-cultured with plasmid independently and subjected to DNA digestion and extraction according to the procedures described in the "Materials and Methods" section. The samples were analyzed via agarose gel electrophoresis, and the results are provided in Figure 1.

[0127] After digestion, all detectable nucleotides were extracted, and it became clear from gel electrophoresis that they were derived from liposomes (i.e., cationic lipid-DNA complexes), indicating that this cationic lipid was capable of encapsulating the plasmid and protecting the plasmid from the action of nucleases (Lanes 25 - 28 and 31 - 34 in Figure 1).

[0128] Furthermore, the encapsulation ratio of this cationic lipid for encapsulating short-chain RNA was investigated. For this purpose, a commercially available cationic lipid and this cationic lipid (i.e., Compound 6 (TGG-linoleic acid) and 22 (TGG-di(palmitoleic acid))) were each mixed with a fluorochrome-conjugated short-chain RNA (i.e., Cy5-mir302a-siRNA, SEQ ID NOs: 6 and 7) according to the preparation procedure described in the "Materials and Methods" section, thereby generating lipid nanoparticles (LNPs) containing short-chain RNA therein. The size, zeta potential, and encapsulation efficiency (EE) of each LNP were analyzed by the procedures described in the "Materials and Methods" section. The results are summarized in Table 1. [Table 1]

[0129] The data in Table 1 revealed that both Compound 6 and 22 formed liposomes successfully and encapsulated siRNA therein. Specifically, compared with the LNPs prepared with the commercially available cationic lipid MC30, the LNPs composed of this Compound 22 had a smaller size and a higher encapsulation efficiency.

[0130] 2.2 Delivery of Nucleic Acids In this example, it was investigated whether this cationic lipid could promote the delivery of nucleic acids into cells. For this purpose, A549 and NCI-H460 lung cancer cell lines were co-cultured with this LNP or control LNP containing various concentrations of siRNA (0.25, 1.25, or 2.5 ng / μL) therein for 24 hours, respectively, and the transfection efficiency was verified by flow cytometry. The results are shown in Figures 2A - 2B.

[0131] After co-culturing with this LNP composed of compound 6 or 22, it becomes clear from the flow cytometry histograms in Figures 2A and 2B that the fluorescence intensity released from lung cancer cells gradually increases, indicating that nucleic acids from the outside were successfully transfected into cancer cells. Furthermore, the peak has shifted to the rightmost histogram of the LNP composed of this compound 22 (TGG-di(palmitoleic acid)), indicating that it has better delivery efficacy than that of the LNP composed of compound 6.

[0132] The above data as a whole show that this novel cationic lipid can encapsulate functional nucleic acids therein. Therefore, the lipid nanoparticles assembled by this cationic lipid can act as carriers for the delivery of therapeutic agents into cells, thereby achieving effective gene editing, cell engineering, and disease treatment.

[0133] It should be understood that the above description of the embodiments is given by way of example only and that various modifications can be made by those skilled in the art. The above specification, examples, and data provide a complete description of the structure and use of the exemplary embodiments of the invention. Although various embodiments of the invention have been described to some degree of particularity or with reference to one or more individual embodiments, those skilled in the art should be able to make numerous changes to the disclosed embodiments without departing from the spirit or scope of the invention.

Claims

1. The cationic lipid of formula (I), 【Chemical 1】 wherein the cationic lipid of formula (I) is a cationic lipid selected from the group consisting of [Chemical Formula 2]

2. Lipid nanoparticles (LNP) comprising the cationic lipid according to claim 1, a non-cationic lipid and a pharmaceutically active ingredient (API).

3. The LNP according to claim 2, wherein the API is a nucleic acid, a peptide, a polypeptide, a protein, a carbohydrate, a proteoglycan, a glycoprotein or a combination thereof.

4. The LNP according to claim 3, wherein the nucleic acid is mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA) or an aptamer.

5. The LNP according to claim 4, wherein the miRNA is a precursor miRNA or a mature miRNA.

6. The LNP according to claim 5, wherein the API is the miRNA having the sequence of SEQ ID NO:

1.

7. The LNP according to claim 6, wherein the miRNA has the sequence of SEQ ID NO: 2, 3, 4 or 5.

8. The cationic lipid is and the API is the siRNA having the sequence of SEQ ID NO: 6 or 7, the LNP according to claim 4. 【Chemical 4】

9. Each of the non-cationic lipids is selected from the group consisting of helper lipids, phospholipids, PEGylated lipids, PEGylated phospholipids and combinations thereof, the LNP according to claim 2.

10. The helper lipid is dioleoylphosphatidylethanolamine (DOPE), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine (DSPE), dimyristoylphosphatidylethanolamine (DMPE) or distearoylphosphatidylcholine (DSPC), the LNP according to claim 9.

11. The helper lipid is DSPC, the LNP according to claim 10.

12. The cationic lipid and the non-cationic lipid are each present in the LNP in a molar ratio of 1:5 to 5:1, the LNP according to claim 2.

13. ​ The LNP according to claim 2, further comprising a steroid selected from the group consisting of cholesterol, lanosterol, ergosterol, phytosterol, stigmasterol, and brassicasterol.

14. The LNP according to claim 13, wherein the steroid is cholesterol.

15. A pharmaceutical composition comprising the LNP according to claim 2 for treating a disease in a subject in need thereof.

16. The pharmaceutical composition according to claim 15, wherein the disease is cancer, an infectious disease, or an autoimmune disease.

17. The pharmaceutical composition according to claim 16, wherein the cancer is selected from the group consisting of bladder cancer, bone cancer, bone marrow cancer, brain cancer, breast cancer, bile duct cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gingival cancer, head and neck cancer, kidney cancer, liver cancer, lung cancer, hypopharyngeal cancer, leukemia, lymphoma, ovarian cancer, prostate cancer, skin cancer, gastric cancer, testicular cancer, tongue cancer, and uterine cancer.

18. The pharmaceutical composition according to claim 17, wherein the API is an miRNA having the sequence of SEQ ID NO:

1.

19. The pharmaceutical composition according to claim 18, wherein the miRNA has the sequence of SEQ ID NO: 2, 3, 4, or 5.

20. The cancer is the lung cancer, and the cationic lipid is 【Chemical Formula 6】 The pharmaceutical composition according to claim 17, wherein the API is an siRNA having the sequence of SEQ ID NO: 6 or 7.

21. The pharmaceutical composition according to claim 15, wherein the subject is a human.

22. A method for delivering an API into cultured cells, comprising the step of contacting the cultured cells with an effective amount of the LNP according to claim 2, wherein the API is a nucleic acid, polypeptide, protein, carbohydrate, proteoglycan, glycoprotein, or a combination thereof.

23. The method according to claim 22, wherein the nucleic acid is mitochondrial DNA (mtDNA), chloroplast DNA (cpDNA), plasmid, messenger RNA (mRNA), short interfering RNA (siRNA), ribosomal RNA (rRNA), transfer RNA (tRNA), microRNA (miRNA), or an aptamer.

24. The method according to claim 23, wherein the miRNA is a precursor miRNA or a mature miRNA.

25. The method according to claim 22, wherein the cultured cells are stem cells, immune cells, or a combination thereof.

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