Cationic lipid and method for preparing the same, and nanoparticle composition for drug delivery comprising the same

Abstract

The present invention relates to a cationic lipid, a method for preparing the same, and a composition of nanoparticles for drug delivery containing the same. More specifically, the invention relates to a cationic lipid useful for drug delivery that forms (or can form) a complex with an anionic drug due to a specific structure, a method for preparing the same, and a composition for drug delivery in which a drug is encapsulated inside a nanoparticle structure formed by the cationic lipid and a polymer, and a method for preparing the same.

Description

Technology Field

[0001] The present invention relates to a cationic lipid, a method for preparing the same, and a composition of nanoparticles for drug delivery containing the same. More specifically, the invention relates to a cationic lipid useful for drug delivery that forms (or can form) a complex with an anionic drug due to a specific structure, a method for preparing the same, and a composition for drug delivery in which a drug is encapsulated inside a nanoparticle structure formed by the cationic lipid and a polymer, and a method for preparing the same. Background Technology

[0002] In the treatment of anionic drugs, including nucleic acids, safe and efficient drug delivery technologies have long been studied, and various carriers and delivery technologies have been developed. Carriers are broadly classified into viral carriers utilizing adenoviruses or retroviruses, and non-viral carriers utilizing cationic lipids and cationic polymers. Viral carriers are known to pose risks such as non-specific immune responses and face significant challenges in commercialization due to their complex production processes. Therefore, recent research trends are moving toward improving these shortcomings by utilizing non-viral carriers. Although non-viral carriers lag behind viral carriers in terms of efficiency, they offer advantages such as fewer side effects in terms of in vivo safety and lower production costs in terms of economic feasibility.

[0003] Representative non-viral delivery vehicles used for the delivery of nucleic acid materials are lipoplexes and polyplexes. These cationic lipids or polycationic polymers have been the subject of extensive research because they stabilize anionic drugs and enhance intracellular delivery by forming complexes through electrostatic interactions (De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).

[0004] However, when administering the necessary amount intravenously to achieve a sufficient effect, polycation polymers present problems for actual use as pharmaceuticals due to cytotoxicity caused by their polyvalent cation charges, although to a lesser extent than viral carriers; furthermore, nucleic acid-cationic lipid complexes are difficult to use in vivo due to their low stability in the blood. Additionally, ionic liposomes containing cationic lipids, neutral lipids, and fusogenic lipids have disadvantages, such as the complex synthesis methods of the cationic lipids used, cytotoxicity, and low efficiency of intracellular nucleic acid delivery. Therefore, there is a need to develop anionic drug delivery technologies that reduce toxicity by minimizing the use of potentially toxic cationic polymers or cationic lipids, while remaining stable in vivo and capable of intracellular delivery to achieve a sufficient effect.

[0005] Accordingly, anionic drug delivery compositions and various manufacturing methods have been disclosed, wherein a complex is formed through electrostatic interaction between a nucleic acid and a cationic lipid, and said complex is encapsulated within the nanoparticle structure of an amphiphilic block copolymer. For example, Korean Published Patent No. 2017-0032858 discloses an anionic drug delivery composition and a method thereof, comprising an anionic drug; a cationic compound; an amphiphilic block copolymer; and a polylactic acid as active ingredients, wherein the anionic drug forms a complex through electrostatic interaction with the cationic compound, and the complex thus formed is encapsulated within the nanoparticle structure formed by the amphiphilic block copolymer and the polylactic acid. However, existing nanoparticle drug delivery systems, including those disclosed in the aforementioned published patent, still lack sufficient in vivo delivery efficiency for drugs (particularly mRNA), such as nucleic acids, polypeptides, or viruses. The problem to be solved

[0006] The objective of the present invention is to provide a cationic lipid of a specific structure that is useful for drug delivery as it can easily form a complex with an anionic drug, and a method for preparing the same.

[0007] Another objective of the present invention is to provide a drug delivery composition and a method for preparing the same, which has significantly improved in vivo delivery efficiency of drugs (especially mRNA), such as nucleic acids, polypeptides, or viruses, compared to previously known nanoparticle drug delivery systems. means of solving the problem

[0008] A first aspect of the present invention provides a cationic lipid having a structure represented by the following chemical formula 1:

[0009] [Chemical Formula 1]

[0010]

[0011] In the above chemical formula 1,

[0012] R1 and R2 are substituted or unsubstituted alkylene groups, and

[0013] R3, R4, and R5 are each independently substituted or unsubstituted alkylene groups, and

[0014] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon groups, and

[0015] R9 and R 10 Each is independently a substituted or unsubstituted alkyl group or a carboncyclic group, or a hydroxyalkyl group or each independently -R 11 -(L4) n -R 12 And,

[0016] R 11 Each is independently a substituted or unsubstituted alkylene group, and

[0017] R 12 Each is independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, and

[0018] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, or alkenylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, and alkenyl, and

[0019] n is 0 or 1, and

[0020] X - is a pharmaceutically acceptable monovalent anion.

[0021] More specifically, in the above chemical formula 1,

[0022] R1 and R2 are substituted or unsubstituted C 1-6 It is an alkylene group, and

[0023] R3, R4, and R5 are each independently substituted or unsubstituted C 2-15 It is an alkylene group, and

[0024] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 It is a hydrocarbon group, and

[0025] R9 and R 10 Each is independently substituted or unsubstituted C 1-6 alkyl group or C 3-6 It is a carbon cyclic group, or C 1-6 hydroxyalkyl group or -R independently, respectively 11 -(L4) n -R 12 And,

[0026] R 11 Each is independently substituted or unsubstituted C 3-12 It is an alkylene group, and

[0027] R 12Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 It is a hydrocarbon group, and

[0028] L1, L2, L3, and L4 are each independently -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, C 2-6 Alkenylene, C 6-20 Arylene, and C 3-20 Selected from the group composed of heteroarylenes, where L' is a direct bond, C 1-13 Alkylene or C 2-13 It is alkenylene, and R' are independently hydrogen atoms, C 1-18 Alkyl and C 2-18 Selected from a group composed of alkenils,

[0029] n is 0 or 1, and

[0030] X - is a monovalent anion of a pharmaceutically acceptable inorganic or organic acid.

[0031] More specifically, in the above chemical formula 1,

[0032] R1 and R2 are substituted or unsubstituted C 1-4 It is an alkylene group, and

[0033] R3, R4, and R5 are each independently substituted or unsubstituted C 2-8 It is an alkylene group, and

[0034] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 6-22 It is a hydrocarbon group, and

[0035] R9 and R 10 Each is independently substituted or unsubstituted C 1-2 It is an alkyl group, or C 2-4 hydroxyalkyl group or -R independently, respectively 11 -(L4)n -R 12 And,

[0036] R 11 Each is independently substituted or unsubstituted C 3-8 It is an alkylene group, and

[0037] R 12 Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 6-22 It is a hydrocarbon group, and

[0038] L1, L2, L3, and L4 are each independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -P(O)(OR')O-, -SS-, and C 2-5 Selected from the group consisting of alkenylenes, where R' is independently a hydrogen atom and C 1-6 Selected from the group consisting of alkyls,

[0039] n is 0 or 1, and

[0040] X - is F - , Cl - , Br - , I - , nitrate anion, benzoate anion, methanesulfonate anion, acetate anion (CH3COO - )(i.e., AcO - ), or trihaloacetate anion (e.g., CF3COO - )am.

[0041] In one specific example, the above R6 to R 10 One or more of the moieties may each independently be further substituted with one or more moieties specifically suitable for a target tissue or organ, and said moieties may be selected from amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof.

[0042] More specifically, the cationic lipid has any one of the following chemical formulas A to V:

[0043]

[0044]

[0045]

[0046]

[0047]

[0048] A second aspect of the present invention provides a method for producing a cationic lipid, comprising: (1-1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; and (1-2) reacting a compound of formula c with a compound of formula d to obtain a cationic lipid of formula 1-1; or (2-1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula e; and (2-2) reacting a compound of formula e with a compound of formula d to obtain a cationic lipid of formula 1-2.

[0049] [Chemical formula a]

[0050] R6-L3-R3-X

[0051] [Chemical formula b]

[0052] H2N-R1-SS-R2-NH2

[0053] [Chemical formula c]

[0054]

[0055] [Chemical formula d]

[0056] R9-X

[0057] [Chemical formula e]

[0058]

[0059] [Chemical Formula 1-1]

[0060]

[0061] [Chemical Formula 1-2]

[0062]

[0063] As mentioned above,

[0064] R1 and R2 are substituted or unsubstituted alkylene groups, and

[0065] R3 is each independently a substituted or unsubstituted alkylene group, and

[0066] R6 is each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, and

[0067] R9 is each independently a substituted or unsubstituted alkyl group or carboncyclic group or hydroxyalkyl group, and

[0068] L3 is each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, or alkenylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, and alkenyl, and

[0069] X - is a pharmaceutically acceptable monovalent anion.

[0070] A third aspect of the present invention provides a drug delivery composition comprising a cationic lipid according to the present invention.

[0071] A fourth aspect of the present invention provides a drug delivery composition comprising: an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; a cationic lipid of the present invention; and a lipid-polymer, an amphiphilic block copolymer, or a combination thereof.

[0072] A fifth aspect of the present invention provides a method for preparing a drug delivery composition, comprising: (a) preparing a solution in which a cationic lipid of the present invention and a lipid-polymer polymer, an amphiphilic block copolymer, or a mixture thereof are dissolved in a water-miscible organic solvent; and (b) adding and mixing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof to the solution prepared in step (a). Effects of the invention

[0073] The cationic lipid according to the present invention can easily form a complex with an anionic drug due to a specific structure having both a quaternary amine and a tertiary amine, and by utilizing this complex, the drug can be efficiently delivered into a target biological tissue.

[0074] The drug delivery composition according to the present invention can significantly improve the in vivo delivery efficiency of drugs (especially mRNA), such as nucleic acids, polypeptides, or viruses, compared to previously known nanoparticle drug delivery systems. Brief explanation of the drawing

[0075] Figure 1 is a schematic diagram of the reaction for the synthesis process of compound A of formula A performed in Example A1. Figure 2 is a schematic diagram of the reaction for the synthesis process of compound B of formula performed in Example A2. Figure 3 is a schematic diagram of the reaction for the synthesis process of the compound of formula C performed in Example A3. Figure 4 is a schematic diagram of the reaction for the synthesis process of compound D of formula performed in Example A4. Figure 5 is a schematic diagram of the reaction for the synthesis process of the compound of formula E performed in Example A5. Figure 6 is a schematic diagram of the reaction for the synthesis process of the compound of formula F performed in Example A6. Figure 7 is a schematic diagram of the reaction for the synthesis process of the compound of formula G performed in Example A7. Figure 8 is a schematic diagram of the reaction for the synthesis process of the compound of formula H performed in Example A8. Figure 9 is a schematic diagram of the reaction for the synthesis process of the compound of formula I performed in Example A9. Figure 10 is a schematic diagram of the reaction for the synthesis process of the compound of formula J performed in Example A10. Figure 11 is a schematic diagram of the reaction for the synthesis process of the compound of formula K performed in Example A11. Figure 12 is a schematic diagram of the reaction for the synthesis process of the compound of formula L performed in Example A12. Figure 13 is a schematic diagram of the reaction for the synthesis process of the compound of formula M performed in Example A13. Specific details for implementing the invention

[0076] The present invention will be described in more detail below.

[0077] [Cationic Lipids and Methods for Manufacturing the Same]

[0078] The cationic lipid provided according to the first aspect of the present invention has a structure represented by the following chemical formula 1:

[0079] [Chemical Formula 1]

[0080]

[0081] In the above chemical formula 1,

[0082] R1 and R2 are substituted or unsubstituted alkylene groups, and

[0083] R3, R4, and R5 are each independently substituted or unsubstituted alkylene groups, and

[0084] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon groups, and

[0085] R9 and R 10 Each is independently a substituted or unsubstituted alkyl group or a carboncyclic group, or a hydroxyalkyl group or each independently -R 11 -(L4)n -R 12 And,

[0086] R 11 Each is independently a substituted or unsubstituted alkylene group, and

[0087] R 12 Each is independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, and

[0088] L1, L2, L3 and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, or alkenylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, and alkenyl, and

[0089] n is 0 or 1, and

[0090] X - is a pharmaceutically acceptable monovalent anion.

[0091] In one embodiment, the above R6 to R 10 One or more of the may be further substituted, each independently, with one or more moieties specifically suitable for a target tissue or organ (e.g., liver, lung, spleen, brain, heart, etc.), preferably selected from amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof. Preferably, R9 and R 10One or more of the may each independently be further substituted with one or more moieties specifically suitable for a target tissue or organ (e.g., liver, lung, spleen, brain, heart, etc.), preferably selected from amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof.

[0092] In one embodiment, the above R 11 and R 12 One or more of the above may be further substituted, each independently, with one or more moieties specifically suitable for a target tissue or organ (e.g., liver, lung, spleen, brain, heart, etc.), preferably selected from amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof.

[0093] In this specification, the expression “substituted or unsubstituted” means that, unless otherwise specified, the group is not substituted, or -OH, a halogen atom, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 1-6 alkyl halide group, C 1-6 Halogenated alkoxy group, C 3-20 Cycloalkyl group, C 3-20 heterocycloalkyl group, C 6-20 aryl group or C 3-20 It means that it is substituted with one or more substituents selected from heteroaryl groups.

[0094] In this specification, “hydroxyalkyl group” means an alkyl group substituted with one or more hydroxyl groups.

[0095] In this specification, the expression that any group (e.g., heteroaryl, heterocycloalkyl, etc.) is a “hetero” group means that, unless otherwise specified, the group has one or more (e.g., 1 to 3) hetero atoms selected from N, O and S.

[0096] In this specification, the “monovalent hydrocarbon group” may be branched or unbranched, annular or non-annular, or aromatic.

[0097] According to one embodiment of the present invention, in the above formula 1,

[0098] R1 and R2 are substituted or unsubstituted C 1-6 It can be an alkylene group, and

[0099] R3, R4, and R5 are each independently substituted or unsubstituted C 2-15 It can be an alkylene group, and

[0100] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 It can be a hydrocarbon group,

[0101] R9 and R 10 Each is independently substituted or unsubstituted C 1-6 alkyl group or C 3-6 It is a carbon cyclic group, or C 1-6 hydroxyalkyl group or -R independently, respectively 11 -(L4) n -R 12 It could be,

[0102] R 11 Each is independently substituted or unsubstituted C 2-15 It can be an alkylene group, and

[0103] R 12 Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 It can be a hydrocarbon group,

[0104] L1, L2, L3, and L4 are each independently -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, C 2-6 Alkenylene, C 6-20 Arylene, and C 3-20 It can be selected from the group consisting of heteroarylenes, where L' is direct bonding, C 1-13 Alkylene or C 2-13 It can be alkenylene, where R' are independently hydrogen atoms, C 1-18 Alkyl and C 2-18 It can be selected from a group composed of alkenils, and

[0105] n is 0 or 1, and

[0106] X - It may be a monovalent anion of a pharmaceutically acceptable inorganic or organic acid.

[0107] More specifically, in the above chemical formula 1,

[0108] R1 and R2 are substituted or unsubstituted C 1-4 It can be an alkylene group, and

[0109] R3, R4, and R5 are each independently substituted or unsubstituted C 2-8 It can be an alkylene group, and

[0110] R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 6-26 It can be a hydrocarbon group,

[0111] R9 and R 10 Each is independently substituted or unsubstituted C 1-2 It is an alkyl group, or C 2-4 hydroxyalkyl group or -R independently, respectively 11 -(L4) n -R 12 It could be,

[0112] R 11 Each is independently substituted or unsubstituted C 2-8 It can be an alkylene group, and

[0113] R 12 Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 6-26 It can be a hydrocarbon group,

[0114] L1, L2, L3, and L4 are each independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -P(O)(OR')O-, -SS-, and C 2-5 It can be selected from the group consisting of alkenylenes, where R' is independently a hydrogen atom and C 1-6 It can be selected from the group consisting of alkyl groups, and

[0115] n is 0 or 1, and

[0116] X - is F - , Cl - , Br - , I - , nitrate anion, benzoate anion, methanesulfonate anion, acetate anion (CH3COO - )(i.e., AcO - ), or trihaloacetate anion (e.g., CF3COO - It can be.

[0117] More specifically, the cationic lipid may have any one of the structures selected from the following chemical formulas A to V:

[0118]

[0119]

[0120]

[0121]

[0122]

[0123] The lipid according to the present invention can form a complex with anionic drugs and has a structure in which various substituents can be introduced, allowing for diverse molecular designs depending on the target tissue or organ, making it useful for specifically delivering drugs to the target tissue or organ. To this end, if necessary, the lipid according to the present invention may include, in part of its structure, a moiety specifically suitable for the target tissue or organ, such as amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, small molecules having pharmaceutical activity, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof, or may bind to such a moiety.

[0124] In one embodiment, the “moiety specifically suited to a target tissue or organ” includes all molecules such as amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof that can interact directly or indirectly with other compounds such as receptors. The sugar may include, but is not limited to, galactose, galactosamine, N-acetylgalactosamine, or combinations thereof. The hormone may include, but is not limited to, estrogen, testosterone, progesterone, glucocortisone, adrenaline, insulin, glucagon, cortisol, vitamin D, thyroid hormone, retinoic acid, growth hormone, or combinations thereof. The neurotransmitter may include growth factors such as VEGF, EGF, NGF, and PDGF; cholesterol; bile acids; It may include, but is not limited to, GABA, glutamate, acetylcholine, or combinations thereof.

[0125] In one embodiment, a “moiety specifically suitable for a target tissue or organ” may be attached to the lipid of the present invention using a linker molecule. The linker molecule may include, but is not limited to, amides, carbonyls, esters, peptides, disulfides, silanes, nucleosides, abasic nucleosides, polyethers, polyamines, polyamides, carbohydrates, lipids, polyhydrocarbons, phosphate esters, phosphoramidates, thiophosphates, alkyl phosphates, biodegradable linkers, photolabile linkers, etc.

[0126] The cationic lipid having the structure represented by Chemical Formula 1 of the present invention can perform both the roles of an ionizable lipid and a helper lipid in a drug delivery composition. The ionizable lipid is a lipid capable of controlling its charge according to pH and is one of the key components of a lipid nanoparticle composition useful for delivering nucleic acids such as RNA and DNA. The helper lipid is a lipid that increases the particle stability and fluidity of lipid nanoparticles and may refer to various types of lipids, such as phospholipids included in lipid nanoparticles, in addition to the ionizable lipid.

[0127] However, even if the cationic lipid having the structure represented by Chemical Formula 1 above can perform both the role of an ionizable lipid and a helper lipid, the use of such additional lipid components is not excluded in the drug delivery composition according to one embodiment of the present invention. Accordingly, the drug delivery composition according to one embodiment of the present invention may further include one or more ionizable lipids of other types other than the cationic lipid having the structure represented by Chemical Formula 1, or one or more helper lipids of other types, as needed.

[0128] A second aspect of the present invention provides a method for producing a cationic lipid, comprising: (1-1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; and (1-2) reacting a compound of formula c with a compound of formula d to obtain a cationic lipid of formula 1-1; or (2-1) reacting a compound of formula a with a compound of formula b to obtain a compound of formula e; and (2-2) reacting a compound of formula e with a compound of formula d to obtain a cationic lipid of formula 1-2.

[0129] [Chemical formula a]

[0130] R6-L3-R3-X

[0131] [Chemical formula b]

[0132] H2N-R1-SS-R2-NH2

[0133] [Chemical formula c]

[0134]

[0135] [Chemical formula d]

[0136] R9-X

[0137] [Chemical formula e]

[0138]

[0139] [Chemical Formula 1-1]

[0140]

[0141] [Chemical Formula 1-2]

[0142]

[0143] In the above,

[0144] R1 and R2 are substituted or unsubstituted alkylene groups, and

[0145] R3 is each independently a substituted or unsubstituted alkylene group, and

[0146] R6 is each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, and

[0147] R9 is each independently a substituted or unsubstituted alkyl group or carboncyclic group or hydroxyalkyl group, and

[0148] L3 is each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, or alkenylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, and alkenyl, and

[0149] X - is a pharmaceutically acceptable monovalent anion.

[0150] The above R1, R2, R3, R6, R9, L3 and X - More specific examples of this have been explained earlier.

[0151] In one embodiment of the method for producing cationic lipids according to the present invention, the reaction between the compound of formula a and the compound of formula b may be carried out in a solvent (e.g., acetonitrile (ACN), tetrahydrofuran (THF), etc.) in the presence of a catalyst (e.g., Na2CO3, K2CO3, etc.) at a temperature (e.g., 60°C to 100°C), but is not limited thereto.

[0152] In one embodiment of the method for producing cationic lipids according to the present invention, the reaction between the compound of formula c and the compound of formula d may be carried out at room temperature (e.g., 20°C to 40°C) in the presence of a catalyst (e.g., Na2CO3, K2CO3, etc.) in a solvent (e.g., tetrahydrofuran (THF), etc.), but is not limited thereto.

[0153] In one embodiment of the method for producing cationic lipids according to the present invention, the reaction between the compound of formula e and the compound of formula d may be carried out at room temperature (e.g., 20°C to 40°C) in the presence of a catalyst (e.g., Na2CO3, K2CO3, etc.) in a solvent (e.g., tetrahydrofuran (THF), etc.), but is not limited thereto.

[0154] [Drug delivery composition and method of manufacturing the same]

[0155] The cationic lipid according to the present invention is useful for drug delivery because it can easily form a complex with anionic drugs by including both quaternary amines and tertiary amines.

[0156] Accordingly, according to a third aspect of the present invention, a drug delivery composition comprising a cationic lipid of the present invention is provided.

[0157] In one embodiment, the drug may be selected from nucleic acids, polypeptides, viruses, or a combination thereof.

[0158] The “nucleic acid” mentioned above may be, for example, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamers, antisense oligonucleotides, or combinations thereof, but is not limited thereto.

[0159] The above “polypeptide” may refer to a protein having activity in the body, such as an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a protein that can be recognized as an antigen through a series of processes in the body, including a polypeptide sequence of an antigen, an analog thereof, or a precursor thereof.

[0160] The “virus” mentioned above may be an oncolytic virus and may be one or more selected from the group consisting of, for example, adenovirus, AAV, vaccinia virus, herpes simplex virus (HSV), and vesicular stomatitis virus (VSV). In one embodiment, the oncolytic virus is an adenovirus. The adenovirus used in an embodiment of the present invention contains a luciferase gene, which can be confirmed through imaging.

[0161] The above-mentioned virus can express various types of therapeutic genes within the body of an individual and is not limited to specific molecular weight, protein, viability, or therapeutic field. The prophylactic virus can induce immunity within the body of an individual against a target disease. A composition containing a disease-preventive virus has the advantage of reducing immunity induction by the virus itself, being able to designate or expand target cells, and reducing the hyperimmune response to the virus upon re-administration, thereby enabling effective effects to be obtained through multiple inoculations.

[0162] In one embodiment of the present invention, the active ingredient is mRNA (messenger RNA).

[0163] The above mRNA may have its backbone, sugar, or base chemically modified or its terminals modified for purposes such as increasing blood stability or weakening the immune response.

[0164] In one embodiment, the lipid of the present invention forms a complex with a drug, and this complex can be encapsulated inside a nanoparticle structure formed by an amphiphilic block copolymer.

[0165] In one embodiment, the amphiphilic block copolymer may be an AB-type block copolymer comprising a hydrophilic A block and a hydrophobic B block. The AB-type block copolymer forms core-shell type polymer nanoparticles in an aqueous solution, wherein the hydrophobic B block forms the core (inner wall) and the hydrophilic A block forms the shell (outer wall).

[0166] In one embodiment, the hydrophilic A block may be one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.

[0167] More specifically, the hydrophilic A block may be one or more selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0168] Additionally, if necessary, a functional group, a ligand capable of reaching specific tissues or cells, or a functional group capable of promoting intracellular delivery can be chemically bonded to the terminal end of the hydrophilic A block to control the distribution of the nanoparticle delivery vehicle in the body or to increase the efficiency of the nanoparticle delivery vehicle being delivered into cells. In one embodiment, the functional group or ligand may be one or more selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies against cell surface receptors. More specifically, the functional group or ligand may be one or more selected from the group consisting of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, and antibodies against transferrin receptors.

[0169] The above hydrophobic B block is a biocompatible biodegradable polymer, and in one embodiment, it may be one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazine.

[0170] More specifically, the hydrophobic B block may be one or more selected from the group consisting of polylactide (PLA), polyglycolide, polycaprolactone, polydioxane-2-one, copolymers of polylactide and glycolide, copolymers of polylactide and polydioxane-2-one, copolymers of polylactide and polycaprolactone, and copolymers of polyglycolide and polycaprolactone.

[0171] In addition, in one embodiment, the hydrophobic B block may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to the hydroxyl group at the end of the hydrophobic B block in order to increase the hydrophobicity of the hydrophobic B block and improve the stability of the nanoparticle.

[0172] In another embodiment, the hydrophobic B block may be a biocompatible biodegradable polymer having repeating units of a structure represented by the following chemical formula 2:

[0173] [Chemical Formula 2]

[0174]

[0175] In the above chemical formula 2,

[0176] R represents a branched alkylene group having 3 or more carbon atoms.

[0177] In one embodiment, the number of repeating units (degree of polymerization) of the hydrophobic block having a repeating unit of the structure represented by the above chemical formula 2 may be, for example, 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more, and may also be 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less, but is not limited thereto.

[0178] In one embodiment, the number of carbon atoms of R in the above formula 2 may be, for example, 3 or more, 4 or more, 5 or more, 6 or more, or 7 or more, and may also be 20 or less, 19 or less, 18 or less, 17 or less, 16 or less, 15 or less, 14 or less, or 13 or less, but is not limited thereto.

[0179] In one embodiment, R in the above formula 2 may represent a branched alkylene group having 3 to 20 carbon atoms, more specifically a branched alkylene group having 3 to 17 carbon atoms, even more specifically a branched alkylene group having 3 to 15 carbon atoms, and even more specifically a branched alkylene group having 3 to 13 carbon atoms, but is not limited thereto.

[0180] In one embodiment, the hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be a biocompatible biodegradable polymer having a repeating unit of the structure selected from the following, but is not limited thereto.

[0181] , , , , , , , , , , , , , , , ,

[0182] In one embodiment, the repeating unit of the structure represented by the above chemical formula 2 can be obtained by ring-opening polymerization of a lactone compound.

[0183] In one embodiment, the number average molecular weight (g / mol) of the hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be 80 or more, 100 or more, 150 or more, 200 or more, 500 or more, 1,000 or more, or 1,700 or more, and may also be 50,000 or less, 20,000 or less, 10,000 or less, or 6,000 or less, but is not limited thereto.

[0184] For example, combinations of number-average molecular weights of a hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be 2,000-6,000, 2,000-4,000, 2,000-3,000, 2,000-1,700, 2,000-1,000, 2,000-800, 2,000-500, etc., but are not limited thereto.

[0185] In addition, in one embodiment, the hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to the hydroxyl group at the end of the hydrophobic block in order to increase the hydrophobicity of the hydrophobic block and improve the stability of the nanoparticle.

[0186] In one embodiment, in an amphiphilic block copolymer comprising a hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2, the compositional ratio of the hydrophilic block to the hydrophobic block may be in the range of 25 to 95 weight% for the hydrophilic block based on the total weight of the copolymer, specifically in the range of 40 to 90 weight%, and more specifically in the range of 50 to 80 weight%. Since it is difficult to form nanoparticles because the solubility of the polymer in water is low when the ratio of the hydrophilic block is less than 25 weight% based on the total weight of the copolymer, it is preferable that the ratio of the hydrophilic block be 25 weight% or more so that the copolymer has sufficient solubility in water to form nanoparticles. Meanwhile, if the ratio of the hydrophilic block (A) exceeds 95% by weight based on the total weight of the copolymer, the hydrophilicity is too high and the stability of the polymer nanoparticles is lowered, making it difficult to use as a solubilizing composition for a composite containing active ingredients. Therefore, considering the stability of the nanoparticles, it is preferable that the ratio of the hydrophilic block be 95% by weight or less.

[0187] A fourth aspect of the present invention provides a drug delivery composition comprising: an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; a cationic lipid of the present invention; and a lipid-polymer, an amphiphilic block copolymer, or a combination thereof.

[0188] The above nucleic acids, polypeptides, and viruses are as described above.

[0189] In one embodiment, the active ingredient may be mRNA (messenger RNA).

[0190] The above mRNA may have its backbone, sugar, or base chemically modified or its terminals modified for purposes such as increasing blood stability or weakening the immune response.

[0191] In one embodiment, the content of the active ingredient may be 0.05 wt% or more, 0.1 wt% or more, 0.2 wt% or more, 0.3 wt% or more, 0.4 wt% or more, or 0.5 wt% or more, based on the dry weight of the total composition, and may also be 10 wt% or less, 9 wt% or less, 8 wt% or less, 7 wt% or less, 6 wt% or less, 5 wt% or less, 4 wt% or less, or 3 wt% or less. If the content of the active ingredient is excessively low, the amount of carrier used is too high compared to the active ingredient, so there may be side effects caused by the carrier, and conversely, if it is excessively high, there will be a large amount of active ingredient that is not encapsulated in the nanoparticles, and efficiency will decrease.

[0192] The above lipid-polymer is a polymer that has both hydrophilic and hydrophobic parts within the polymer molecule.

[0193] In one embodiment, the lipid-polymer may be a polymer in which one or more saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms are introduced as hydrophobic parts into a hydrophilic block which is a hydrophilic part.

[0194] The above hydrophilic block may be one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.

[0195] More specifically, the hydrophilic block may be one or more selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0196] In one embodiment, the hydrophilic block may have a number average molecular weight (g / mol) of 200 or more, 500 or more, 1,000 or more, or 2,000 or more, and may also be 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less, but is not limited thereto.

[0197] Additionally, if necessary, a functional group, a ligand capable of reaching specific tissues or cells, or a functional group capable of promoting intracellular delivery can be chemically attached to the end of the hydrophilic block to control the in vivo distribution of the nanoparticle delivery vehicle or increase the efficiency of the nanoparticle delivery vehicle being delivered into cells. The functional group or ligand may be one or more selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies against cell surface receptors. More specifically, the functional group or ligand may be one or more selected from the group consisting of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, and antibodies against transferrin receptors.

[0198] In one embodiment, the saturated and unsaturated hydrocarbon group having 11 to 25 carbon atoms introduced as a hydrophobic portion into the hydrophilic block, which is a hydrophilic portion, may independently be selected from the group consisting of myristoyl, dimyristoyl, lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, and docosahexaenyl.

[0199] In addition, in one embodiment, the compositional ratio of the hydrophilic portion to the hydrophobic portion in the lipid-polymer may be in the range of 10 to 90 weight% based on the weight of the polymer, specifically 20 to 80 weight%, more specifically 30 to 80 weight%, and even more specifically 40 to 80 weight%. If the proportion of the hydrophilic portion is excessively small, the solubility of the polymer in water is low, making it difficult to form nanoparticles; conversely, if it is excessively large, the hydrophilicity is too high, which may lower the stability of the nanoparticles.

[0200] In one embodiment of the present invention, the lipid-polymer may be a polyalkylene glycol (e.g., polyethylene glycol) into which saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms (e.g., myristyl groups) have been introduced. For example, the lipid-polymer may be a PEGylated lipid. The PEG lipid refers to a polyethylene glycol (PEG)-modified lipid, which is a type of PEG derivative with a lipid moiety attached, such as DMG (dimyristoylglycerol) or DSPE (distearoylglycerophosphoethanolamine), and the PEG lipid may be used to improve the circulation time of active ingredients encapsulated in lipid nanoparticles and to reduce non-specific absorption. The above PEG lipid may be one or more combinations selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. More specifically, the PEG lipid may include 1,2-dimyristoyl-sn-glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)](PEG-DSPE), PEG-distearyl glycerol (PEG-DSG), PEG-dipalmitoyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxypropyl-3-amine (PEG-c-DMA).

[0201] The above amphiphilic block copolymer may be an AB-type block copolymer comprising a hydrophilic A block and a hydrophobic B block. The above AB-type block copolymer forms core-shell type polymer nanoparticles in an aqueous solution, wherein the hydrophobic B block forms the core (inner wall) and the hydrophilic A block forms the shell (outer wall).

[0202] The above hydrophilic A block and the above hydrophobic B block are as described above.

[0203] In one embodiment, the number average molecular weight (g / mol) of the hydrophilic A block may be 200 or more, 500 or more, 1,000 or more, or 2,000 or more, and may also be 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less, but is not limited thereto.

[0204] In one embodiment, the number average molecular weight (g / mol) of the hydrophobic B block may be 200 or more, 500 or more, 1,000 or more, or 1,700 or more, and may also be 50,000 or less, 20,000 or less, 10,000 or less, or 6,000 or less, but is not limited thereto.

[0205] For example, the number average molecular weight combination of the hydrophilic A block and hydrophobic B block may be 2,000 (A block)-6,000 (B block), 2,000-4,000, 2,000-3,000, 2,000-1,700, 2,000-1,300, etc., but is not limited thereto.

[0206] In one embodiment, in the amphiphilic block copolymer, the compositional ratio of the hydrophilic block (A) and the hydrophobic block (B) may be in the range of 20 to 70 weight% for the hydrophilic block (A) based on the total weight of the copolymer, and more specifically, in the range of 30 to 60 weight%. If the ratio of the hydrophilic block (A) is less than 20 weight% based on the total weight of the copolymer, the solubility of the polymer in water is low, making it difficult to form nanoparticles; therefore, it is preferable that the ratio of the hydrophilic block (A) be 20 weight% or more so that the copolymer has sufficient solubility in water to form nanoparticles. On the other hand, if the ratio of the hydrophilic block (A) exceeds 70 weight% based on the total weight of the copolymer, the hydrophilicity is too high, which lowers the stability of the polymer nanoparticles and makes it difficult to use as a solubilizing composition for an active ingredient / lipid complex; therefore, considering the stability of the nanoparticles, it is preferable that the ratio of the hydrophilic block (A) be 70 weight% or less.

[0207] In one embodiment, the amphiphilic block copolymer may be a biocompatible biodegradable polymer comprising the hydrophilic block described above; and a hydrophobic block having repeating units of the structure represented by Formula 2.

[0208] In one embodiment, the number average molecular weight (g / mol) of the hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be 80 or more, 100 or more, 150 or more, 200 or more, 500 or more, 1,000 or more, or 1,700 or more, and may also be 50,000 or less, 20,000 or less, 10,000 or less, or 6,000 or less, but is not limited thereto.

[0209] For example, combinations of number-average molecular weights of a hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2 may be 2,000-6,000, 2,000-4,000, 2,000-3,000, 2,000-1,700, 2,000-1,000, 2,000-800, 2,000-500, etc., but are not limited thereto.

[0210] In one embodiment, in an amphiphilic block copolymer comprising a hydrophobic block having a repeating unit of the structure represented by Chemical Formula 2, the compositional ratio of the hydrophilic block to the hydrophobic block may be in the range of 25 to 95 weight% for the hydrophilic block based on the total weight of the copolymer, specifically in the range of 40 to 90 weight%, and more specifically in the range of 50 to 80 weight%. Since it is difficult to form nanoparticles because the solubility of the polymer in water is low when the ratio of the hydrophilic block is less than 25 weight% based on the total weight of the copolymer, it is preferable that the ratio of the hydrophilic block be 25 weight% or more so that the copolymer has sufficient solubility in water to form nanoparticles. Meanwhile, if the ratio of the hydrophilic block (A) exceeds 95% by weight based on the total weight of the copolymer, the hydrophilicity is too high and the stability of the polymer nanoparticles is lowered, making it difficult to use as a solubilizing composition for a composite containing active ingredients. Therefore, considering the stability of the nanoparticles, it is preferable that the ratio of the hydrophilic block be 95% by weight or less.

[0211] In one embodiment, the content of the polymer, which is a lipid-polymer, an amphiphilic block copolymer, or a combination thereof, in the drug delivery composition of the present invention may be 5% by weight or more, 7% by weight or more, 10% by weight or more, 12% by weight or more, 15% by weight or more, 17% by weight or more, or 20% by weight or more, based on the dry weight of the total composition, and may also be 90% by weight or less, 80% by weight or less, 70% by weight or less, 60% by weight or less, 50% by weight or less, or 40% by weight or less. If the content of the polymer is too low, the size of the nanoparticles becomes too large, which may reduce the stability of the nanoparticles and increase the loss rate during filter sterilization; conversely, if it is too high, there is a risk that the content of the active ingredient that can be incorporated will become too low.

[0212] In the drug delivery composition of the present invention, the active ingredient is maintained in a state of being encapsulated within a nanoparticle structure formed by the lipid-polymer polymer, the amphiphilic block copolymer or a combination thereof, and the cationic lipid of Formula 1, thereby improving stability in blood or body fluids.

[0213] In one embodiment, the particle size of the nanoparticle may be defined by a Z-average value and, for example, may be 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less, and may also be 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, or 100 nm or more. In one embodiment, the particle size of the nanoparticle defined by a Z-average value may be, for example, 10 to 800 nm, 20 to 600 nm, 30 to 500 nm, 40 to 400 nm, or 50 to 300 nm.

[0214] In one embodiment, the relative amount of the lipid-polymer, amphiphilic block copolymer, or a combination thereof polymer relative to the cationic lipid of Formula 1 may be 0.01 parts by weight or more, 0.02 parts by weight or more, 0.03 parts by weight or more, 0.04 parts by weight or more, or 0.05 parts by weight or more, based on 1 part by weight of the cationic lipid of Formula 1, and may also be 50 parts by weight or less, 49 parts by weight or less, 47 parts by weight or less, 45 parts by weight or less, 43 parts by weight or less, 41 parts by weight or less, 40 parts by weight or less, 39 parts by weight or less, or 37 parts by weight or less, but is not limited thereto.

[0215] More specifically, the relative amount of the lipid-polymer polymer used relative to the cationic lipid of Formula 1 may be 0.01 parts by weight or more, 0.02 parts by weight or more, 0.03 parts by weight or more, 0.04 parts by weight or more, or 0.05 parts by weight or more, based on 1 part by weight of the cationic lipid of Formula 1, and may also be 5 parts by weight or less, 2 parts by weight or less, 1 part by weight or less, 0.5 parts by weight or less, or 0.1 parts by weight or less, but is not limited thereto.

[0216] More specifically, the relative amount of the amphiphilic block copolymer relative to the cationic lipid of Formula 1 may be 0.1 parts by weight or more, 0.5 parts by weight or more, 1 part by weight or more, 2 parts by weight or more, or 3 parts by weight or more, based on 1 part by weight of the cationic lipid of Formula 1, and may also be 50 parts by weight or less, 40 parts by weight or less, 30 parts by weight or less, 20 parts by weight or less, or 10 parts by weight or less, but is not limited thereto.

[0217] Arbitrary additional components

[0218] In one embodiment, the drug delivery composition of the present invention may further include a fused lipid to increase the efficiency of delivery of an active ingredient into the body.

[0219] In one embodiment, the fusion lipid may be one or more combinations selected from the group consisting of phospholipids, cholesterol, and tocopherol.

[0220] Specifically, the phospholipid may be one or more selected from the group consisting of phosphatidylethanolamin (PE), phosphatidylcholine (PC), and phosphatidic acid. The phosphatidylethanolamin (PE), phosphatidylcholine (PC), and phosphatidic acid may be in a form combined with one or two C10-24 fatty acids. The cholesterol and tocopherol include analogs, derivatives, and metabolites of cholesterol and tocopherol, respectively.

[0221] Specifically, the fused lipid is dilauroyl phosphatidylethanolamine, dimyristoyl phosphatidylethanolamine, dipalmitoyl phosphatidylethanolamine, distearoyl phosphatidylethanolamine, dioleoyl phosphatidylethanolamine, dilinoleoyl phosphatidylethanolamine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, dilauroyl phosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoyl phosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroyl phosphatidic acid,It may be one or more combinations selected from the group consisting of dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidic acid, 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol, and tocopherol.

[0222] More specifically, the fused lipid is dioleoyl phosphatidylethanolamine (DOPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPPC), distearoyl phosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), 1,2-dioleoyl-3-dimethylammonium-propane (1,2-dioleoyl-3-dimethylammonium propane, DODAP), 1,2-dioleoyl- sn -glycero-3-phosphate(1,2-dioleoyl- snIt may be one or more combinations selected from the group consisting of -glycero-3-phosphate, 18PA), cholesterol and tocopherol.

[0223] In one embodiment, the fused lipid may be distearoyl phosphatidylcholine, cholesterol, or a combination thereof.

[0224] In one embodiment, the fused lipid content may be 1 wt% or more, 2 wt% or more, 3 wt% or more, 4 wt% or more, or 5 wt% or more based on the dry weight of the total composition, and may also be 40 wt% or less, 35 wt% or less, 30 wt% or less, 25 wt% or less, or 20 wt% or less.

[0225] In one embodiment, the relative amount of the fused lipid to the cationic lipid of Formula 1 may be 0.05 parts by weight or more, 0.06 parts by weight or more, 0.07 parts by weight or more, 0.08 parts by weight or more, 0.09 parts by weight or more, or 0.1 parts by weight or more, based on 1 part by weight of the cationic lipid of Formula 1, and may also be 6 parts by weight or less, 5.5 parts by weight or less, 5 parts by weight or less, 4.5 parts by weight or less, 4 parts by weight or less, or 3.8 parts by weight or less, but is not limited thereto.

[0226] In one embodiment, when a phospholipid is used as the fused lipid, the relative amount used may be 0.03 parts by weight or more, 0.04 parts by weight or more, 0.05 parts by weight or more, or 0.06 parts by weight or more based on 1 part by weight of the lipid of Formula 1, and may also be 4 parts by weight or less, 3.9 parts by weight or less, 3.7 parts by weight or less, 3.5 parts by weight or less, 3.3 parts by weight or less, 3.1 parts by weight or less, 3 parts by weight or less, 2.9 parts by weight or less, or 2.7 parts by weight or less, but is not limited thereto.

[0227] In one embodiment, when cholesterol is used as the fused lipid, the relative amount used may be 0.02 parts by weight or more, 0.03 parts by weight or more, or 0.04 parts by weight or more based on 1 part by weight of the cationic lipid of Formula 1, and may also be 2 parts by weight or less, 1.9 parts by weight or less, 1.7 parts by weight or less, 1.5 parts by weight or less, 1.3 parts by weight or less, or 1.1 parts by weight or less, but is not limited thereto.

[0228] In addition, in one embodiment, the drug delivery composition of the present invention may further include one or more additive components (hereinafter, “optional additive components”) that are typically included in drug delivery compositions.

[0229] In one embodiment, the optional additive component may be one or more selected from, for example, pH modifiers (e.g., acidifying agents, alkalizing agents, buffering agents), tonicity modifiers, bulking agents (e.g., sugars, polyols, amino acids, polymers, proteins, etc.), wetting agents, solubilizing agents, surfactants, antioxidants, antimicrobial agents, chelating agents, complexing agents, etc., but is not limited thereto.

[0230] In one embodiment, the pH adjuster may be one or more selected from acetate, citrate, tartrate, histidine, glutamate, phosphate, Tris, glycine, bicarbonate, succinate, sulfate, nitrate, etc., but is not limited thereto.

[0231] In one embodiment, the intestinal regulator may be one or more selected from mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, potassium chloride, glycerol, glycerin, propylene glycol, etc., but is not limited thereto.

[0232] In one embodiment, the bulking agent may be one or more selected from sugars and polyols including sucrose, trehalose, glucose, lactose sorbitol, mannitol, glycerol, etc.; amino acids including arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine, methionine, alanine, etc.; polymers and proteins including gelatin, polyvinylpyrrolidone (PVP), polylactate-co-glycolate (PLGA), polyethylene glycol (PEG), dextran, cyclodextran, or derivatives thereof, starch derivatives, hydroxylamine sulfate (HAS), bovine serum albumin (BSA), etc., or combinations thereof, but is not limited thereto.

[0233] In one embodiment, the wetting agent and / or solubilizing agent is lecithin, PEG 300, PEG 600, PEG 1000, polyoxyethylene lauryl ether (e.g., Brij 30, Brij 35, Brij 56, Brij 76, Brij 97), polypropylene glycol (PPG) 2000, glucoside alkyl ethers, polyoxyethylene glycol octylphenol ethers, polyoxyethylene glycol alkylphenol ethers, glycerol alkyl esters, polysorbates 20, polysorbates 40, polysorbates 60, polysorbates 80, sorbitan monolaurate (Span It may be one or more selected from, but not limited to, sorbitan monooleate (Span 80), sorbitan trioleate (Span 85), cocamide monoethanolamine (cocamide MEA), cocamide diethanolamine (cocamide DEA), dodecyldimethylamine oxide, poloxamer, polyvinylpyrrolidone K25, polyvinyl alcohol, oligolactic acid, sodium dioctyl sulfosuccinate, diethylene glycol monolaurate, triethanolamine oleate, sodium oleate, potassium oleate, ethyl oleate, oleic acid, ethyl laurate, sodium lauryl sulfate, etc.

[0234] In one embodiment, the antioxidant may be one or more selected from tocopherol (vitamin E), tocopherol alpha, alpha tocopherol hydrogen succinate, ascorbic acid, accorbyl palmitate, butylated hydroxy anisole (BHA), butylated hydroxy toluene (BHT), monothioglycerol, potassium metabisulfite, propionic acid, propyl gallate, sodium ascorbate, sodium bisulfite, sodium metabisulfite, sodium sulfite, histamine, methionine, glutathione, poly(ethylamine), etc., but is not limited thereto.

[0235] In one embodiment, the antimicrobial agent may be one or more selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimid, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, metacresol, ethyl alcohol, glycerin, hexatidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercury nitrate, propylene glycol, thimerosal, etc., but is not limited thereto.

[0236] In one embodiment, the chelating agent may be one or more selected from ethylenediaminetetraacetic acid (EDTA), disodium edetate, dipotassium edetate, edetic acid, fumaric acid, malic acid, phosphoric acid, sodium edetate, tartaric acid, diethylenetriaminepentaacetic acid (DPTA), citric acid, hexaphosphate, thiolglycolic acid, zinc, etc., but is not limited thereto.

[0237] When any of the above additive components are used in the drug delivery composition of the present invention, the content of each additive may be, for example, 0.01 wt% or more, 0.05 wt% or more, or 0.1 wt% or more based on the dry weight of the total composition, and may also be 10 wt% or less, 5 wt% or less, or 1 wt% or less, but is not limited thereto.

[0238] Composition and method of manufacturing the same

[0239] The drug delivery composition according to the present invention may be administered through routes of administration such as blood vessels, muscles, mucous membranes, subcutaneous, intradermal, oral, bone, transdermal, or local tissues, and may be formulated into various oral or parenteral formulations suitable for such routes of administration. Examples of oral formulations include tablets, capsules, powders, liquids, etc., and examples of parenteral formulations include ophthalmic drops, injections, etc. In one embodiment, the composition may be an injectable formulation. For example, when the composition according to the present invention is freeze-dried, it may be reconstituted with injectable distilled water, 0.9% physiological saline, and a 5% aqueous dextrose solution to produce an injectable formulation.

[0240] Accordingly, the fifth aspect of the present invention provides a method for preparing a drug delivery composition comprising: (a) preparing a solution in which a cationic lipid represented by Formula 1; and a lipid-polymer polymer, an amphiphilic block copolymer, or a mixture thereof; is dissolved in a water-miscible organic solvent; and (b) adding and mixing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof to the solution prepared in step (a).

[0241] In one embodiment, the water-miscible organic solvent of step (a) may be ethanol.

[0242] In one embodiment, step (b) may be performed in a solution under acidic conditions.

[0243] In one embodiment, the step (b) may include: (b-1) a step of preparing a buffer solution containing the active ingredient; and (b-2) a step of adding and mixing the buffer solution of the active ingredient prepared in step (b-1) to the solution prepared in step (a).

[0244] In one embodiment, the mixing ratio of the buffer solution of the active ingredient prepared in step (b-1) to the solution prepared in step (a) may be 1:1 to 1:5 in volume ratio, and more specifically, 1:2 to 1:4.

[0245] In another embodiment, the step (b) may include: (b-1) a step of adding the active ingredient to the solution prepared in step (a); and (b-2) a step of adding and mixing a buffer solution to the result of step (b-1).

[0246] In one embodiment, the method for preparing the drug delivery composition may further include the step of adding a pH adjusting buffer, water for injection, or a combination thereof to the product of step (b).

[0247] In another embodiment, the method for preparing the drug delivery composition may further include the step of removing the solvent from the product of step (b) and then adding a freeze-drying aid to freeze-dry.

[0248] The freeze-drying aid is added to help the freeze-dried composition maintain a cake shape or to help the composition melt uniformly within a short period of time during the reconstitution process after freeze-drying, and specifically, it may be one or more selected from the group consisting of sugars, amino acids, polymers, and proteins. For example, it may be one or more selected from the group consisting of lactose, mannitol, sorbitol, and sucrose. The content of the freeze-drying aid may be 1 to 90 weight% based on the total dry weight of the freeze-dried composition, and more specifically, 10 to 60 weight%.

[0249] The present invention will be explained in more detail below based on the following examples, but these are for the purpose of explaining the invention and do not limit the scope of the invention in any way.

[0250] [Example]

[0251] Example A: Preparation of lipid compounds and preparation of drug delivery composition Example I

[0252] Example A1

[0253] A compound of the following chemical formula A (compound A) was prepared according to the synthesis outline shown in Fig. 1.

[0254] [Chemical Formula A]

[0255]

[0256] (1) Synthesis of Compound A-1

[0257] [Compound A-1]

[0258]

[0259] A reaction solution was prepared by dissolving 2,2'-disulfanediylbis(ethan-1-amine) (1.00 g, 6.57 mmol, 1.00 eq), 6-bromohexyl 2-hexyldecanoate (5.51 g, 13.1 mmol, 2.00 eq), and K2CO3 (2.72 g, 19.7 mmol, 3.00 eq) in acetonitrile (ACN) (10.0 mL) in a 500 mL 3-neck round-bottom flask (RBF). This mixture was purged with N2 three times and stirred for 16 hours at 70°C under a nitrogen atmosphere. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (dichloromethane (DCM):methanol (MeOH) = 100:1 → 0:1). Compound A-1 (1.50 g, 1.28 mmol, 19.5% yield) was obtained in the form of a yellow oil.

[0260] (2) Synthesis of Compound A

[0261] A reaction solution was prepared by dissolving compound A-1 (1.00 g, 856 μmol, 1.00 eq), methyl iodide (MeI) (243 mg, 1.71 mmol, 2.00 eq), and K2CO3 (236 mg, 1.71 mmol, 2.00 eq) in tetrahydrofuran (THF) (10.0 mL) in a 250 mL 3-neck RBF. This mixture was purged with N2 three times and stirred for 3 hours under a nitrogen atmosphere at 25°C. The reaction was stopped by adding a saturated aqueous solution of ammonium chloride (NH4Cl) (20.0 mL) to the reaction mixture at 25°C, and then diluted with ethyl acetate (EtOAc) (20.0 mL). The reaction mixture was extracted with EtOAc (20.0 mL × 3), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.05% HCl)-ACN]; gradient: 50% → 80% B, 15.0 min). The purified product was concentrated under reduced pressure to remove ACN, after which the aqueous layer was neutralized with K2CO3 solution (10.0 mL) and stirred for 12 hours. The mixture was extracted with DCM (5.00 mL × 2), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound A (0.15 g, 125 μmol, 14.6% yield) was obtained in the form of yellow wax.

[0262] 1 1H NMR (400 MHz, CHLOROFORM- d ): δ11.68 - 11.95 (m, 1H), 3.99 - 4.20 (m, 8H), 3.27 - 3.77 (m, 14H), 2.97 - 3.24 (m, 4H), 2.20 - 2.45 (m, 3H), 1.76 - 1.91 (m, 6H), 1.54 - 1.68 (m, 14H), 1.44 (br s, 18H), 1.22 - 1.32 (m, 58H), 0.88 (t, J = 6.75 Hz, 18H)

[0263] Example A2

[0264] A compound of the following chemical formula B (compound B) was prepared according to the synthesis outline shown in Figure 2.

[0265] [Chemical Formula B]

[0266]

[0267] (1) Synthesis of Compound B-1

[0268] [Compound B-1]

[0269]

[0270] A reaction solution was prepared by dissolving 2,2'-disulfanediylbis(ethane-1-amine) (1.00 g, 6.57 mmol, 1.00 eq), heptadecan-9-yl 8-bromooctanoate (6.06 g, 13.1 mmol, 2.00 eq), and K2CO3 (2.72 g, 19.7 mmol, 3.00 eq) in ACN (10.0 mL) in a 250 mL 3-neck RBF. This mixture was purged with N2 three times and stirred for 16 hours at 70°C under a nitrogen atmosphere. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (DCM:MeOH=100:1 → 0:1). Compound B-1 (1.00 g, 772 μmol, 11.7% yield) was obtained in the form of a yellow oil.

[0271] (2) Synthesis of Compound B

[0272] Compound B-1 (1.00 g, 772 μmol, 1.00 eq) was dissolved in THF (10.0 mL) in a 250 mL 3-neck RBF. K2CO3 (213 mg, 1.55 mmol, 2.00 eq) was added to the reaction solution at 25°C, followed by the addition of MeI (219 mg, 1.55 mmol, 2.00 eq) at 0°C. The mixture was stirred at 25°C for 3 hours under an N2 atmosphere. The reaction was stopped by adding NH4Cl (20.0 mL) to the reaction mixture at 25°C, after which it was diluted with EtOAc (20.0 mL). The reaction mixture was extracted with EtOAc (20.0 mL × 3), filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.05% HCl)-ACN]; gradient: 50% → 80% B, 15.0 min). The purified product was concentrated under reduced pressure to remove ACN, after which the aqueous layer was neutralized with K2CO3 solution (10.0 mL) and stirred for 12 hours. The mixture was extracted with DCM (5.00 mL × 2), dried with Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound B (0.13 g, 94.3 μmol, 12.2% yield, 96.0% purity) was obtained in the form of yellow wax.

[0273] 1 1H NMR (400 MHz, CHLOROFORM- d ): δ4.86 (t, J = 6.19 Hz, 3H), 3.80 - 3.93 (m, 2H), 3.35 - 3.47 (m, 4H), 3.18 - 3.30 (m, 10H), 3.00 - 3.12 (m, 4H), 2.25 - 2.33 (m, 6H), 1.47 - 1.56 (m, 18H), 1.18 - 1.39 (m, 96H), 0.89 (t, J = 6.82 Hz, 18H)

[0274] Example A3

[0275] A compound of the following chemical formula C (compound C) was prepared according to the synthesis outline shown in Fig. 3.

[0276] [Chemical Formula C]

[0277]

[0278] (1) Synthesis of compound C-1

[0279] [Compound C-1]

[0280]

[0281] 6-bromohexyl 2-hexyl decanoate (5.00 g, 11.9 mmol, 4.50 eq) was dissolved in THF (15.0 mL) in a 250 mL 3-neck RBF, and then K2CO3 (2.93 g, 21.2 mmol, 8.00 eq) was added. 2,2'-disulfanediylbis(ethane-1-amine) (403 mg, 2.65 mmol, 1.00 eq) was added to the reaction solution, and the mixture was stirred at 90°C for 16 hours. The reaction mixture was filtered, and the solvent was removed under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50 mm×7 μm, mobile phase: [H2O(0.1% trifluoroacetic acid (TFA))-ACN]; gradient: 70% → 100% B, 15.0 min). As a result, compound C-1 (1.20 g, 796 μmol, 30.0% yield) was obtained as a pale yellow oil.

[0282] (2) Synthesis of Compound C

[0283] Compound C-1 (1.00 g, 664 μmol, 1.00 eq) was dissolved in THF (2.00 mL) in a 100 mL 3-neck RBF. MeI (118 mg, 830 μmol, 51.7 μL, 1.25 eq) and K2CO3 (138 mg, 996 μmol, 1.50 eq) were added to the reaction solution, and the mixture was stirred at 25°C for 24 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (Column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, Mobile phase: [H2O(0.05% HCl)-ACN]; Gradient: 68% → 98% B, 15.0 min). Compound C (0.10 g, 65.7 μmol, 9.90% yield) was obtained in the form of a yellow oil.

[0284] 1 1H NMR (400 MHz, CHLOROFORM- d ): δ 4.06 (dt, J = 3.4, 6.6 Hz, 8H), 3.92 (br dd, J = 2.9, 5.3 Hz, 2H), 3.68 - 3.39 (m, 10H), 3.35 (br s, 3H), 3.23 - 3.02 (m, 4H), 2.38 - 2.26 (m, 4H), 1.88 (br s, 12H), 1.82 - 1.69 (m, 8H), 1.66 (br s, 8H), 1.61 - 1.52 (m, 8H), 1.50 - 1.47 (m, 4H), 1.43 (br s, 8H), 1.26 (br s, 80H), 0.88 (t, J = 6.8 Hz, 24H)

[0285] Example A4

[0286] A compound of the following chemical formula D (compound D) was prepared according to the synthesis outline shown in Fig. 4.

[0287] [Chemical Formula D]

[0288]

[0289] (1) Synthesis of compound D-1

[0290] [Compound D-1]

[0291]

[0292] 3-bromopropyl 2-hexyltridecanoate (4.00 g, 9.54 mmol, 3.00 eq), 2,2'-disulfanediylbis(ethane-1-amine) (HCl salt, 599 mg, 3.18 mmol, 1.00 eq), and N,N-diisopropylethylamine (DIEA) (3.29 g, 25.4 mmol, 4.43 mL, 8.00 eq) were mixed in EtOH (40 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 70°C for 16 hours under an N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.1% TFA)-ACN]; gradient: 70% → 100% B, 20.0 min). Compound D-1 (2.00 g, 1.71 mmol, 53.8% yield) was obtained as a yellow oil.

[0293] (2) Synthesis of Compound D

[0294] Compound D-1 (1.00 g, 856 μmol, 1.00 eq) was dissolved in THF (10.0 mL) in a 250 mL 3-neck RBF. K2CO3 (236 mg, 1.71 mmol, 2.00 eq) was added to the reaction solution at 25°C, followed by the addition of MeI (243 mg, 1.71 mmol, 106 μL, 2.00 eq) at 0°C. The mixture was stirred at 25°C for 3 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; gradient: 60% → 90% B, 15.0 min). Compound D (0.30 g, 250 μmol, 29.2% yield) was obtained as a yellow oil.

[0295] 1 H NMR (400 MHz, CHLOROFORM-d): δ12.58-12.02 (m, 1H), 4.36-4.18 (m, 6H), 4.18-4.05 (m, 2H), 3.67 (br s, 2H), 3.59-3.47 (m, 6H), 3.46 (br s, 6H), 3.36-3.13 (m, 4H), 2.38-2.30 (m, 3H), 2.29-2.08 (m, 6H), 1.67-1.37 (m, 16H), 1.26 (s, 74H), 0.89 (t, J = 6.8 Hz, 18H)

[0296] Example A5

[0297] A compound of the following chemical formula E (compound E) was prepared according to the synthesis outline shown in Fig. 5.

[0298] [Chemical Formula E]

[0299]

[0300] (1) Synthesis of compound E-1

[0301] [Compound E-1]

[0302]

[0303] 2,2'-Disulfanediylbis(ethane-1-amine) (HCl salt, 1.80 g, 9.54 mmol, 1.00 eq), 6-Bromohexyl 2-hexyldecanoate (8.00 g, 19.0 mmol, 2.00 eq), and Na2CO3 (4.04 g, 38.1 mmol, 4.00 eq) were mixed in ACN (80 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 50°C for 48 hours under an N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.1% TFA) - ACN]; gradient: 70% → 100% B, 20.0 min). Compound E-1 (1.00 g, 1.21 mmol, 12.6% yield) was obtained as a yellow oil.

[0304] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.07 (t, J = 6.6 Hz, 4H), 3.02 (t, J = 6.2 Hz, 2H), 2.85-2.70 (m, 6H), 2.53-2.39 (m, 4H), 2.36-2.25 (m, 2H), 1.70-1.57 (m, 12H), 1.45-1.20 (m, 54H), 0.88 (t, J = 6.7 Hz, 12H)

[0305] (2) Synthesis of Compound E-2

[0306] [Compound E-2]

[0307]

[0308] Compound E-1 (1.00 g, 1.21 mmol, 1.00 eq), undecan-3-yl 8-bromooctanoate (1.37 g, 3.62 mmol, 3.00 eq), and Na2CO3 (511 mg, 4.82 mmol, 4.00 eq) were mixed in ACN (4.0 mL) in a 100 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 95°C for 16 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; gradient: 60% → 90% B, 15.0 min). Compound E-2 (0.60 g, 421.83 μmol, 34.9% yield) was obtained as a yellow oil.

[0309] (3) Synthesis of Compound E

[0310] Compound E-2 (0.60 g, 703 μmol, 1.00 eq) was dissolved in THF (10.0 mL) in a 100 mL 3-neck RBF. K2CO3 (194 mg, 1.41 mmol, 2.00 eq) was added to the reaction solution at 25°C, followed by the addition of MeI (199 mg, 1.41 mmol, 2.00 eq) at 0°C. The mixture was stirred at 25°C for 3 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (Column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, Mobile phase: [H2O(0.05% HCl) - ACN]; Gradient: 60% → 90% B, 15.0 min). Compound E (0.065 g, 35.2 μmol, 10.4% yield, 97% purity) was obtained in the form of a yellow oil.

[0311] 1 H NMR (400 MHz, CHLOROFORM-d): δ11.86 (br dd, J = 2.4, 4.6 Hz, 1H), 4.81 (quin, J = 6.2 Hz, 2H), 4.06 (dt, J = 3.4, 6.5 Hz, 4H), 3.99-3.86 (m, 2H), 3.72-3.40 (m, 10H), 3.38 (br s, 3H), 3.25-2.98 (m, 4H), 2.40-2.20 (m, 6H), 1.91-1.69 (m, 7H), 1.63-1.51 (m, 16H), 1.51-1.36 (m, 24H), 1.26 (br s, 68H), 1.00-0.75 (m, 24H)

[0312] Example A6

[0313] A compound of the following chemical formula F (compound F) was prepared according to the synthesis outline shown in Fig. 6.

[0314] [Chemical Formula F]

[0315]

[0316] (1) Synthesis of compound F-1

[0317] [Compound F-1]

[0318]

[0319] Na2CO3 (2.26 g, 21.3 mmol, 8.00 eq) was added at 25°C to a solution prepared by dissolving 2,2'-disulfanediylbis(ethane-1-amine) (2HCl, 0.60 g, 2.66 mmol, 1.00 eq) and 7-bromoheptyl 2-hexyldecanoate (5.20 g, 11.9 mmol, 4.50 eq) in THF (3.00 mL) and ACN (3.00 mL). The mixture was stirred at 70°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was dissolved in THF (3.00 mL) and ACN (3.00 mL), and after adding Na2CO3 (2.26 g, 21.3 mmol, 8.00 eq) at 25°C, the mixture was stirred at 70°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue, which was then dissolved in DCM (20.0 mL) and H2O (50.0 mL) and extracted with DCM (30.0 mL × 3, total 90.0 mL). After combining the organic layers, the residue was obtained by drying with anhydrous Na2SO4, filtering, and concentrating under reduced pressure. The residue was first purified by silica gel column chromatography (DCM / MeOH = 100 / 1 → 0 / 1), followed by purification using prep-HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.1% TFA) - ACN]; gradient: 70% → 100% B, 20.0 min). Compound F-1 (2.20 g, 1.41 mmol, 52.8% yield, 100% purity) was obtained in the form of a yellow oil.

[0320] 1H NMR (400 MHz, CHLOROFORM-d): δ4.08 (t, J = 6.6 Hz, 8H), 2.97 (br d, J = 5.8 Hz, 2H), 2.90-2.77 (m, 7H), 2.69-2.62 (m, 2H), 2.48 (br d, J = 2.0 Hz, 5H), 2.38-2.27 (m, 4H), 1.62 (br s, 32H), 1.50-1.41 (m, 14H), 1.36 (br s, 18H), 1.27 (br s, 72H), 0.90 (t, J = 6.5 Hz, 24H)

[0321] (2) Synthesis of Compound F

[0322] Compound F-1 (1.00 g, 639 μmol, 1.00 eq) was dissolved in DCM (10.0 mL) in a 50 mL 3-neck RBF. A mixed solution of MeI (90.8 mg, 639 μmol, 39.8 μL, 1.00 eq) and DCM (1.00 mL) was added at 15°C, and the mixture was stirred at 20°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using prep-HPLC (Column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, Mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; Gradient: 60% → 90% B, 15.0 min). Compound F (139 mg, 85.7 μmol, 13.4% yield, 97.1% purity) was obtained in the form of yellow gum.

[0323] 1H NMR (400 MHz, CHLOROFORM-d): δ4.06 (br s, 8H), 3.66-2.97 (m, 14H), 2.39-2.22 (m, 4H), 1.91-1.70 (m, 7H), 1.67-1.62 (m, 16H), 1.56 (br s, 14H), 1.48-1.37 (m, 26H), 1.33-1.22 (m, 78H), 0.89 (t, J = 6.7 Hz, 24H)

[0324] Example A7

[0325] A compound of the following chemical formula G (compound G) was prepared according to the synthesis outline shown in Fig. 7.

[0326] [Chemical Formula G]

[0327]

[0328] (1) Synthesis of compound G-1

[0329] [Compound G-1]

[0330]

[0331] 3-bromopropyl 2-hexyltridecanoate (4.00 g, 9.54 mmol, 4.00 eq), 2,2'-disulfanediylbis(ethane-1-amine) (HCl salt, 449 mg, 2.38 mmol, 1.00 eq), and Na2CO3 (1.52 g, 14.3 mmol, 6.00 eq) were mixed in ACN (40.0 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 95°C for 16 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 100 / 1 → 0 / 1). Compound G-1 (2.00 g, 1.33 mmol, 55.6% yield) was obtained in the form of a yellow oil.

[0332] (2) Synthesis of Compound G

[0333] Compound G-1 (1.00 g, 856 μmol, 1.00 eq) was dissolved in THF (10.0 mL) in a 100 mL 3-neck RBF. K2CO3 (91.7 mg, 1.71 mmol, 1.00 eq) was added to the reaction solution at 25°C, followed by the addition of MeI (94.21 mg, 1.71 mmol, 106 μL, 1.00 eq) at 0°C. The mixture was stirred at 25°C for 3 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; gradient: 70% → 100% B, 15.0 min). Compound G (0.10 g, 65.72 μmol, 9.90% yield) was obtained as a yellow oil.

[0334] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.24 (br t, J = 5.3 Hz, 8H), 4.10 (br s, 2H), 3.81-3.59 (m, 4H), 3.56-3.48 (m, 4H), 3.45 (br s, 3H), 3.39-3.13 (m, 4H), 2.38-2.29 (m, 4H), 2.26-2.15 (m, 6H), 1.60-1.42 (m, 16H), 1.26 (s, 108H), 0.89 (t, J = 6.8 Hz, 24H)

[0335] Example A8

[0336] A compound of the following chemical formula H (compound H) was prepared according to the synthesis outline shown in Fig. 8.

[0337] [Chemical Formula H]

[0338]

[0339] (1) Synthesis of compound H-1

[0340] [Compound H-1]

[0341]

[0342] Heptadecan-9-yl 8-bromooctanoate (5.00 g, 10.83 mmol, 4.50 eq) and 2,2'-disulfanediylbis(ethane-1-amine) (HCl salt, 454.36 mg, 2.41 mmol, 1.00 eq) were dissolved in ACN (50.0 mL) in a 3-neck RBF. After adding K2CO3 (1.66 g, 12.04 mmol, 5.00 eq) to the reaction solution, the mixture was stirred at 90°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 10 / 1). Compound H-1 (2.00 g, 1.19 mmol, 49.60% yield) was obtained in the form of a colorless oil.

[0343] (2) Synthesis of Compound H

[0344] Compound H-1 (0.92 g, 549.30 μmol, 1.00 eq) was dissolved in DCM (10.0 mL) in a 3-neck RBF, and methyl triflate (MeOTf) (90.14 mg, 549.30 μmol, 60.25 μL, 1.00 eq) was added at 25°C. The mixture was stirred at 25°C for 16 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue, and the residue was purified by prep-HPLC (Column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, Mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; Gradient: 70% → 100% B, 15.0 min). Subsequently, to obtain compound H, the residue was prepared using chiral prep-HPLC (Column: Chiral-IC-30-DAICEL CHIRALPAK IC, 250×30 mm, 10 μm; Mobile phase: CO₂-MeOH:ACN = 7:3 (0.1% NH3·H2O) Compound H (0.25 g, 147.94 μmol, 26.93% yield) was obtained in the form of a yellow oil by purifying with an isocratic solvent (B% = 70%), and then repurifying with prep-HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; gradient: 70% → 100% B, 15.0 min).

[0345] 1H NMR (400 MHz, CHLOROFORM-d): δ4.86 (quin, J = 6.1 Hz, 4H), 4.16-3.83 (m, 2H), 3.68-3.40 (m, 10H), 3.38 (br s, 3H), 3.21-2.98 (m, 4H), 2.33-2.24 (m, 8H), 1.91-1.79 (m, 4H), 1.69 (br s, 12H), 1.63 (br d, J = 6.0 Hz, 8H), 1.50 (br s, 8H), 1.45-1.34 (m, 24H), 1.32-1.22 (m, 96H), 0.88 (t, J = 6.8 Hz, 24H)

[0346] Example A9

[0347] A compound of the following chemical formula I (compound I) was prepared according to the synthesis outline shown in Fig. 9.

[0348] [Chemical Formula I]

[0349]

[0350] (1) Synthesis of Compound I-1

[0351] [Compound I-1]

[0352]

[0353] Undecane-3-yl 8-bromooctanoate (10.0 g, 26.5 mmol, 2.00 eq), tert-butyl (2-((2-aminoethyl)disulfaneyl)ethyl)carbamate (3.34 g, 13.2 mmol, 1.00 eq), and Na2CO3 (4.21 g, 39.7 mmol, 3.00 eq) were mixed in ACN (50.0 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 90°C for 16 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 100 / 1 → 0 / 1). Compound I-1 (9.50 g, 11.2 mmol, 84.8% yield) was obtained in the form of a yellow oil.

[0354] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.82 (t, J = 5.9 Hz, 2H), 3.46 (br d, J = 5.4 Hz, 2H), 2.88-2.67 (m, 6H), 2.42 (br t, J = 7.0 Hz, 4H), 2.29 (t, J = 7.6 Hz, 4H), 1.71-1.48 (m, 14H), 1.45 (s, 9H), 1.43-1.21 (m, 38H), 1.05-0.71 (m, 12H)

[0355] (2) Synthesis of Compound I-2

[0356] [Compound I-2]

[0357]

[0358] Compound I-1 (9.50 g, 11.2 mmol, 1.00 eq) and HCl / EtOAc (2 M, 95.0 mL) were mixed in DCM (95.0 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 25°C for 3 hours under an N2 atmosphere. The reaction mixture was stopped by adding an aqueous Na2CO3 solution (25 mL) at 0°C, and extracted with ethyl acetate (EtOAc, EA) (10 mL × 3, total 30 mL). The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound I-2 (7.00 g, 9.39 mmol, 83.5% yield) was obtained in the form of a yellow oil without a separate purification process.

[0359] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.81 (t, J = 6.1 Hz, 2H), 3.03 (t, J = 6.2 Hz, 2H), 2.83-2.78 (m, 4H), 2.59-2.43 (m, 4H), 2.29 (br t, J = 7.5 Hz, 6H), 1.69-1.45 (m, 16H), 1.37-1.19 (m, 38H), 0.96-0.74 (m, 12H)

[0360] (3) Synthesis of Compound I-3

[0361] [Compound I-3]

[0362]

[0363] Compound I-2 (2.00 g, 2.68 mmol, 1.00 eq), heptadecan-9-yl 8-bromooctanoate (1.24 g, 2.68 mmol, 1.00 eq), and Na2CO3 (568 mg, 5.37 mmol, 2.00 eq) were mixed in ACN (40.0 mL) in a 250 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 50°C for 16 hours under an N2 atmosphere. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 100 / 1 → 0 / 1). Compound I-3 (0.90 g, 799 μmol, 29.7% yield) was obtained as a yellow oil.

[0364] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.97-4.72 (m, 3H), 3.00-2.89 (m, 2H), 2.88-2.80 (m, 2H), 2.76 (s, 4H), 2.62 (t, J = 7.3 Hz, 2H), 2.48-2.35 (m, 4H), 2.34-2.21 (m, 6H), 1.70-1.53 ​​(m, 18H), 1.45-1.18 (m, 72H), 0.99-0.80 (m, 18H)

[0365] (4) Synthesis of Compound I

[0366] Compound I-3 (0.900 g, 799 μmol, 1.00 eq) and K2CO3 (220 mg, 1.60 mmol, 2.00 eq) were mixed in THF (9.00 mL) at 25°C in a 100 mL 3-neck RBF. After adding MeI (226 mg, 1.60 mmol, 99.5 μL, 2.00 eq) at 0°C, the mixture was stirred at 25°C for 3 hours under an N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (Column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, Mobile phase: [H2O(0.05% HCl) - ACN]; Gradient: 48% → 78% B, 15.0 min). Compound I (0.130 g, 109 μmol, 13.6% yield, HCl salt) was obtained in the form of a white solid.

[0367] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.83 (td, J = 6.1, 19.0 Hz, 3H), 4.10 (br d, J = 2.1 Hz, 2H), 3.68-3.48 (m, 4H), 3.42 (br s, 10H), 3.20-2.98 (m, 4H), 2.36-2.25 (m, 6H), 1.79 (br d, J = 5.8 Hz, 6H), 1.62-1.50 (m, 14H), 1.43-1.18 (m, 70H), 0.95-0.82 (m, 18H)

[0368] Example A10

[0369] A compound of the following chemical formula J (compound J) was prepared according to the synthesis outline shown in Fig. 10.

[0370] [Chemical Formula J]

[0371]

[0372] (1) Synthesis of compound J-1

[0373] [Compound J-1]

[0374]

[0375] 2,2'-Disulfanediylbis(ethane-1-amine) (2HCl, 0.60 g, 2.66 mmol, 1.00 eq) and 7-Bromoheptyl 2-hexyldecanoate (4.04 g, 9.32 mmol, 3.50 eq) were dissolved in EtOH (6.0 mL) in a 3-neck RBF, and DIEA (2.75 g, 21.3 mmol, 3.71 mL, 8.00 eq) was added at 25°C. The mixture was stirred at 70°C for 48 hours. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified by silica gel column chromatography (SiO₂, DCM / MeOH = 100 / 1 → 0 / 1) and then purified using prep-HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.1% TFA) - ACN]; gradient: 70% → 100% B, 25.0 min). Compound J-1 (0.70 g, 578 μmol, 21.7% yield) was obtained in the form of a yellow oil.

[0376] (2) Synthesis of Compound J

[0377] Compound J-1 (0.70 g, 578 μmol, 1.00 eq) was dissolved in DCM (7.0 mL) in a 50 mL 3-neck RBF, and a mixed solution of K2CO3 (159 mg, 1.16 mmol, 2.00 eq) and MeI (205 mg, 1.45 mmol, 90.0 μL, 2.50 eq) and DCM (1.0 mL) was added at 10–15°C. The mixture was stirred at 20°C for 3 hours. The reaction mixture was filtered, and the filtrate was concentrated under reduced pressure to obtain the residue. The residue was first purified by prep-HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN]; gradient: 55% → 85% B, 15.0 min), and then re-purified by prep-HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN:THF = 2:1]; gradient: 55% → 85% B, 15.0 min). Compound J (99.63 mg, 78.80 μmol, 13.62% yield, 98.0% purity) was obtained in the form of a milky white gum.

[0378] 1 H NMR (400 MHz, CHLOROFORM-d): δ11.83 (br d, J = 0.8 Hz, 1H), 4.17-4.01 (m, 8H), 3.64-3.33 (m, 14H), 3.21-3.01 (m, 4H), 2.37-2.24 (m, 3H), 1.78 (br d, J = 3.1 Hz, 8H), 1.66-1.55 (m, 12H), 1.48-1.36 (m, 26H), 1.31-1.22 (m, 56H), 0.88 (t, J = 6.7 Hz, 18H)

[0379] Example A11

[0380] A compound of the following chemical formula K (compound K) was prepared according to the synthesis outline shown in Fig. 11.

[0381] [Chemical Formula K]

[0382]

[0383] (1) Synthesis of compound K-1

[0384] [Compound K-1]

[0385]

[0386] 6-bromohexyl 2-hexyl decanoate (11.0 g, 26.2 mmol, 2.20 eq) and tert-butyl (2-((2-aminoethyl)disulfanyl)ethyl)carbamate (3.01 g, 11.9 mmol, 1.00 eq) were dissolved in ACN (110 mL) in a 3-neck RBF. Na2CO3 (3.16 g, 29.8 mmol, 2.50 eq) was added to the mixed solution, and the mixture was stirred at 90°C for 24 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 10 / 1 → 1 / 100). Compound K-1 (3.00 g, 3.07 mmol, 25.7% yield, 95% purity) was obtained in the form of a yellow oil.

[0387] 1 H NMR (400 MHz, CHLOROFORM-d, EC22615-205-P1_A1): δ4.96 (br s, 1H), 4.07 (t, J = 6.7 Hz, 4H), 3.46 (br d, J = 5.4 Hz, 2H), 2.93-2.63 (m, 6H), 2.43 (br t, J = 7.0 Hz, 4H), 2.37-2.24 (m, 2H), 1.69-1.59 (m, 6H), 1.50-1.38 (m, 18H), 1.37-1.18 (m, 49H), 0.88 (t, J = 6.6 Hz, 12H)

[0388] (2) Synthesis of Compound K-2

[0389] [Compound K-2]

[0390]

[0391] Compound K-1 (3.00 g, 3.23 mmol, 1.00 eq) was dissolved in EtOAc (3.0 mL) in a 50 mL 3-neck RBF. HCl / EtOAc (2 M, 6.45 mL, 4.00 eq) was added to this, and the mixture was stirred at 20°C for 3 hours. After spin-drying the organic layer, an aqueous Na2CO3 solution (100 mL) was added to the reaction mixture at 25°C to stop the reaction, and the mixture was extracted with DCM (100 mL × 3, total 300 mL). The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. Compound K-2 (2.50 g, 3.01 mmol, 93.4% yield) was obtained in the form of a yellow oil without a separate purification process.

[0392] 1 H NMR (400 MHz, CHLOROFORM-d, EC22615-212-P1_A1): δ4.07 (t, J = 6.7 Hz, 4H), 3.03 (t, J = 6.2 Hz, 2H), 2.89-2.60 (m, 6H), 2.55-2.39 (m, 4H), 2.38-2.24 (m, 2H), 1.67-1.55 (m, 12H), 1.52-1.39 (m, 10H), 1.36-1.23 (m, 44H), 0.88 (br t, J = 6.6 Hz, 12H)

[0393] (3) Synthesis of Compound K-3

[0394] [Compound K-3]

[0395]

[0396] Compound K-2 (1.00 g, 1.21 mmol, 1.00 eq) and heptadecan-9-yl 8-bromooctanoate (445 mg, 965 μmol, 0.80 eq) were dissolved in ACN (16.0 mL) and THF (4.0 mL) in a 100 mL 3-neck RBF. After adding Na2CO3 (153 mg, 1.45 mmol, 1.20 eq) to the reaction solution, the mixture was stirred at 70°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD30-WePure Biotech Phenyl-Hexyl 250×50mm×7μm, mobile phase: [H2O(0.1% TFA) - ACN]; gradient: 50% → 80% B, 25.0 min). Compound K-3 (0.55 g, 455 μmol, 37.7% yield) was obtained as a yellow oil.

[0397] (4) Synthesis of Compound K

[0398] Compound K-3 (0.900 g, 799 μmol, 1.00 eq) and K2CO3 (220 mg, 1.60 mmol, 2.00 eq) were mixed in THF (9.00 mL) at 25°C in a 100 mL 3-neck RBF. After adding MeI (226 mg, 1.60 mmol, 99.5 μL, 2.00 eq) at 0°C, the mixture was stirred for 3 hours at 0-25°C under an N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using reverse-phase HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25mm×7μm, mobile phase: [H2O(0.05% HCl) - ACN]; gradient: 48% → 78% B, 15.0 min). Compound K (0.130 g, 109 μmol, 13.6% yield, HCl salt) was obtained as a white solid.

[0399] 1H NMR (400 MHz, CHLOROFORM-d): δ4.83 (td, J = 6.1, 19.0 Hz, 3H), 4.10 (br d, J = 2.1 Hz, 2H), 3.68-3.48 (m, 4H), 3.42 (br s, 10H), 3.20-2.98 (m, 4H), 2.36-2.25 (m, 6H), 1.79 (br d, J = 5.8 Hz, 6H), 1.62-1.50 (m, 14H), 1.43-1.18 (m, 70H), 0.95-0.82 (m, 18H)

[0400] Example A12

[0401] A compound of the following chemical formula L (compound L) was prepared according to the synthesis outline shown in Fig. 12.

[0402] [Chemical Formula L]

[0403]

[0404] (1) Synthesis of compound L-1

[0405] [Compound L-1]

[0406]

[0407] 6-bromohexyl 2-hexyldecanoate (11.0 g, 26.2 mmol, 2.20 eq) and tert-butyl (2-((2-aminoethyl)disulfaneyl)ethyl)carbamate (3.01 g, 11.9 mmol, 1.00 eq) were dissolved in ACN (110 mL) in a 250 mL 3-neck RBF. Na2CO3 (3.16 g, 29.8 mmol, 2.50 eq) was added to the mixed solution, and the mixture was stirred at 90°C for 24 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 10 / 1 → 1 / 100). Compound L-1 (3.00 g, 3.07 mmol, 25.7% yield, 95% purity) was obtained in the form of a yellow oil.

[0408] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.96 (br s, 1H), 4.07 (t, J = 6.7 Hz, 4H), 3.46 (br d, J = 5.4 Hz, 2H), 2.93-2.63 (m, 6H), 2.43 (br t, J = 7.0 Hz, 4H), 2.37-2.24 (m, 2H), 1.69-1.59 (m, 6H), 1.50-1.38 (m, 18H), 1.37-1.18 (m, 49H), 0.88 (t, J = 6.6 Hz, 12H)

[0409] (2) Synthesis of Compound L-2

[0410] [Compound L-2]

[0411]

[0412] Compound L-1 (3.00 g, 3.23 mmol, 1.00 eq) was dissolved in EtOAc (3.0 mL) in a 50 mL 3-neck RBF. Subsequently, HCl / EtOAc (2 M, 6.45 mL, 4.00 eq) was added, and the mixture was stirred at 20°C for 3 hours. After spin-drying the organic layer, an aqueous Na2CO3 solution (100 mL) was added to the reaction mixture at 25°C to stop the reaction. The mixture was then extracted with DCM (100 mL × 3, total 300 mL), the organic layer was dried with anhydrous Na2SO4, and the residue was obtained by filtration and vacuum concentration. Compound L-2 (2.50 g, 3.01 mmol, 93.4% yield) was obtained in the form of a yellow oil without a separate purification process.

[0413] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.07 (t, J = 6.7 Hz, 4H), 3.03 (t, J = 6.2 Hz, 2H), 2.89-2.60 (m, 6H), 2.55-2.39 (m, 4H), 2.38-2.24 (m, 2H), 1.67-1.55 (m, 12H), 1.52-1.39 (m, 10H), 1.36-1.23 (m, 44H), 0.88 (br t, J = 6.6 Hz, 12H)

[0414] (3) Synthesis of Compound L-3

[0415] [Compound L-3]

[0416]

[0417] Undecane-3-yl 8-bromooctanoate (364 mg, 965 μmol, 0.80 eq) and compound L-2 (1.00 g, 1.21 mmol, 1.00 eq) were dissolved in ACN (16.0 mL) and THF (4.0 mL) in a 100 mL 3-neck RBF. After adding Na2CO3 (153 mg, 1.45 mmol, 1.20 eq) to the reaction solution, the mixture was stirred at 70°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. Compound L-3 (0.55 g, 489 μmol, 40.5% yield) was obtained in the form of a yellow oil.

[0418] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.82 (br t, J = 6.2 Hz, 1H), 4.07 (t, J = 6.7 Hz, 4H), 3.02-2.90 (m, 2H), 2.88-2.81 (m, 2H), 2.77 (s, 4H), 2.63 (t, J = 7.2 Hz, 2H), 2.53-2.38 (m, 4H), 2.36-2.21 (m, 4H), 1.67-1.41 (m, 30H), 1.37-1.23 (m, 61H), 0.88 (br t, J = 6.3 Hz, 18H)

[0419] (4) Synthesis of Compound L

[0420] Compound L-3 (0.50 g, 444 μmol, 1.00 eq) was dissolved in DCM (5.0 mL) in a 50 mL 3-neck RBF, and CH₃I (75.6 mg, 533 μmol, 1.20 eq) was slowly added using a syringe while maintaining the temperature at 25°C. The mixture was stirred at 25°C for 16 hours. The reaction mixture was filtered and concentrated under reduced pressure to obtain the residue. 0.50 g of the crude material was purified by reverse-phase HPLC (column: CD12-WePure Biotech Phenyl-Hexyl 150×25 mm×7 μm, mobile phase: [H₂O(0.05% HCl) - ACN:THF = 2:1]; gradient: 45% → 75% B, 15.0 min) to obtain 0.16 g of a sample with a purity of 84%. This sample was re-purified using reverse-phase HPLC under the same conditions to obtain 0.16 g of a sample with 93% purity. Then, 0.12 g of the sample with 93% purity was purified using reverse-phase HPLC (column: same; mobile phase: [H2O(0.05% HCl) - ACN:THF = 1:1]; gradient: 50% → 80% B, 15.0 min) to finally obtain 0.07 g of compound L with 97% purity (58.8 μmol, 13.2% yield, 97% purity, HCl salt) in the form of a yellow oil.

[0421] 1 H NMR (400 MHz, CHLOROFORM-d): δ11.81 (br s, 1H), 4.80 (quin, J = 6.2 Hz, 1H), 4.05 (br t, J = 6.6 Hz, 6H), 3.59-3.03 (m, 18H), 2.38-2.25 (m, 4H), 1.83 (br s, 12H), 1.68-1.50 (m, 14H), 1.43 (br s, 16H), 1.26 (br s, 49H), 0.95-0.81 (m, 18H)

[0422] Example A13

[0423] A compound of the following chemical formula M (compound M) was prepared according to the synthesis outline shown in Fig. 13.

[0424] [Chemical Formula M]

[0425]

[0426] (1) Synthesis of henicosan-9-ol

[0427] [Henicosan-9-ol]

[0428]

[0429] 1-bromooctane (19.5 g, 100 mmol, 1.00 eq), Mg (7.35 g, 303 mmol, 3.00 eq), and I₂ (1.28 g, 5.04 mmol, 0.05 eq) were mixed in THF (400 mL) in a 1000 mL 3-neck RBF, and separately, tridecanal (20.0 g, 100 mmol, 1.00 eq) was dissolved in THF (100 mL). After degassing and purging the reaction mixture with N₂ three times, the tridecanal solution was added and stirred for 16 hours at 0-50°C under an N₂ atmosphere. The reaction mixture was combined, quenched by slowly adding 3 N HCl (600 mL), and extracted with EtOAc (300 mL × 3, total 900 mL). After concentrating the organic layer under reduced pressure, it was triturated with commercial hexane at 20°C for 30 minutes. Henicosan-9-ol (40.0 g, 128 mmol, 63.5% yield) was obtained as a white solid.

[0430] 1 H NMR (400 MHz, CHLOROFORM-d): δ3.59 (br dd, J = 6.57, 4.32 Hz, 1H), 1.46-1.26 (m, 36H), 0.89 (t, J = 6.75 Hz, 6H)

[0431] (2) Synthesis of henicosan-9-yl 4-bromobutanoate

[0432] [Henicosan-9-il 4-bromobutanoate]

[0433]

[0434] Henicosan-9-ol (15.0 g, 48.0 mmol, 1.00 eq), 4-bromobutanoic acid (16.0 g, 96.0 mmol, 2.00 eq), N,N'-diisopropylcarbodiimide (DIC) (15.1 g, 120 mmol, 2.50 eq), 4-dimethylaminopyridine (DMAP) (293 mg, 2.40 mmol, 0.05 eq) and triethylamine (TEA) (7.44 g, 57.6 mmol, 1.20 eq) were mixed in DCM (150 mL) in a 500 mL 3-neck RBF. The reaction mixture was degassed and substituted with N2 three times, and then stirred at 20°C for 16 hours under an N2 atmosphere. An aqueous solution of Na2CO3 (100 mL) was added to the reaction mixture at 25°C to stop the reaction, and the mixture was extracted with DCM (100 mL × 3, total 300 mL). The organic layer was dried with anhydrous Na2SO4, filtered, and concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, commercial hexane / EtOAc = 100 / 1 → 10 / 1). Henicosan-9-yl 4-bromobutanoate (15.0 g, 32.5 mmol, 67.7% yield) was obtained in the form of a colorless oil.

[0435] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.90 (quin, J = 6.22 Hz, 1H), 3.49 (t, J = 6.44 Hz, 2H), 2.51 (t, J = 7.19 Hz, 2H), 2.20 (quin, J = 6.85 Hz, 2H), 1.54 (br d, J = 5.38 Hz, 4H), 1.28 (s, 32H), 0.90 (t, J = 6.75 Hz, 6H)

[0436] (3) Synthesis of Compound M-1

[0437] [Compound M-1]

[0438]

[0439] Henicosan-9-yl 4-bromobutanoate (1.89 g, 4.10 mmol, 1.00 eq), 2,2'-disulfanediylbis(ethane-1-amine) (0.25 g, 1.64 mmol, 0.33 eq), and DIEA (1.49 g, 11.5 mmol, 7.00 eq) were mixed in EtOH (19.0 mL) in a 100 mL 3-neck RBF. The reaction mixture was degassed and purged with N2 three times, and then stirred at 75°C for 16 hours under an N2 atmosphere. The reaction mixture was concentrated under reduced pressure to obtain the residue. The residue was purified using silica gel column chromatography (SiO₂, DCM / MeOH = 100 / 0 → 100 / 2). Compound M-1 (1.30 g, 1.02 mmol, 61.8% yield) was obtained in the form of a yellow oil.

[0440] (4) Synthesis of Compound M

[0441] Compound M-1 (0.80 g, 625 μmol, 1.00 eq) and K2CO3 (173 mg, 1.25 mmol, 2.00 eq) were dissolved in THF (12 mL) in a 250 mL 3-neck RBF and degassed and replaced with N2 three times. Then, MeI (177 mg, 1.25 mmol, 2.00 eq) was slowly dropwise added using a syringe at 25°C. The mixture was stirred for 16 hours under an N2 atmosphere at 25°C. The product of the reaction mixture was concentrated under reduced pressure and purified by reverse-phase HPLC. Primary purification was performed using a CD12-WePure Biotech Phenyl-Hexyl 150×25 mm, 7 μm column with a mobile phase of [H2O (0.05% HCl) - ACN] and a gradient of 70%→B (15.0 min). Secondary purification was performed in the same column under [H2O (0.05% HCl) - ACN:THF = 2:1], gradient: 68%→B (15.0 min). Finally, compound M (0.14 g, 106 μmol, 8.47% yield) was obtained in the form of a yellow oil.

[0442] ¹H NMR (400 MHz, CHLOROFORM-d): δ11.41-12.61 (m, 1H), 4.84 (quin, J = 6.10 Hz, 3H), 4.04-4.19 (m, 2H), 3.66-3.79 (m, 2H), 3.34-3.64 (m, 12H), 3.11-3.31 (m, 4H), 2.35-2.63 (m, 6H), 2.00-2.25 (m, 6H), 1.52 (br d, J = 6.00 Hz, 12H), 1.26 (s, 96H), 0.89 (t, J = 6.82 Hz, 18H)

[0443] [Preparation Example I of a Drug Delivery Composition ]

[0444] 1. Preparation of raw materials

[0445] As shown in the table below, the substances required for formulation preparation were dissolved in each dilution solvent to the required concentrations. When dissolving, the substances were brought to room temperature before adding the solvent and dissolving them.

[0446]

[0447] 2. Mixing of raw materials

[0448] The required amounts of raw materials were taken and mixed according to the ratio of compounds A to S:DOPE:cholesterol:DMG-PEG = 50:10:38.5:1.5, with the NP ratio (amine group of lipid component: phosphate group of mRNA) set to 6. Ethanol was added to the ethanol layer so that the sum of all raw materials was within 12.5 mM, and the aqueous phase and ethanol phase were mixed while maintaining a volume ratio of 3:1. Buffer exchange was performed as follows to lower the total ethanol content after mixing: the mixture was concentrated by centrifuging at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL), and then the process of diluting with PBS and centrifuging to concentrate was repeated to perform buffer exchange.

[0449] The specific process sequence is as follows.

[0450] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0451] 2) Each of compounds A to S and DOPE, cholesterol, and DMG-PEG were added in sequence to Tube (A) in molar amounts calculated according to experimental conditions, and mixed by vortexing after addition.

[0452] 3) Ethanol was added as needed in the ethanol phase so that the total of all raw materials was within 6.25-12.5 mM.

[0453] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in Tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0454] 5) Mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, Precision Nanosystem) instrument. The Microfluidics operating conditions were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 12 mL / min.

[0455] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K) to concentrate it, then diluted with PBS and centrifuged to concentrate it again to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

[0456] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0457] 3. Evaluation of Physical Properties of the Formulation

[0458] 1) For the prepared formulations, particle characteristics (i.e., zeta-average particle size, polydispersity index (PDI), and zeta-potential) were determined using a particle size analyzer (Dynamic Light Scattering, DLS), and the results are shown in Table 1 below.

[0459] 2) For the prepared formulations, mRNA encapsulation efficiency was confirmed through a Ribo-green assay, and the results are shown in Table A below.

[0460] [Table A]

[0461]

[0462] Example B: Preparation Example II of a drug delivery composition and drug delivery test

[0463] Example B1

[0464] (1) Preparation of solutions by component

[0465] The components listed in Table 1 below were dissolved in each dilution solvent to prepare the compositions at the concentrations shown in Table 1 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0466] [Table 1]

[0467]

[0468] (2) Mixing of raw materials

[0469] Each component was taken and mixed according to a weight ratio of Compound A : DOPE : Cholesterol : DMG-PEG = 100 : 21.1 : 24.2 : 6.1, adjusting the N / P ratio (amine groups of lipid components / phosphate groups of mRNA) to be between 5 and 20. Ethanol was added to the ethanol layer so that the total molecular weight of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0470] The specific process sequence is as follows.

[0471] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0472] 2) The moles of compound A, DOPE, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0473] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0474] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0475] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0476] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0477] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0478] 8) The concentration was measured using a Ribo-green assay and used.

[0479] (3) Evaluation of physical properties of the formulation

[0480] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 2 below.

[0481] [Table 2]

[0482]

[0483] (4) Administration of composition

[0484] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 3 below.

[0485] [Table 3]

[0486]

[0487] Example B2

[0488] (1) Preparation of solutions by component

[0489] The components listed in Table 4 below were dissolved in each diluting solvent to prepare the compositions at the concentrations shown in Table 4 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0490] [Table 4]

[0491]

[0492] (2) Mixing of raw materials

[0493] Each component was taken and mixed according to a weight ratio of Compound A : DSPC : Cholesterol : DMG-PEG = 100 : 12.8 : 24.2 : 6.1, with an N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5 and 20. Ethanol was added to the ethanol layer so that the total molecular weight of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0494] The specific process sequence is as follows.

[0495] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0496] 2) The moles of compound A, DSPC, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0497] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0498] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0499] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0500] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0501] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0502] 8) The concentration was measured using a Ribo-green assay and used.

[0503] (3) Evaluation of the physical properties of the formulation

[0504] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 5 below.

[0505] [Table 5]

[0506]

[0507] (4) Administration of composition

[0508] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 6 below.

[0509] [Table 6]

[0510]

[0511] Example B3

[0512] (1) Preparation of solutions by component

[0513] The components listed in Table 7 below were dissolved in each dilution solvent to prepare the compositions at the concentrations shown in Table 7 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0514] [Table 7]

[0515]

[0516] (2) Mixing of raw materials

[0517] Each component was taken and mixed according to a weight ratio of Compound B : DOPE : Cholesterol : DMG-PEG = 100 : 11.6 : 21.9 : 5.5, with an N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5 and 20. Ethanol was added to the ethanol layer so that the total molecular weight of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0518] The specific process sequence is as follows.

[0519] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0520] 2) The moles of compound B, DOPE, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0521] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0522] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0523] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0524] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0525] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0526] 8) The concentration was measured using a Ribo-green assay and used.

[0527] (3) Evaluation of the physical properties of the formulation

[0528] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 8 below.

[0529] [Table 8]

[0530]

[0531] (4) Administration of composition

[0532] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 9 below.

[0533] [Table 9]

[0534]

[0535] Example B4

[0536] (1) Preparation of solutions by component

[0537] The components listed in Table 10 below were dissolved in each dilution solvent to prepare the compositions at the concentrations shown in Table 10 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used to prepare the compositions after visually confirming that there were no undissolved particles.

[0538] [Table 10]

[0539]

[0540] (2) Mixing of raw materials

[0541] Each component was taken and mixed according to a weight ratio of Compound B : DSPC : Cholesterol : DMG-PEG = 100 : 11 : 21.9 : 5.5, with an N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5 and 20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0542] The specific process sequence is as follows.

[0543] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0544] 2) The moles of compound B, DSPC, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0545] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0546] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0547] 5) Mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0548] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0549] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0550] 8) The concentration was measured using a Ribo-green assay and used.

[0551] (3) Evaluation of the physical properties of the formulation

[0552] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 11 below.

[0553] [Table 11]

[0554]

[0555] (4) Administration of composition

[0556] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 12 below.

[0557] [Table 12]

[0558]

[0559] Example B5

[0560] (1) Preparation of solutions by component

[0561] The components listed in Table 13 below were dissolved in each diluting solvent to prepare the compositions at the concentrations shown in Table 13 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0562] [Table 13]

[0563]

[0564] (2) Mixing of raw materials

[0565] Each component was taken and mixed according to a weight ratio of Compound C : DOPE : Cholesterol : DMG-PEG = 100 : 9.6 : 19.1 : 4.8, adjusting the N / P ratio (amine groups of lipid components / phosphate groups of mRNA) to be between 5 and 20. Ethanol was added to the ethanol layer so that the total molecular weight of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0566] The specific process sequence is as follows.

[0567] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0568] 2) The moles of compound C, DOPE, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0569] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0570] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0571] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0572] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0573] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0574] 8) The concentration was measured using a Ribo-green assay and used.

[0575] (3) Evaluation of the physical properties of the formulation

[0576] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 14 below.

[0577] [Table 14]

[0578]

[0579] (4) Administration of composition

[0580] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 15 below.

[0581] [Table 15]

[0582]

[0583] Example B6

[0584] (1) Preparation of solutions by component

[0585] The components listed in Table 16 below were dissolved in each diluting solvent to prepare the compositions at the concentrations shown in Table 16 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0586] [Table 16]

[0587]

[0588] (2) Mixing of raw materials

[0589] Each component was taken and mixed according to a weight ratio of Compound C : DSPC : Cholesterol : DMG-PEG = 100 : 10.1 : 19.1 : 4.8, with an N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5 and 20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0590] The specific process sequence is as follows.

[0591] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0592] 2) The moles of compound C, DSPC, cholesterol, and DMG-PEG calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0593] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0594] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0595] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0596] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0597] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0598] 8) The concentration was measured using a Ribo-green assay and used.

[0599] (3) Evaluation of the physical properties of the formulation

[0600] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 17 below.

[0601] [Table 17]

[0602]

[0603] (4) Administration of composition

[0604] The prepared formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice at a dose of 2 μg mRNA per mouse. Four hours after administration, whole-body and organ-specific protein expression was confirmed using the IVIS spectrum in vivo imaging system, and the results are shown in Table 18 below.

[0605] [Table 18]

[0606]

[0607] Example B7

[0608] (1) Preparation of solutions by component

[0609] The components listed in Table 19 below were dissolved in each dilution solvent to prepare the compositions at the concentrations shown in Table 19 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used to prepare the compositions after visually confirming that there were no undissolved particles.

[0610] [Table 19]

[0611]

[0612] (2) Mixing of raw materials

[0613] Each component was taken and mixed according to a weight ratio of Compound A : DOPE : Cholesterol : mPEG-PLA (2K-4K) = 100 : 9.2 : 11.7 : 375.4, adjusting the N / P ratio (amine groups of lipid components / phosphate groups of mRNA) to be between 5 and 20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0614] The specific process sequence is as follows.

[0615] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0616] 2) The moles of compound A, DOPE, cholesterol, and mPEG-PLA (2K-4K) calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0617] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0618] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0619] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0620] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0621] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0622] 8) The concentration was measured using a Ribo-green assay and used.

[0623] (3) Evaluation of the physical properties of the formulation

[0624] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 20 below.

[0625] [Table 20]

[0626]

[0627] Example B8

[0628] (1) Preparation of solutions by component

[0629] The components listed in Table 21 below were dissolved in each diluting solvent to prepare the compositions at the concentrations shown in Table 21 below. When dissolving, the substances were heated to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the compositions were used after visually confirming that there were no undissolved particles.

[0630] [Table 21]

[0631]

[0632] (2) Mixing of raw materials

[0633] Each component was taken and mixed according to a weight ratio of Compound K : DOPE : Cholesterol : mPEG-PLA (2K-4K) = 100 : 8.9 : 11.4 : 363, adjusting the N / P ratio (amine groups of lipid components / phosphate groups of mRNA) to be between 5 and 20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25–12.5 mM, and mRNA was diluted in the aqueous phase layer using 20 mM sodium acetate buffer (pH 4.6). The aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, to reduce the total ethanol content, the mixture was diluted with DPBS (Dulbecco's phosphate-buffered saline) so that the % ethanol was 5% or less, and then buffered and concentrated as follows: The mixture was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K or 100K, volume 0.5 mL or 4 mL or 15 mL) to undergo the dilution and concentration process, followed by buffer exchange.

[0634] The specific process sequence is as follows.

[0635] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0636] 2) Molaries of compound K, DOPE, cholesterol, and mPEG-PLA (2K-4K) calculated according to experimental conditions were added sequentially to tube (A) and mixed by vortexing.

[0637] 3) Ethanol was added as needed so that the molecular sum of all components in the ethanol phase was within 6.25-12.5 mM.

[0638] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). The ratio was calculated so that the volume of the aqueous phase was three times the volume of the ethanol phase.

[0639] 5) The mixing of tube (A) and tube (B) was performed using a Microfluidics (Ignite, precision Nanosystem) instrument or by vortexing. The operating conditions for the Microfluidics were a Flow Rate Ratio (FRR) of C:R=3:1 and a Total Flow Rate (TRR) of 3-12 mL / min. When vortexing, the solution from tube (B) was added to tube (A), and the mixture was mixed at the fastest and most constant speed possible to ensure a uniform formulation.

[0640] 6) The resulting mixture from step 5) was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K or 100K), and the process of concentration and dilution was repeated to remove excess ethanol so that the ethanol content was 0.1% or less, and then concentrated to the final target concentration.

[0641] 7) Once concentrated to the desired concentration, it was sterilized using a 0.22 µm pore size filter.

[0642] 8) The concentration was measured using a Ribo-green assay and used.

[0643] (3) Evaluation of the physical properties of the formulation

[0644] For the manufactured formulations, particle characteristics were confirmed using a dynamic light scattering (DLS) analyzer, and the results are shown in Table 22 below.

[0645] [Table 22]

[0646]

Claims

Claim 1 Cationic lipid having a structure represented by the following chemical formula 1: [Chemical Formula 1] In the above Chemical Formula 1, R1 and R2 are substituted or unsubstituted alkylene groups, R3, R4, and R5 are each independently substituted or unsubstituted alkylene groups, R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon groups, and R9 and R 10 Each is independently a substituted or unsubstituted alkyl group or a carboncyclic group, or a hydroxyalkyl group or each independently -R 11 -(L4) n -R 12 and, R 11 ≡ are each independently substituted or unsubstituted alkylene groups, and R 12 is each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, and L1, L2, L3, and L4 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, where L' is a direct bond, alkylene, or alkenylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, and alkenyl, n is 0 or 1, and X - is a pharmaceutically acceptable monovalent anion. Claim 2 In paragraph 1, R1 and R2 are substituted or unsubstituted C 1-6 It is an alkylene group, and R3, R4, and R5 are each independently substituted or unsubstituted C 2-15 It is an alkylene group, and R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 It is a hydrocarbon group, and R9 and R 10 Each is independently substituted or unsubstituted C 1-6 alkyl group or C 3-6 It is a carbon cyclic group, or C 1-6 hydroxyalkyl group or -R independently, respectively 11 -(L4) n -R 12 and, R 11 Each is independently substituted or unsubstituted C 3-12 It is an alkylene group, and R 12 Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 3-26 They are hydrocarbon groups, and L1, L2, L3, and L4 are each independently -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, C 2-6 Alkenylene, C 6-20 Arylene, and C 3-20 Selected from the group composed of heteroarylenes, where L' is a direct bond, C 1-13 Alkylene or C 2-13 It is alkenylene, and R' are independently hydrogen atoms, C 1-18 Alkyl and C 2-18 Selected from the group consisting of alkenyls, n is 0 or 1, and X - is a cationic lipid that is a monovalent anion of a pharmaceutically acceptable inorganic or organic acid. Claim 3 In paragraph 1, R1 and R2 are substituted or unsubstituted C 1-4 It is an alkylene group, and R3, R4, and R5 are each independently substituted or unsubstituted C 2-8 It is an alkylene group, and R6, R7, and R8 are each independently substituted or unsubstituted saturated or unsaturated monovalent C 6-22 It is a hydrocarbon group, and R9 and R 10 Each is independently substituted or unsubstituted C 1-2 It is an alkyl group, or C 2-4 hydroxyalkyl group or -R independently, respectively 11 -(L4) n -R 12 and, R 11 Each is independently substituted or unsubstituted C 3-8 It is an alkylene group, and R 12 Each is independently substituted or unsubstituted saturated or unsaturated monovalent C 6-22 They are hydrocarbon groups, and L1, L2, L3 and L4 are each independently -C(O)O-, -OC(O)-, -C(O)N(R')-, -N(R')C(O)-, -P(O)(OR')O-, -SS-, and C 2-5 Selected from the group consisting of alkenylenes, where R' is independently a hydrogen atom and C 1-6 Selected from the group consisting of alkyls, n is 0 or 1, and X - is F - , Cl - , Br - , I - , cationic lipids that are nitrate anions, benzoate anions, methanesulfonate anions, acetate anions, or trihaloacetate anions. Claim 4 In paragraph 1, the above R6 to R 10 One or more of the above are each independently further substituted with one or more moieties specifically suitable for a target tissue or organ, said moieties being cationic lipids selected from amino acids, sugars, vitamins, peptides, proteins, hormones, antibodies, neurotransmitters, pharmaceutically active small molecules, endosome-degrading agents, cell membrane permeability agents, charge blockers, drugs, nucleic acids, or derivatives thereof. Claim 5 A cationic lipid having any one of the following chemical formulas A to V, wherein, in claim 1: Claim 6 A method for preparing a cationic lipid, comprising: (1-1) a step of reacting a compound of formula a with a compound of formula b to obtain a compound of formula c; and (1-2) a step of reacting a compound of formula c with a compound of formula d to obtain a cationic lipid of formula 1-1; or (2-1) a step of reacting a compound of formula a with a compound of formula b to obtain a compound of formula e; and (2-2) a step of reacting a compound of formula e with a compound of formula d to obtain a cationic lipid of formula 1-2; [Formula a]R6-L3-R3-X[Formula b]H2N-R1-SS-R2-NH2[Formula c] [Chemical Formula d]R9-X[Chemical Formula e] [Chemical Formula 1-1] [Chemical Formula 1-2] In the above, R1 and R2 are substituted or unsubstituted alkylene groups, R3 is each independently a substituted or unsubstituted alkylene group, R6 is each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, R9 is each independently a substituted or unsubstituted alkyl group or carboncyclic group or hydroxyalkyl group, and L3 is each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-L'-C(O)O-, -C(O)N(R')-, -N(R')C(O)-, -C(O)-, -C(S)-, -C(S)S-, -SC(S)-, -CH(OH)-, -P(O)(OR')O-, -S(O)2-, -SS-, alkenylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene or It is alkenylene, where R' is each independently selected from the group consisting of hydrogen atoms, alkyl and alkenyl, and X - is a pharmaceutically acceptable monovalent anion. Claim 7 A drug delivery composition comprising a cationic lipid according to any one of claims 1 to 5. Claim 8 A drug delivery composition comprising: an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; a cationic lipid of any one of claims 1 to 5; and a lipid-polymer, an amphiphilic block copolymer, or a combination thereof. Claim 9 A drug delivery composition according to claim 8, wherein the active ingredient is mRNA. Claim 10 A drug delivery composition according to claim 8, wherein the lipid-polymer polymer is a polymer in which one or more saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms are introduced as hydrophobic portions into a hydrophilic block which is a hydrophilic portion. Claim 11 A drug delivery composition according to claim 10, wherein the hydrophilic block is one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof. Claim 12 A drug delivery composition according to claim 10, wherein the saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms are independently selected from the group consisting of lauryl, myristoyl, dimyristoyl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, cerotyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, and docosahexaenyl. Claim 13 A drug delivery composition according to claim 8, wherein the amphiphilic block copolymer is an AB-type diblock copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof, and the hydrophobic B block is one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine. Claim 14 A drug delivery composition according to claim 13, wherein the terminal hydroxyl group of the hydrophobic B block is modified by one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. Claim 15 A drug delivery composition according to claim 8, further comprising a fusion lipid. Claim 16 A drug delivery composition according to claim 15, wherein the fusion lipid is one or more combinations selected from the group consisting of phospholipids, cholesterol, and tocopherol. Claim 17 (a) a step of preparing a solution in which a cationic lipid of any one of claims 1 to 5; and a lipid-polymer polymer, an amphiphilic block copolymer, or a mixture thereof; is dissolved in a water-miscible organic solvent; and (b) a step of adding and mixing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof to the solution prepared in step (a); comprising a method for preparing a drug delivery composition. Claim 18 A method for preparing a drug delivery composition according to claim 17, wherein the water-miscible organic solvent of step (a) is ethanol. Claim 19 A method for preparing a drug delivery composition according to claim 17, wherein step (b) comprises: (b-1) a step of preparing a buffer solution containing the active ingredient; and (b-2) a step of adding and mixing the buffer solution of the active ingredient prepared in step (b-1) to the solution prepared in step (a). Claim 20 A method for preparing a drug delivery composition according to claim 17, wherein step (b) comprises: (b-1) a step of adding an active ingredient to the solution prepared in step (a); and (b-2) a step of adding a buffer solution to the product of step (b-1) and mixing.

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