Nanoparticle composition for drug delivery

Abstract

The present invention relates to a composition for drug delivery, and a preparation method therefor, and, more specifically, to a composition for drug delivery, and a preparation method therefor, the composition having a form such that a drug is encapsulated inside a nanoparticle structure formed by a polymer and a cationic lipid with a specific structure.

Description

Composition of nanoparticles for drug delivery

[0001] The present invention relates to a drug delivery composition and a method for producing the same, and more particularly, to a drug delivery composition in which a drug is encapsulated inside a nanoparticle structure formed by a cationic lipid of a specific structure and a polymer, and a method for producing the same.

[0002] Safe and efficient drug delivery technologies for the treatment of anionic drugs, including nucleic acids, have long been studied, and various carriers and delivery technologies have been developed. Carriers are broadly divided into viral carriers, such as those using adenoviruses or retroviruses, and non-viral carriers, such as those using cationic lipids and cationic polymers. Viral carriers are known to be susceptible to risks such as non-specific immune responses. Therefore, recent research is focused on improving these drawbacks by utilizing non-viral carriers. While non-viral carriers are less efficient than viral carriers, they offer the advantage of fewer side effects in terms of in vivo safety.

[0003] Among non-viral delivery vehicles, the most representative ones are complexes of cationic lipids and nucleic acids (lipoplexes) and polycationic polymers and nucleic acids (polyplexes). These cationic lipids or polycationic polymers have been extensively studied for their ability to stabilize anionic drugs and enhance intracellular delivery by forming complexes with them through electrostatic interactions. However, when administered intravenously at the doses necessary to achieve sufficient efficacy, they induce significant toxicity, although less than viral vectors, making them unsuitable for pharmaceutical use. Therefore, the development of anionic drug delivery technologies that minimize the use of potentially toxic cationic polymers or cationic lipids, thereby reducing toxicity while ensuring stability in vivo and enabling intracellular delivery for sufficient efficacy is needed.

[0004] Accordingly, anionic drug delivery compositions and various manufacturing methods have been disclosed, which form a complex through electrostatic interaction between a nucleic acid and a cationic lipid, and encapsulate the complex within the nanoparticle structure of an amphiphilic block copolymer. For example, Korean Patent Publication No. 2017-0032858 discloses an anionic drug delivery composition and a manufacturing method thereof, which comprises an anionic drug as an active ingredient; a cationic compound; an amphiphilic block copolymer; and a polylactic acid salt, 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 salt. However, existing nanoparticle drug delivery systems, including those disclosed in the above-mentioned patent, still lack the efficiency of delivering drugs (particularly, mRNA) such as nucleic acids, polypeptides, or viruses into the body.

[0005] The present invention aims to provide a drug delivery composition and a method for producing the same, which have significantly improved delivery efficiency of drugs (particularly, mRNA) such as nucleic acids, polypeptides or viruses into the body compared to existing known nanoparticle drug delivery vehicles.

[0006] A first aspect of the present invention provides a drug delivery composition comprising: an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; a cationic lipid having a structure represented by the following chemical formula 1; and a lipid-polymer, an amphiphilic block copolymer, or a combination thereof:

[0007] [Chemical Formula 1]

[0008]

[0009] Here,

[0010] R1 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0011] R2, R3 and R4 are each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0012] R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0013] R8 and R9 are each independently a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group or carbon cyclic group, or each independently -R 10 -(L4) n -R 11 And,

[0014] R 10 are each independently a substituted or unsubstituted alkylene group,

[0015] R 11 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0016] 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, alkynylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl,

[0017] n is 0 or 1,

[0018] X - is a pharmaceutically acceptable monovalent anion.

[0019] A second aspect of the present invention provides a method for preparing a composition for drug delivery, comprising: (a) preparing a solution in which a cationic lipid represented by the above chemical formula 1; and a lipid-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 a nucleic acid, a polypeptide, a virus, or a combination thereof to the solution prepared in step (a).

[0020] The drug delivery composition according to the present invention can significantly improve the delivery efficiency of drugs (particularly, mRNA) such as nucleic acids, polypeptides or viruses into the body compared to existing nanoparticle drug delivery vehicles.

[0021] Figure 1 is a reaction schematic diagram for the synthesis process of the compound of chemical formula A performed in Manufacturing Example 1.

[0022] Figure 2 is a reaction schematic diagram for the synthesis process of the compound of chemical formula B performed in Manufacturing Example 2.

[0023] Figure 3 is a reaction schematic diagram for the synthesis process of the compound of chemical formula C performed in Manufacturing Example 3.

[0024] Figure 4 is a reaction schematic diagram for the synthesis process of compounds of chemical formula D and E performed in Manufacturing Example 4.

[0025] Figure 5 is a reaction schematic diagram for the synthesis process of the chemical formula F compound performed in Manufacturing Example 5.

[0026] Figure 6 is a reaction schematic diagram for the synthesis process of the compound of chemical formula G performed in Manufacturing Example 6.

[0027] Figure 7 is a reaction schematic diagram for the synthesis process of the chemical formula H compound performed in Manufacturing Example 7.

[0028] Hereinafter, the present invention will be described in more detail.

[0029] Active ingredients

[0030] The active ingredient included in the drug delivery composition of the present invention is selected from nucleic acids, polypeptides, viruses, or a combination thereof.

[0031] The above “nucleic acid” may be, for example, but is not limited to, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof.

[0032] The above “polypeptide” may mean a protein that is active in the body, such as an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a polypeptide sequence of an antigen, an analogue or precursor thereof, which can be recognized as an antigen through a series of processes in the body.

[0033] The above “virus” may be an oncolytic virus, and may be, for example, one or more selected from the group consisting of adenovirus, AAV, vaccinia virus, herpes simplex virus (HSV), and vesicular stomatitis virus (VSV). In one specific embodiment, the oncolytic virus is an adenovirus. The adenovirus used in a specific embodiment of the present invention comprises a luciferase gene, which can be detected by imaging.

[0034] The above virus can express various therapeutic genes in a subject's body, and is not limited to a specific molecular weight, protein, bioactivity, or therapeutic area. The prophylactic virus can induce immunity in a subject's body against the target disease. A composition containing the disease-preventing virus has the advantages of reducing the immune induction caused by the virus itself, targeting or expanding target cells, and reducing hyperimmune responses to the virus upon re-administration, allowing for effective effects with multiple vaccinations.

[0035] In one specific embodiment of the present invention, the active ingredient is messenger RNA (mRNA).

[0036] The mRNA may have its backbone, sugars or bases chemically modified or its ends modified for purposes such as increasing blood stability or weakening the immune response.

[0037] In one specific example, 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, 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, based on the dry weight of the entire composition. If the content of the active ingredient is too small, the amount of carrier used is too large compared to the drug, which may cause side effects due to the carrier, and conversely, if it is too large, the amount of drug that is not encapsulated in the nanoparticles increases, which reduces the efficiency.

[0038] cationic lipids

[0039] The cationic lipid included in the nanoparticle composition of the present invention is represented by the following chemical formula 1:

[0040] [Chemical Formula 1]

[0041]

[0042] Here,

[0043] R1 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0044] R2, R3 and R4 are each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group,

[0045] R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0046] R8 and R9 are each independently a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group or carbon cyclic group, or each independently -R 10 -(L4) n -R 11 And,

[0047] R 10 are each independently a substituted or unsubstituted alkylene group,

[0048] R 11 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group,

[0049] 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, alkynylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl,

[0050] n is 0 or 1,

[0051] X - is a pharmaceutically acceptable monovalent anion.

[0052] In this specification, the expression that any group is “substituted or unsubstituted” means that the group is unsubstituted or substituted with —OH, a halogen atom, C 1-6 Alkyl group, C 1-6 Alkoxy group, C 1-6 Halogenated alkyl 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 among heteroaryl groups.

[0053] In this specification, the expression that any group (e.g., heteroaryl, heterocycloalkyl, etc.) is a “hetero” group means that the group has one or more (e.g., 1 to 3) heteroatoms selected from N, O, and S, unless otherwise specified.

[0054] In this specification, “monohydric hydrocarbon group” may be branched or unbranched, cyclic or acyclic, or aromatic.

[0055] In this specification, “alkyl”, “alkenyl”, “alkynyl”, “alkylene”, “alkenylene” and “alkynylene” can each independently be branched or unbranched, or cyclic or acyclic.

[0056] According to one specific example of the present invention,

[0057] The above R1 is substituted or unsubstituted C 1-6 alkylene group, C 2-6 Alkenylene group or C 2-6 It may be an alkynylene group,

[0058] The above R2, R3 and R4 are each independently substituted or unsubstituted C 3-12 alkylene group, C 3-12 Alkenylene group or C 3-12 It may be an alkynylene group,

[0059] The above R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 3-20 It may be a hydrocarbon group,

[0060] The above R8 and R9 are each independently substituted or unsubstituted C 1-6 Alkyl group, C 2-6 Alkenyl group, C 2-6 Alkynyl group or C 3-6 A carbon cyclic group, or each independently -R 10 -(L4) n -R 11 It can be,

[0061] The above R 10 are independently substituted or unsubstituted C 3-12 It may be an alkylene group,

[0062] The above R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 3-20 It may be a hydrocarbon group,

[0063] The above 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 2-6 Alkynylene, C 6-20 Arylene, and C 3-20 may be selected from the group consisting of heteroarylene, wherein L' is a direct bond, C 1-13 Alkylene, C 2-13 Alkenylene or C 2-13 It can be alkynylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 may be selected from the group consisting of alkynyl,

[0064] n is 0 or 1,

[0065] X - may be a monovalent anion of a pharmaceutically acceptable inorganic acid or organic acid.

[0066] More specifically,

[0067] The above R1 is substituted or unsubstituted C 3-4 alkylene group, C 3-4 Alkenylene group or C 3-4 It may be an alkynylene group,

[0068] The above R2, R3 and R4 are each independently substituted or unsubstituted C 6-8 alkylene group, C 6-8 Alkenylene group or C 6-8 It may be an alkynylene group,

[0069] The above R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It may be a hydrocarbon group,

[0070] The above R8 and R9 are each independently substituted or unsubstituted C 1-2 Alkyl group, C 2-3 Alkenylene group or C 2-3 Alkynylene group, or each independently -R 10 -(L4) n -R 11 It can be,

[0071] R 10 are independently substituted or unsubstituted C 6-8 It may be an alkylene group,

[0072] R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It may be a hydrocarbon group,

[0073] The above 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-, C 2-5 Alkenylene and C 2-5 may be selected from the group consisting of alkynylene, wherein R' is each independently a hydrogen atom, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 may be selected from the group consisting of alkynyl,

[0074] n is 0 or 1,

[0075] X - is a halide (F - , Cl - , Br - , I - ), nitrate anion (NO3 - ), benzoic acid anion (C6H5COO - ), methanesulfonic acid anion, acetate anion (CH3COO - )(=AcO - ), or trihaloacetate anion (CF3COO - ) may be.

[0076] More specifically,

[0077] The above R1 is substituted or unsubstituted C 3-4 It may be an alkylene group,

[0078] The above R2, R3 and R4 are each independently substituted or unsubstituted C 6-8 It may be an alkylene group,

[0079] The above R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It may be a hydrocarbon group,

[0080] The above R8 and R9 are each independently substituted or unsubstituted C 1-2 An alkyl group, or each independently -R 10 -(L4) n -R 11 It can be,

[0081] R 10 are independently substituted or unsubstituted C 6-8 It may be an alkylene group,

[0082] R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It may be a hydrocarbon group,

[0083] The above 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 may be selected from the group consisting of alkenylene, wherein R' is each independently a hydrogen atom and C 1-6 may be selected from the group consisting of alkyl,

[0084] n is 0 or 1,

[0085] X - is Cl - , Br - , or acetate anion (CH3COO - )(=AcO - ) may be.

[0086] More specifically, the cationic lipid may have a structure selected from any one of the following chemical formulae A to Q:

[0087]

[0088]

[0089]

[0090] In one specific embodiment, the content of the cationic lipid in the drug delivery composition of the present invention may be 5 wt% or more, 10 wt% or more, 15 wt% or more, 20 wt% or more, 25 wt% or more, 30 wt% or more, or 35 wt% or more, and may also be 95 wt% or less, 90 wt% or less, 85 wt% or less, 80 wt% or less, 75 wt% or less, 70 wt% or less, or 65 wt% or less, based on the dry weight of the entire composition. If the content of the cationic lipid is too low, it may not be sufficient to form nanoparticles, and conversely, if it is too high, the size of the nanoparticles may become too large, which may lower the stability of the nanoparticles and increase the loss rate during filter sterilization.

[0091] The cationic lipid having the structure represented by the above chemical formula 1 can function as both an ionizable lipid and a helper lipid in a drug delivery composition. The ionizable lipid is a lipid that can control its charge depending on 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.

[0092] However, even if the cationic lipid having the structure represented by the above chemical formula 1 can perform the roles of both an ionizable lipid and a helper lipid, the use of such an additional lipid component is not excluded in the present invention. Therefore, the drug delivery composition of the present invention may further include one or more types of ionizable lipids or one or more types of helper lipids other than the cationic lipid having the structure represented by the above chemical formula 1, as needed.

[0093] polymer

[0094] The drug delivery composition of the present invention comprises a lipid-polymer, an amphiphilic block copolymer, or a combination thereof as a polymer component.

[0095] The above lipid-polymer polymer is a polymer having both hydrophilic and hydrophobic portions within the polymer molecule.

[0096] In one specific example, 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 a hydrophobic moiety into a hydrophilic block, which is a hydrophilic moiety.

[0097] The above hydrophilic block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof.

[0098] More specifically, the hydrophilic block may be at least one selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0099] In one specific example, 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 have a number average molecular weight (g / mol) of 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less, but is not limited thereto.

[0100] In addition, if necessary, a functional group, ligand, or functional group capable of reaching a specific tissue or cell, or a functional group capable of promoting intracellular delivery may be chemically bonded to the end of the hydrophilic block to control the distribution of the nanoparticle carrier in the body or increase the efficiency of delivery of the nanoparticle carrier into the cell. The functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies to cell surface receptors. More specifically, the functional group or ligand may be at least one 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, antibodies to transferrin receptors, etc.

[0101] In one specific example, the saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms introduced as a hydrophobic moiety in the hydrophilic block, which is a hydrophilic moiety, may be independently 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. there is.

[0102] In addition, in one specific example, in the lipid-polymer polymer, the composition ratio of the hydrophilic portion and the hydrophobic portion may be in the range of 10 to 90 wt% of the hydrophilic portion, specifically 20 to 80 wt%, more specifically 30 to 80 wt%, and even more specifically 40 to 80 wt%, based on the weight of the polymer. If the ratio of the hydrophilic portion is too small, the solubility of the polymer in water may be low, making it difficult to form nanoparticles, and conversely, if it is too large, the hydrophilicity may be too high, lowering the stability of the nanoparticles.

[0103] In one specific embodiment of the present invention, the lipid-polymer may be a polyalkylene glycol (e.g., polyethylene glycol) having saturated and unsaturated hydrocarbon groups (e.g., myristyl groups) having 11 to 25 carbon atoms introduced therein. For example, the lipid-polymer may be a PEG lipid (PEGylated lipid). The PEG lipid refers to a polyethylene glycol (PEG)-modified lipid, which is a type of PEG derivative having a lipid moiety such as dimyristoylglycerol (DMG) or distearoylglycerophosphoethanolamine (DSPE) attached thereto. The PEG lipid can be used to improve the circulation time of an active ingredient encapsulated in a lipid nanoparticle and to reduce nonspecific absorption. The above PEG lipid may be a combination of one or more 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-dimyristyloxylpropyl-3-amine (PEG-c-DMA).

[0104] The above amphiphilic block copolymer may be an AB type block copolymer including a hydrophilic A block and a hydrophobic B block. The AB type block copolymer forms core-shell type polymer nanoparticles in an aqueous solution, in which the hydrophobic B block forms a core (inner wall) and the hydrophilic A block forms a shell (outer wall).

[0105] In one specific example, the hydrophilic A block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof.

[0106] More specifically, the hydrophilic A block may be at least one selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0107] In one specific example, 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.

[0108] In addition, if necessary, a functional group, ligand, or functional group capable of promoting intracellular delivery that can reach a specific tissue or cell can be chemically bonded to the terminal of the hydrophilic A block to control the distribution in the body of the polymer nanoparticle carrier formed of the amphiphilic block copolymer and polylactic acid, or to increase the efficiency of delivery of the nanoparticle carrier into cells. In one specific example, the functional group or ligand may be at least one selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies to cell surface receptors. More specifically, the functional group or ligand may be at least one 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, antibodies to transferrin receptors, etc.

[0109] The above hydrophobic B block is a biocompatible biodegradable polymer, and in one specific example, it may be at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazine.

[0110] More specifically, the hydrophobic B block may be at least one selected from the group consisting of polylactide (PLA), polyglycolide, polycaprolactone, polydioxan-2-one, a copolymer of polylactide and glycolide, a copolymer of polylactide and polydioxan-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

[0111] In one specific example, 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.

[0112] For example, the number average molecular weight combination of the hydrophilic A block-hydrophobic B block may be 2,000-6,000, 2,000-4,000, 2,000-3,000, 2,000-1,700, 2,000-1,300, etc., but is not limited thereto.

[0113] Additionally, in one specific example, the hydrophobic B block may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to a hydroxyl group at the end of the hydrophobic B block to increase the hydrophobicity of the hydrophobic B block and thereby improve the stability of the nanoparticle.

[0114] In one specific example, in the amphiphilic block copolymer, the composition ratio of the hydrophilic block (A) and the hydrophobic block (B) may be in the range of 20 to 70 wt% of the hydrophilic block (A), and more specifically, in the range of 30 to 60 wt%, based on the total weight of the copolymer. If the ratio of the hydrophilic block (A) is less than 20 wt% based on the total weight of the copolymer, the polymer has low solubility in water, making it difficult to form nanoparticles. Therefore, it is preferable that the ratio of the hydrophilic block (A) is 20 wt% or more in order for the copolymer to have sufficient solubility in water to form nanoparticles. On the other hand, if the ratio of the hydrophilic block (A) exceeds 70 wt% based on the total weight of the copolymer, the hydrophilicity is too high, lowering the stability of the polymer nanoparticles, making it difficult to use them as a solubilizing composition of an active ingredient / lipid complex. Therefore, it is preferable that the ratio of the hydrophilic block (A) is 70 wt% or less in consideration of nanoparticle stability.

[0115] In one specific example, the amphiphilic block copolymer may be a biocompatible biodegradable polymer comprising the hydrophilic block described above; and a hydrophobic block having repeating units of a structure represented by the following chemical formula 2:

[0116] [Chemical Formula 2]

[0117]

[0118] In the above chemical formula 2,

[0119] R represents a branched alkylene group having 3 or more carbon atoms.

[0120] In one specific example, the number of repeating units (degree of polymerization) of the hydrophobic block having repeating units 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, but is not limited to, 25 or less, 24 or less, 23 or less, 22 or less, 21 or less, or 20 or less.

[0121] In one specific example, the number of carbon atoms of R in the above chemical 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.

[0122] In one specific example, R in the above chemical formula 2 may represent a branched alkylene group having 3 to 20 carbon atoms, more specifically, may represent a branched alkylene group having 3 to 17 carbon atoms, even more specifically, may represent a branched alkylene group having 3 to 15 carbon atoms, and even more specifically, may represent a branched alkylene group having 3 to 13 carbon atoms, but is not limited thereto.

[0123] In one specific example, the hydrophobic block having a repeating unit of the structure represented by the above chemical formula 2 may be a biocompatible biodegradable polymer having a repeating unit of a structure selected from, but not limited to:

[0124] , , , , , , , , , , , , , , ,

[0125] In one specific example, the repeating unit of the structure represented by the above chemical formula 2 can be obtained by ring-opening polymerization of a lactone compound.

[0126] In one specific example, the number average molecular weight (g / mol) of the hydrophobic block having a repeating unit of the structure represented by the above 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.

[0127] For example, the number average molecular weight combinations of the hydrophilic block and the hydrophobic block having repeating units of the structure represented by the chemical formula 2 may be, but are not limited to, 2,000-6,000, 2,000-4,000, 2,000-3,000, 2,000-1,700, 2000-1000, 2000-800, 2000-500, etc.

[0128] In addition, in one specific example, the hydrophobic block having a repeating unit of the structure represented by the above chemical formula 2 may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to a hydroxyl group at the end of the hydrophobic block to increase the hydrophobicity of the hydrophobic block and thereby improve the stability of the nanoparticle.

[0129] In one specific example, in the amphiphilic block copolymer including a hydrophobic block having a repeating unit of the structure represented by the above chemical formula 2, the composition ratio of the hydrophilic block and the hydrophobic block may be in the range of 25 to 95 wt% of the hydrophilic block, specifically 40 to 90 wt%, and more specifically 50 to 80 wt%, based on the total weight of the copolymer. If the ratio of the hydrophilic block is less than 25 wt% based on the total weight of the copolymer, the polymer has low solubility in water, making it difficult to form nanoparticles. Therefore, it is preferable that the ratio of the hydrophilic block is 25 wt% or more in order for the copolymer to have sufficient solubility in water to form nanoparticles. On the other hand, if the ratio of the hydrophilic block (A) exceeds 95 wt% based on the total weight of the copolymer, the hydrophilicity becomes too high, lowering the stability of the polymer nanoparticles, making it difficult to use it as a solubilizing composition of a complex containing an effective ingredient. Therefore, considering the stability of the nanoparticles, it is preferable that the ratio of the hydrophilic block be 95 wt% or less.

[0130] In one specific 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 wt% or more, 7 wt% or more, 10 wt% or more, 12 wt% or more, 15 wt% or more, 17 wt% or more, or 20 wt% or more, based on the dry weight of the entire composition, and may also be 90 wt% or less, 80 wt% or less, 70 wt% or less, 60 wt% or less, 50 wt% or less, or 40 wt% or less. If the content of the polymer is too low, the size of the nanoparticles may become too large, which may lower the stability of the nanoparticles and increase the loss rate during filter sterilization. On the other hand, if the content is too high, the content of the active ingredient that can be incorporated may become too low.

[0131] 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, amphiphilic block copolymer or a polymer that is a combination thereof, and the cationic lipid of the chemical formula 1, thereby improving stability in blood or body fluid.

[0132] In one embodiment, the particle size of the nanoparticles can be defined by the Z-average value and can be, for example, 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 can 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 nanoparticles defined by the Z-average value can be, for example, 10 to 800 nm, 20 to 600 nm, 30 to 500 nm, 40 to 400 nm, or 50 to 300 nm.

[0133] In one specific example, the relative amount of the lipid-polymer, amphiphilic block copolymer, or a combination thereof, relative to the lipid of the formula 1 may be 0.01 part by weight or more, 0.02 part by weight or more, 0.03 part by weight or more, 0.04 part by weight or more, or 0.05 part by weight or more, 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, based on 1 part by weight of the lipid of the formula 1, but is not limited thereto.

[0134] More specifically, the relative amount of the lipid-polymer polymer used compared to the lipid of the above chemical formula 1 may be 0.01 part by weight or more, 0.02 part by weight or more, 0.03 part by weight or more, 0.04 part by weight or more, or 0.05 part by weight or more, based on 1 part by weight of the lipid of the above chemical 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 part by weight or less, or 0.1 part by weight or less, but is not limited thereto.

[0135] More specifically, the relative amount of the amphiphilic block copolymer used compared to the lipid of the above chemical formula 1 may be 0.1 parts by weight or more, 0.5 parts by weight or more, 1 parts 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 lipid of the above chemical 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.

[0136] Any additional ingredients

[0137] In one specific embodiment, the drug delivery composition of the present invention may further comprise a fusible lipid to increase the delivery efficiency of the active ingredient into the body.

[0138] In one specific embodiment, the fusion lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.

[0139] Specifically, the phospholipid may be at least one selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid may be in a form bound to one or two C10-24 fatty acids. The cholesterol and tocopherol include analogs, derivatives, and metabolites of cholesterol and tocopherol, respectively.

[0140] Specifically, the above-mentioned fusion lipids are 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 a combination of two or more 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.

[0141] More specifically, the fusion lipids include 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), It may be one or a combination of two or more selected from the group consisting of 1,2-dioleoyl-3-dimethylammonium propane (DODAP), 1,2-dioleoyl-sn-glycero-3-phosphate (18PA), cholesterol, and tocopherol.

[0142] In one embodiment of the present invention, the fusion lipid may be distearoyl phosphatidylcholine, cholesterol, or a combination thereof.

[0143] In one specific embodiment, the content of the fusion lipid may be at least 1 wt%, at least 2 wt%, at least 3 wt%, at least 4 wt%, or at least 5 wt%, and may also be at most 40 wt%, at most 35 wt%, at most 30 wt%, at most 25 wt%, or at most 20 wt%, based on the dry weight of the entire composition.

[0144] In one specific example, the relative amount of the fusion lipid used compared to the cationic lipid of the chemical 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 the chemical 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.

[0145] In one specific example, when a phospholipid is used as the fusion 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 Chemical 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.

[0146] In one specific example, when cholesterol is used as the fusion 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 Chemical 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.

[0147] In addition, in one specific embodiment, the drug delivery composition of the present invention may additionally include one or more additive components (hereinafter, “optional additive components”) that are typically included in drug delivery compositions.

[0148] In one specific embodiment, the optional additive component may be, but is not limited to, one or more selected from, for example, a pH modifier (e.g., an acidifying agent, an alkalizing agent, a buffering agent), a tonicity modifier, a bulking agent (e.g., a sugar, a polyol, an amino acid, a polymer, a protein, etc.), a wetting agent, a solubilizing agent, a surfactant, an antioxidant, an antimicrobial agent, a chelating agent, a complexing agent, etc.

[0149] In one specific example, the pH adjusting agent may be one or more selected from acetate, citrate, tartrate, histidine, glutamate, phosphate, Tris, glycine, bicarbonate, succinate, sulfate, nitrate, and the like, but is not limited thereto.

[0150] In one specific example, the tonicity regulator may be one or more selected from, but is not limited to, mannitol, sorbitol, lactose, dextrose, trehalose, sodium chloride, potassium chloride, glycerol, glycerin, propylene glycol, and the like.

[0151] In one embodiment, the bulking agent may be one or more selected from, but is not limited to, sugars and polyols including sucrose, trehalose, glucose, lactose sorbitol, mannitol, glycerol, and the like; amino acids including arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine, methionine, alanine, and the like; 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), and the like; or combinations thereof.

[0152] In one embodiment, the humectant and / or solubilizer is selected from the group consisting of lecithin, PEG 300, PEG 600, PEG 1000, polyoxyethylene lauryl ethers (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, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, sorbitan monolaurate (Span 20), sorbitan monooleate (Span 80), sorbitan trioleate (Span 85), cocamide monoethanolamine (cocamide MEA), cocamide diethanolamine (cocamide DEA), dodecyldimethylamine oxide, poloxamer, polyvinyl pyrrolidone 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., but are not limited thereto.

[0153] In one specific example, the antioxidant may be one or more selected from, but is not limited to, tocopherol (vitamin E), alpha tocopherol, alpha tocopherol hydrogen succinate, ascorbic acid, acorbyl 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), and the like.

[0154] In one specific example, the antimicrobial agent may be one or more selected from benzalkonium chloride, benzethonium chloride, benzyl alcohol, bronopol, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, cresol, macresol, ethyl alcohol, glycerin, hexetidine, imidurea, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric nitrate, propylene glycol, thimerosal, and the like, but is not limited thereto.

[0155] In one specific example, 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, and the like, but is not limited thereto.

[0156] When any of the above additive components is 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, and may also be 10 wt% or less, 5 wt% or less, or 1 wt% or less, based on the dry weight of the entire composition, but is not limited thereto.

[0157] Composition and method for producing the same

[0158] The drug delivery composition according to the present invention can be administered via routes of administration such as blood vessels, muscles, mucous membranes, subcutaneous, intradermal, oral, bone, transdermal, or local tissues, and can be formulated into various oral or parenteral administration preparations suitable for such routes of administration. Examples of the oral administration preparations include tablets, capsules, powder preparations, and liquid preparations, and examples of the parenteral administration preparations include various preparations such as eye drops and injections. In one specific example, the composition may be an injectable preparation. For example, when the composition according to the present invention is lyophilized, it can be reconstituted with distilled water for injection, 0.9% saline solution, and 5% dextrose aqueous solution, and thus can be prepared in the form of an injectable preparation.

[0159] The present invention also provides a method for preparing a drug delivery composition, comprising: (a) preparing a solution in which a cationic lipid represented by the above chemical formula 1; and a lipid-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 a nucleic acid, a polypeptide, a virus, or a combination thereof to the solution prepared in step (a).

[0160] In one specific example, the water-miscible organic solvent of step (a) may be ethanol.

[0161] In one specific embodiment, the step (b) may be performed in a solution under acidic conditions.

[0162] In one specific example, the step (b) may include: (b-1) preparing a buffer solution containing the active ingredient; and (b-2) adding and mixing the buffer solution of the active ingredient prepared in step (b-1) to the solution prepared in step (a).

[0163] In one specific example, 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 by volume, and more specifically, 1:2 to 1:4.

[0164] In another specific example, the step (b) may include: (b-1) adding the active ingredient to the solution prepared in step (a); and (b-2) adding and mixing a buffer solution to the resultant product of step (b-1).

[0165] In one specific example, the method for preparing the drug delivery composition may further include a step of adding a pH adjusting buffer, water for injection, or a combination thereof to the resultant product of step (b).

[0166] In another specific example, the method for preparing the drug delivery composition may further include a step of removing the solvent from the resultant product of step (b) and then adding a freeze-drying aid to freeze-dry the product.

[0167] The freeze-drying aid is added to enable the freeze-dried composition to maintain a cake shape or to help the composition melt evenly within a short period of time during the reconstitution process after freeze-drying, and specifically, may be at least one selected from the group consisting of sugars, amino acids, polymers, and proteins. For example, it may be at least one selected from the group consisting of lactose, mannitol, sorbitol, and sucrose. The content of the freeze-drying aid may be 1 to 90 wt%, and more specifically, 10 to 60 wt%, based on the total dry weight of the freeze-dried composition.

[0168] Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for explaining the present invention and the scope of the present invention is not limited in any way by these examples.

[0169] [Example]

[0170] Manufacturing Example 1

[0171] A compound of the following chemical formula A was prepared according to the synthetic outline shown in Figure 1.

[0172] [Chemical Formula A]

[0173]

[0174] (1) Synthesis of 6-bromohexyl 2-hexyldecanoate

[0175] A 2000 mL 3-neck round-bottom flask (RBF) was charged with 2-hexyldecanoic acid (100 g, 390 mmol, 1.00 eq), 6-bromohexan-1-ol (91.8 g, 507 mmol, 1.30 eq), and toluene (1000 mL), and H2SO4 (7.65 g, 78.0 mmol, 0.20 eq) was added to the mixture. The mixture was degassed and purged with N2 gas, and stirred at 120°C for 16 h. After cooling to room temperature, the mixture was concentrated under reduced pressure to remove the solvent. The residue was purified by silica column chromatography (petroleum ether: ethyl acetate (EtOAc) = 10:1 → 1:100) to obtain 6-bromohexyl 2-hexyldecanoate (135 g, 322 mmol, 82.5% yield) as a pale yellow oil.

[0176] (2) Synthesis of the compound of chemical formula A

[0177] 6-Bromohexyl 2-hexyldecanoate (15.0 g, 35.8 mmol, 3.50 eq) and N were added to a 500 mL 3-neck RBF. 1 ,N 1 -Dimethylpropane-1,3-diamine(N 1 ,N 1-dimethylpropane-1,3-diamine) and ethanol (EtOH) (150 mL) were added, and Na2CO3 (3.25 g, 30.7 mmol, 3.00 eq) was added to the mixture. After purging with N2 gas three times, the mixture was stirred at 90°C for 48 hours. After cooling to room temperature, the solvent was removed under reduced pressure. The residue was extracted with a saturated aqueous solution of Na2CO3 and dichloromethane (DCM), the DCM layer was collected, and the solvent was removed under reduced pressure to obtain residue A. After dissolving residue A in EtOH (60 mL), H2O (60 mL) and excess Na2CO3 were added, and the mixture was stirred at room temperature for 16 hours. After filtering the mixture, the filtrate was removed under reduced pressure to remove EtOH. The residual solution was extracted with DCM, the DCM layer was collected, and the solvent was removed under reduced pressure to obtain residue B. Residue B was purified by reversed-phase HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 55%-85% for 20 minutes) to obtain the compound of formula A (12.0 g, 10.7 mmol, 35.5% yield) as a yellow solid.

[0178] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.07-4.04 (m, 4H), 3.45 (td, 2H), 3.28 (s, 6H), 3.08 (br, 4H), 2.72 (br, H), 2.33-2.27 (m, 3H), 1.84 (br, 6H), 1.68-1.45 (m, 20H), 1.45-1.42 (m, 18H), 1.32-1.25 (m, 62H), 0.89 (t, 18H)

[0179] Manufacturing Example 2

[0180] A compound of the following chemical formula B was prepared according to the synthetic outline shown in Fig. 2.

[0181] [Chemical Formula B]

[0182]

[0183] (1) Synthesis of heptadecan-9-yl 8-bromooctanoate

[0184] To a 1000 mL 3-neck RBF were added heptadecan-9-ol (43.00 g, 168.00 mmol, 1.00 eq), 8-bromooctanoic acid (44.90 g, 201.00 mmol, 1.20 eq), 4-dimethylaminopyridine (DMAP) (20.50 g, 168.00 mmol, 1.00 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (48.20 g, 252.00 mmol, 1.50 eq), and triethylamine (TEA) (17.00 g, 168.00 mmol, 23.30 mL, 1.00 eq), followed by the addition of DCM (430 mL). After replacing the mixture with N2 three times, it was stirred under N2 at 25℃ for 16 hours. The reaction mixture was quenched by adding 500 mL of water at 20℃, and extracted with 900 mL of DCM (300 mL*3). The combined organic layer was concentrated under reduced pressure to obtain a residue, which was then purified by silica column chromatography (petroleum ether: EtOAc = 100:1→1:1) to obtain heptadecan-9-yl 8-bromooctanoate (36.00 g, 78.00 mmol, 46.5% yield) as a pale yellow oil.

[0185] (2) Synthesis of the compound of chemical formula B

[0186] In a 100 mL 3-neck RBF was added heptadecan-9-yl 8-bromooctanoate (3.16 g, 6.85 mmol, 3.50 eq), N 1 ,N 1-Dimethylpropane-1,3-diamine (0.20 g, 1.96 mmol, 1.00 eq), N,N-diisopropylethylamine (DIEA) (1.26 g, 9.79 mmol, 1.70 mL, 5.00 eq) were added, followed by EtOH (5 mL). The mixture was replaced with N2 three times and stirred at 95°C under N2 conditions for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to remove the solvent. The mixture was extracted with saturated Na2CO3 and 300 mL (100 mL*3) of DCM, and the combined organic layer was concentrated under reduced pressure to obtain residue A. After the residue A was dissolved in EtOH (50 mL), 60 mL of water and excess Na2CO3 were added to the mixture, and the mixture was stirred at 25°C for 16 h. The mixture was filtered and concentrated under reduced pressure to remove EtOH, and the aqueous layer was extracted with 300 mL of DCM (100 mL*3). The combined organic layer was concentrated under reduced pressure to obtain residue B. The residue B was purified by prep-HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 55%-85% for 20 min) and concentrated under reduced pressure to remove the solvent to obtain the compound of formula B (1.52 g, 1.19 mmol, 60.6% yield) in the form of a pale yellow syrup.

[0187] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.86 (t,J=6.2 Hz, 3H), 4.15 (br d,J=1.3 Hz, 2H), 3.44 - 3.32 (m, 4H), 3.28 (s, 6H), 3.14 - 2.94 (m, 4H), 2.72 - 2.58 (m, 2H), 2.28 (dt,J=2.1, 7.4 Hz, 6H), 1.96 - 1.74 (m, 12H), 1.65 - 1.58 (m, 6H), 1.51 (br d,J=5.8 Hz, 12H), 1.41 - 1.25 (m, 84H), 0.94 - 0.83 (m, 18H)

[0188] Manufacturing Example 3

[0189] A compound of the following chemical formula C was prepared according to the synthetic outline shown in Fig. 3.

[0190] [Chemical Formula C]

[0191]

[0192] (1) Synthesis of 1-cyclopropyloctyl 5-bromopentanoate

[0193] 1-Cyclopropyloctan-1-ol (15.00 g, 88.90 mmol, 1.00 eq), DMAP (2.15 g, 17.60 mmol, 0.20 eq), and TEA (17.80 g, 176.00 mmol, 24.50 mL, 2.00 eq) were added to a 500 mL 3-neck RBF, followed by DCM (150 mL). The mixture was purged three times with N2, and 5-bromopentanoyl chloride (26.40 g, 132.00 mmol, 17.70 mL, 1.50 eq) was added to the solution at 0 °C. The solution was stirred at 25 °C for 16 h, and then the reaction mixture was quenched by adding 100 mL of H2O at 25 °C. Extracted with 1500 mL of DCM (500 mL*3), dried over Na2SO4, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1) to obtain 1-cyclopropyloctyl 5-bromopentanoate (20.0 g, 60.0 mmol, 68.1% yield) as a colorless oil.

[0194] (2) Synthesis of 1-cyclopropyloctyl 5-iodopentanoate

[0195] 1-Cyclopropyloctyl 5-bromopentanoate (20.00 g, 60.00 mmol, 1.00 eq) and KI (19.90 g, 120.00 mmol, 2.00 eq) were added to a 500 mL 3-neck RBF, followed by acetonitrile (ACN) (200 mL). The mixture was purged with N2 three times and stirred at 90°C under N2 conditions for 16 h. The mixture was filtered and concentrated under reduced pressure to obtain 1-cyclopropyloctyl 5-iodopentanoate (18.00 g, 47.30 mmol, 78.9% yield) as a yellow oil.

[0196] (3) Synthesis of the compound of chemical formula C

[0197] N in a 50 mL 3-neck RBF 1 ,N 1 -Dimethylpropane-1,3-diamine (0.20 g, 1.96 mmol, 0.33 eq), 1-cyclopropyloctyl 5-iodopentanoate (2.23 g, 5.87 mmol, 1.00 eq), and K2CO3 (812.00 mg, 5.87 mmol, 3.00 eq) were added, followed by EtOH (2 mL) and ACN (2 mL). The mixture was replaced with N2 three times and stirred at 80°C under N2 conditions for 12 h. The mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 55%-85% for 20 min) to obtain the compound of formula C (0.19 g, 221.00 μmol, 11.3% yield) in the form of a yellow syrup.

[0198] 1H NMR (400 MHz, CHLOROFORM-d): δ0.26 (dt,J=9.57, 4.85 Hz, 3 H), 0.34 (dq,J=9.54, 4.87 Hz, 3 H), 0.40 - 0.49 (m, 3 H), 0.51 - 0.60 (m, 3 H), 0.83 - 0.92 (m, 9 H), 0.92 - 1.01 (m, 3 H), 1.27 (br s, 30 H), 1.39 - 1.47 (m, 4 H), 1.55 - 1.69 (m, 10 H), 1.70 - 1.78 (m, 2 H), 1.78 - 1.92 (m, 4 H), 2.32 (t,J=7.00 Hz, 4 H), 2.36 - 2.46 (m, 6 H), 2.50 (br t,J=5.88 Hz, 2 H), 3.38 (s, 6 H), 3.48 - 3.60 (m, 2 H), 3.60 - 3.74 (m, 2 H), 4.14 - 4.33 (m, 3H)

[0199] Manufacturing Example 4

[0200] Compounds of the following chemical formulas D and E were prepared according to the synthetic outline shown in Fig. 4.

[0201] [Chemical Formula D]

[0202]

[0203] [Chemical Formula E]

[0204]

[0205] (1) Synthesis of undecan-3-yl 8-bromooctanoate

[0206] Undecan-3-ol (25.00 g, 145.00 mmol, 1.00 eq) dissolved in toluene (250 mL) was added to a 500 mL 3-neck RBF, followed by 8-bromooctanoic acid (48.60 g, 218.00 mmol, 1.50 eq). Then, H2SO4 (2.13 g, 21.8 mmol, 0.15 eq) was added. The mixture was purged with N2 three times and stirred at 120°C for 16 h. The mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain undecane-3-yl 8-bromooctanoate (40.00 g, 106.00 mmol, 73.1% yield) as a yellow oil.

[0207] (2) Synthesis of heptadecan-9-yl 8-bromooctanoate

[0208] Heptadecan-9-ol (235.00 g, 916.00 mmol, 1.00 eq) dissolved in toluene (2.35 L) was placed in a 5000 mL 3-neck RBF, and 8-bromooctanoic acid (245.00 g, 1.10 mol, 1.20 eq) was added. Then, H2SO4 (18.00 g, 183.00 mmol, 0.20 eq) was added. The mixture was purged with N2 three times and stirred at 120°C for 16 h. The mixture was concentrated under reduced pressure to obtain a residue. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain heptadecan-9-yl 8-bromooctanoate (470.00 g, 1.02 mol, 55.6% yield) as a yellow oil.

[0209] (3) Synthesis of 8-(heptadecan-9-yloxy)-N-(3-((8-(heptadecan-9-yloxy)-8-oxooctyl)amino)propyl)-N,N-dimethyl-8-oxooctan-1-aminium chloride

[0210] Heptadecan-9-yl 8-bromooctanoate (10.00 g, 21.70 mmol, 2.20 eq) dissolved in EtOH (100 mL) in a 250 mL 3-neck RBF, N 1 ,N 1 -Dimethylpropane-1,3-diamine (1.01 g, 9.85 mmol, 1.00 eq), Na2CO3 (2.09 g, 19.70 mmol, 2.00 eq), and DIEA (3.82 g, 29.50 mmol, 3.00 eq) were added. The mixture was stirred at 90°C for 16 hours, and then the reaction mixture was concentrated under reduced pressure. The residue was purified by silica column chromatography (DCM:MeOH=10:1→1:100), and 6 g of the purified product was further purified by reverse phase HPLC to obtain 8-(heptadecan-9-yloxy)-N-(3-((8-(heptadecan-9-yloxy)-8-oxooctyl)amino)propyl)-N,N-dimethyl-8-oxooctane-1-aminum chloride (2.00 g, 2.31 mmol, 23.5% yield) as a yellow solid.

[0211] (4) Synthesis of compounds of chemical formula D and E

[0212] To a 50 mL 3-neck RBF were added undecan-3-yl 8-bromooctanoate (314.00 mg, 833.00 μmol, 1.20 eq), 8-(heptadecan-9-yloxy)-N-(3-((8-(heptadecan-9-yloxy)-8-oxooctyl)amino)propyl)-N,N-dimethyl-8-oxooctan-1-aminium chloride (0.60 g, 694.00 μmol, 1.00 eq), and K2CO3 (115.00 mg, 833.00 μmol, 1.20 eq) dissolved in ACN (6 mL). The mixture was stirred at 90°C for 24 h, and then the reaction mixture was concentrated under reduced pressure. The product was purified by reverse phase HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 45%-75% for 25 minutes) to obtain a compound of formula D (0.20 g, 167.00 μmol, 24.1% yield) as a yellow oil and a compound of formula E (0.30 g, 258.00 μmol, 37.2% yield) as a white solid.

[0213] [Compound of chemical formula D]

[0214] 1 H NMR (400 MHz, CHLOROFORM-d):δ11.60 (br s, 1H), 4.92 - 4.75 (m, 3H), 4.16 (br s, 2H), 3.48 - 3.33 (m, 4H), 3.29 (s, 6H), 3.14 - 2.96 (m, 4H), 2.66 (br s, 2H), 2.28 (tt,J=2.3, 7.3 Hz, 6H), 2.21 (br s, 2H), 1.82 (br s, 6H), 1.65 - 1.58 (m, 6H), 1.57 - 1.46 (m, 12H), 1.42 - 1.34 (m, 16H), 1.26 (br s, 54H), 0.88 (t,J=6.6 Hz, 18H)

[0215] [Compound of chemical formula E]

[0216] 1H NMR (400 MHz, CHLOROFORM-d):δ11.60 (br s, 1H), 4.90 - 4.76 (m, 3H), 4.14 (br d,J=7.1 Hz, 2H), 3.48 - 3.32 (m, 4H), 3.29 (s, 6H), 3.14 - 2.97 (m, 4H), 2.65 (br s, 2H), 2.32 - 2.25 (m, 8H), 1.82 (br s, 6H), 1.65 - 1.58 (m, 6H), 1.57 - 1.46 (m, 12H), 1.43 - 1.34 (m, 16H), 1.32 - 1.24 (m, 59H), 0.93 - 0.82 (m, 18H)

[0217] Manufacturing Example 5

[0218] A compound of the following chemical formula F was prepared according to the synthetic outline shown in Fig. 5.

[0219] [Chemical formula F]

[0220]

[0221] (1) Synthesis of di(heptadecan-9-yl) 8,8'-(propane-1,3-diylbis(azanediyl))dioctanoate

[0222] To a 100 mL 3-neck RBF was added heptadecan-9-yl 8-bromooctanoate (2.00 g, 4.33 mmol, 2.00 eq), propane-1,3-diamine (160.00 mg, 2.17 mmol, 1.00 eq), and K2CO3 (598.00 mg, 4.33 mmol, 2.00 eq), followed by ACN (20 mL). The mixture was purged three times with N2, and the solution was stirred at 45°C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by silica column chromatography (DCM:MeOH=10:1) to obtain di(heptadecan-9-yl) 8,8'-(propane-1,3-diylbis(azanediyl))dioctanoate (0.80 g, 0.96 mmol, 11.0% yield) as a white solid.

[0223] (2) Synthesis of di(heptadecan-9-yl) 8,8'-(9,33-diethyl-11,31-dioxo-10,32-dioxa-19,23-diazahentetracontane-19,23-diyl)dioctanoate

[0224] Di(heptadecan-9-yl) 8,8'-(propane-1,3-diylbis(azanediyl))dioctanoate (0.80 g, 0.96 mmol, 1.00 eq), undecan-3-yl 8-bromooctanoate (722.00 mg, 1.92 mmol, 1.00 eq), and K2CO3 (264.00 mg, 1.92 mmol, 2.00 eq) were added to a 50 mL 3-neck RBF, followed by ACN (8 mL). The solution was stirred at 55°C for 16 h. The reaction mixture was filtered and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (water(HCl)-ACN) and concentrated under reduced pressure to remove ACN. NaHCO3 aqueous solution (10 mL) was added to the aqueous solution from which ACN had been removed, and neutralized overnight. Then, extraction was performed with DCM (5 mL * 2), dried over Na2SO4, filtered, and the mixture was vacuum-treated to obtain di(heptadecan-9-yl) 8,8'-(9,33-diethyl-11,31-dioxo-10,32-dioxa-19,23-diazahentetracontane-19,23-diyl)dioctanoate (0.40 g, 0.28 mmol, 29.2% yield) as a yellow oil.

[0225] (3) Synthesis of the compound of chemical formula F

[0226] To a 50 mL 3-neck RBF was added di(heptadecan-9-yl) 8,8'-(9,33-diethyl-11,31-dioxo-10,32-dioxa-19,23-diazahentetracontane-19,23-diyl)dioctanoate (0.40 g, 0.28 mmol, 1.00 eq) dissolved in THF (4 mL), K2CO3 (42.50 mg, 0.31 mmol, 1.10 eq), followed by CH3I (39.70 mg, 0.28 mmol, 1.00 eq). The solution was stirred at 25°C for 16 h. The reaction mixture was quenched with 2 M HCl (10 mL), diluted with distilled water (DW) (5 mL), and extracted with EtOAc (5 mL*2). The combined organic layer was concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 45%-75% for 25 min) and concentrated under reduced pressure to remove ACN. NaHCO3 aqueous solution (10 mL) was added to the aqueous solution from which ACN had been removed, and the mixture was neutralized overnight. Afterwards, it was extracted with DCM (5 mL*2), dried over Na2SO4, filtered, and purified under reduced pressure to obtain the compound of formula F (0.40 g, 0.28 mmol, 29.2% yield) as a yellow oil.

[0227] 1 HNMR: (400 MHz, CHLOROFORM-d)δ0.74 - 1.00 (m, 24 H) 1.18 - 1.44 (m, 98 H) 1.47 - 1.56 (m, 14 H) 1.57 (br s, 8 H) 1.64 - 1.88 (m, 10 H) 2.18 - 2.38 (m, 9 H) 2.65 (ddd,J=10.98, 5.16, 1.88 Hz, 1 H) 2.90 - 3.15 (m, 3 H) 3.20 - 3.48 (m, 8 H) 3.92 - 4.12 (m, 2 H) 4.84 (dt,J=19.35, 6.14 Hz, 4 H)

[0228] Manufacturing Example 6

[0229] A compound of the following chemical formula G was prepared according to the synthetic outline shown in Fig. 6.

[0230] [Chemical formula G]

[0231]

[0232] (1) Synthesis of 2-octyldecanoic acid

[0233] N2 in 1000 mL 3-neck RBF, 0°C Nonanoic acid (60.00 g, 348.00 mmol, 1.00 eq) and THF (100 mL) were added under conditions. To the mixture, lithium diisopropylamide (LDA) (2 M, 383.00 mL, 2.20 eq) was added N2, 0℃ and stirred at 0°C for 30 minutes. Afterwards, 1-iodooctane (92.00 g, 383.00 mmol, 1.00 eq) was added at room temperature and stirred at 45°C for 16 hours. The reaction mixture was quenched by adding 1N HCl (1 L), extracted with EtOAc (2 L), dried over Na2SO4, filtered, and concentrated to obtain a residue. After replacing the mixture three times with N2, the solution was stirred at 45°C for 16 hours. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 100:1 → 10:1) to obtain 2-octyldecanoic acid (67.00 g, 236.00 mmol, 67.6% yield) as a yellow oil.

[0234] (2) Synthesis of 7-bromoheptyl 2-octyldecanoate

[0235] 7-Bromoheptan-1-ol (5.14 g, 26.40 mmol, 1.50 eq), octyldecanoic acid (5.00 g, 17.60 mmol, 1.00 eq), and H2SO4 (259 mg, 2.64 mmol, 0.15 eq) dissolved in toluene (50 mL) were added to a 250 mL 3-neck RBF, and then the mixture was replaced three times with N2. The mixture was stirred at 120 °C for 16 h and concentrated under reduced pressure to obtain a residue. The residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain 7-bromoheptyl 2-octyldecanoate (5.50 g, 11.90 mmol, 67.8% yield) as a yellow oil.

[0236] (3) Synthesis of 7-iodoheptyl 2-octyldecanoate

[0237] 7-Bromoheptyl 2-octyldecanoate (5.50 g, 11.90 mmol, 1.00 eq) was placed in a 250 mL 3-neck RBF, and ACN (55 mL) was added. KI (3.96 g, 23.80 mmol, 2.00 eq) was then added, and the mixture was purged three times with N2. The mixture was stirred at 90 °C for 24 h, filtered, and concentrated under reduced pressure to obtain 7-iodoheptyl 2-octyldecanoate (5.00 g, 9.83 mmol, 82.5% yield) as a yellow oil.

[0238] (4) Synthesis of the compound of chemical formula G

[0239] To a 250 mL 3-neck RBF was added 7-iodoheptyl 2-octyldecanoate (5.00 g, 9.83 mmol, 4.00 eq), 2,2'-((3-aminopropyl)azanediyl)bis(ethanol-1-ol) (399.00 mg, 2.46 mmol, 1.00 eq), and K2CO3 (42.50 mg, 0.31 mmol, 1.10 eq) dissolved in ACN (50 mL), followed by purging three times with N2. The solution was stirred at 80°C for 16 h, filtered, and concentrated under reduced pressure to obtain a residue. The residue was purified by prep-HPLC (column: C1 250×80 mm, 10 μm; mobile phase: [HCl aqueous solution-ACN]; gradient: 60%-90% for 25 min) to obtain the compound of formula G (0.25 g, 0.19 mmol, 99.2% yield) as a gray oil.

[0240] 1 H NMR (400 MHz, CHLOROFORM-d):δ5.04 - 4.62 (m, 2H), 4.06 (t,J=6.8 Hz, 6H), 3.95 - 3.86 (m, 2H), 3.70 - 3.61 (m, 4H), 3.35 - 3.21 (m, 6H), 2.70 (br s, 2H), 2.61 (br s, 4H), 2.31 (tt,J=5.4, 8.8 Hz, 3H), 1.85 - 1.69 (m, 10H), 1.66 - 1.53 (m, 12H), 1.46 - 1.36 (m, 24H), 1.26 (s, 65H), 0.93 - 0.84 (m, 18H)

[0241] Manufacturing Example 7

[0242] A compound of the following chemical formula H was prepared according to the synthetic outline shown in Fig. 7.

[0243] [Chemical formula H]

[0244]

[0245] (1) Synthesis of 6-((3-(dimethylamino)propyl)amino)hexyl 2-hexyldecanoate)

[0246] N in a 100 mL 3-neck round bottom flask (RBF) 1 ,N 1 -Dimethylpropane-1,3-diamine (1.0 g, 9.79 mmol, 1.22 mL, 1 eq), 6-bromohexyl 2-hexyldecanoate (4.11 g, 9.79 mmol, 1 eq), and ethanol (20 mL) were added. N,N-diisopropylethylamine (6.32 g, 48.93 mmol, 8.52 mL, 5.0 eq) was added to the mixture. The mixture was stirred at 90 °C for 3 days. After cooling to room temperature, silica (1 g) was added to the mixture, and the solvent was removed in vacuo. The residue was purified using a silica column with petroleum ether:EtOAc=1:1→2:1 to obtain 6-((3-(dimethylamino)propyl)amino)hexyl 2-hexyldecanoate) (1.08 g, 25.0% yield) as a light yellow oil.

[0247] (2) Synthesis of the compound of chemical formula H

[0248] A 50 mL 3-neck RBF was charged with 6-((3-(dimethylamino)propyl)amino)hexyl 2-hexyldecanoate) (1.08 g, 2.45 mmol, 1 eq), 6-bromohexyl 2-hexyldecanoate (1.03 g, 2.45 mmol, 1 eq), and ethanol (10 mL). N,N-Diisopropylethylamine (633.40 mg, 4.90 mmol, 853.63 μL, 2 eq) was added to the mixture, and the mixture was stirred at 90 °C for 3 days. After cooling to room temperature, silica (1 g) was added to the mixture, and the solvent was removed under vacuum. The residue was purified using a silica column with petroleum ether:EtOAc=3:1→1:1 to obtain a compound of chemical formula H (61.94 mg, 2.3% yield) in the form of a light yellow oil.

[0249] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.07-4.04 (m, 4H), 3.45 (td, 2H), 3.28 (s, 6H), 3.08 (br, 4H), 2.72 (br, H), 2.33-2.27 (m, 3H), 1.84 (br, 6H), 1.68-1.45 (m, 20H), 1.45-1.42 (m, 18H), 1.32-1.25 (m, 62H), 0.89 (t, 18H)

[0250] Example 1: Preparation of a drug delivery composition using the lipid of Preparation Example 2 (compound of chemical formula B) and drug delivery test

[0251] (1) Preparation of solutions by component

[0252] The components shown in Table 1 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 1 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0253]

[0254] (2) Mixing of raw materials

[0255] Each component was taken according to the weight ratio of lipid: DOTMA: cholesterol: DMG-PEG = 100:10.5:23.2:5.9 of Manufacturing Example 2 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-10. Ethanol was added to the ethanol layer so that the sum of the molecules of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0256] The specific process sequence is as follows.

[0257] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0258] 2) The lipids, DOTMA, cholesterol, and DMG-PEG of Manufacturing Example 2 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0259] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0260] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0261] 5) Mixing of tubes (A) and (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. The microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0262] 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.

[0263] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0264] 8) The concentration was measured using the Ribo-green assay.

[0265] (3) Evaluation of physical properties of the formulation

[0266] For the manufactured formulation, particle characteristics were confirmed using a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 2 below.

[0267]

[0268] (4) Administration of the composition

[0269] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 3.

[0270]

[0271] Example 2: Preparation of a drug delivery composition using the lipid of Preparation Example 2 (compound of chemical formula B) and drug delivery test

[0272] (1) Preparation of solutions by component

[0273] The components shown in Table 4 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 4 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0274]

[0275] (2) Mixing of raw materials

[0276] Each component was taken according to the weight ratio of Lipid: 18:0 Lyso PC: Cholesterol: mPEG-PLA (2K-4K) = 100:8.2:24.2:468.5 in Manufacturing Example 2 to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5-10 and mixed. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0277] The specific process sequence is as follows.

[0278] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0279] 2) According to the experimental conditions, the moles of mPEG-PLA (2K-4K), lipid of Manufacturing Example 2, 18:0 Lyso PC, and cholesterol were sequentially added to tube (A) and mixed by vortexing.

[0280] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0281] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0282] 5) Mixing of tube (A) and tube (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. Microfluidics operating conditions were FRR (Flow Rate Ratio) C:R=3:1, TRR (Total Flow Rate) 3-12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0283] 6) The resulting mixture from step 5 was diluted with DPBS and 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.

[0284] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0285] 8) The concentration was measured using the Ribo-green assay.

[0286] (3) Evaluation of physical properties of the formulation

[0287] For the manufactured formulation, particle characteristics were confirmed using a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 5 below.

[0288]

[0289] (4) Administration of the composition

[0290] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 6.

[0291]

[0292] Example 3: Preparation of a drug delivery composition using the lipid of Preparation Example 4 (compound of chemical formula D) and drug delivery test

[0293] (1) Preparation of solutions by component

[0294] The components shown in Table 7 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 7 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0295]

[0296] (2) Mixing of raw materials

[0297] Each component was mixed according to the weight ratio of lipid: 18:0 Lyso PC: cholesterol: DMG-PEG = 100:8.8:24.9:6.3 of Manufacturing Example 4 to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) between 5-10. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0298] The specific process sequence is as follows.

[0299] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0300] 2) The lipids of Manufacturing Example 4, 18:0 Lyso PC, cholesterol, and DMG-PEG in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0301] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0302] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0303] 5) Mixing of tubes (A) and (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. The microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0304] 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.

[0305] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0306] 8) The concentration was measured using the Ribo-green assay.

[0307] (3) Evaluation of physical properties of the formulation

[0308] For the manufactured formulation, particle characteristics were confirmed through a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 8 below.

[0309]

[0310] (4) Administration of the composition

[0311] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 9 below.

[0312]

[0313] Example 4: Preparation of a drug delivery composition using the lipid of Preparation Example 4 (compound of chemical formula D) and drug delivery test

[0314] (1) Preparation of solutions by component

[0315] The components shown in Table 10 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 10 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0316]

[0317] (2) Mixing of raw materials

[0318] Each component was taken according to the weight ratio of lipid: DDAB: cholesterol: mPEG-PLA (2K-4K) = 100:10.5:25.9:501.5 of Manufacturing Example 4 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-10. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0319] The specific process sequence is as follows.

[0320] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0321] 2) According to the experimental conditions, the moles of mPEG-PLA (2K-4K), lipid of Manufacturing Example 4, DDAB, and cholesterol were sequentially added to tube (A) and mixed by vortexing.

[0322] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0323] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0324] 5) Mixing of tube (A) and tube (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. Microfluidics operating conditions were FRR (Flow Rate Ratio) C:R=3:1, TRR (Total Flow Rate) 3-12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0325] 6) The resulting mixture from step 5 was diluted with DPBS and 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.

[0326] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0327] 8) The concentration was measured using the Ribo-green assay.

[0328] (3) Evaluation of physical properties of the formulation

[0329] For the manufactured formulation, particle characteristics were confirmed using a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 11 below.

[0330]

[0331] (4) Administration of the composition

[0332] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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.

[0333]

[0334] Example 5: Preparation of a drug delivery composition using the lipid of Preparation Example 5 (compound of chemical formula F) and drug delivery test

[0335] (1) Preparation of solutions by component

[0336] The components shown in Table 13 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 13 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0337]

[0338] (2) Mixing of raw materials

[0339] Each component was taken according to the weight ratio of lipid: DPPC: cholesterol: DMG-PEG = 100:9.9:20.1:5.1 of Manufacturing Example 5 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-10. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0340] The specific process sequence is as follows.

[0341] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0342] 2) The lipids, DPPC, cholesterol, and DMG-PEG of Manufacturing Example 5 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0343] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0344] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0345] 5) Mixing of tubes (A) and (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. The microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0346] 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.

[0347] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0348] 8) The concentration was measured using the Ribo-green assay.

[0349] (3) Evaluation of physical properties of the formulation

[0350] For the manufactured formulation, particle characteristics were confirmed through a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 14 below.

[0351]

[0352] (4) Administration of the composition

[0353] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 15 below.

[0354]

[0355] Example 6: Preparation of a drug delivery composition using the lipid of Preparation Example 5 (compound of chemical formula F) and drug delivery test

[0356] (1) Preparation of solutions by component

[0357] The components shown in Table 16 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 16 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0358]

[0359] (2) Mixing of raw materials

[0360] Each component was taken according to the weight ratio of lipid: DDAB: cholesterol: mPEG-PLA (2K-4K) = 100:8.5:20.9:405.7 of Manufacturing Example 5 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-10. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0361] The specific process sequence is as follows.

[0362] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0363] 2) According to the experimental conditions, the moles of mPEG-PLA (2K-4K), lipid of Manufacturing Example 5, DDAB, and cholesterol were sequentially added to tube (A) and mixed by vortexing.

[0364] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0365] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0366] 5) Mixing of tube (A) and tube (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. Microfluidics operating conditions were FRR (Flow Rate Ratio) C:R=3:1, TRR (Total Flow Rate) 3-12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0367] 6) The resulting mixture from step 5 was diluted with DPBS and 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.

[0368] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0369] 8) The concentration was measured using the Ribo-green assay.

[0370] (3) Evaluation of physical properties of the formulation

[0371] For the manufactured formulation, the particle characteristics were confirmed through a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 17 below.

[0372]

[0373] (4) Administration of the composition

[0374] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 18 below.

[0375]

[0376] Example 7: Preparation of a drug delivery composition using the lipid (compound of chemical formula H) of Preparation Example 7 and drug delivery test

[0377] (1) Preparation of solutions by component

[0378] The components shown in Table 19 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 19 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0379]

[0380] (2) Mixing of raw materials

[0381] Each component was taken according to the weight ratio of lipid: DSPC: cholesterol: DMG-PEG = 100:14.1:26.6:6.7 of Manufacturing Example 7 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0382] The specific process sequence is as follows.

[0383] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0384] 2) The lipids, DSPC, cholesterol, and DMG-PEG of Manufacturing Example 7 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0385] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0386] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0387] 5) Mixing of tubes (A) and (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. The microfluidics operating conditions were FRR (Flow Rate Ratio) of C:R=3:1 and TRR (Total Flow Rate) of 12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0388] 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.

[0389] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0390] 8) The concentration was measured using the Ribo-green assay.

[0391] (3) Evaluation of physical properties of the formulation

[0392] For the manufactured formulation, particle characteristics were confirmed through a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 20 below.

[0393]

[0394] (4) Administration of the composition

[0395] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 21 below.

[0396]

[0397] Example 8: Preparation of a drug delivery composition using the lipid (compound of chemical formula H) of Preparation Example 7 and drug delivery test

[0398] (1) Preparation of solutions by component

[0399] The components shown in Table 22 below were dissolved in each dilution solvent to prepare the concentrations shown in Table 22 below. When dissolving, the substances were warmed to room temperature, the solvent was added, and after dissolving to an appropriate concentration, the absence of undissolved particles was confirmed with the naked eye before use in the preparation of the composition.

[0400]

[0401] (2) Mixing of raw materials

[0402] Each component was taken according to the weight ratio of lipid: DOPE: cholesterol: mPEG-PLA (2K-4K) = 100:13.3:27.6:536.2 of Manufacturing Example 7 and mixed to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 5-20. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and 20 mM sodium acetate buffer (pH 4.6) was used to dilute the mRNA in the aqueous phase layer. The aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, the total ethanol content was lowered by diluting with DPBS (Dulbecco's phosphate-buffered saline) to 5% or less of % ethanol, and buffer exchange and concentration were performed as follows: The mixed solution 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 dilution and concentration, and buffer exchange was performed.

[0403] The specific process sequence is as follows.

[0404] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0405] 2) According to the experimental conditions, the moles of mPEG-PLA (2K-4K), lipid of Manufacturing Example 7, DOPE, and cholesterol were sequentially added to tube (A) and mixed by vortexing.

[0406] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0407] 4) mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed in tube (B). At this time, the ratio was calculated so that the volume of the aqueous phase was three times the total volume of the ethanol phase, and added.

[0408] 5) Mixing of tube (A) and tube (B) was performed using a microfluidics (Ignite, precision Nanosystem) device or by vortexing. Microfluidics operating conditions were FRR (Flow Rate Ratio) C:R=3:1, TRR (Total Flow Rate) 3-12 mL / min. When vortexing, the solution in tube (B) was added to tube (A) and mixed as quickly and consistently as possible to ensure a uniform formulation.

[0409] 6) The resulting mixture from step 5 was diluted with DPBS and 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.

[0410] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0411] 8) The concentration was measured using the Ribo-green assay.

[0412] (3) Evaluation of physical properties of the formulation

[0413] For the manufactured formulation, particle characteristics were confirmed through a particle size analyzer (dynamic light scattering, DLS), and the results are shown in Table 23 below.

[0414]

[0415] (4) Administration of the composition

[0416] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravascularly to mice, administering 2 μg of 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 presented in Table 24 below.

[0417]

Claims

1. An active ingredient selected from nucleic acids, polypeptides, viruses or a combination thereof; A cationic lipid having a structure represented by the following chemical formula 1; and comprising a lipid-polymer polymer, an amphiphilic block copolymer or a combination thereof; Compositions for drug delivery: [Chemical Formula 1] Here, R1 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R2, R3 and R4 are each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, R8 and R9 are each independently a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group or carbon cyclic group, or each independently -R 10 -(L4) n -R 11 And, R 10 are each independently a substituted or unsubstituted alkylene group, R 11 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, 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, alkynylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl, n is 0 or 1, X - is a pharmaceutically acceptable monovalent anion.

2. In paragraph 1, R1 is substituted or unsubstituted C 1-6 Alkylene group, C 2-6 Alkenylene group or C 2-6 It is an alkynylene group, R2, R3 and R4 are each independently substituted or unsubstituted C 3-12 Alkylene group, C 3-12 Alkenylene group or C 3-12 It is an alkynylene group, R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 3-20 It is a hydrocarbon group, R8 and R9 are each independently substituted or unsubstituted C 1-6 Alkyl group, C 2-6 Alkenyl group, C 2-6 Alkynyl group or C 3-6 A carbon cyclic group, or each independently -R 10 -(L4) n -R 11 And, R 10 are each independently substituted or unsubstituted C 3-12 It is an alkylene group, R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 3-20 It is a hydrocarbon group, 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 2-6 Alkynylene, C 6-20 Arylene, and C 3-20 Selected from the group consisting of heteroarylene, wherein L' is a direct bond, C 1-13 Alkylene, C 2-13 Alkenylene or C 2-13 It can be alkynylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl, C 2-18 Alkenyl and C 2-18 Selected from the group consisting of alkynyl, n is 0 or 1, X - is a monovalent anion of a pharmaceutically acceptable inorganic or organic acid, Composition for drug delivery.

3. In paragraph 1, R1 is substituted or unsubstituted C 3-4 Alkylene group, C 3-4 Alkenylene group or C 3-4 It is an alkynylene group R2, R3 and R4 are each independently substituted or unsubstituted C 6-8 Alkylene group, C 6-8 Alkenylene group or C 6-8 It is an alkynylene group, R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It is a hydrocarbon group, R8 and R9 are each independently substituted or unsubstituted C 1-2 Alkyl group, C 2-3 Alkenylene group or C 2-3 Alkynylene group, or each independently -R 10 -(L4) n -R 11 And, R 10 are each independently substituted or unsubstituted C 6-8 It is an alkylene group, R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It is a hydrocarbon group, 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-, C 2-5 Alkenylene and C 2-5 selected from the group consisting of alkynylene, wherein R' is each independently a hydrogen atom, C 1-6 Alkyl, C 2-6 Alkenyl and C 2-6 Selected from the group consisting of alkynyl, n is 0 or 1, X - is a halide, nitrate anion (NO3 - ), benzoic acid anion (C6H5COO - ), methanesulfonic acid anion, acetate anion (CH3COO - )(=AcO - ), or trihaloacetate anion (CF3COO - )person, Composition for drug delivery.

4. In paragraph 1, R1 is substituted or unsubstituted C 3-4 It is an alkylene group, R2, R3 and R4 are each independently substituted or unsubstituted C 6-8 It is an alkylene group, R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It is a hydrocarbon group, R8 and R9 are each independently substituted or unsubstituted C 1-2 An alkyl group, or each independently -R 10 -(L4) n -R 11 And, R 10 are each independently substituted or unsubstituted C 6-8 It is an alkylene group, R 11 are independently substituted or unsubstituted saturated or unsaturated monovalent C 5-15 It is a hydrocarbon group, 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 alkenylene, wherein R' is each independently a hydrogen atom and C 1-6 Selected from the group consisting of alkyl, n is 0 or 1, X - is Cl - , Br - , or acetate anion (CH3COO - )(=AcO - )person, Composition for drug delivery.

5. A drug delivery composition according to claim 1, wherein the cationic lipid has any one structure selected from the following chemical formulas A to Q:

6. A drug delivery composition according to claim 1, wherein the effective ingredient is mRNA.

7. A drug delivery composition in the first paragraph, wherein the lipid-polymer is a polymer in which at least one saturated and unsaturated hydrocarbon group having 11 to 25 carbon atoms is introduced as a hydrophobic moiety into a hydrophilic block as a hydrophilic moiety.

8. A drug delivery composition in claim 7, wherein the hydrophilic block is at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof.

9. A drug delivery composition in claim 7, 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.

10. A drug delivery composition in the first paragraph, wherein the amphiphilic block copolymer is an AB type double block copolymer composed of a hydrophilic A block and a hydrophobic B block, wherein the hydrophilic A block is at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyacrylamide, and derivatives thereof, and the hydrophobic B block is at least one selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphazine.

11. A drug delivery composition in claim 10, wherein the terminal hydroxyl group of the hydrophobic B block is modified with at least one selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms.

12. A drug delivery composition according to any one of claims 1 to 11, further comprising a fusion lipid.

13. A composition for drug delivery in claim 12, wherein the fusion lipid is one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol. 14.(a) a step of preparing a solution in which a cationic lipid represented by the following chemical formula 1; and a lipid-polymer, an amphiphilic block copolymer or a mixture thereof; are dissolved in a water-miscible organic solvent; and (b) a step of adding and mixing an effective ingredient selected from a nucleic acid, a polypeptide, a virus or a combination thereof to the solution prepared in step (a); Method for preparing a composition for drug delivery: [Chemical Formula 1] Here, R1 is a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R2, R3 and R4 are each independently a substituted or unsubstituted alkylene group, alkenylene group or alkynylene group, R5, R6 and R7 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, R8 and R9 are each independently a substituted or unsubstituted alkyl group, alkenyl group, alkynyl group or carbon cyclic group, or each independently -R 10 -(L4) n -R 11 And, R 10 are each independently a substituted or unsubstituted alkylene group, R 11 are each independently a substituted or unsubstituted saturated or unsaturated monovalent hydrocarbon group, 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, alkynylene, arylene, and heteroarylene, wherein L' is a direct bond, alkylene, alkenylene or alkynylene, and R' is each independently selected from the group consisting of a hydrogen atom, alkyl, alkenyl and alkynyl, n is 0 or 1, X - is a pharmaceutically acceptable monovalent anion.

15. A method for producing a drug delivery composition in claim 14, wherein the water-miscible organic solvent of step (a) is ethanol.

16. In paragraph 14, step (b) (b-1) a step of preparing a buffer solution containing the above effective ingredient; and (b-2) a step of adding and mixing the buffer solution of the effective ingredient prepared in step (b-1) to the solution prepared in step (a); A method for preparing a composition for drug delivery.

17. In paragraph 14, step (b) (b-1) a step of adding an effective ingredient to the solution prepared in step (a); and (b-2) a step of adding a buffer solution to the result of step (b-1) and mixing; including; A method for preparing a composition for drug delivery.

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