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 systems, 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 maintaining 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 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] In the above chemical formula 1,

[0010] M1 and M2 are each independently a divalent linker group,

[0011] R1 and R2 are each independently a substituted or unsubstituted carbon cyclic group or heterocyclic group,

[0012] R3 is a hydrogen atom or a substituted or unsubstituted organic group optionally containing one or more heteroatoms,

[0013] R4 to R7 are each independently a hydrogen atom or a substituted or unsubstituted saturated or unsaturated hydrocarbon group,

[0014] a and b are each independently an integer from 1 to 20.

[0015] 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 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).

[0016] 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.

[0017] Hereinafter, the present invention will be described in more detail.

[0018] Active ingredients

[0019] The active ingredient included in the drug delivery composition of the present invention is selected from nucleic acids, polypeptides, viruses, or a combination thereof.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] In one specific embodiment of the present invention, the active ingredient is messenger RNA (mRNA).

[0025] 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.

[0026] 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.

[0027] Geology

[0028] The lipid included in the nanoparticle composition of the present invention is represented by the following chemical formula 1:

[0029] [Chemical Formula 1]

[0030]

[0031] In the above chemical formula 1,

[0032] M1 and M2 are each independently a divalent linker group,

[0033] R1 and R2 are each independently a substituted or unsubstituted carbon cyclic group or heterocyclic group,

[0034] R3 is a hydrogen atom or a substituted or unsubstituted organic group optionally containing one or more heteroatoms,

[0035] R4 to R7 are each independently a hydrogen atom or a substituted or unsubstituted saturated or unsaturated hydrocarbon group,

[0036] a and b are each independently an integer from 1 to 20.

[0037] The range of lipids included in the drug delivery composition of the present invention includes not only those having the structure of the above chemical formula 1 but also their cationic forms.

[0038] 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 1-8 Alkyl group (more specifically C 3-7 alkyl group) or C 1-8 Halogenated alkyl group (more specifically C 3-7 It means that it is substituted with one or more substituents selected from among halogenated alkyl groups.

[0039] According to one specific example of the present invention, in the above chemical formula 1, M1 and M2 are each independently -C(O)O-, -OC(O)-, -OC(O)-M'-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-, arylene (more specifically, C 6-20 Arylene, more specifically C 6-10 arylene), and heteroarylene (more specifically, C having one or more (e.g., 1 to 3) heteroatoms selected from N, O and S 3-20 Heteroarylene, more specifically C 3-10 heteroarylene), wherein M' is a direct bond, C 1-13 Alkylene (more specifically C 1-6 alkylene) or C 2-13 Alkenylene (more specifically C 2-6 alkenylene), and R' is each independently a hydrogen atom, C 1-18 Alkyl (more specifically C 1-10 Alkyl, more specifically C 1-6 alkyl) and C 2-18 Alkenyl (more specifically C 2-10Alkenyl, more specifically C 2-6 It can be selected from the group consisting of alkenyl).

[0040] According to one specific example of the present invention, in the chemical formula 1, R1 and R2 are each independently substituted or unsubstituted C 3-20 Cycloalkyl (more specifically C 3-15 Cycloalkyl, more specifically C 6-15 cycloalkyl), substituted or unsubstituted C 3-20 Cycloalkenyl (more specifically C 3-15 Cycloalkenyl, more specifically C 6-15 cycloalkenyl), substituted or unsubstituted C 6-20 Aryl (more specifically C 6-14 Aryl), substituted or unsubstituted C 3-20 Heterocycloalkyl (more specifically C 3-15 heterocycloalkyl), substituted or unsubstituted C 3-20 Heterocycloalkenyl (more specifically C 3-15 heterocycloalkenyl), and substituted or unsubstituted C 3-20 Heteroaryl (more specifically C 3-15 may be selected from the group consisting of heterocycloalkyl, heterocycloalkenyl and heteroaryl, wherein each of the heterocycloalkyl, heterocycloalkenyl and heteroaryl may independently have one or more (e.g., 1 to 3) heteroatoms selected from N, O and S.

[0041] According to one specific example of the present invention, in the above chemical formula 1, R3 is a hydrogen atom, a substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 Carbocyclic group, -(CH2) n Q, - (CH2) n may be selected from the group consisting of CHQR, -CHQR and -CQ(R)2, wherein each R is independently a hydrogen atom, C 1-3 Alkyl and C 2-3may be selected from the group consisting of alkenyl; Q is a carbocyclic group, a heterocyclic group, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R 12 , N(R)S(O)2R 12 , -O(CH2) n OR, -N(R)C(=NR 13 )N(R)2, -N(R)C(=CHR 13 )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR 13 )N(R)2, -N(OR)C(=CHR 13 )N(R)2, -C(=NR 13 )N(R)2, - C(=NR 13 )R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, wherein each n is independently an integer from 1 to 5, and R 12 is C 3-6 Selected from the group consisting of carbon cyclic groups and heterocyclic groups, R 13 Silver H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 Selected from the group consisting of a carbon cyclic group and a heterocyclic group, each R is independently a hydrogen atom, C 1-3 Alkyl and C 2-3 is selected from the group consisting of alkenyl, each X is independently selected from the group consisting of F, CI, Br and I, provided that R3 is -(CH2) n Q, -(CH2)n When CHQR, -CHQR or -CQ(R)2, (i) if n is 1, 2, 3, 4 or 5, Q is not -N(R)2, or (ii) if n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl.

[0042] According to one specific example of the present invention, in the chemical formula 1, R4 to R7 are each independently a hydrogen atom, C 1-3 Alkyl and C 2-3 It can be selected from the group consisting of alkenyl.

[0043] According to one specific example of the present invention, in the chemical formula 1, a and b can each independently be an integer from 1 to 15, and more specifically, an integer from 3 to 13.

[0044] More specifically, in the above chemical formula 1, M1 and M2 can each be independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')-, and -N(R')C(O)-, wherein R' is as defined above.

[0045] More specifically, in the above chemical formula 1, R1 and R2 are each independently substituted or unsubstituted C 3-15 It may be cycloalkyl.

[0046] More specifically, in the above chemical formula 1, R3 is a hydrogen atom, or a substituted or unsubstituted C 1-3 It could be an alkyl.

[0047] More specifically, in the above chemical formula 1, R4 to R7 may be hydrogen atoms.

[0048] More specifically, in the above chemical formula 1, a and b can each independently be an integer from 5 to 11, and more specifically, an integer from 5 to 9.

[0049] In one specific example, R1 and R2 are different from each other, wherein R1 is substituted or unsubstituted C 6-15 It may be cycloalkyl, and R2 is substituted or unsubstituted C 3-6 It may be cycloalkyl.

[0050] More specifically, the lipid may have any one structure selected from the following chemical formulas A to R:

[0051]

[0052] In one specific embodiment, the lipid content in the drug delivery composition of the present invention may be 1 wt% or more, 3 wt% or more, 5 wt% or more, 7 wt% or more, or 9 wt% or more, and may also be 95 wt% or less, 90 wt% or less, 85 wt% or less, 80 wt% or less, or 75 wt% or less, based on the dry weight of the entire composition. If the lipid content 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.

[0053] The 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. However, even if the lipid having the structure represented by the above chemical formula 1 can function as both an ionizable lipid and a helper lipid, the use of such additional lipid components 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 other than the lipid having the structure represented by the above chemical formula 1, or one or more types of helper lipids, as needed.

[0054] polymer

[0055] The drug delivery composition of the present invention comprises a lipid-polymer, an amphiphilic block copolymer, or a combination thereof as a polymer component.

[0056] The above lipid-polymer polymer is a polymer having both hydrophilic and hydrophobic portions within the polymer molecule.

[0057] 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.

[0058] The above hydrophilic block may be at least one selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide and derivatives thereof.

[0059] 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.

[0060] 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.

[0061] 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.

[0062] 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.

[0063] In addition, in one specific example, in the lipid-polymer, the composition ratio of the hydrophilic portion and the hydrophobic portion may be in the range of 10 to 90 wt%, specifically in the range of 20 to 80 wt%, more specifically in the range of 30 to 80 wt%, and even more specifically in the range of 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.

[0064] 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).

[0065] 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).

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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 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.

[0070] 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.

[0071] 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.

[0072] 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.

[0073] 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.

[0074] 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.

[0075] 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.

[0076] 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:

[0077] [Chemical Formula 2]

[0078]

[0079] In the above chemical formula 2,

[0080] R represents a branched alkylene group having 3 or more carbon atoms.

[0081] 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.

[0082] 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.

[0083] 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.

[0084] 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:

[0085] , , , , , , , , , , , , , , , ,

[0086] 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.

[0087] 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.

[0088] 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.

[0089] 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.

[0090] 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.

[0091] 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 1 wt% or more, 3 wt% or more, 5 wt% or more, 7 wt% or more, or 9 wt% or more, and may also be 95 wt% or less, 93 wt% or less, 90 wt% or less, 88 wt% or less, or 85 wt% or less, based on the dry weight of the entire composition. 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. Conversely, if the content of the polymer is too high, there is a concern that the content of the active ingredient that can be incorporated may become too low.

[0092] 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.

[0093] 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, 50 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, 50 to 400 nm, or 80 to 300 nm.

[0094] 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.

[0095] Any additional ingredients

[0096] 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.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] 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.

[0101] In one embodiment of the present invention, the fusion lipid may be distearoyl phosphatidylcholine, cholesterol, or a combination thereof.

[0102] 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.

[0103] In one specific example, the relative amount of the fusion lipid used compared to the lipid of the above 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 lipid of the above 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.

[0104] 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.

[0105] 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 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.

[0106] Composition and method for producing the same

[0107] 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.

[0108] The present invention also provides a method for preparing a drug delivery composition, comprising: (a) preparing a solution in which a 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).

[0109] In one specific example, the water-miscible organic solvent of step (a) may be ethanol.

[0110] In one specific embodiment, the step (b) may be performed in a solution under acidic conditions.

[0111] 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).

[0112] 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.

[0113] 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).

[0114] 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).

[0115] 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.

[0116] 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.

[0117] 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.

[0118] [Example]

[0119] Lipid preparation example 1

[0120] A compound of the following chemical formula A was prepared.

[0121] [Chemical Formula A]

[0122]

[0123] 1-2. Synthesis of 4-pentylcyclohexyl 8-bromooctanoate

[0124] 8-Bromooctanoic acid (2.00 g, 8.96 mmol, 1.00 eq), dichloromethane (DCM) (40 mL), and dimethylformamide (DMF) (0.5 mL) were placed together in a 250 mL 3-neck round bottom flask (RBF), and oxalyl chloride (2.28 g, 17.9 mmol, 2.00 eq) was added thereto at 0°C under a nitrogen atmosphere. The resulting mixture was stirred at 25°C for 4 h under a nitrogen atmosphere, then 4-pentylcyclohexan-1-ol (2.29 g, 13.5 mmol, 1.50 eq) and triethylamine (TEA) (1.36 g, 13.5 mmol, 1.50 eq) were added, and the mixture was stirred at 25°C for an additional 12 h under a nitrogen atmosphere. The mixture in the reactor was concentrated in vacuo, and the concentrated residue was purified using a silica column with petroleum ether:ethyl acetate (EtOAc) = 1:0→50:1 to obtain 4-pentylcyclohexyl 8-bromooctanoate (2.46 g, 6.55 mmol, 73.1% yield) as a pale yellow oil.

[0125] 1H NMR (400 MHz, CHLOROFORM-d):δ0.76 - 0.86 (m, 3 H) 0.88 - 1.94 (m, 28 H) 2.21 (dt, 2 H) 3.33 (td, 2 H) 4.52 - 4.66 (m, 1 H) 4.87 - 4.95 (m, 1 H)

[0126] 1-3. Synthesis of 4-pentylcyclohexyl 8-((2-hydroxyethyl)amino)octanoate

[0127] A 100 mL 3-neck RBF was charged with 4-pentylcyclohexyl 8-bromooctanoate (2.46 g, 6.55 mmol, 1.00 eq), 2-aminoethan-1-ol (2.00 g, 32.8 mmol, 5.00 eq), and ethanol (EtOH) (50 mL) and stirred at 80°C for 16 h under a nitrogen atmosphere. The mixture in the reactor was concentrated under vacuum, and the concentrated residue was purified using a silica column with a ratio of petroleum ether:EtOAc = 1:0→50:1 to obtain 4-pentylcyclohexyl 8-((2-hydroxyethyl)amino)octanoate (2.00 g, 5.62 mmol, 85.8% yield) as a yellow solid.

[0128] 1 H NMR (400 MHz, CHLOROFORM-d):δ0.76 - 0.86 (m, 3 H) 0.88 - 1.94 (m, 27 H) 2.32 (t, 2 H) 2.51 (t, 2 H) 2.71 (t, 2H), 3.54 (d, 2 H) 3.91 - 4.00 (m, 1 H) 4.11 - 4.31 (m, 1 H)

[0129] 1-4. Synthesis of cyclopentadecyl 8-bromooctanoate

[0130] A 250 mL 3-neck RBF was charged with cyclopentadecanol (5.00 g, 22.1 mmol, 1.00 eq), 8-bromooctanoic acid (4.93 g, 22.1 mmol, 1 eq), sulfuric acid (H2SO4) (217 mg, 2.21 mmol, 0.10 eq), and toluene (100 mL) and stirred at 120°C for 16 h under a nitrogen atmosphere. The solvent was evaporated, and the residue was purified using a silica column with a ratio of petroleum ether:EtOAc = 1:0→50:1 to obtain cyclopentadecyl 8-bromooctanoate (2.60 g, 6.03 mmol, 27.3% yield) as a colorless oil.

[0131] 1 H NMR (400 MHz, CHLOROFORM-d): δ 4.89 (quin, 1H), 3.41 (dt, 2H), 2.28 (t, 2H), 1.85 (quin, 2H), 1.73 - 1.16 (m, 36H)

[0132] 1-5. Synthesis of the compound of chemical formula A

[0133] A 100 mL 3-neck RBF was charged with cyclopentadecyl 8-bromooctanoate (1.60 g, 3.71 mmol, 1.00 eq), 4-pentylcyclohexyl 8-((2-hydroxyethyl)amino)octanoate (1.32 g, 3.71 mmol, 1.00 eq), N,N-diisopropylethylamine (DIEA) (527 mg, 4.08 mmol, 1.10 eq), and EtOH (30 mL) and stirred at 80°C for 48 h. The solvent was evaporated, and the residue was purified using a silica column with a ratio of petroleum ether:EtOAc = 10:1→1:1. Subsequently, the mixture was purified again by prep-HPLC (Folic Acid condition), washed with NaHCO3 aqueous solution (300 mL), concentrated the organic layer, and extracted with DCM (200 mL x 2). The organic layer was dried over anhydrous Na2SO4, filtered, and the filtrate was concentrated to obtain the compound of chemical formula A (0.240 g, 340 μmol, 9.16% yield) as a yellow oil.

[0134] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.91 (br s, 1H), 4.82 (quin, 1H), 3.46 (br t, 2H), 2.51 (br d, 2H), 2.37 (br t, 4H), 2.21 (td, 4H), 1.58 - 1.42 (m, 14H), 1.33 - 1.17 (m, 52H), 0.83 - 0.80 (m, 3H).

[0135] Lipid manufacturing example 2

[0136] A compound of the following chemical formula B was prepared.

[0137] [Chemical Formula B]

[0138]

[0139] 2-2. Synthesis of cyclopentadecyl 6-bromohexanoate

[0140] In a 100 mL 3-neck RBF, add cyclopentadecanol (5.00 g, 22.1 mmol, 1.00 eq), 6-bromohexanoic acid (6.46 g, 33.1 mmol, 1.50 eq), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDCI) (5.08 g, 26.5 mmol, 1.20 eq), 4-dimethylaminopyridine (DMAP) (540 mg, 4.42 mmol, 0.20 eq), TEA (4.47 g, 44.2 mmol, 6.15 mL, 2.00 eq), and DCM (50 mL), and the mixture was stirred for 3 times with nitrogen gas. Purged. The resulting mixture was stirred under a nitrogen atmosphere at 25°C for 16 h, quenched by adding 200 mL of water at 20°C, and extracted with 600 mL of DCM (200 mL x 3). The organic layers were combined, dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo. The concentrated residue was purified using a silica column with petroleum ether:EtOAc = 100:1 → 1:1 to give cyclopentadecyl 6-bromohexanoate (2.20 g, 5.45 mmol, 24.7% yield) as a yellow oil.

[0141] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.83 (quin, 1H), 3.50 - 3.30 (m, 2H), 2.23 (t, 2H), 1.87 - 1.68 (m, 2H), 1.63 - 1.37 (m, 10H), 1.26 (br s, 22H)

[0142] 2-3. Synthesis of 4-propylcyclohexyl 6-bromohexanoate

[0143] A 250 mL 3-neck RBF was charged with 4-propylcyclohexan-1-ol (10.0 g, 70.3 mmol, 1.00 eq), 6-bromohexanoic acid (16.5 g, 84.4 mmol, 1.20 eq), EDCI (20.2 g, 105 mmol, 1.50 eq), DMAP (8.59 g, 70.3 mmol, 1.00 eq), and TEA (7.11 g, 70.3 mmol, 9.79 mL, 1.00 eq) in DCM (100 mL), purged with nitrogen three times, and stirred at 25 °C for 16 h in a nitrogen environment. The reaction mixture was neutralized by adding 100 mL of water at 20°C, extracted with 600 mL of DCM (200 mL x 3), the organic layers were combined and concentrated in vacuo, and the concentrated residue was purified using a silica column with petroleum ether:EtOAc = 100:1 → 10:1 to obtain 4-propylcyclohexyl 6-bromohexanoate (6.80 g, 21.3 mmol, 30.3% yield) as a yellow oil.

[0144] 1 H NMR (400 MHz, CHLOROFORM-d): δ 0.76 - 0.86 (m, 3 H) 0.88 - 1.94 (m, 19 H) 2.21 (dt, 2 H) 3.33 (td, 2 H) 4.52 - 4.66 (m, 1 H)

[0145] 2-4. Synthesis of 4-propylcyclohexyl 6-((2-hydroxyethyl)amino)hexanoate

[0146] A 250 mL 3-neck RBF was charged with 4-propylcyclohexyl 6-bromohexanoate (3.00 g, 9.40 mmol, 1.00 eq), 2-aminoethanol-1-ol (2.87 g, 47.0 mmol, 2.84 mL, 5.00 eq), and EtOH (60 mL), purged with nitrogen three times, and stirred at 85 °C for 16 h in a nitrogen environment. After cooling the reaction mixture to room temperature, it was concentrated to remove the solvent, and the residue was purified by column chromatography (SiO2, DCM:MeOH=100:1→1:1) to give 4-propylcyclohexyl 6-((2-hydroxyethyl)amino)hexanoate (0.70 g, 2.34 mmol, 24.9% yield) as a yellow oil.

[0147] 1 H NMR (400 MHz, CHLOROFORM-d): δ 5.04 - 4.60 (m, 1H), 3.70 - 3.61 (m, 2H), 2.79 (t, 2H), 2.64 (dt, 2H), 2.35 - 2.24 (m, 2H), 2.01 - 1.94 (m, 2H), 1.87 - 1.74 (m, 2H), 1.65 (qd, 2H), 1.56 - 1.48 (m, 4H), 1.41 - 1.16 (m, 9H), 1.07 - 0.94 (m, 1H), 0.89 (dt, 3H)

[0148] 2-5. Synthesis of the compound of chemical formula B

[0149] A 50 mL 3-neck RBF was charged with 4-propylcyclohexyl 6-((2-hydroxyethyl)amino)hexanoate (371 mg, 1.24 mmol, 1.00 eq), 4-propylcyclohexyl 6-bromohexanoate (0.50 g, 1.24 mmol, 1.00 eq), and N,N-diisopropylethylamine (DIEA) (800 mg, 6.20 mmol, 1.08 mL, 5 eq) together with 1,4-dioxane (10 mL), purged with nitrogen three times, and stirred at 95°C for 16 h in a nitrogen environment. After the reaction mixture was cooled to room temperature, the solvent was removed by concentrating under reduced pressure, and the residue was purified through column chromatography (SiO2, DCM:MeOH=100:1→10:1) to obtain the compound of chemical formula B (0.21 g, 337.64 μmol, 27.2% yield) as a yellow oil.

[0150] 1 H NMR (400 MHz, CHLOROFORM-d): δ 4.94 - 4.52 (m, 2H), 3.79 (br s, 2H), 3.06 - 2.63 (m, 6H), 2.29 - 2.17 (m, 4H), 1.93 - 1.81 (m, 1H), 1.79 - 1.37 (m, 17H), 1.36 - 1.09 (m, 34H), 0.99 - 0.86 (m, 1H), 0.85 - 0.76 (m, 3H)

[0151] Lipid manufacturing example 3

[0152] A compound of the following chemical formula C was prepared.

[0153] [Chemical Formula C]

[0154]

[0155] 3-2. Synthesis of cyclopentadecyl 10-bromodecanoate

[0156] In a 100 mL 3-neck RBF, cyclopentadecanol (5.00 g, 22.1 mmol, 1.00 eq), 10-bromodecanoic acid (8.32 g, 33.1 mmol, 1.50 eq), DMAP (540 mg, 4.42 mmol, 0.20 eq), EDCI (5.08 g, 26.5 mmol, 1.20 eq), and TEA (4.47 g, 44.2 mmol, 2.00 eq) were added together with DCM (50 mL), purged with nitrogen three times, and stirred at 50°C for 16 h in a nitrogen environment. The reaction mixture was cooled to 20°C, water was added to terminate the reaction, and the mixture was extracted with 600 mL of DCM (200 mL x 3). The organic layer was collected, dried over Na2SO4, filtered, and the filtrate was concentrated. The residue obtained was purified by column chromatography (SiO2, petroleum ether: EtOAc = 100:1 → 1:1) to obtain cyclopentadecyl 10-bromodecanoate (2.60 g, 5.66 mmol, 25.6% yield) as a yellow oil.

[0157] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.90 (quin, 1H), 3.58 - 3.36 (m, 2H), 2.27 (t, 2H), 1.90 - 1.72 (m, 2H), 1.64 - 1.53 (m, 6H), 1.43 - 1.28 (m, 34H)

[0158] 3-3. Synthesis of 4-heptylcyclohexan-1-ol

[0159] LiAlH4 (2.50 M in THF, 61.1 mL, 1.20 eq) was placed in a 500 mL 3-neck RBF with THF (250 mL), cooled to 0°C, and a solution of 4-heptylcyclohexan-1-one (25.0 g, 127 mmol, 1.00 eq) in THF (250 mL) was slowly added thereto over 20 min under a nitrogen atmosphere. The temperature of the mixture solution was then increased to 25°C and stirred for 3 h under a nitrogen atmosphere. The reaction mixture was cooled to 0°C, and water (120 mL) was added thereto under a nitrogen atmosphere, paying attention to the formation of a large amount of foam. Then, at 0°C, 15% aq. NaOH (12 mL) was added to the reaction mixture, and after 5 minutes, water (36 mL) was added at the same temperature, and the temperature was slowly raised to 25°C and stirred for 15 minutes. The resulting mixture was filtered, and the filtered cake was concentrated under vacuum to obtain 4-heptylcyclohexan-1-ol (50.0 g, 252 mmol, 98.9% yield) as a colorless powder.

[0160] 1 H NMR (400 MHz, CHLOROFORM-d): δ3.47 (tt, H), 1.95 - 1.84 (m, 2H), 1.75 - 1.65 (m, 2H), 1.42 (s, 1H), 1.24 - 1.14 (m, 14H), 1.11 - 1.08 (m, 2H), 0.83 - 0.80 (m, 3H)

[0161] 3-4. Synthesis of 4-heptylcyclohexyl 10-bromodecanoate

[0162] In a 250 mL 3-neck RBF, 4-heptylcyclohexan-1-ol (10.0 g, 50.4 mmol, 1.00 eq), 10-bromodecanoic acid (15.2 g, 60.5 mmol, 1.20 eq), EDCI (14.5 g, 75.6 mmol, 1.50 eq), DMAP (6.16 g, 50.4 mmol, 1.00 eq), and TEA (5.10 g, 50.4 mmol, 1.00 eq) were added with DCM (100 mL), purged with nitrogen three times, and stirred at 25°C for 16 h in a nitrogen environment. The reaction mixture was neutralized by adding 200 mL of water at 20°C, extracted with 600 mL of DCM (200 mL x 3), the organic layer was collected, concentrated under vacuum, and the concentrated residue was purified by column chromatography (SiO2, petroleum ether: EtOAc = 100:1 → 1:1) to obtain 4-heptylcyclohexyl 10-bromodecanoate (6.60 g, 15.3 mmol, 30.3% yield) as a white solid.

[0163] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.59 (tt, 1H), 3.46 (t, 1H), 3.33 (t, 1H), 2.19 (t, 2H), 1.87 (br dd, 2H), 1.82 - 1.67 (m, 4H), 1.58 - 1.50 (m, 2H), 1.38 - 1.32 (m, 2H), 1.21 (br d, 23H), 0.97 - 0.86 (m, 2H), 0.81 (t, 3H)

[0164] 3-5. Synthesis of 4-heptylcyclohexyl 10-((2-hydroxyethyl)amino)decanoate

[0165] A 250 mL 3-neck RBF was charged with 4-heptylcyclohexyl 10-bromodecanoate (2.30 g, 5.33 mmol, 1.00 eq) and 2-aminoethanol-1-ol (1.63 g, 26.6 mmol, 5 eq) together with EtOH (60 mL), purged with nitrogen three times, and stirred at 95 °C for 16 h in a nitrogen environment. The reaction mixture was concentrated in vacuo, and the concentrated residue was purified by column chromatography (SiO2, DCM:MeOH=100:1→1:1) to afford 4-heptylcyclohexyl 10-((2-hydroxyethyl)amino)decanoate (1.30 g, 3.16 mmol, 59.2% yield) as a white solid.

[0166] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.66 (tt, 1H), 3.83 - 3.73 (m, 2H), 2.96 - 2.88 (m, 2H), 2.81 - 2.73 (m, 2H), 2.26 (t, 2H), 1.95 (br dd, 2H), 1.78 (br d, 2H), 1.62 (td, 4H), 1.28 (br d, 24H), 1.18 (br d, 2H), 1.04 - 0.96 (m, 2H), 0.89 (t, 3H)

[0167] 3-6. Synthesis of the compound of chemical formula C

[0168] A 50 mL 3-neck RBF was charged with 4-heptylcyclohexyl 10-((2-hydroxyethyl)amino)decanoate (0.80 g, 1.94 mmol, 1.00 eq), cyclopentadecyl 10-bromodecanoate (893 mg, 1.94 mmol, 1.00 eq), and DIEA (1.26 g, 9.72 mmol, 1.69 mL, 5.00 eq) together with EtOH (3 mL), purged with nitrogen three times, and stirred at 95°C for 16 h in a nitrogen environment. After the reaction mixture was cooled to room temperature, concentrated under vacuum, and the concentrated residue was purified by chromatography (SiO2, DCM:MeOH=100:1→10:1) to obtain a compound of chemical formula C (0.35 g, 447 μmol, 22.9% yield) as a white solid.

[0169] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.96 - 4.61 (m, 2H), 3.77 - 3.52 (m, 2H), 2.84 - 2.37 (m, 6H), 2.27 (dt, 4H), 2.01 - 1.92 (m, 2H), 1.79 (br d, 2H), 1.68 - 1.46 (m, 18H), 1.37 - 1.25 (m, 56H), 1.00 (br d, 1H), 0.89 (br t, 3H)

[0170] Lipid preparation example 4

[0171] A compound of the following chemical formula D was prepared.

[0172] [Chemical Formula D]

[0173]

[0174] 4-2. Synthesis of (E)-2-(hept-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane

[0175] In a 500 mL 3-neck RBF, hept-1-yne (64.4 g, 670 mmol, 1.00 eq), TEA (Et3N, 6.78 g, 670 mmol, 0.10 eq), 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (90.0 g, 703 mmol, 1.05 eq), chlorozirconium; cyclopentane (17.9 g, 670 mmol, 0.10 eq) were added and the mixture was purged with nitrogen three times, and stirred at 60°C for 16 h in a nitrogen environment. After the reaction mixture was cooled to room temperature, filtered, and the filtrate was concentrated under vacuum, the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1) to obtain (E)-2-(hept-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (102 g, 455 mmol, 67.9% yield) as a yellow oil.

[0176] 1 H NMR: (400 MHz, CHLOROFORM-d): δ 6.64 (td, 1H), 5.43 (td, 1H), 2.19 - 2.12 (m, 2H), 1.44 - 1.39 (m, 2H), 1.30 (br d, 4H), 1.27 (s, 12H), 0.90 - 0.87 (m, 3H)

[0177] 4-3. Synthesis of 4,4,5,5-tetramethyl-2-((1R,2R)-2-pentylcyclopropyl)-1,3,2-dioxaborolane

[0178] Diethyl zinc (1 M, 178.5 mL, 2.00 eq) was added to a 1000 mL 3-neck RBF together with distilled purified DCM (80 mL) under a nitrogen atmosphere, and a solution of trifluoroacetic acid (TFA) (20.4 g, 178 mmol, 2.00 eq) dissolved in DCM (40 mL) was added little by little at 0°C. In addition, a solution of diiodomethane (47.8 g, 178 mmol, 2.00 eq) dissolved in DCM (40 mL) was added while stirring at 0°C for 30 minutes. After the reaction mixture was further stirred for 30 minutes, a solution of (E)-2-(hept-1-en-1-yl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (20.0 g, 89.2 mmol, 1.00 eq) dissolved in DCM (40 mL) was added thereto at 0°C, and the mixture was stirred at 25°C for 2 hours in a nitrogen environment. After the reaction mixture was neutralized with water, extracted with DCM (1000 mL x 3), the organic layers were collected, dried over Na2SO4, filtered, the filtrate was concentrated in vacuo, and the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain 4,4,5,5-tetramethyl-2-((1R,2R)-2-pentylcyclopropyl)-1,3,2-dioxaborolane (14.0 g, 58.8 mmol, 65.9% yield) as a yellow oil.

[0179] 1 H NMR (400 MHz, CHLOROFORM-d): δ 1.42 - 1.37 (m, 2H), 1.32 - 1.26 (m, 6H), 1.26 - 1.24 (m, 2H), 1.22 (s, 10H), 0.89 (br t, 4H), 0.67 (dt, 1H), 0.43 - 0.33 (m, 1H), -0.42 (td, 1H)

[0180] 4-4. Synthesis of (1R,2R)-2-pentylcyclopropan-1-ol

[0181] 4,4,5,5-Tetramethyl-2-((1R,2R)-2-pentylcyclopropyl)-1,3,2-dioxaborolane (8.00 g, 33.6 mmol, 1.00 eq) and THF (160 mL) were added to a 500 mL 3-neck RBF, and NaOH (2.69 g, 67.2 mmol, 2.00 eq) was slowly added to the mixture at 0°C, followed by H2O2 (7.87 g, 69.4 mmol, 30% purity, 2.07 eq) at 0°C, and stirred at 25°C for 16 h. Afterwards, the reaction was terminated by pouring Na2SO3 (2 eq) ice water into the reaction mixture, and then extracted with DCM (30 mL x 3), and the organic layer was collected, dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo, and the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain (1R,2R)-2-pentylcyclopropan-1-ol (2.50 g, 19.5 mmol, 58.1% yield) as a yellow oil.

[0182] 1 H NMR (400 MHz, CHLOROFORM-d):δ3.20 (td, 1H), 1.84 - 1.67 (m, 1H), 1.41 - 1.35 (m, 2H), 1.33 - 1.26 (m, 4H), 1.23 - 1.06 (m, 2H), 0.94 - 0.87 (m, 4H), 0.68 (ddd, 1H), 0.31 (q, 1H)

[0183] 4-5. Synthesis of (1R,2R)-2-pentylcyclopropyl 8-bromooctanoate

[0184] (1R,2R)-2-pentylcyclopropan-1-ol (2.50 g, 19.5 mmol, 1.00 eq) was added to a 100 mL 3-neck RBF with DCM (25 mL), and 8-bromooctanoic acid (5.22 g, 23.4 mmol, 1.20 eq), EDCI (4.49 g, 23.4 mmol, 1.20 eq), DMAP (476 mg, 3.90 mmol, 0.20 eq), and Et3N (3.95 g, 39.00 mmol, 2.00 eq) were added. After purging with nitrogen three times, the mixture was stirred at 25°C for 16 h in a nitrogen environment. The reaction mixture was filtered, the filtrate was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:100) to obtain (1R,2R)-2-pentylcyclopropyl 8-bromooctanoate (3.50 g, 10.5 mmol, 53.9% yield) as a yellow oil.

[0185] 1 H NMR (400 MHz, CHLOROFORM-d): δ 3.85 - 3.79 (m, 1H), 3.41 (t, 2H), 2.26 (t, 2H), 1.86 (quin, 2H), 1.65 - 1.58 (m, 2H), 1.46 - 1.38 (m, 4H), 1.36 - 1.27 (m, 10H), 1.00 (ddd, 1H), 0.89 (br t, 3H), 0.78 (ddd, 1H), 0.52 (q, 1H)

[0186] 4-6. Synthesis of cyclohexyl 8-bromooctanoate

[0187] 8-Bromooctanoic acid (15.0 g, 67.2 mmol, 1.00 eq) was placed in a 1000 mL 3-neck RBF with DCM (150 mL), and trifluoroacetic anhydride (TFAA) (14.1 g, 67.2 mmol, 1.00 eq) was added under nitrogen, and the mixture was stirred at 0-25 °C for 2.5 h under nitrogen. Cyclohexanol (33.7 g, 336 mmol, 35.1 mL, 5.00 eq) was added to the mixture at 0 °C, and the mixture was stirred at 25 °C for 16 h under nitrogen, and the reaction mixture was neutralized with 100 mL of water at 20 °C and extracted with 600 mL of DCM (200 mL x 3). The organic layers were collected, dried over Na2SO4, filtered, the filtrate was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (SiO2, petroleum ether:EtOAc=100:1→10:1) to obtain cyclohexyl 8-bromooctanoate (7.10 g, 23.3 mmol, 34.6% yield) as a colorless oil.

[0188] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.77 (br d, 1H), 3.42 (t, 2H), 2.30 (t, 2H), 1.93 - 1.82 (m, 4H), 1.74 (br dd, 2H), 1.67 - 1.55 (m, 4H), 1.47 - 1.31 (m, 10H)

[0189] 4-7. Synthesis of cyclohexyl 8-((2-hydroxyethyl)amino)octanoate

[0190] In a 250 mL 3-neck RBF, cyclohexyl 8-bromooctanoate (3.00 g, 9.83 mmol, 1.00 eq), 2-aminoethanol-1-ol (3.00 g, 49.1 mmol, 5.00 eq), and Na2CO3 (1.04 g, 9.83 mmol, 1.00 eq) were added together with 1,4-dioxane (60 mL), purged with nitrogen three times, and stirred at 80°C for 16 h in a nitrogen environment. The reaction mixture was filtered, the filtrate was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (DCM:MeOH=100:1→10:1) to obtain cyclohexyl 8-((2-hydroxyethyl)amino)octanoate (2.80 g, 9.81 mmol, 99.8% yield) as a yellow oil.

[0191] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.77 (td, 1H), 3.75 - 3.67 (m, 2H), 2.91 - 2.80 (m, 5H), 2.68 (t, 2H), 2.29 (t, 2H), 1.88 - 1.82 (m, 2H), 1.77 - 1.70 (m, 2H), 1.66 - 1.60 (m, 2H), 1.55(br d, 2H), 1.46 - 1.28 (m, 12H)

[0192] 4-8. Synthesis of the compound of chemical formula D

[0193] In a 50 mL 3-neck RBF, cyclohexyl 8-((2-hydroxyethyl)amino)octanoate (942 mg, 3.30 mmol, 1.10 eq) was added together with EtOH (10 mL), and (1R,2R)-2-pentylcyclopropyl 8-bromooctanoate (1.00 g, 3.00 mmol, 1.00 eq) and DIEA (1.94 g, 15.00 mmol, 5.00 eq) were added, and the mixture was stirred at 90°C for 16 h under a nitrogen atmosphere. The reaction mixture was filtered, the filtrate was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (DCM:MeOH=10:1→1:100) to obtain the compound of chemical formula D (0.15 g, 279 μmol, 9.30% yield) as a yellow oil.

[0194] 1 H NMR (400 MHz, CHLOROFORM-d):δ5.23 - 4.94 (m, 2H), 4.63 - 4.56 (m, 1H), 4.12 (br s, 2H), 3.68 - 3.57 (m, 2H), 3.56 - 3.37 (m, 4H), 2.40 - 2.27 (m, 2H), 2.04 (br d, 6H), 1.91 - 1.76 (m, 2H), 1.74 - 1.55 (m, 17H), 1.54 - 1.46 (m, 2H), 1.43 - 1.25 (m, 5H), 1.21 (s, 2H), 0.90 (d, 6H)

[0195] Lipid preparation example 5

[0196] A compound of the following chemical formula E was prepared.

[0197] [Chemical Formula E]

[0198]

[0199] 5-2. Synthesis of cyclopentadecanecarbonitrile

[0200] In a 2000 mL 3-neck RBF, cyclopentadecanone (25.0 g, 111 mmol, 1.00 eq), potassium; 2-methylpropan-2-olate (25.0 g, 223 mmol, 2.00 eq), 2-methylpropan-2-ol (250 mL) were added together with THF (500 mL), and the mixture was cooled to 0°C. 1-(isocyanomethylsulfonyl)-4-methyl-benzene (32.6 g, 167 mmol, 1.50 eq) was slowly added thereto at 0°C over 1 hour, and the mixture was stirred at 20°C for 11 hours. The reaction mixture was diluted with H2O (400 mL) and extracted with EtOAc (400 mL). The organic layer was washed with H2O (400 mL), dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo. The concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 1:0 → 5:1) to obtain cyclopentadecanecarbonitrile (20 g, 84.96 mmol, 76.25% yield) as a colorless oil.

[0201] 1 H NMR (400 MHz, CHLOROFORM-d):δ1.33 (br s, 20 H) 1.44 - 1.53 (m, 4 H) 1.67 (q, 4 H) 2.59 (quin, 1 H)

[0202] 5-3. Synthesis of cyclopentadecanecarboxylic acid

[0203] Cyclopentadecanecarbonitrile (14.0 g, 59.5 mmol, 1.00 eq), KOH (6.00 M, 69.4 mL, 7.00 eq), and EtOH (70 mL) were placed together in a 250 mL 3-neck RBF, purged with nitrogen three times, and stirred at 100 °C for 16 h in a nitrogen environment. The reaction mixture was concentrated under vacuum, and the concentrated residue was acidified to pH 4 with 4 M aqueous hydrochloric acid solution (50 mL), and extracted with EtOAc (50 mL x 3). The organic layers were collected, dried over Na2SO4, filtered, and the filtrate was concentrated in vacuo. The concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1 → 1:1) to obtain cyclopentadecanecarboxylic acid (1.20 g, 4.72 mmol, 7.93% yield) as a white solid.

[0204] 1 H NMR (400 MHz, CHLOROFORM-d):δppm 1.27 - 1.46 (m, 24 H) 1.55 - 1.72 (m, 4 H) 2.44 (quin, 1 H) 10.15 - 11.75 (m, 1 H)

[0205] 5-4. Synthesis of 7-bromoheptyl cyclopentadecanecarboxylate

[0206] Cyclopentadecanecarboxylic acid (1.20 g, 4.72 mmol, 1.10 eq) was added to a 50 mL 3-neck RBF with toluene (12 mL), and 7-bromoheptan-1-ol (837 mg, 4.29 mmol, 1.00 eq) was added, followed by H2SO4 (84.1 mg, 858 μmol, 45.7 μL, 0.20 eq), and the mixture was purged with nitrogen three times, and stirred at 120°C for 16 h in a nitrogen environment. The reaction mixture was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1) to obtain 7-bromoheptyl cyclopentadecanecarboxylate (0.90 g, 2.09 mmol, 48.6% yield) as a colorless oil.

[0207] 1 H NMR (400 MHz, CHLOROFORM-d):δ1.29 - 1.40 (m, 30 H) 1.56 - 1.64 (m, 6 H) 1.87 (quin2 H) 2.34 - 2.46 (m, 1 H) 3.41 (t, 2 H) 4.07 (t, 2 H)

[0208] 5-5. Synthesis of 7-bromoheptyl 4-pentylcyclohexane-1-carboxylate

[0209] 4-Pentylcyclohexane-1-carboxylic acid (4.47 g, 22.9 mmol, 1.00 eq) was added to a 250 mL 3-neck RBF together with toluene (50 mL), 7-bromoheptan-1-ol (5.00 g, 25.2 mmol, 1.00 eq) was added, and then H2SO4 (450 mg, 4.58 mmol, 244 μL, 0.20 eq) was added, and the mixture was purged with nitrogen three times and stirred at 120°C for 16 h in a nitrogen environment. The reaction mixture was concentrated under vacuum, and the concentrated residue was purified by silica column chromatography (petroleum ether: EtOAc = 10:1) to obtain 7-bromoheptyl 4-pentylcyclohexane-1-carboxylate (7.00 g, 18.7 mmol, 81.4% yield) as a colorless oil.

[0210] 1 H NMR (400 MHz, CHLOROFORM-d):δ0.86 - 0.90 (m, 3 H) 1.18 - 1.32 (m, 10 H) 1.32 - 1.40 (m, 5 H) 1.41 - 1.59 (m, 6 H) 1.59 - 1.68 (m, 2 H) 1.81 - 1.90 (m, 2 H) 1.91 - 2.02 (m, 2 H) 2.46 - 2.54 (m, 1 H) 3.41 (t, 2 H) 4.02 - 4.12 (m, 2 H)

[0211] 5-6. Synthesis of 7-((2-hydroxyethyl)amino)heptyl 4-pentylcyclohexane-1-carboxylate

[0212] A 250 mL 3-neck RBF was charged with 7-bromoheptyl 4-pentylcyclohexane-1-carboxylate (3.00 g, 7.99 mmol, 1.00 eq), 2-aminoethanol-1-ol (2.44 g, 40.0 mmol, 2.41 mL, 5.00 eq) and 1,4-dioxane (90 mL), purged with nitrogen three times, and stirred at 100 °C for 16 h in a nitrogen environment. The reaction mixture was concentrated in vacuo, and the concentrated residue was purified by silica column chromatography (DCM:MeOH=10:1) to give 7-((2-hydroxyethyl)amino)heptyl 4-pentylcyclohexane-1-carboxylate (1.70 g, 4.78 mmol, 59.8% yield) as a yellow oil.

[0213] 1 H NMR (400 MHz, CHLOROFORM-d):δ0.88 (t, 3 H) 1.19 - 1.28 (m, 8 H) 1.34 (br s, 6 H) 1.41 - 1.71 (m, 8 H) 1.84 - 2.01 (m, 6 H) 2.42 - 2.54 (m, 1 H) 2.63 (t, 2 H) 2.74 - 2.86 (m, 2 H) 3.50 - 3.79 (m, 2 H) 3.99 - 4.13 (m, 2 H)

[0214] 5-7. Synthesis of the compound of chemical formula E

[0215] 7-((2-Hydroxyethyl)amino)heptyl 4-pentylcyclohexane-1-carboxylate (90 mg, 209 μmol, 1.00 eq) was placed in a 50 mL 3-neck RBF with 1,4-dioxane (5 mL). 7-Bromoheptyl cyclopentadecanecarboxylate (74.2 mg, 209 μmol, 1.00 eq) and DIEA (135 mg, 1.04 mmol, 5.00 eq) were added, and the mixture was purged with nitrogen three times and stirred at 105 °C for 16 h in a nitrogen environment. The reaction mixture was cooled to 25 °C, water (10 mL) was added to quench the reaction, and extracted with 30 mL of EtOAc (10 mL x 3). The organic layers were collected, dried over Na2SO4, filtered, the filtrate was concentrated in vacuo, and the concentrated residue was purified by silica column chromatography (DCM:MeOH=10:1) to obtain the compound of chemical formula E (70 mg, 99.1 μmol, 47.5% yield) as a yellow oil.

[0216] 1 H NMR (400 MHz, CHLOROFORM-d):δ0.88 (t, 3 H) 1.23 - 1.40 (m, 46 H) 1.45 - 1.50 (m, 4 H) 1.52 - 1.66 (m, 12 H) 1.72 - 2.05 (m, 4 H) 2.37 - 2.55 (m, 6 H) 2.63 (br t, 2 H) 3.57 (br t, 2 H) 4.02 - 4.09 (m, 4 H)

[0217] Lipid preparation example 6

[0218] A compound of the following chemical formula F was prepared.

[0219] [Chemical formula F]

[0220]

[0221] Synthesis of 6-2 cyclopentadecyl 10-bromodecanoate

[0222] Synthesis was performed using the method described in Manufacturing Example 3-2.

[0223] 6-3. Synthesis of 4-propylcyclohexyl 6-bromohexanoate

[0224] Synthesis was performed using the method described in Manufacturing Example 2-3.

[0225] 6-4. Synthesis of 4-propylcyclohexyl 6-((2-hydroxyethyl)amino)hexanoate

[0226] Synthesis was performed by the method described in Manufacturing Example 2-4.

[0227] 6-5. Synthesis of the compound of chemical formula F

[0228] A 50 mL 3-neck RBF was charged with 4-propylcyclohexyl 6-((2-hydroxyethyl)amino)hexanoate (0.10 g, 1.00 eq), cyclopentadecyl 10-bromodecanoate (153 mg, 1.00 eq), and DIEA (44 mg, 1.00 eq) along with EtOH (5 mL), purged with nitrogen three times, and stirred at 95°C for 16 h in a nitrogen environment. The reaction mixture was cooled to room temperature, concentrated in vacuo, and the concentrated residue was purified by silica column chromatography (DCM:MeOH=100:1→10:1) to obtain the compound of formula F (70 mg, 30.9% yield) as a yellow oil.

[0229] 1 H NMR (400 MHz, CHLOROFORM-d):δ5.03 - 4.62 (m, 2H), 4.01 - 3.72 (m, 2H), 3.10 - 2.75 (m, 6H), 2.35 - 2.25 (m, 4H), 1.95 (br d, 1H), 1.59 (br s, 18H), 1.40 - 1.17 (m, 42H), 1.05 - 0.96 (m, 1H), 0.92 - 0.87 (m, 3H)

[0230] Lipid preparation example 7

[0231] A compound of the following chemical formula G was prepared in the same manner as in Preparation Example 1, except that 3-pentylcyclopentan-1-ol was used instead of 4-pentylcyclohexan-1-ol.

[0232] [Chemical formula G]

[0233]

[0234] 1 H NMR (400 MHz, CHLOROFORM-d): δ5.03 - 4.77 (m, 2H), 4.09 - 3.88 (m, 2H), 3.04 - 2.88 (br, 6H), 2.41 - 2.31 (m, 4H), 1.89 - 1.56 (br, 14H), 1.51 - 0.96 (br, 53H), 0.88 (t, 3H)

[0235] Lipid preparation example 8

[0236] A compound of the following chemical formula H was prepared in the same manner as in Preparation Example 1, except that 6-bromohexanoic acid was used instead of 8-bromooctanoic acid and 3-pentylcyclopentan-1-ol was used instead of 4-pentylcyclohexan-1-ol.

[0237] [Chemical formula H]

[0238]

[0239] 1 H NMR (400 MHz, CHLOROFORM-d): δ4.68 (m, 1H), 4.48 (m, 1H), 3.91 (m, 2H), 3.02 - 2.89 (br, 6H), 2.35 - 2.29 (m, 4H), 1.92 - 1.61 (m, 14H), 1.58 - 1.01 (m, 45H), 0.90 (t, 3H)

[0240] Lipid preparation example 9

[0241] A compound of the following chemical formula I was prepared in the same manner as in Preparation Example 1, except that 10-bromodecanoic acid was used instead of 8-bromooctanoic acid and 3-pentylcyclopentan-1-ol was used instead of 4-pentylcyclohexan-1-ol.

[0242] [Chemical Formula I]

[0243]

[0244] 1H NMR (400 MHz, CHLOROFORM-d): δ4.70 (m, 1H), 4.47 (m, 1H), 3.90 (m, 2H), 2.99 - 2.79 (br, 6H), 2.31 - 2.26 (m, 4H), 1.92 - 1.61 (m, 14H), 1.58 - 1.01 (m, 61H), 0.85 (t, 3H)

[0245] Lipid preparation example 10

[0246] A compound of the following chemical formula J was prepared in the same manner as in Preparation Example 1, except that cyclohexanol was used instead of cyclopentadecanol and 3-pentylcyclopentan-1-ol was used instead of 4-pentylcyclohexan-1-ol.

[0247] [Chemical formula J]

[0248]

[0249] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.68 - 4.61 (m, 2H), 3.94 (m, 2H), 3.12 - 2.94 (br, 6H), 2.44 (m, 4H), 1.91 - 1.10 (m 45H), 0.88 (t, 3H)

[0250] Lipid preparation example 11

[0251] A compound of the following chemical formula K was prepared in the same manner as in Preparation Example 1, except that aminocyclopentadecane was used instead of cyclopentadecanol.

[0252] [Chemical formula K]

[0253]

[0254] 1H NMR (400 MHz, CHLOROFORM-d):δ8.10 (br, 1H), 4.67 - 4.63 (m, 1H), 3.95 - 3.88 (m, 2H), 3.64 - 3.59 (m, 1H), 3.11 - 2.89 (br, 6H), 2.31 - 2.19 (m, 4H), 1.95 - 1.25 (m, 65H), 0.88 (t, 3H)

[0255] Lipid preparation example 12

[0256] A compound of the following chemical formula L was prepared in the same manner as in Preparation Example 1, except that 1-amino-4-pentylcyclohexane was used instead of 4-pentylcyclohexan-1-ol.

[0257] [chemical formula L]

[0258]

[0259] 1 H NMR (400 MHz, CHLOROFORM-d):δ8.12 (br, 1H), 4.50 - 4.43 (m, 1H), 3.88 - 3.76 (m, 2H), 3.49 - 3.40 (m, 2H), 3.36 - 3.33 (m, 1H), 3.03 - 2.90 (br, 6H), 2.31 - 2.90 (m, 4H), 1.92 - 1.20 (m, 65H), 0.90 (t, 3H)

[0260] Lipid preparation example 13

[0261] A compound of the following chemical formula M was prepared in the same manner as in Preparation Example 1, except that aminocyclopentadecane was used instead of cyclopentadecanol and 1-amino-4-pentylcyclohexane was used instead of 4-pentylcyclohexan-1-ol.

[0262] [Chemical formula M]

[0263]

[0264] 1H NMR (400 MHz, CHLOROFORM-d):δ8.10 (br, 2H), 3.84 - 3.78 (m, 2H), 3.69 - 3.61 (m, 2H), 3.11 - 2.98 (br, 6H), 2.33 - 2.19 (m, 4H), 1.90 - 1.22 (m, 65H), 0.91 (t, 3H)

[0265] Lipid preparation example 14

[0266] A compound of the following chemical formula N was prepared in the same manner as in Preparation Example 1, except that (1R,2R)-2-pentylcyclopropan-1-ol prepared in Example 4-4 was used instead of 4-pentylcyclohexan-1-ol.

[0267] [Chemical formula N]

[0268]

[0269] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.56 - 4.45 (m, 1H), 3.56 - 3.52 (m, 2H), 3.42 (br, 1H), 3.11 - 3.05 (br, 4H), 2.77 - 2.70 (br, 2H), 2.44 - 2.38 (m, 4H), 1.91 - 1.21 (m, 57H), 0.9 (t, 3H), 0.62 - 0.55 (m, 2H)

[0270] Lipid Preparation Example 15

[0271] A compound of the following chemical formula O was prepared in the same manner as in Preparation Example 1, except that methylamine (CH3-NH2) was used instead of 2-aminoethanol-1-ol.

[0272] [chemical formula O]

[0273]

[0274] 1H NMR (400 MHz, CHLOROFORM-d):δ4.41 - 4.38 (m, 1H), 4.22 - 4.18 (m, 1H), 3.00 - 2.82 (m, 4H), 2.75 (s, 3H), 2.42 - 2.39 (m, 4H), 1.92 - 1.20 (m, 65H), 0.90 (t, 3H)

[0275] Lipid preparation example 16

[0276] A compound of the following chemical formula P was prepared in the same manner as in Preparation Example 1, except that methylamine (CH3-NH2) was used instead of 2-aminoethanol-1-ol, 10-bromodecanoic acid was used instead of 8-bromooctanoic acid, and 4-heptylcyclohexan-1-ol was used instead of 4-pentylcyclohexan-1-ol.

[0277] [chemical formula P]

[0278]

[0279] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.43 - 4.40 (m, 1H), 4.21 - 4.18 (m, 1H), 3.12 - 2.92 (m, 4H), 2.80 (s, 3H), 2.42 - 2.39 (m, 4H), 1.93 - 1.19 (m, 73H), 0.90 (t, 3H)

[0280] Lipid Preparation Example 17

[0281] A compound of the following chemical formula Q was prepared in the same manner as in Preparation Example 1, except that cyclodecanol was used instead of cyclopentadecanol.

[0282] [Chemical formula Q]

[0283]

[0284] 1H NMR (400 MHz, CHLOROFORM-d): δ4.48 - 4.41 (m, 1H), 4.19 - 4.17 (m, 1H), 3.54 - 3.49 (m, 2H), 3.02 - 2.92 (br, 4H), 2.78 - 2.71 (br, 2H), 2.35 - 2.31 (m, 4H), 1.91 - 1.19 (m, 55H), 0.88 (t, 3H)

[0285] Lipid preparation example 18

[0286] A compound of the following chemical formula R was prepared in the same manner as in Preparation Example 1, except that cyclodecanol was used instead of cyclopentadecanol and methylamine (CH3-NH2) was used instead of 2-aminoethanol-1-ol.

[0287] [Chemical formula R]

[0288]

[0289] 1 H NMR (400 MHz, CHLOROFORM-d):δ4.48 - 4.41 (m, 1H), 4.19 - 4.17 (m, 1H), 3.01 - 2.93 (br, 4H), 2.75 (br, 3H), 2.34 - 2.30 (m, 4H), 1.90 - 1.21 (m, 55H), 0.90 (t, 3H)

[0290] [Manufacturing of composition and delivery test of active ingredients into tissue]

[0291] Example 1: Preparation and drug delivery test of a drug delivery composition using each of the lipids and lipid-polymers of Preparation Examples 1, 2, 3, 5, or 6

[0292] (1) Preparation of solutions by component

[0293] The components shown in Table 1 below were dissolved in each dilution solvent and prepared at the concentrations shown in Table 1 below. After dissolving to an appropriate concentration, the composition was used for manufacturing after confirming with the naked eye that there was no undissolved precipitation.

[0294] [Table 1]

[0295]

[0296] (2) Mixing of raw materials

[0297] The lipid:DOPE or DSPC:cholesterol:DMG-PEG ratios of each of Manufacturing Examples 1, 2, 3, 5, or 6 were taken according to the ratios shown in Table 2 below to match the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6, and then each component was mixed. Ethanol was added to the ethanol layer so that the molecular sum of all components was 6.25-12.5 mM, and the aqueous phase and the ethanol phase were mixed while maintaining a ratio of 3:1. To reduce the total ethanol content after mixing, buffer exchange was 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), and the buffer was exchanged through concentration and PBS dilution.

[0298] The specific process sequence is as follows.

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

[0300] 2) Each of the lipids, DOPE or DSPC, cholesterol, and DMG-PEG of Manufacturing Examples 1, 2, 3, 5, or 6 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) In tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed. 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 (aqueous phase:ethanol phase volume ratio = 3:1).

[0303] 5) The prepared tube (A) and tube (B) were mixed.

[0304] 6) The resulting mixture from step 5 was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K), and the process of concentration and dilution was repeated to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

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

[0306] [Table 2]

[0307]

[0308] (3) Evaluation of physical properties of the formulation

[0309] 1) 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 3 below.

[0310] 2) For the manufactured formulation, the mRNA encapsulation efficiency was confirmed through the Ribo-green assay, and the results are shown in Table 3 below.

[0311] (4) Administration of the composition

[0312] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravenously to mice so that 2 μg of mRNA was administered per mouse. Four hours after administration, luciferin dissolved in sterile water was prepared at a concentration of 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g mouse. Fifteen minutes after intraperitoneal administration of luciferin, protein expression results by organ were measured using a luminescence measurement imaging system and are shown in Table 3 below.

[0313] As confirmed from Table 3, the drug delivery formulation according to the present invention had excellent drug delivery efficiency to the target organ upon administration.

[0314] Meanwhile, the formulation of Comparative Example 1 was intravenously administered to mice so that the same amount of mRNA (2 μg / 200 μL) as in the example was administered, and the results of imaging 4 hours later using the same method as in the example are shown in Table 3. The formulation of Comparative Example showed delivery to the target organ upon administration.

[0315] [Table 3]

[0316]

[0317] Example 2: Preparation of a drug delivery composition using the lipid and amphiphilic block copolymer of Preparation Example 1 and drug delivery test

[0318] (1) Preparation of solutions by component

[0319] Using the ingredients shown in Table 4 below, solutions for each ingredient were prepared in the same manner as in step (1) of Example 1, with the concentrations shown in Table 4 below.

[0320] [Table 4]

[0321]

[0322] (2) Mixing of raw materials

[0323] The lipid:DOPE:cholesterol:DMG-PEG ratio of Manufacturing Example 1 was adjusted to the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6 to 20 according to the ratio shown in Table 5 below, and each component was mixed. Ethanol was added to the ethanol layer so that the sum of the molecules of all components was 6.25-12.5 mM, and the aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, a buffer exchange was performed as follows to lower the total ethanol content: 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), and the buffer was exchanged through concentration and PBS dilution.

[0324] The specific process sequence is as follows.

[0325] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0326] 2) The lipids, DSPC, cholesterol, and MPEG-PLA (2K-4K) of Manufacturing Example 1 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0327] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0328] 4) In tube (B), mRNA and sodium acetate buffer adjusted to pH 4.6 were mixed. 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 (aqueous phase:ethanol phase volume ratio = 3:1).

[0329] 5) The prepared tube (A) and tube (B) were mixed.

[0330] 6) The resulting mixture from step 5 was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K), and the process of concentration and dilution was repeated to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

[0331] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0332] [Table 5]

[0333]

[0334] (3) Evaluation of physical properties of the formulation

[0335] The physical properties of the manufactured formulation were evaluated in the same manner as in step (3) of Example 1, and the results are shown in Table 6 below.

[0336] (4) Administration of the composition

[0337] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravenously (IV) to mice so that 2 μg of mRNA was administered per mouse. Four hours after administration, luciferin dissolved in sterile water was prepared at a concentration of 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g mouse. Fifteen minutes after intraperitoneal administration of luciferin, protein expression results by organ were measured using a luminescence measurement imaging system and are shown in Table 6 below.

[0338] As confirmed from Table 6, the drug delivery formulation according to the present invention had excellent drug delivery efficiency to the target organ upon administration.

[0339] Meanwhile, each formulation of Comparative Examples 2-1 and 2-2 was intravenously administered to mice so that the same amount of mRNA (2 μg / 200 μL) as in the Example was administered, and the results of imaging 4 hours later using the same method as in the Example are shown in Table 6. The Comparative Example formulation showed delivery to the target organ upon administration.

[0340] [Table 6]

[0341]

[0342] Example 3: Preparation of a drug delivery composition using the lipid and amphiphilic block copolymer of Preparation Example 3 and drug delivery test

[0343] (1) Preparation of solutions by component

[0344] Using the ingredients shown in Table 7 below, solutions for each ingredient were prepared in the same manner as in step (1) of Example 1, with the concentrations shown in Table 7 below.

[0345] [Table 7]

[0346]

[0347] (2) Mixing of raw materials

[0348] In accordance with the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6, the lipid:DOPE or DSPC:cholesterol:MPEG-PLA(2K-4K) ratio of Manufacturing Example 3 was taken according to the ratio shown in Table 8 below, and each component was mixed. Ethanol was added to the ethanol layer so that the sum of the molecules of all components was 6.25-12.5 mM, and the aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, a buffer exchange was performed as follows to lower the total ethanol content: 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), and the buffer was exchanged through concentration and PBS dilution.

[0349] The specific process sequence is as follows.

[0350] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0351] 2) The lipids, DOPE or DSPC, cholesterol, and MPEG-PLA (2K-4K) of Manufacturing Example 3 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0352] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0353] 4) In tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed. 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 (aqueous phase:ethanol phase volume ratio = 3:1).

[0354] 5) The prepared tube (A) and tube (B) were mixed.

[0355] 6) The resulting mixture from step 5 was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K), and the process of concentration and dilution was repeated to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

[0356] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0357] [Table 8]

[0358]

[0359] (3) Evaluation of physical properties of the formulation

[0360] The physical properties of the manufactured formulation were evaluated in the same manner as in step (3) of Example 1, and the results are shown in Table 9 below.

[0361] (4) Administration of the composition

[0362] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravenously (IV) to mice so that 2 μg of mRNA was administered per mouse. Four hours after administration, luciferin dissolved in sterile water was prepared at a concentration of 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g mouse. Fifteen minutes after intraperitoneal administration of luciferin, protein expression results by organ were measured using a luminescence measurement imaging system and are shown in Table 9 below.

[0363] As confirmed from Table 9, the drug delivery formulation according to the present invention had excellent drug delivery efficiency to the target organ upon administration.

[0364] Meanwhile, the formulation of Comparative Example 3-1 was intravenously administered to mice so that the same amount of mRNA (2 μg / 200 μL) as in the example was administered, and the results of imaging 4 hours later using the same method as in the example are shown in Table 9. The formulation of Comparative Example showed delivery to the target organ upon administration.

[0365] [Table 9]

[0366]

[0367] Example 4: Preparation of a drug delivery composition using the lipid and amphiphilic block copolymer of Preparation Example 5 and drug delivery test

[0368] (1) Preparation of solutions by component

[0369] Using the ingredients shown in Table 10 below, solutions for each ingredient were prepared in the same manner as in step (1) of Example 1, with the concentrations shown in Table 10 below.

[0370] [Table 10]

[0371]

[0372] (2) Mixing of raw materials

[0373] In accordance with the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6, the lipid:DOPE:cholesterol:MPEG-PLA(2K-4K) ratio of Manufacturing Example 5 was taken according to the ratio shown in Table 11 below, and each component was mixed. Ethanol was added to the ethanol layer so that the sum of the molecules of all components was 6.25-12.5 mM, and the aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, a buffer exchange was performed as follows to lower the total ethanol content: 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), and the buffer was exchanged through concentration and PBS dilution.

[0374] The specific process sequence is as follows.

[0375] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0376] 2) The lipids, DOPE, cholesterol, and MPEG-PLA (2K-4K) of Manufacturing Example 5 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0377] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0378] 4) In tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed. 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 (aqueous phase:ethanol phase volume ratio = 3:1).

[0379] 5) The prepared tube (A) and tube (B) were mixed.

[0380] 6) The resulting mixture from step 5 was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K), and the process of concentration and dilution was repeated to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

[0381] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0382] [Table 11]

[0383]

[0384] (3) Evaluation of physical properties of the formulation

[0385] The physical properties of the manufactured formulation were evaluated in the same manner as in step (3) of Example 1, and the results are shown in Table 12 below.

[0386] (4) Administration of the composition

[0387] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravenously (IV) to mice so that 2 μg of mRNA was administered per mouse. Four hours after administration, luciferin dissolved in sterile water was prepared at a concentration of 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g mouse. Fifteen minutes after intraperitoneal administration of luciferin, protein expression results by organ were measured using a luminescence measurement imaging system and are shown in Table 12 below.

[0388] As confirmed from Table 12, the drug delivery formulation according to the present invention had excellent drug delivery efficiency to the target organ upon administration.

[0389] Meanwhile, each formulation of Comparative Examples 4-1 and 4-2 was intravenously administered to mice so that the same amount of mRNA (2 μg / 200 μL) as in the Example was administered, and the results of imaging 4 hours later using the same method as in the Example are shown in Table 12. The Comparative Example formulation showed delivery to the target organ upon administration.

[0390] [Table 12]

[0391]

[0392] Example 5: Preparation of a drug delivery composition using the lipid and amphiphilic block copolymer of Preparation Example 6 and drug delivery test

[0393] (1) Preparation of solutions by component

[0394] Using the ingredients shown in Table 13 below, solutions for each ingredient were prepared in the same manner as in step (1) of Example 1, with the concentrations shown in Table 13 below.

[0395] [Table 13]

[0396]

[0397] (2) Mixing of raw materials

[0398] In accordance with the N / P ratio (amine group of lipid component / phosphate group of mRNA) of 6, the lipid:DOPE or DSPC:cholesterol:MPEG-PLA(2K-4K) ratio of Manufacturing Example 6 was taken according to the ratio shown in Table 14 below, and each component was mixed. Ethanol was added to the ethanol layer so that the sum of the molecules of all components was 6.25-12.5 mM, and the aqueous phase and ethanol phase were mixed while maintaining a ratio of 3:1. After mixing, a buffer exchange was performed as follows to lower the total ethanol content: 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), and the buffer was exchanged through concentration and PBS dilution.

[0399] The specific process sequence is as follows.

[0400] 1) Two autoclaved tubes were prepared (tubes (A), (B)).

[0401] 2) The lipids, DOPE or DSPC, cholesterol, and MPEG-PLA (2K-4K) of Manufacturing Example 6 in moles calculated according to the experimental conditions were sequentially added to tube (A) and mixed by vortexing.

[0402] 3) Ethanol was added when necessary so that the molecular sum of all components was within 6.25-12.5 mM.

[0403] 4) In tube (B), mRNA and 20 mM sodium acetate buffer (pH 4.6) were mixed. 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 (aqueous phase:ethanol phase volume ratio = 3:1).

[0404] 5) The prepared tube (A) and tube (B) were mixed.

[0405] 6) The resulting mixture from step 5 was centrifuged at 4,000 rpm using an Amicon-Ultra tube filter (50K), and the process of concentration and dilution was repeated to remove excess ethanol, and then concentrated to a final x mg / mL (theoretical concentration).

[0406] 7) Once concentrated to the desired concentration, sterilize using a 0.22 ㎛ pore size filter.

[0407] [Table 14]

[0408]

[0409] (3) Evaluation of physical properties of the formulation

[0410] The physical properties of the manufactured formulation were evaluated in the same manner as in step (3) of Example 1, and the results are shown in Table 15 below.

[0411] (4) Administration of the composition

[0412] The manufactured formulation was prepared at a concentration of 10 μg / mL and administered intravenously (IV) to mice so that 2 μg of mRNA was administered per mouse. Four hours after administration, luciferin dissolved in sterile water was prepared at a concentration of 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g mouse. Fifteen minutes after intraperitoneal administration of luciferin, protein expression results by organ were measured using a luminescence measurement imaging system and are shown in Table 15 below.

[0413] As confirmed from Table 15, the drug delivery formulation according to the present invention had excellent drug delivery efficiency to the target organ upon administration.

[0414] Meanwhile, the formulation of Comparative Example 5-1 was intravenously administered to mice so that the same amount of mRNA (2 μg / 200 μL) as in the example was administered, and the results of imaging 4 hours later using the same method as in the example are shown in Table 15. The formulation of Comparative Example showed delivery to the target organ upon administration.

[0415] [Table 15]

[0416]

Claims

1. An active ingredient selected from nucleic acids, polypeptides, viruses or a combination thereof; A 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] In the above chemical formula 1, M1 and M2 are each independently a two-valent linker, R1 and R2 are each independently a substituted or unsubstituted carbon cyclic group or heterocyclic group, R3 is a hydrogen atom or a substituted or unsubstituted organic group optionally containing one or more heteroatoms, R4 to R7 are each independently a hydrogen atom or a substituted or unsubstituted saturated or unsaturated hydrocarbon group, a and b are each independently an integer from 1 to 20.

2. In paragraph 1, M1 and M2 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-M'-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-, arylene, and heteroarylene, wherein M' is a direct bond, C 1-13 Alkylene or C 2-13 Alkenylene, and R' is each independently a hydrogen atom, C 1-18 Alkyl and C 2-18 Selected from the group consisting of alkenyl, R1 and R2 are each independently, C 3-20 Cycloalkyl, C 3-20 Cycloalkenyl, C 6-20 Aryl, C 3-20 Heterocycloalkyl, C 3-20 Heterocycloalkenyl, and C 3-20 Selected from the group consisting of heteroaryl, each of which is independently unsubstituted or C 1-18 Alkyl or C 2-18 substituted with alkenyl, R3 is a hydrogen atom, substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-6 Carbocyclic group, -(CH2) n Q, - (CH2) n selected from the group consisting of CHQR, -CHQR and -CQ(R)2, wherein each R is independently a hydrogen atom, C 1-3 Alkyl and C 2-3 is selected from the group consisting of alkenyl; Q is a carbocyclic group, a heterocyclic group, -OR, -O(CH2) n N(R)2, -C(O)OR, -OC(O)R, -CX3, -CX2H, -CXH2, -CN, -N(R)2, -C(O)N(R)2, -N(R)C(O)R, -N(R)S(O)2R, -N(R)C(O)N(R)2, -N(R)C(S)N(R)2, -N(R)R 12 , N(R)S(O)2R 12 , -O(CH2) n OR, -N(R)C(=NR 13 )N(R)2, -N(R)C(=CHR 13 )N(R)2, -OC(O)N(R)2, -N(R)C(O)OR, -N(OR)C(O)R, -N(OR)S(O)2R, -N(OR)C(O)OR, -N(OR)C(O)N(R)2, -N(OR)C(S)N(R)2, -N(OR)C(=NR 13 )N(R)2, -N(OR)C(=CHR 13 )N(R)2, -C(=NR 13 )N(R)2, - C(=NR 13 )R, -C(O)N(R)OR and -C(R)N(R)2C(O)OR, wherein each n is independently an integer from 1 to 5, and R 12 is C 3-6 Selected from the group consisting of carbon cyclic groups and heterocyclic groups, R 13 Silver H, CN, NO2, C 1-6 Alkyl, -OR, -S(O)2R, -S(O)2N(R)2, C 2-6 Alkenyl, C 3-6 Selected from the group consisting of a carbon cyclic group and a heterocyclic group, each R is independently a hydrogen atom, C 1-3 Alkyl and C 2-3 is selected from the group consisting of alkenyl, each X is independently selected from the group consisting of F, CI, Br and I, provided that R3 is -(CH2) n Q, -(CH2) n When CHQR, -CHQR or -CQ(R)2, (i) if n is 1, 2, 3, 4 or 5, Q is not -N(R)2, or (ii) if n is 1 or 2, Q is not a 5-, 6- or 7-membered heterocycloalkyl, R4 to R7 are each independently a hydrogen atom, C 1-3 Alkyl and C 2-3 Selected from the group consisting of alkenyl, a and b are each independently an integer from 1 to 15, Composition for drug delivery.

3. In paragraph 2, M1 and M2 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -OC(O)-M'-C(O)O-, -C(O)N(R')-, -N(R')C(O)- and -C(O)-, wherein M' and R' are as defined in clause 2, R1 and R2 are each independently C 3-20 Cycloalkyl and C 3-20 Selected from the group consisting of heterocycloalkyl, each of which is independently unsubstituted or C 1-18 Alkyl or C 2-18 substituted with alkenyl, R3 is a hydrogen atom, substituted or unsubstituted C 1-6 Alkyl and substituted or unsubstituted C 3-6 Selected from the group consisting of carbon cyclic groups, R4 to R7 are each independently a hydrogen atom or C 1-3 It is alkyl, a and b are each independently an integer from 3 to 13, Composition for drug delivery.

4. In paragraph 3, M1 and M2 are each independently selected from the group consisting of -C(O)O-, -OC(O)-, -C(O)N(R')- and -N(R')C(O)-, wherein R' is each independently a hydrogen atom, C 1-18 Alkyl and C 2-18 Selected from the group consisting of alkenyl, R1 and R2 are each independently substituted or unsubstituted C 3-15 It is cycloalkyl, R3 is a hydrogen atom, or a substituted or unsubstituted C 1-3 It is alkyl, R4 to R7 are hydrogen atoms, a and b are each independently an integer from 5 to 11, Composition for drug delivery.

5. A composition for drug delivery, wherein the lipid has any one structure selected from the following chemical formulas A to R in paragraph 4:

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 according to 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 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] In the above chemical formula 1, M1 and M2 are each independently a two-valent linker, R1 and R2 are each independently a substituted or unsubstituted carbon cyclic group or heterocyclic group, R3 is a hydrogen atom or a substituted or unsubstituted organic group optionally containing one or more heteroatoms, R4 to R7 are each independently a hydrogen atom or a substituted or unsubstituted saturated or unsaturated hydrocarbon group, a and b are each independently an integer from 1 to 20.

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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