Nanoparticle Compositions for Pulmonary Drug Delivery - Patent application

Abstract

The present invention relates to a nanoparticle composition for pulmonary drug delivery, and more specifically, to a drug delivery composition useful for specifically delivering a drug to the lungs by comprising a drug-containing nanoparticle in which a complex of an anionic drug and a specific lipid is encapsulated within a nanoparticle formed by an amphiphilic block copolymer.

Description

[Technical field]

[0001] The present invention relates to a nanoparticle composition for pulmonary drug delivery, and more specifically, to a drug delivery composition useful for specifically delivering a drug to the lungs by comprising a drug-containing nanoparticle in which a complex of an anionic drug and a specific lipid is encapsulated within a nanoparticle formed by an amphiphilic block copolymer. [Background technology]

[0002] In the treatment using anionic drugs including nucleic acids, techniques for safely and efficiently delivering drugs have been studied for a long time, and various delivery vehicles and delivery technologies have been developed. Delivery vehicles are mainly divided into viral delivery vehicles using adenoviruses, retroviruses, etc., and non-viral delivery vehicles using cationic lipids and cationic polymers, etc. It is known that viral delivery vehicles are exposed to risks such as non-specific immune reactions and have many problems in commercialization due to the complex manufacturing process. Therefore, in recent research, research has been conducted in the direction of using non-viral delivery vehicles to improve such disadvantages. Non-viral delivery vehicles have the advantages of fewer side effects in terms of safety in the body and lower manufacturing costs in terms of economy compared to viral delivery vehicles.

[0003] Representative non-viral delivery vehicles for delivering nucleic acid substances include cationic lipid / nucleic acid complexes (lipoplexes) and polycationic polymer / nucleic acid complexes (polyplexes). Such cationic lipids or polycationic polymers form complexes with anionic drugs through electrostatic interactions to stabilize the anionic drugs and increase their intracellular delivery, and thus have been the subject of various studies (Non-Patent Documents 1 and 2).

[0004] Meanwhile, in order to provide a mixed polymer nanoparticle composition that can solubilize a large amount of a poorly soluble drug and has excellent stability in an aqueous solution, Patent Document 1 discloses a mixed polymer nanoparticle composition that contains an amphiphilic block copolymer of a hydrophilic block and a hydrophobic block, and a polylactic acid derivative containing a carboxylic acid end group, and that can form polymer nanoparticles in a body fluid or an aqueous solution, and a pharmaceutical composition in which a poorly soluble drug is contained in the polymer nanoparticles formed by the mixed polymer nanoparticle composition. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Korea Patent No. 2003-0032897 [Non-patent literature]

[0006] [Non-Patent Document 1] De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56 [Non-Patent Document 2] Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectives on the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73 Summary of the Invention [Problem to be solved by the invention]

[0007] It is an object of the present invention to provide a drug delivery composition that is useful for specifically delivering drugs to the lungs. [Means for solving the problem]

[0008] In order to achieve the above object, a first aspect of the present invention includes a drug-containing nanoparticle, the drug-containing nanoparticle comprising a drug as an active ingredient; an amphiphilic block copolymer; and a compound represented by the following formula (1): [ka] (wherein n is an integer from 1 to 6; R 1 and R 2 are each independently selected from the group consisting of unsaturated hydrocarbon groups having 12 to 26 carbon atoms.) wherein the drug forms a complex with the lipid, and the complex is encapsulated within the nanoparticle structure formed by the amphiphilic block copolymer.

[0009] In one embodiment, in the formula (1), n ​​may be an integer of 2 to 4.

[0010] In one embodiment, in formula (1), R 1 and R 2 are each independently -(C 5 -C 12 alkylene)-CH=CH-(C 5 -C 12 It may be alkyl). More specifically, in the formula (1), R 1 and R 2 are each independently -(C 6 -C 10 alkylene)-CH=CH-(C 6 -C 10 It may be alkyl). More specifically, in the formula (1), R 1 and R 2 is -(CH 2 ) 8 CH=CH(CH 2 ) 7 CH 3 may be also possible.

[0011] More specifically, the lipid may have the following structure: [ka]

[0012] In one embodiment, the lipid is prepared by a process comprising the steps of: (1) reacting a compound of formula (a) with methyl acrylate to obtain a compound of formula (b): (2) reacting a compound of the following formula (b) with a compound of the following formula (c-1) or with a compound of the following formula (c-1) and a compound of the following formula (c-2) in sequence to obtain a compound of the following formula (d-1) or (d-2), respectively; and (3) a step of deprotecting a compound of the following formula (d-1) or (d-2): [ka] (wherein P is a protecting group, n is an integer of 1 to 6, and R 1 and R 2 are each independently selected from the group consisting of unsaturated hydrocarbon groups having 12 to 26 carbon atoms.)

[0013] In one embodiment, the drug-containing nanoparticles may further comprise polylactate. Effect of the Invention

[0014] The compositions of the present invention are highly useful for specifically delivering drugs to the lungs encapsulated within nanoparticles formed by amphiphilic block copolymers. [Brief description of the drawings]

[0015] [Figure 1]NMR data of the lipid [2OA-PAMAM: (3-((2-aminoethyl)(3-(((Z)-nonadec-10-en-1-yl)amino)-3-oxopropyl)amino)-N-((Z)-octadec-9-en-1-yl)propanamide)] produced in the examples of the present invention. [Diagram 2] 1 shows the results of a drug delivery experiment carried out in a test example of the present invention, and photographs of the results measured using a luminescence measurement imaging system. BEST MODE FOR CARRYING OUT THEINVENTION

[0016] The present invention will now be described in more detail.

[0017] The composition of the present invention comprises drug-containing nanoparticles, the drug-containing nanoparticles comprising a drug as an active ingredient; an amphiphilic block copolymer; and a compound represented by the following formula (1): [ka] (In the formula, n is an integer of 1 to 6, more specifically, an integer of 2 to 6; R 1 and R 2 are each independently selected from the group consisting of unsaturated hydrocarbon groups having 12 to 26 carbon atoms, more specifically selected from the group consisting of unsaturated hydrocarbon groups having 14 to 22 carbon atoms, and even more specifically selected from the group consisting of unsaturated hydrocarbon groups having 16 to 20 carbon atoms; The drug forms a complex with the lipid, and the complex is encapsulated within the nanoparticle structure formed by the amphiphilic block copolymer.

[0018] In one embodiment, in the formula (1), n ​​may be an integer of 2 to 4.

[0019] In one embodiment, in formula (1), R 1 and R 2 are each independently -(C 5 -C 12 alkylene)-CH=CH-(C5 -C 12 It may be alkyl).

[0020] More specifically, in the formula (1), R 1 and R 2 are each independently -(C 6 -C 10 alkylene)-CH=CH-(C 6 -C 10 It may be alkyl).

[0021] The "alkylene" and "alkyl" may be independently unsubstituted or substituted, and in this case, the substituent may be, for example, a hydroxy group, a halogen group, or a C 1 -C 4 Examples include, but are not limited to, alkyl groups.

[0022] More specifically, in the formula (1), R 1 and R 2 is -(CH 2 ) 8 CH=CH(CH 2 ) 7 CH 3 may be also possible.

[0023] More specifically, the lipid may have the following structure: [ka]

[0024] In one embodiment, the lipid is prepared by a process comprising the steps of: (1) reacting a compound of formula (a) with methyl acrylate to obtain a compound of formula (b): (2) reacting a compound of the following formula (b) with a compound of the following formula (c-1) or with a compound of the following formula (c-1) and a compound of the following formula (c-2) in sequence to obtain a compound of the following formula (d-1) or (d-2), respectively; and (3) a step of deprotecting a compound of the following formula (d-1) or (d-2): [ka] (wherein P is a protecting group, more specifically, a t-butoxycarbonyl group (t-BOC), n, R 1 and R 2 is defined as above in formula (1).

[0025] The overall synthetic scheme of the method for producing lipids of the present invention is illustrated as follows: [ka]

[0026] In one embodiment, the drug-containing nanoparticles may further comprise polylactate, and the complex may be encapsulated within a nanoparticle structure formed by the amphiphilic block copolymer and polylactate.

[0027] In the nanoparticles, in an aqueous environment, the hydrophilic portion of the amphiphilic block copolymer forms the outer wall of the nanoparticle, and the hydrophobic portion of the amphiphilic block copolymer (and polylactate, if present) forms the inner wall of the nanoparticle, and the complex of the drug and the lipid of the present invention can be encapsulated inside the nanoparticles thus formed. Such a nanoparticle structure improves the stability of the active ingredient in blood or body fluids.

[0028] In one embodiment, the drug may be selected from a nucleic acid, a polypeptide, a virus, or a combination thereof.

[0029] The "nucleic acid" may be, for example, but is not limited to, DNA, RNA, siRNA, shRNA, miRNA, mRNA, an aptamer, an antisense oligonucleotide, or a combination thereof.

[0030] The "polypeptide" may mean a protein that has activity in the body, such as an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a protein that can be recognized as an antigen through a series of processes in the body, including a polypeptide sequence of an antigen, an analog thereof, or a precursor thereof.

[0031] In one embodiment, the particle size of the nanoparticles can be defined by a Z-average value, and may 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, or 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 may be, for example, 10 to 800 nm, 20 to 600 nm, 30 to 500 nm, 50 to 400 nm, or 80 to 300 nm.

[0032] The "Z-average" may refer to the average hydrodynamic diameter of a particle distribution measured using dynamic light scattering (DSL). The nanoparticles may have a monodisperse particle distribution, the polydispersity index of which may be, for example, 0.01 to 0.50, 0.05 to 0.455, or 0.1 to 0.40.

[0033] In one embodiment, the surface charge of the nanoparticles may be, for example, 0 mV or more, 1 mV or more, 5 mV or more, or 10 mV or more, and may be 80 mV or less, 70 mV or less, or 60 mV or less. In one embodiment, the surface charge of the nanoparticles may be, for example, 0 to 80 mV, 1 to 70 mV, or 5 to 60 mV. The surface charge may be measured in an environment close to a biological environment, for example, in 8 to 12 mM HEPES buffer (pH 7.0 to 7.5).

[0034] Maintaining the particle size and surface charge of the nanoparticles at the above levels is preferable in terms of the stability of the nanoparticle structure, the amount of the component and the absorbability in the body, and the ease of sterilization as a pharmaceutical composition. For example, when the active ingredient is a nucleic acid, one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms include analogs, derivatives, and metabolites of cholesterol, tocopherol, and fatty acids.

[0035] In one embodiment, when the active ingredient is RNA, the amount of the active ingredient may be, for example, 0.05-30% by weight, 0.1-25% by weight, 0.25-20% by weight, 0.5-15% by weight, 1-10% by weight, or 1-5% by weight based on the total weight of the nanoparticles. If the amount of the active ingredient is less than the above range, the amount of the delivery agent is too large relative to the drug, and side effects due to the delivery agent may occur. If the amount of the active ingredient is more than the above range, the size of the nanoparticles may become too large, reducing the stability of the nanoparticles and increasing the loss rate during filter sterilization.

[0036] In one embodiment, the amount of the lipid in the composition may be, for example, 500 parts by weight or less, 400 parts by weight or less, 300 parts by weight or less, 200 parts by weight or less, 100 parts by weight or less, 80 parts by weight or less, or 60 parts by weight or less, or 1 part by weight or more, 2 parts by weight or more, 3 parts by weight or more, 4 parts by weight or more, or 5 parts by weight or more, relative to 1 part by weight of the active ingredient. In one embodiment, the amount of the lipid in the composition may be 1 to 500 parts by weight, 2 to 400 parts by weight, 3 to 300 parts by weight, 4 to 200 parts by weight, or 5 to 80 parts by weight, relative to 1 part by weight of the active ingredient. If the amount of the lipid in the composition is less than the above range, it may not be possible to form a stable complex with the active ingredient, and if it is more than the above range, the size of the nanoparticles may become too large, resulting in reduced stability and increased loss rate during filter sterilization.

[0037] When the active ingredient is a nucleic acid, the lipid and the nucleic acid are bound to each other by electrostatic interaction to form a complex. In one embodiment, the charge ratio of the nucleic acid (P) and the lipid (N) (N / P; the ratio of the positive charge of the lipid to the negative charge of the nucleic acid) may be 0.5 or more, 1 or more, 2 or more, or 5 or more, and may be 200 or less, 150 or less, 100 or less, or 60 or less, for example, 0.5 to 200, 1 to 150, 2 to 100, or 5 to 60. If the ratio (N / P) is smaller than the above range, it may be difficult to form a complex containing a sufficient amount of nucleic acid, and if the ratio (N / P) is larger than the above range, toxicity may be induced. In addition, the N / P ratio may play an important role in expressing an active ingredient specifically in the spleen.

[0038] In a specific embodiment, the amphiphilic block copolymer may be an AB type block copolymer comprising a hydrophilic A block and a hydrophobic B block. The AB type block copolymer forms a core-shell type polymeric nanoparticle in an aqueous environment, in which the hydrophobic B block forms the core (inner wall) and the hydrophilic A block forms the shell (outer wall).

[0039] In one embodiment, the hydrophilic A block may be one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyacrylamide, and derivatives thereof.

[0040] More specifically, the hydrophilic A block may be one or more selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0041] In one embodiment, the number average molecular weight (g / mol) of the hydrophilic A block may be 200 or more, 500 or more, 1,000 or more, or 2,000 or more, and may be 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less, but is not limited thereto.

[0042] In addition, if necessary, a functional group capable of reaching a specific tissue or cell, a ligand, or a functional group capable of promoting intracellular delivery can be chemically bonded to the end of the hydrophilic A block to regulate the distribution in the body of the polymeric nanoparticle delivery body formed of the amphiphilic block copolymer and polylactate, or to increase the delivery efficiency of the nanoparticle delivery body into cells. In one embodiment, the functional group or ligand may be one or more selected from the group consisting of monosaccharides, polysaccharides, vitamins, peptides, proteins, and antibodies against cell surface receptors. More specifically, the functional group or ligand may be one or more selected from the group consisting of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, and antibodies against transferrin receptors.

[0043] The hydrophobic B block is a biocompatible, biodegradable polymer, which in one embodiment may be one or more selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazines.

[0044] More specifically, the hydrophobic B block may be one or more selected from the group consisting of polylactide (PLA), polyglycolide, polycaprolactone, polydioxane-2-one, copolymers of polylactide and glycolide, copolymers of polylactide and polydioxane-2-one, copolymers of polylactide and polycaprolactone, and copolymers of polyglycolide and polycaprolactone.

[0045] In one embodiment, the number average molecular weight (g / mol) of the hydrophobic B block may be 200 or more, 500 or more, 1,000 or more, or 1,700 or more, and may be 50,000 or less, 20,000 or less, 10,000 or less, or 6,000 or less, but is not limited thereto.

[0046] For example, the combination of the number average molecular weight of the hydrophilic A block-hydrophobic B block may be, but is not limited to, 2,000 to 6,000, 2,000 to 4,000, 2,000 to 3,000, or 2,000 to 1,700.

[0047] In one embodiment, the hydrophobic B block can be modified by chemically binding the terminal hydroxy group of the hydrophobic B block with tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms in order to increase the hydrophobicity of the hydrophobic B block and thereby improve the stability of the nanoparticles.

[0048] In one embodiment, the polylactate is distributed in the core (inner wall) of the nanoparticle, strengthening the hydrophobicity of the core to stabilize the nanoparticle, while effectively avoiding the reticuloendothelial system (RES) in the body. That is, the carboxylate anion in the polylactate binds to the cation complex more effectively than polylactate, reducing the surface potential of the polymeric nanoparticle. As a result, the positive charge of the surface potential of the polymeric nanoparticle is smaller than that of the polymeric nanoparticle without polylactate, making it difficult to be captured by the reticuloendothelial system, and it may be efficiently delivered to the target site (e.g., cancer cells, inflammatory cells, etc.).

[0049] In one embodiment, the number average molecular weight (g / mol) of the polylactate may be 500 to 50,000, more specifically, 1,000 to 10,000. If the number average molecular weight of the polylactate is less than 500, the hydrophobicity may be too low and the polylactate may not exist in the core (inner wall) of the nanoparticle, and if it exceeds 50,000, the size of the polymer nanoparticle may become too large.

[0050] In one embodiment, the terminal opposite to the terminal metal carboxylate (e.g., sodium carboxylate) of the polylactate (e.g., sodium salt of polylactate) may be substituted with one selected from the group consisting of hydroxy, acetoxy, benzoyloxy, decanoyloxy, palmitoyloxy, and alkoxy having 1 to 2 carbon atoms.

[0051] In one embodiment, the polylactate may be one or more selected from the group consisting of compounds of the following formulas (2) to (7) (wherein "COO" means a carboxyl group, i.e., "C(=O)O"): RO-CHZ-[A] m -[B] n -COOM (2) (Wherein, A is -COO-CHZ-; B is -COO-CHY-, -COO-CH 2 CH 2 CH 2 CH 2 CH 2 -or-COO-CH 2 CH 2 OCH 2 -; R is a hydrogen atom, or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; Z and Y are each a hydrogen atom, or a methyl or phenyl group; M is Na, K, or Li; n is an integer from 1 to 30; and m is an integer from 0 to 20. RO-CHZ-[COO-CHX] p -[COO-CHY'] q -COO-CHZ-COOM (3) (In the formula, X is a methyl group; Y' is a hydrogen atom or a phenyl group; p is an integer of 0 to 25, q is an integer of 0 to 25, with the proviso that p+q is an integer of 5 to 25; R is a hydrogen atom, or an acetyl, benzoyl, decanoyl, palmitoyl, methyl or ethyl group; M is Na, K or Li; and Z is a hydrogen atom, methyl or phenyl group.) RO-PAD-COO-WM' (4) (Wherein, W-M' is [ka] PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, copolymers of D,L-lactic acid and glycolic acid, copolymers of D,L-lactic acid and mandelic acid, copolymers of D,L-lactic acid and caprolactone, and copolymers of D,L-lactic acid and 1,4-dioxane-2-one; R is a hydrogen atom, or an acetyl, benzoyl, decanoyl, palmitoyl, methyl, or ethyl group; M is independently Na, K, or Li. SO-PAD-COO-Q (5) [In the formula, S is [ka] {where L is -NR 1 - or -O- (where R 1 is a hydrogen atom or C 1 - 10 a is an integer of 0 to 4; b is an integer of 1 to 10; M is Na, K, or Li}; PAD is one or more selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, a copolymer of D,L-lactic acid and caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxane-2-one; Q is CH 3 , C.H. 2 CH 3 , C.H. 2 CH 2 CH 3 , C.H. 2 CH 2 CH 2 CH 3 , or C.H. 2 C 6 H 5 It is.] [ka] [In the formula, R' is -PAD-OC(O)-CH 2 CH 2-C(O)-OM (where PAD is selected from the group consisting of D,L-polylactic acid, D-polylactic acid, polymandelic acid, a copolymer of D,L-lactic acid and glycolic acid, a copolymer of D,L-lactic acid and mandelic acid, a copolymer of D,L-lactic acid and caprolactone, and a copolymer of D,L-lactic acid and 1,4-dioxan-2-one, and M is Na, K, or Li); a is an integer from 1 to 4. YO-[-C(O)-(CHX) a -O-] m -C(O)-R-C(O)-[-O-(CHX') b -C(O)-] n -OZ (7) [In the formula, X and X' are independently hydrogen, alkyl having 1 to 10 carbon atoms, or aryl having 6 to 20 carbon atoms; Y and Z are independently Na, K, or Li; m and n are independently integers from 0 to 95, provided that 5 < m + n < 100; a and b are independently integers from 1 to 6; R is -(CH 2 ) k -(where k is an integer from 0 to 10), divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof.

[0052] In one embodiment, the polylactate may be the compound of formula (2) or formula (3).

[0053] In one embodiment, the nanoparticles of the present invention can further contain a fusogenic lipid in order to enhance the efficiency of intracellular delivery of a drug (e.g., mRNA).

[0054] When a drug (e.g., mRNA) is mixed with the complex of the lipid of the present invention, the fusogenic lipid forms a drug (e.g., mRNA) / lipid of the present invention / fusogenic lipid complex by hydrophobic interaction, and the complex containing the fusogenic lipid is encapsulated inside the nanoparticle structure of the amphiphilic block copolymer.

[0055] More specifically, the fusogenic lipid may be one or more phospholipids 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 their respective analogs, derivatives and metabolites.

[0056] More specifically, the fusogenic lipid may be dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE), dilinoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dilauroylphosphatidylethanolamine, The phosphatidylcholine may be one or a combination of two or more selected from the group consisting of dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroylphosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, dilinoleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidic acid, 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol, and tocopherol.

[0057] More specifically, the fusogenic lipid may be one or more selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), distearoylphosphatidylcholine (DSPC) and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0058] In one embodiment, the amount of the amphiphilic block copolymer used may be 1 to 500 parts by weight, 1 to 400 parts by weight, 1 to 300 parts by weight, 2 to 500 parts by weight, 2 to 400 parts by weight, 2 to 300 parts by weight, 3 to 500 parts by weight, 3 to 400 parts by weight, 3 to 300 parts by weight, 4 to 500 parts by weight, 4 to 400 parts by weight, or 4 to 300 parts by weight, relative to 1 part by weight of the lipid.

[0059] In one embodiment, the amount of the polylactate used may be 0.1 to 100 parts by weight, 0.1 to 80 parts by weight, 0.1 to 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 10 parts by weight, 0.1 to 5 parts by weight, 0.5 to 100 parts by weight, 0.5 to 80 parts by weight, 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 1 to 100 parts by weight, 1 to 80 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 2 to 100 parts by weight, 2 to 80 parts by weight, 2 to 50 parts by weight, or 2 to 30 parts by weight, relative to 1 part by weight of the lipid of the present invention.

[0060] In one embodiment, the composition of the present invention may further include an aqueous solution, a water-miscible organic solvent, or a combination thereof. The "aqueous solution" may be used in the same sense as an aqueous solution, and may refer to, for example, water, a sterile solution, a buffer solution, an injection solution, or the like, and may further include a buffer solution containing an organic acid. The aqueous solution may be, for example, a citrate buffer solution, a PBS buffer solution, or the like, but is not limited thereto. The "water-miscible organic solvent" may be, for example, a C1-C4 lower alcohol, acetone, acetonitrile, a mixture of these with water, or a mixture of these, or the like, but is not limited thereto.

[0061] In one embodiment, the composition of the present invention may further include a stabilizer suitable for improving the stability of the active ingredient. The stabilizer may include, but is not limited to, a pH regulator, an inorganic salt, a sugar, a surfactant, a chelating agent, etc. The "sugar" refers to monosaccharides, disaccharides, sugar alcohols which are reduced sugars, polymers of single or mixed polysaccharides, etc., and the polysaccharide refers to trisaccharides or higher. The monosaccharides may include, for example, mannose, glucose, arabinose, fructose, galactose, etc.; the disaccharides may include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, melibiose, etc.; the sugar alcohols may include mannitol, sorbitol, xylitol, erythritol, maltitol, etc.; the polysaccharides may include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, oligosaccharides, etc. The "regulating agent" may be, but is not limited to, Tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate, or a combination thereof, and the "surfactant" may be, but is not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroyl sarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, sodium glycodeoxycholic acid, benzalkonium chloride, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polysorbate 20, 40, 60, 65, and 80, or a combination thereof. The "chelating agent" may be, but is not limited to, citric acid, polyphenolic acid, EDTA, DTPA, EDDHA, or a combination thereof. The "inorganic salt" refers to a salt of a monovalent or divalent metal, such as NaCl, KCl, MgCl 2 , CaCl 2 , MgSO 4 , CaSO4 , CaCO 3 , MgCO 3 It may be, but is not limited to, the following.

[0062] The present invention will now be described in more detail with reference to the following examples. However, these examples are for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention in any way.

[0063] Working Example Synthesis of lipid of formula (1) (2OA-PAMAM) (1) Preparation of dimethyl 3,3'-((2-((tert-butoxycarbonyl)amino)ethyl)azanediyl)dipropionate (Boc-2MeO-PAMAM) [Boc-2MeO-PAMAM] [ka] In a 100 mL 3-neck round bottom flask (RBF) (reactor 1), N-tert-butoxycarbonyl (N-Boc)ethylenediamine (4 g, 24.97 mmol, 1 eq) and methanol (MEOH) (30 mL) were added and cooled to 0° C., after which methyl acrylate (8.60 g, 99.87 mmol, 4 eq) was added dropwise to reactor 1. The mixture was then stirred at room temperature for 24 h and concentrated under vacuum [yield: 8.25 g (99%)].

[0064] (2) Preparation of tert-butyl (2-(bis(3-(((Z)-octadec-9-en-yl-1-yl)amino)-3-oxopropyl)amino)ethyl)carbamate (Boc-2OA-PAMAM) [Boc-2OA-PAMAM] [ka] Boc-2MeO-PAMAM (4 g, 12.04 mmol, 1 eq) and oleylamine (9.66 g, 36.12 mmol, 3 eq) prepared in (1) were added to a 100 mL three-necked round-bottom flask (RBF) (reactor 1), and reacted with stirring at 80° C. for 6 days. Next, the developing solvent was changed from 100% ethyl acetate (EA) to EA:MEOH (20:1) using a silica gel column for purification [yield: 4.6 g (48%)].

[0065] (3) Preparation of 3,3'-((2-aminoethyl)azanediyl)bis(N-((Z)-octadec-9-en-yl-1-yl)propanamide) (2OA-PAMAM) [2OA-PAMAM] [ka] Into a 100 mL three-neck round-bottom flask (RBF) (Reactor 1), Boc-2OA-PAMAM (2.2 g, 2.74 mmol, 1 eq) prepared in (2) above and dichloromethane (MC) (18 mL) were added and cooled to 0° C. Trifluoroacetic acid (2 mL) was added dropwise to Reactor 1, and the mixture was stirred at 25° C. for 12 hours. The mixture was then diluted with MC (200 mL) and saturated NaHCO 3 The organic layer was removed and extracted with Na 2 SO 4 The water was removed by evaporating the solvent. The final product was then purified using a silica gel column with EA:MEOH (1:2) as the developing solvent. The NMR data of the obtained final product is shown in Figure 1 [Yield: 0.79g (41%)].

[0066] Preparation of the Composition and Pulmonary Delivery Testing (1) Preparation of solutions of each component The components shown in Table 1 below were dissolved in each dilution solvent to prepare solutions with the concentrations shown in Table 1 below. The dissolution was performed using an ultrasonic generator for about 5 to 10 minutes, and the absence of undissolved particles was confirmed visually before use. For dioleoylphosphatidylethanolamine (DOPE) and cholesterol, the solutions were incubated in a 65°C oven for about 5 minutes, and the absence of precipitation was confirmed visually before use in the experiment.

[0067] [Table 1] [mPEG-PLA(2K-4K): A copolymer of monomethoxypolyethylene glycol (mPEG) block with a number average molecular weight of 2,000 and polylactic acid (PLA) block with a number average molecular weight of 4,000]

[0068] (2) Preparation of the composition The necessary amounts of each component were mixed so that the N / P ratio (amine group of lipid component / phosphate group of mRNA) was 20, 2OA-PAMAM:mPEG-PLA(2K-4K):DOPE:cholesterol = 5:5:1:4. Ethanol was added to the ethanol layer so that the total molecular weight of all components was 6.25-12.5 mM, and the aqueous phase and the ethanol phase were mixed in a ratio of 3:1. After mixing, the total amount was diluted 20 times with PBS to reduce the total ethanol content, and concentrated using an Amicon-Ultra tube filter (Merk Millipore, UFC505096 or UFC805024, pore size: 50K, volume: 0.5 mL or 4 mL). The buffer used for the aqueous phase was 20 mM sodium acetate buffer (pH 4.6) (this was prepared by diluting 3 M sodium acetate buffer to 20 mM and titrating to pH 4.6 with 1 M HCl). A more specific procedure for preparing the composition is as follows: 1) Two autoclaved tubes were prepared (tubes (A) and (B)). 2) To tube (A), 2OA-PAMAM, DOPE, cholesterol, and mPEG-PLA (2K-4K) were added in that order in molar amounts calculated according to the experimental conditions, and mixed by vortexing. 3) In the ethanol phase, ethanol was added as necessary so that the total molecular weight of all components was within 12.5 mM (in the case of mPEG-PLA, at least 3% water is required for complete dissolution, so if the solution became cloudy after adding ethanol, a minimum amount of water was added to dissolve it). 4) In tube (B), mRNA was mixed with 20 mM sodium acetate buffer (pH 4.6), with the aqueous phase added in such a way that the total volume was three times that of the ethanol phase. 5) The solution in tube (B) was transferred to tube (A) and mixed. At this time, mixing was performed as quickly as possible to obtain a homogenous preparation, and then the mixture was mixed with a vortex. 6) The prepared preparation was diluted with PBS to a total volume of 20 times, so that the ethanol content was 5% or less. 7) After dilution, the mixture was centrifuged using an Amicon Ultra Centrifugal Filter Unit (50K) and concentrated to the desired volume depending on the purpose.

[0069] (3) Administration of the composition The preparation prepared according to the above method was adjusted to 2 μg / 200 μL based on mRNA and administered intravenously to mice. After 4 hours, luciferin dissolved in sterile water was adjusted to 15 μg / μL and administered intraperitoneally so that 3 mg of luciferin was administered per 20 g of mouse. 15 minutes after intraperitoneal administration of luciferin, the results of protein expression were measured using a luminescence measurement imaging system and are shown in Figure 2.

[0070] As can be seen from FIG. 2, the drug-containing nanoparticle formulation according to the present invention had excellent pulmonary delivery efficiency when administered intravenously.

[0071] On the other hand, as a comparative example, LNP was produced using SM102 [heptadecan-9-yl 8-((2-hydroxyethyl)(6-oxo-6-(undecyloxy)hexyl)amino)octanoate, purchased from SINOPEG] and D-Lin-MC3-DMA (dilinoleylmethyl-4-dimethylaminobutyrate, purchased from MedChemExpress), and the same amount of mRNA (2 μg / 200 μL) as in the example was intravenously administered to a mouse. After 4 hours, imaging was performed in the same manner as in the example, and the results are shown in FIG. 2. As can be seen from FIG. 2, the formulation of the comparative example was delivered to the liver when administered intravenously.

Claims

1. The drug-containing nanoparticles include A drug as an active ingredient; an amphiphilic block copolymer; and 【Chemistry 1】 (wherein n is an integer from 1 to 6; R 1 and R 2 are each independently selected from the group consisting of unsaturated hydrocarbon groups having 12 to 26 carbon atoms; Including, A nanoparticle composition for pulmonary drug delivery, wherein the drug is complexed with the lipid, and the complex is encapsulated within a nanoparticle structure formed by the amphiphilic block copolymer.

2. The nanoparticle composition for pulmonary drug delivery according to claim 1, wherein n is an integer from 2 to 4.

3. R 1 and R 2 Each independently represents -(C 5 -C 12 alkylene)-CH=CH-(C 5 -C 12 The nanoparticle composition for pulmonary drug delivery according to claim 1, wherein the aryl group is aryl, aryl, or aryl.

4. R 1 and R 2 Each independently represents -(C 6 -C 10 alkylene)-CH=CH-(C 6 -C 10 The nanoparticle composition for pulmonary drug delivery according to claim 1, wherein the aryl group is aryl, aryl, or aryl.

5. R 1 and R 2 is -(CH 2 ) 8 CH=CH(CH 2 ) 7 CH 3 2. The nanoparticle composition for pulmonary drug delivery according to claim 1, wherein

6. 2. The nanoparticle composition for pulmonary drug delivery of claim 1, wherein the lipid has the following structure: 【Chemistry 2】

7. Lipids, (1) reacting a compound of the following formula (a) with methyl acrylate to obtain a compound of the following formula (b): (2) reacting a compound of the following formula (b) with a compound of the following formula (c-1) or with a compound of the following formula (c-1) and a compound of the following formula (c-2) in sequence to obtain a compound of the following formula (d-1) or (d-2), respectively; and (3) a step of deprotecting a compound represented by the following formula (d-1) or (d-2):

2. The nanoparticle composition for pulmonary drug delivery of claim 1, which is produced by a method comprising: 【Chemistry 3】 (wherein P is a protecting group, n is an integer from 1 to 6, and R 1 and R 2 are each independently selected from the group consisting of unsaturated hydrocarbon groups having 12 to 26 carbon atoms.

8. The nanoparticle composition for pulmonary drug delivery according to any one of claims 1 to 7, wherein the drug-containing nanoparticle further comprises a polylactate.

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