Nanoparticle compositions for pulmonary drug delivery

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 lung by including drug-containing nanoparticles in the form of nanoparticles formed by an amphiphilic block copolymer, and encapsulating a complex of an anionic drug and a specific lipid within the nanoparticles. [Background technology]

[0002] In the treatment of anionic drugs, including nucleic acids, safe and efficient drug delivery technologies have long been studied, 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, cationic polymers, etc. Viral delivery vehicles are known to pose many challenges to commercialization due to risks such as nonspecific immune responses and complex manufacturing processes. Therefore, recent research has focused on the use of non-viral delivery vehicles to address these drawbacks. Non-viral delivery vehicles have the advantages of fewer side effects in terms of in vivo safety and lower manufacturing costs compared to viral delivery vehicles.

[0003] Typical non-viral delivery vehicles for delivering nucleic acid substances are 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, stabilizing the anionic drugs and increasing their intracellular delivery, and thus have been the subject of extensive research (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 body fluids or aqueous solutions, and a pharmaceutical composition in which a poorly soluble drug is contained within polymer nanoparticles formed from 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] An object of the present invention is to provide a drug delivery composition useful for specifically delivering drugs to the lungs. [Means for solving the problem]

[0008] 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 of 1 to 6, and R1 and R2 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 a nanoparticle structure formed by the amphiphilic block copolymer.

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

[0010] In one embodiment, in the formula (1), R and R are each independently -(C-C 12 alkylene)-CH=CH-(C5-C 12 alkyl). More specifically, in the formula (1), R1 and R2 are each independently -(C6-C 10 alkylene)-CH=CH-(C6-C 10 alkyl). More specifically, in the formula (1), R1 and R2 may be -(CH2)8CH=CH(CH2)7CH3.

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

[0012] In one embodiment, the lipid is prepared by a process comprising: (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 succession 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] (In the formula, P is a protecting group, n is an integer of 1 to 6, and R1 and R2 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. [Effects of the Invention]

[0014] The compositions of the present invention are highly useful for delivering drugs specifically to the lungs encapsulated within nanoparticles formed by amphiphilic block copolymers. [Brief explanation 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 an example of the present invention. [Figure 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 THE INVENTION

[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] (wherein n is an integer of 1 to 6, more specifically an integer of 2 to 6, and R1 and R2 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, n may be an integer of 2 to 4 in the formula (1).

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

[0020] More specifically, in the formula (1), R1 and R2 each independently represent -(C6-C 10 alkylene)-CH=CH-(C6-C 10 alkyl).

[0021] The "alkylene" and "alkyl" may each independently be unsubstituted or substituted, and in this case, examples of the substituent include, but are not limited to, a hydroxy group, a halogen group, or a C1-C4 alkyl group.

[0022] More specifically, in the formula (1), R1 and R2 may be -(CH2)8CH=CH(CH2)7CH3.

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

[0024] In one embodiment, the lipid is prepared by a process comprising: (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 succession 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] (In the formula, P is a protecting group, more specifically, a t-butoxycarbonyl group (t-BOC), and n, R1, and R2 are defined as in formula (1).)

[0025] The overall synthetic scheme for the method of 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 drug-lipid complex 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 term "polypeptide" may refer to a protein that has activity in vivo, 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 the 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 the 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, and the polydispersity index 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, or 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 similar 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 these levels is preferable in terms of the stability of the nanoparticle structure, the amount of ingredients and bioavailability, and 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, respectively.

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

[0037] When the active ingredient is a nucleic acid, the lipid and nucleic acid bind to each other through electrostatic interaction to form a complex. In one embodiment, the charge ratio between 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, or 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. If the ratio (N / P) is larger than the above range, toxicity may be induced. Furthermore, the N / P ratio may play an important role in spleen-specific expression of the active ingredient.

[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, which forms, in an aqueous environment, core-shell polymeric nanoparticles 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, a copolymer 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, but is not limited to, 200 or more, 500 or more, 1,000 or more, or 2,000 or more, and 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less.

[0042] Furthermore, if necessary, functional groups, ligands, or functional groups capable of targeting specific tissues or cells can be chemically bonded to the ends of the hydrophilic A block to regulate the biodistribution of polymeric nanoparticle delivery vehicles formed from an amphiphilic block copolymer and polylactate, or to enhance the intracellular delivery efficiency of the nanoparticle delivery vehicles. In one embodiment, the functional groups or ligands 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 groups or ligands 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, a copolymer of polylactide and glycolide, a copolymer of polylactide and polydioxane-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

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

[0046] For example, the number average molecular weight combination of the hydrophilic A block and the 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 bonding the terminal hydroxy group of the hydrophobic B block with tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms 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 and stabilizing the nanoparticle while also effectively evading the reticuloendothelial system (RES) in the body. That is, the carboxylate anions in polylactate bind to cation complexes more effectively than polylactic acid, reducing the surface potential of the polymeric nanoparticles. This reduces the positive charge on the surface potential of the polymeric nanoparticles compared to polymeric nanoparticles without polylactate, making them less likely to be captured by the reticuloendothelial system and potentially more efficiently delivered to target sites (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 be present in the core (inner wall) of the nanoparticle, whereas if it exceeds 50,000, the size of the polymer nanoparticle may become too large.

[0050] In one embodiment, the terminal end of the polylactate (e.g., sodium salt of polylactic acid) opposite the terminal metal carboxylate (e.g., sodium carboxylate) 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) (In the formula, A is -COO-CHZ-; B is -COO-CHY-, -COO-CHCHCHCHCH-, or -COO-CHCHOCH-; 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 of 1 to 30; and m is an integer of 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] wherein 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; and M is independently Na, K, or Li. SO-PAD-COO-Q (5) [Wherein S is [ka] {wherein L is -NR1- or -O- (wherein R1 is a hydrogen atom or C1- 10is alkyl.) where a is an integer from 0 to 4; b is an integer from 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-dioxan-2-one; Q is CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, or CH2C6H5. [Chemical formula] [In the formula, R' is -PAD-O-C(O)-CH2CH2-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 -(CH2) 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 comprise a fusogenic lipid to increase the efficiency of intracellular delivery of a drug (eg, mRNA).

[0054] When mixed with a drug (e.g., mRNA) and a lipid complex of the present invention, the fusogenic lipid forms a drug (e.g., mRNA) / lipid of the present invention / fusogenic lipid complex through hydrophobic interactions, and the complex containing the fusogenic lipid is encapsulated within 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 bound to one or two C10-24 fatty acids. The cholesterol and tocopherol include their analogs, derivatives, and metabolites.

[0056] More specifically, the fusogenic lipid is selected from the group consisting of 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, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, 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, dioleo ... The phosphatidylcholine may be one or a combination of two or more selected from the group consisting of dilauroylphosphatidic acid, dimyristoylphosphatidic acid, 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 comprise an aqueous solution, a water-miscible organic solvent, or a combination thereof. The term "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 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 to these. The "water-miscible organic solvent" may be, for example, a C1-C4 lower alcohol, acetone, acetonitrile, a water mixture thereof, or a mixture thereof, but is not limited to these.

[0061] In one embodiment, the composition of the present invention may further contain a stabilizer suitable for improving the stability of the active ingredient. Examples of the stabilizer include, but are not limited to, pH adjusters, inorganic salts, sugars, surfactants, and chelating agents. The term "saccharides" refers to monosaccharides, disaccharides, their reducing sugars, sugar alcohols, and polymers of single or mixed polysaccharides. Polysaccharides refer to trisaccharides or higher. Examples of monosaccharides include mannose, glucose, arabinose, fructose, and galactose. Examples of disaccharides include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, and melibiose. Examples of sugar alcohols include mannitol, sorbitol, xylitol, erythritol, and maltitol. Examples of polysaccharides include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharides. The "modulating agent" may be, but is not limited to, Tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate, or a combination thereof. The "surfactant" may be, but is not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acid sodium salt, 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, and may be, but is not limited to, NaCl, KCl, MgCl2, CaCl2, MgSO4, CaSO4, CaCO3, MgCO3, etc.

[0062] The present invention will be described in more detail below with reference to the following examples, but these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0063] Example Synthesis of lipid (2OA-PAMAM) of formula (1) (1) Preparation of dimethyl 3,3'-((2-((tert-butoxycarbonyl)amino)ethyl)azanediyl)dipropionate (Boc-2MeO-PAMAM) [Boc-2MeO-PAMAM] [ka] N-tert-butoxycarbonyl (N-Boc)ethylenediamine (4 g, 24.97 mmol, 1 eq) and methanol (MEOH) (30 mL) were added to a 100 mL three-necked round-bottom flask (RBF) (Reactor 1). After cooling to 0° C., 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 hours and concentrated under vacuum [yield: 8.25 g (99%)].

[0064] (2) Preparation of tert-butyl (2-(bis(3-(((Z)-octadec-9-en-yl1-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) above were added to a 100 mL three-necked round-bottom flask (RBF) (Reactor 1), and the mixture was allowed to react with stirring at 80°C for 6 days. The mixture was then purified using a silica gel column, changing the developing solvent from 100% ethyl acetate (EA) to EA:MEOH (20:1) [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] A 100 mL three-neck round-bottom flask (RBF) (Reactor 1) was charged with Boc-2OA-PAMAM (2.2 g, 2.74 mmol, 1 eq) prepared in (2) above and dichloromethane (MC) (18 mL) 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 h. The mixture was then diluted with MC (200 mL) and extracted three times with saturated aqueous NaHCO (200 mL). The organic layer was removed, dehydrated with NaSO, and the solvent was evaporated. 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 resulting product are shown in Figure 1 [Yield: 0.79 g (41%)].

[0066] Composition Preparation 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 approximately 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 approximately 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) blocks with a number-average molecular weight of 2,000 and polylactic acid (PLA) blocks with a number-average molecular weight of 4,000]

[0068] (2) Preparation of the composition The necessary amounts of each component were mixed to achieve an N / P ratio (amine groups in lipid components / phosphate groups in mRNA) of 20, with 2OA-PAMAM:mPEG-PLA(2K-4K):DOPE:cholesterol = 5:5:1:4. Ethanol was added to the ethanol layer to adjust the total molecular weight of all components to 6.25–12.5 mM, and the aqueous and ethanol phases were mixed at a 3:1 ratio. After mixing, the total volume was diluted 20-fold with PBS to reduce the total ethanol content, and then concentrated using an Amicon-Ultra tube filter (Merck 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) (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 sterilized autoclaved tubes were prepared (tubes (A) and (B)). 2) 2OA-PAMAM, DOPE, cholesterol, and mPEG-PLA (2K-4K) were added in the molar amounts calculated according to the experimental conditions to tube (A) in that order, 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 carried out as quickly as possible to obtain a uniform preparation, and then the mixture was mixed using a vortex mixer. 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 solution 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 formulation prepared according to the above method was adjusted to 2 μg / 200 μL based on mRNA and administered intravenously to mice. Four hours later, luciferin dissolved in sterile water was adjusted to 15 μg / μL and administered intraperitoneally to give 3 mg of luciferin per 20 g of mouse. 15 minutes after intraperitoneal administration of luciferin, protein expression was measured using a luminescence imaging system and the results 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, LNPs were prepared 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 administered intravenously to mice. After 4 hours, imaging was performed using the same method as in the example, and the results are shown in Figure 2. As can be seen from Figure 2, the comparative formulation was delivered to the liver upon intravenous administration.

Claims

1. The drug-containing nanoparticles include: A drug as an active ingredient; an amphiphilic block copolymer; and a compound represented by the following formula (1): 【Chemistry 1】 (wherein 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; Including, A nanoparticle composition for pulmonary drug delivery, wherein the drug forms a complex with the lipid, and the complex is encapsulated within a nanoparticle structure formed by the amphiphilic block copolymer.

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

3. R 1 and R 2 are each independently -(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, ...

4. R 1 and R 2 are each independently -(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, ...

5. R 1 and R 2 is -(CH 2 ) 8 CH=CH(CH 2 ) 7 CH 3 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. The nanoparticle composition for pulmonary drug delivery according to any one of claims 1 to 6, wherein the drug-containing nanoparticles further comprise a polylactate.

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