Kit for producing nanoparticle composition containing polylactate for drug delivery

Abstract

The present invention relates to a kit for producing a nanoparticle composition for drug delivery, and more specifically, to a kit for producing a nanoparticle composition for drug delivery that is designed to easily form nanoparticles having a drug encapsulated therein by simply mixing the kit components, which are an amphiphilic block copolymer, a cationic compound, a polylactate, and a drug.

Description

[Technical Field]

[0001] The present invention relates to a kit for producing a nanoparticle composition for drug delivery, and more specifically, to a kit for producing a nanoparticle composition for drug delivery that is designed to easily form nanoparticles having a drug encapsulated therein by simply mixing the kit components, which are an amphiphilic block copolymer, a cationic compound, a polylactate, and a drug. [Background technology]

[0002] Safe and efficient drug delivery technologies for treatment using anionic drugs, including nucleic acids, have long been studied, and various delivery vehicles and delivery technologies have been developed. Delivery vehicles are broadly divided into viral delivery vehicles using adenoviruses or retroviruses, etc., and non-viral delivery vehicles using cationic lipids and cationic polymers, etc. Technologies using viral delivery vehicles are known to pose many challenges to commercialization due to risks such as non-specific immune responses and complex production processes. Therefore, recent research has focused on non-viral delivery vehicles to overcome these disadvantages. Compared to viral delivery vehicles, non-viral delivery vehicles have the advantages of fewer side effects in terms of in vivo safety and lower production costs in terms of economy.

[0003] The most representative examples of non-viral delivery vehicles used for nucleic acid delivery include cationic lipid-nucleic acid complexes (lipoplexes) and polycationic polymer-nucleic acid complexes (polyplexes). These cationic lipids or polycationic polymers form complexes with anionic drugs through electrostatic interactions, thereby stabilizing the anionic drugs and promoting their delivery into cells, and thus have been the subject of much research (Non-Patent Documents 1 and 2).

[0004] However, nanoparticles formed by such complexes often lose stability easily depending on the storage environment, making them vulnerable to long-term storage and risking quality damage during transportation.Furthermore, they require complex manufacturing processes to ensure sufficient stability, making them very difficult to manufacture.

[0005] Therefore, there is a need for the development of a kit for producing nanoparticle compositions for drug delivery that is not significantly affected by the storage environment and that can be easily used by end consumers. [Prior art documents] [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] The object of the present invention is to provide a kit for producing a nanoparticle composition for drug delivery, which can be easily used by the end consumer because drug-containing nanoparticles can be easily formed by simply mixing the components of the kit, and which can effectively deliver drugs into the body without being affected by the storage or transportation environment because drug-containing nanoparticles can be easily formed just before use. [Means for solving the problem]

[0008] To solve the above problem, the present invention provides a kit for producing a nanoparticle composition, comprising: a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof. In one embodiment, the kit is for forming nanoparticles that deliver intracellular active ingredients. In one embodiment, one or more of the first and second chambers further comprises an additional solvent. In one embodiment, the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof. In one embodiment, the second chamber further comprises a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent, or a combination thereof. In one embodiment, the amount of the amphiphilic block copolymer may be 0.01 to 50 parts by weight relative to 1 part by weight of the cationic compound. In one embodiment, the amount of the polylactate may be 0.1 to 100 parts by weight relative to 1 part by weight of the cationic compound. In one embodiment, the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactate in the first chamber may be filtered one or more times with a hydrophilic filter after mixing. [Effects of the Invention]

[0009] The kit for preparing a nanoparticle composition according to the present invention contains components for forming drug-containing nanoparticles separated in separate chambers, and therefore, unlike pre-formed nanoparticles, is not affected by the storage or transportation environment. By using the kit, the end user can successfully form nanoparticles with effective drug delivery effects simply by mixing the components in the chambers. [Brief explanation of the drawings]

[0010] [Figure 1] 1 is a graph showing the experimental results of intracellular delivery efficiency of nanoparticles carried out in Experimental Example 2 of the present invention. [Figure 2] 10 is an image showing the results of confirming the formation of nanoparticles by agarose gel analysis in Experimental Example 3 of the present invention. [Figure 3] 1 is a graph showing the results of evaluating the formation of nanoparticles carried out in Experimental Example 3 of the present invention using a dynamic light scattering method. [Figure 4] 1 is a graph showing the experimental results of intracellular delivery efficiency of nanoparticles carried out in Experimental Example 4 of the present invention. [Figure 5] 1 is a graph showing the experimental results of intracellular delivery efficiency before and after an accelerated test carried out in Experimental Example 5 of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0011] The present invention will now be described in more detail. The kit for producing a nanoparticle composition according to the present invention comprises a first chamber containing an amphiphilic block copolymer, a cationic compound and a polylactate; and a second chamber containing an active ingredient selected from a nucleic acid, a polypeptide, a virus or a combination thereof.

[0012] The kit of the present invention is composed of two or more chambers, and the end user can easily form nanoparticles by simply mixing the chambers. The term "simple mixing" can include any act of "mixing" and means that the act of mixing to form nanoparticles is not affected by specific conditions. The mixing can be achieved by various methods, such as, but not limited to, dropping, vortexing, and decanting. According to one embodiment, when using the kit of the present invention, 90% or more, 95% or more, or 99% or more of the theoretically formable amount of nanoparticles can be formed quickly, for example, within 1 minute, 30 seconds, or 15 seconds.

[0013] The active ingredient of the nanoparticles, which are formed by simple mixing by the end user, can form a complex with the cationic compound through electrostatic interaction, and the complex can be encapsulated inside the nanoparticle structure formed by the amphiphilic block copolymer and polylactate.

[0014] In the nanoparticles, in an aqueous environment, the hydrophilic portion of the amphiphilic block copolymer forms the outer wall of the nanoparticle, while the hydrophobic portion of the amphiphilic block copolymer and the polylactate salt contained as a separate component from the amphiphilic block copolymer form the inner wall of the nanoparticle, thereby encapsulating a complex of an active ingredient and a cationic compound within the formed nanoparticle. Such a nanoparticle structure improves the stability of the active ingredient in blood or body fluids.

[0015] 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.

[0016] The term "polypeptide" refers to 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 the polypeptide sequence of an antigen, an analog thereof, or a precursor thereof. The "virus" may be an oncolytic virus, such as one or more selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus (HSV), and vesicular stomatitis virus (VSV). In one embodiment, the oncolytic virus is an adenovirus. The adenovirus used in the embodiments of the present invention contains a luciferase gene that can be confirmed by imaging.

[0017] The virus can express several types of therapeutic genes in the body of an individual and is not limited to a specific molecular weight, protein, biological activity, or therapeutic field. The prophylactic virus can induce immunity in the subject's body against the target disease. A composition containing a disease-preventing virus has the advantages of reducing immune induction by the virus itself, specifying or expanding target cells, and reducing hyperimmune responses to the virus upon re-administration, resulting in effective effects with multiple inoculations.

[0018] In one embodiment, the particle size of the nanoparticles can be defined as a Z-average value. For example, the particle size may be 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 150 nm or less, or 10 nm or more, 50 nm or more, or 100 nm or more. In one embodiment, the particle size of the nanoparticles defined as a Z-average value may be, for example, 10 to 800 nm, 10 to 600 nm, 10 to 500 nm, 10 to 400 nm, 10 to 300 nm, 10 to 200 nm, or 10 to 150 nm.

[0019] 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 simple disperse particle distribution, with a polydispersity index of, for example, 0.01 to 0.30, 0.05 to 0.25, or 0.1 to 0.2.

[0020] In one embodiment, the surface charge of the nanoparticles may be, for example, -40 mV or more, -30 mV or more, -20 mV or more, or -10 mV or more, or may be 40 mV or less, 30 mV or less, 20 mV or less, or 10 mV or less. In one embodiment, the surface charge of the nanoparticles may be, for example, -40 to 40 mV, -30 to 30 mV, -20 to 20 mV, or -10 to 10 mV. The surface charge can 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).

[0021] Maintaining the above-mentioned particle size and surface charge of the nanoparticles is preferable in terms of the stability of the nanoparticle structure, the content of the constituent components, the degree of absorption in the body, and the convenience 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.

[0022] The active ingredient may be present in an amount of, for example, 30% by weight or less, 25% by weight or less, 20% by weight or less, 15% by weight or less, 10% by weight or less, or 5% by weight or less, or 0.001% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, 0.25% by weight or more, 0.5% by weight or more, or 1% by weight or more, based on the weight of the total composition. In one embodiment, the active ingredient may be present in an amount of, 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 weight of the total composition. If the content of the active ingredient is less than the above range, the amount of delivery vehicle used relative to the drug may be too high, which may result in side effects due to the delivery vehicle. If the content exceeds the above range, the size of the nanoparticles may become too large, reducing their stability and increasing the loss rate during filter sterilization. When the active ingredient is a virus, the nanoparticles contain 1×10 6 ~1×10 14 VP (viral particle), 1 × 10 7 ~1×10 13 VP, 1×10 8 ~1×10 12 VP or 1 x 10 9 ~1×10 11 It can contain VPs.

[0023] In certain embodiments, the cationic compound may be a cationic lipid or a cationic polymer, more particularly a cationic lipid.

[0024] In one embodiment, the cationic lipid is N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), N,N,N-trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA), 1,2-diacyl-3-trimethylammonium-propane (TAP), 1,2-diacyl-3-dimethylammonium-propane (DAP), 3β-[N-(N',N',N'-trimethylaminoethoxy)propyl]-N,N,N-trimethylaminoethoxy] ...

[0039] The present invention may be directed to a combination of one or more selected from the group consisting of 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), cholesteryloxypropan-1-amine (COPA), N-(N'-aminoethane)carbamoylpropanoic acid tocopherol (AC-tocopherol), and N-(N'-methylaminoethane)carbamoylpropanoic acid tocopherol (MC-tocopherol).

[0025] When using such cationic lipids, it is preferable to use fewer polycationic lipids with a high intramolecular charge density in order to reduce toxicity induced by the cationic lipids. More specifically, it is preferable to use cationic lipids having one functional group per molecule that can exhibit a positive charge in aqueous solution.

[0026] Thus, in a preferred embodiment, the cationic lipid may be one or more selected from the group consisting of 3β-[N-(N',N',N'-trimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), N-(1-(2,3-dioleoyloxy)propyl-N,N,N-trimethylammonium chloride (DOTAP), N,N-dimethyl-(2,3-dioleoyloxy)propylamine (DODMA), and N,N,N-trimethyl-(2,3-dioleoyloxy)propylamine (DOTMA).

[0027] Meanwhile, in one embodiment, the cationic polymer may be selected from the group consisting of chitosan, glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethyleneimine, dextran, hyaluronic acid, albumin, high molecular weight polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm), more specifically, one or more selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).

[0028] In one embodiment, the cationic lipid has the following formula (1): [ka] (wherein n and m are each independently 0 to 12, provided that 2≦n+m≦12; a and b are each independently 1 to 6; and R1 and R2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms).

[0029] More specifically, in the formula (1), n ​​and m are each independently 1 to 9, and may be 2≦n+m≦10.

[0030] More specifically, in the formula (1), a and b may each independently be 2 to 4.

[0031] More specifically, in the formula (1), R1 and R2 can each independently be selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, celloyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and celloyl.

[0032] In one embodiment, the cationic lipid is 1,6-dioleoyltriethylenetetramide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))oleamide), 1,8-dilinoleoyltetraethylenepentamide ((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyristoleoyl and 1,10-dioleoylpentaethylenehexamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)dioleamide).

[0033] The content of the cationic compound in the composition formed by the kit of the present invention may be, for example, 25 parts by weight or less, 20 parts by weight or less, 18 parts by weight or less, 15 parts by weight or less, 12 parts by weight or less, 10 parts by weight or less, or 8 parts by weight or less, or may be 1 part by weight or more, 1.5 parts by weight or more, 2 parts by weight or more, 2.5 parts by weight or more, 3 parts by weight or more, or 3.5 parts by weight or more, per part by weight of the active ingredient. In one embodiment, the content of the cationic compound in the composition may be 1 to 25 parts by weight, 1.5 to 10 parts by weight, 2 to 15 parts by weight, 2.5 to 10 parts by weight, or 3 to 8 parts by weight, per part by weight of the active ingredient. On the other hand, when a virus, more specifically an adenovirus, is used as the active ingredient, the content of the cationic compound in the composition may be 1 to 25 parts by weight, 1.5 to 10 parts by weight, 2 to 15 parts by weight, 2.5 to 10 parts by weight, or 3 to 8 parts by weight, per part by weight of the active ingredient. 10 The content of the cationic compound relative to the VP may be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, or 150 μg or less, 100 μg or less, 50 μg or less, or 30 μg or less, for example, 1 μg to 150 μg, 5 μg to 100 μg, 10 μg to 50 μg, or 15 μg to 30 μg. If the content of the cationic compound in the composition is less than the above range, a stable complex with the active ingredient may not be formed. If the content is greater than the above range, the size of the nanoparticles may be too large, reducing stability and increasing the loss rate during filter sterilization.

[0034] When the active ingredient is a nucleic acid, the cationic compound and the 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 cationic compound (N) (N / P; the ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) may be 0.5 or more, 1 or more, 2 or more, or 3.5 or more, or 100 or less, 50 or less, or 20 or less, for example, 0.5 to 100, 1 to 50, 2 to 20, 5 to 15, or 7 to 12. If the ratio (N / P) is less than 0.5, it may be difficult to form a complex containing a sufficient amount of nucleic acid. If the ratio (N / P) is more than 100, there is a risk of inducing toxicity. The N / P value may also play an important role in the specific expression of the active ingredient in the spleen.

[0035] In one 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 solution, 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).

[0036] 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.

[0037] More specifically, the hydrophilic A block may be one or more selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0038] In one embodiment, the hydrophilic A block may have a number average molecular weight of 200 to 50,000 daltons, more specifically 1,000 to 20,000 daltons, and even more specifically 1,000 to 5,000 daltons.

[0039] If necessary, functional groups or ligands capable of reaching specific tissues or cells, or functional groups capable of promoting intracellular delivery, can be chemically bound to the ends of the hydrophilic A block to regulate the biodistribution of polymeric nanoparticles formed from amphiphilic block copolymers and polylactate or to increase the efficiency of intracellular delivery of the nanoparticles. 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.

[0040] In one embodiment, the hydrophobic B block may be a biocompatible biodegradable polymer, which may be one or more selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazines.

[0041] More specifically, the hydrophobic B block may be at least one selected from the group consisting of polylactide, 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.

[0042] In one embodiment, the hydrophobic B block may have a number average molecular weight of 50 to 50,000 daltons, more specifically 200 to 20,000 daltons, and even more specifically 1,000 to 5,000 daltons.

[0043] In one embodiment, to increase the hydrophobicity of the hydrophobic B block and thereby improve the stability of the nanoparticles, the hydroxyl group at the end of the hydrophobic B block can be modified by chemically binding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms.

[0044] In one embodiment, the composition ratio of the hydrophilic block (A) to the hydrophobic block (B) of the amphiphilic block copolymer may be in the range of 40 to 70 wt %, more specifically, 50 to 60 wt %, of the hydrophilic block (A) relative to the total weight of the copolymer. If the ratio of the hydrophilic block (A) is less than 40 wt %, the polymer's solubility in water is low, making it difficult to form nanoparticles. To ensure that the copolymer has sufficient water solubility to form nanoparticles, it is advantageous for the ratio of the hydrophilic block (A) to be 40 wt % or more. On the other hand, if the ratio of the hydrophilic block (A) exceeds 70 wt %, the hydrophilicity becomes too high, reducing the stability of the polymer nanoparticles and making them difficult to use as a solubilized composition for an active ingredient / cationic compound complex. Therefore, considering the stability of the nanoparticles, it is advantageous for the ratio of the hydrophilic block (A) to be 70 wt % or less.

[0045] In one embodiment, the polylactate in the composition formed by the kit of the present invention is distributed in the core (inner wall) of the nanoparticles, strengthening the hydrophobicity of the core and thereby stabilizing the nanoparticles, while at the same time effectively evading the reticuloendothelial system (RES) in the body. That is, the carboxylate anion of polylactate binds to cationic complexes more effectively than polylactic acid, reducing the surface potential of the polymer nanoparticles. As a result, the surface potential of the polymer nanoparticles is less positively charged than that of polymer nanoparticles without polylactate. Therefore, they are less likely to be captured by the reticuloendothelial system, resulting in more efficient delivery to target sites (e.g., cancer cells, inflammatory cells, etc.).

[0046] In one embodiment, polylactate, which is included as a component separate from the amphiphilic block copolymer, is a nanoparticle inner wall component and may have a number-average molecular weight of 500 to 50,000 daltons, more specifically, 1,000 to 10,000 daltons. If the number-average molecular weight of polylactate is less than 500 daltons, the hydrophobicity may be too low, making it difficult for polylactate to reside in the core (inner wall) of the nanoparticles. If the number-average molecular weight of polylactate is more than 50,000 daltons, the polymer nanoparticles may have too many particles.

[0047] In one embodiment, the terminal of the polylactate (e.g., polylactate sodium salt) opposite the 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.

[0048] 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 carboxy group, i.e., "C(=O)O"):

[0049] RO-CHZ-[A] m -[B] n -COOM (2) (wherein 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.)

[0050] RO-CHZ-[COO-CHX] p -[COO-CHY'] q -COO-CHZ-COOM (3) (wherein 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.)

[0051] RO-PAD-COO-W-M' (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.

[0052] SO-PAD-COO-Q (5) [Wherein S is [ka] (L is -NR1- or -0-, where R1 is a hydrogen atom or C 1-10 alkyl; a is an integer of 0 to 4; b is an integer of 1 to 10; M is Na, K, or Li); Q is CH3, CH2CH3, CH2CH2CH3, CH2CH2CH2CH3, or CH2C6H5; and PAD is at least one 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.

[0053] [ka] (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; M is Na, K, or Li; and a is an integer from 1 to 4)

[0054] 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; and R is -(CH2) k -, divalent alkenyl having 2 to 10 carbon atoms, divalent aryl having 6 to 20 carbon atoms, or a combination thereof, where k is an integer from 0 to 10)

[0055] In one embodiment, the polylactate may be the compound of formula (2) or formula (3).

[0056] In one embodiment, the kit of the present invention may further contain 0.01 to 50% by weight, more specifically 0.1 to 10% by weight, of a fusogenic lipid based on the total weight of the composition formed by the kit of the present invention in order to enhance the intracellular delivery efficiency of mRNA.

[0057] When the fusogenic lipid is mixed with the complex of mRNA and cationic lipid, it binds to the complex through hydrophobic interactions to form a complex of mRNA, cationic lipid, and fusogenic lipid, and the complex containing the fusogenic lipid is encapsulated inside the nanoparticle structure of the amphiphilic block copolymer.

[0058] In one embodiment, the fusogenic lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.

[0059] More specifically, the phospholipid may be one or more selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid may each have one or two C 10-24 It may be in a form bound to a fatty acid. The cholesterol and tocopherol mentioned above include analogs, derivatives, and metabolites of cholesterol and tocopherol, respectively.

[0060] More specifically, the fusogenic lipid may be dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoleoylphosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine; DPPE), dilinoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; DPPC), dioleoylphosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine; DPPC), or dioleoylphosphocholine (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine; DPPE). 1-palmitoyl-2-oleoylphosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, 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 (and a combination of one or more selected from the group consisting of cholesterol and tocopherol).

[0061] More specifically, the fusogenic lipid may be one or more selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine; DPPC), dioleoylphosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), and dipalmitoleoylphosphoethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine; DPPE).

[0062] In one embodiment, among the components in the composition formed by the kit of the present invention, the content of the amphiphilic block copolymer comprising the hydrophilic block (A) and the hydrophobic block (B) may be 0.01 to 50 parts by weight, 0.01 to 30 parts by weight, 0.01 to 15 parts by weight, 0.01 to 12 parts by weight, 0.1 to 50 parts by weight, 0.1 to 30 parts by weight, 0.1 to 15 parts by weight, 0.1 to 12 parts by weight, 0.5 to 50 parts by weight, 0.5 to 30 parts by weight, 0.5 to 15 parts by weight, 0.5 to 12 parts by weight, 1 to 50 parts by weight, 1 to 30 parts by weight, 1 to 15 parts by weight, or 1 to 12 parts by weight, relative to 1 part by weight of the cationic compound.

[0063] More specifically, the content of the amphiphilic block copolymer can be adjusted within the above range depending on the active ingredient. For example, in one embodiment, when the active ingredient is a virus, the content of the amphiphilic block copolymer can be 3 to 7 parts by weight per 1 part by weight of the cationic compound. In another embodiment, when the active ingredient is a nucleic acid, the content of the amphiphilic block copolymer can be 0.7 to 7 parts by weight, 1 to 5 parts by weight, or 1 to 3 parts by weight per 1 part by weight of the cationic compound.

[0064] In one embodiment, the content of the polylactate among the components in the composition formed by the kit of the present invention 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 cationic compound.

[0065] More specifically, the content of the polylactate can be adjusted within the above range depending on the active ingredient. For example, in one embodiment, when a virus is used as the active ingredient, the content of the polylactate can be 3 to 15 parts by weight per part by weight of the cationic compound. In another embodiment, when a nucleic acid is used as the active ingredient, the content of the polylactate can be 0.2 to 4 parts by weight, 1 to 4 parts by weight, or 0.2 to 0.8 parts by weight per part by weight of the cationic compound.

[0066] In one embodiment, the first chamber and / or the second chamber may further contain an aqueous solution, a water-miscible organic solvent, or a combination thereof. The term "aqueous solution" may be used interchangeably with "aqueous solution," and may refer to, for example, water, sterile water, buffer, injection solution, etc., and may also be a buffer further containing an organic acid. The aqueous solution may be, for example, but is not limited to, citrate buffer, PBS buffer, etc. The "water-miscible organic solvent" may be, for example, but is not limited to, a C1-C4 lower alcohol, acetone, acetonitrile, an aqueous mixture thereof, or a mixture thereof.

[0067] In one embodiment, the mixture of the amphiphilic block copolymer, cationic compound, and polylactate contained in the first chamber can be filtered one or more times through a hydrophilic filter before use. Examples of hydrophilic filter materials include, but are not limited to, nylon, mixed cellulose ester (MCE), polyethylsulfone (PES), polyvinylidene difluoride (PVDF), cellulose acetate (CA), polytetrafluoroethylene (PTFE), and mixtures thereof. When subjected to hydrophilic filtration, the active ingredient can be effectively captured by the nanoparticles, potentially improving the stability of the nanoparticles.

[0068] In one embodiment, the second chamber may further contain a stabilizer suitable for improving the stability of the active ingredient. Examples of stabilizers 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 monosaccharides or mixed polysaccharides, while 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; and examples of polysaccharides include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharides. The "pH-adjusting 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, polyoxyethylene 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, which may be, but is not limited to, NaCl, KCl, MgCl, CaCl, MgSO, CaSO, CaCO, MgCO, etc.

[0069] For example, when a virus is used as the active ingredient, the second chamber may further contain 5-15 mM Tris, 5-15 mM histidine, 50-90 mM NaCl, 2-8% sucrose (w / v), 0.5-1.5 mM MgCl2, 0.005-0.05% (w / v) PS-80, 0.05-0.15 mM EDTA, and 0.1-1.0% ethanol (v / v), and the pH may be 7.0-8.0. In another embodiment, when a nucleic acid is used as the active ingredient, the second chamber may further contain PBS buffer, e.g., a solution containing 2.0-3.5 mM KCl, 1.0-2.5 mM KH2PO4, 125-145 mM NaCl, and 7.5-9.5 mM Na2HPO4 at pH 7.0-8.0.

[0070] The "chamber" may be, but is not limited to, glass, plastic, paper, pack, etc. suitable for containing the nanoparticle material or the solvent containing them.

[0071] The present invention will be described in detail with reference to the following examples, but the examples are for illustrative purposes only and the scope of the present invention is not limited thereby in any way. [Example]

[0072] Preparation Example 1: Preparation of a first chamber composition containing dioTETA / mPEG-PLA-tocopherol (2k-1.7k) / PLANa (1.7k) and formation of nanoparticles (1) Preparation of the First Chamber Composition 20 mg of 1,6-dioleoyltriethylenetetramine (dioTETA) was dissolved in 1 mL of ethanol, 50 mg of monomethoxypolyethylene glycol-polylactide-tocopherol copolymer (mPEG-PLA-tocopherol) (2k-1.7k) was dissolved in 1 mL of 90% ethanol, and 50 mg of polylactic acid sodium salt (PLANa) (1.7k) was dissolved in 50% ethanol. dioTETA, mPEG-PLA-tocopherol (2k-1.7k), and PLANa (1.7k) were mixed according to the ratios shown in Table 1 below, followed by addition of 30x PBS to prepare a complex emulsion. The resulting composition was filtered through a 0.22 μm hydrophilic filter (see Table 1).

[0073] [Table 1]

[0074] (2) Preparation of the second chamber composition containing the oncolytic virus VQAd CMV Luc virus (ViraQuest, lot no. 33088) in the form of an aliquot in A195 buffer (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose (w / v), 1 mM MgCl, 0.02% (w / v) PS-80, 0.1 mM EDTA, 0.5% ethanol (v / v), pH 7.4) was added to 1 × 10 10 I counted and prepared it so that it would be VP.

[0075] (3) Nanoparticle production The first chamber composition and the second chamber composition were vortexed for 10 to 15 seconds immediately before use to mix them together and form nanoparticles.

[0076] Experimental Example 1: Confirmation of the formation of nanoparticles containing oncolytic viruses By mixing the first chamber composition and the second chamber composition immediately before use, it was confirmed whether the nanoparticles of Examples 1 to 4 were formed normally. As a result, in all of Examples 1 to 4, no precipitate was observed even with simple mixing, and it was confirmed that nanoparticles were formed normally.

[0077] Experimental Example 2: Confirmation of intracellular delivery efficiency of nanoparticles containing oncolytic viruses To evaluate the intracellular delivery efficiency of nanoparticles, MDA-MB435 cells with low CAR expression, suitable for evaluating viral delivery efficiency, were prepared. The nanoparticles of Examples 1 to 4 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and then dispensed into the cells in an amount corresponding to 500 moi based on the virus. After further incubation for 15 to 24 hours, luciferin was added to the cells, and the amount of expressed luciferase was measured. A virus (naked Ad; Ad) that was not a nanoparticle was used as a control. The results are shown in Figure 1. As a result, it was observed that all of the nanoparticles of the tested examples showed higher intracellular delivery efficiency compared to the control group.

[0078] Preparation Example 2: Preparation of a first chamber composition containing (dioTETA) / mPEG-PLA(2k-1.7k) / PLANa(1.7k) and formation of nanoparticles (1) Preparation of the First Chamber Composition 20 mg of dioTETA was dissolved in 1 mL of 20 mM sodium acetate buffer (pH 4.6), 10 mg of monomethoxypolyethylene glycol-polylactide copolymer (mPEG-PLA) (2k-1.7k) was dissolved in 1 mL of water, and 10 mg of PLANa (1.7k) was dissolved in 1 mL of water. dioTETA, mPEG-PLA (2k-1.7k), and PLANa (1.7k) were mixed according to the ratios in Table 2 below, and the resulting composition was then filtered through a 0.22 μm hydrophilic filter (see Table 2).

[0079] [Table 2]

[0080] (2) Preparation of the second chamber composition containing mRNA The composition of the second chamber was prepared by dissolving 10 μg CleanCap® Fire Fly Luciferase mRNA (5-methoxyuridine) (TriLink, catalog L-7202) in PBS (pH 7.4, 2.67 mM KCl, 1.47 mM KH2PO4, 136.9 mM NaCl, 8.1 mM Na2HPO4).

[0081] (3) Nanoparticle production The first chamber composition and the second chamber composition were mixed by vortexing for 10 to 15 seconds immediately before use to form nanoparticles.

[0082] Experimental Example 3: Confirmation of mRNA nanoparticle formation The first chamber composition and second chamber composition were mixed immediately before use to confirm whether the nanoparticles of Examples 5 to 8 were formed by binding with mRNA. mRNA complexation was measured using an agarose gel retardation assay (1.5% agarose; DyneGelSafe Kit), and the results are shown in Figure 2. Additionally, particle size measurements using DLS to confirm nanoparticle formation are shown in Figure 3. Based on the gel retardation assay and DLS results compared to free RNA, it was confirmed that mRNA-complexed nanoparticles were formed in all of Examples 5 to 8.

[0083] Experimental Example 4: Confirmation of intracellular delivery efficiency of nanoparticles containing mRNA A549 human lung cancer cells were prepared to evaluate the intracellular delivery efficiency of the nanoparticles. The first and second chamber compositions were mixed immediately before intracellular injection to form nanoparticles corresponding to Examples 6 to 8. These nanoparticles were then dispensed into the cells in an amount corresponding to 250 ng of mRNA per 96-well plate. After an additional 6 hours of incubation, luciferin was added to the cells, and the amount of luciferase expressed was measured. The TransIT®-mRNA Kit (MirusBio) reagent was used as a control. The results are shown in Figure 4. Compared to the control group, the nanoparticles of the experimental examples exhibited similar levels of intracellular delivery efficiency.

[0084] Experimental Example 5: Comparison of intracellular delivery efficiency depending on the stability of nanoparticles containing mRNA To confirm the high intracellular delivery efficiency of nanoparticles prepared using the kit immediately prior to administration due to nanoparticle stability, HepG2 cells were prepared and intracellular delivery efficiency was compared using an accelerated test. Nanoparticles corresponding to Example 6 were formed by mixing the first and second chamber compositions immediately prior to intracellular injection. These nanoparticles were then dispensed into cells in an amount equivalent to 250 ng of RNA per 96-well plate. As a comparison group, nanoparticles prepared using the same method and nanoparticles prepared using the conventional transfection agent L3K were stored at 42°C for 1 hour and then dispensed into cells in the same manner. After a further 15 hours of incubation, luciferin was added to the cells, and the amount of luciferase expressed was measured. The results are shown in Figure 5. The accelerated test after preparation confirmed that stability decreased and intracellular delivery efficiency decreased dramatically.

Claims

1. a first chamber containing an amphiphilic block copolymer, a cationic compound, and a polylactate; and a second chamber containing an active ingredient selected from a nucleic acid, a virus, or a combination thereof; Including, for forming nanoparticles having a particle size of 50 to 400 nm immediately before use; the amount of the amphiphilic block copolymer is 0.5 to 12 parts by weight per 1 part by weight of the cationic compound; the amount of the polylactate is 0.1 to 30 parts by weight relative to 1 part by weight of the cationic compound; A kit for producing a nanoparticle composition, wherein the cationic compound is a cationic lipid represented by the following formula (1): 【Chemistry 1】 During the ceremony, n and m are each independently 0 to 9, with 2≦n+m≦10; a and b are each independently 2 to 4; R 1 and R 2 are each independently selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, celloyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and celloyl.

2. The kit for producing a nanoparticle composition according to claim 1, which is for forming nanoparticles that deliver an intracellular active ingredient.

3. The kit for producing a nanoparticle composition according to claim 1 , wherein at least one selected from the group consisting of the first chamber and the second chamber further contains an additional solvent.

4. The kit for producing a nanoparticle composition according to claim 3 , wherein the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.

5. The kit for producing a nanoparticle composition according to claim 1 , wherein the second chamber further contains a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent, or a combination thereof.

6. The kit for producing a nanoparticle composition according to claim 1 , wherein the mixture of the amphiphilic block copolymer, the cationic compound, and the polylactate in the first chamber is filtered one or more times through a hydrophilic filter after mixing.

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