Kit for manufacturing nanoparticle compositions for drug delivery

Abstract

The present invention relates to a kit for producing a nanoparticle composition for drug delivery. More specifically, the kit does not contain polylactate and is designed to easily form nanoparticles containing a drug encapsulated therein by simply mixing the components of the kit: an amphiphilic block copolymer, a cationic compound, and a drug.

Description

[Technical Field]

[0001] The present invention relates to a kit for producing drug delivery nanoparticle compositions, and more specifically, to a kit for producing drug delivery nanoparticle compositions that does not contain polylactic acid and is designed so that nanoparticles containing a drug can be easily formed by simply mixing the components of the kit: an amphiphilic block copolymer, a cationic compound, and a drug. [Background technology]

[0002] For the treatment of anionic drugs containing nucleic acids, safe and efficient drug delivery technologies have been studied for a long time, and various delivery bodies and technologies have been developed. Delivery bodies are broadly divided into viral delivery bodies, which use adenoviruses or retroviruses, and non-viral delivery bodies, which use cationic lipids and cationic polymers. Technologies using viral delivery bodies are known to have many problems for commercialization due to risks such as nonspecific immune responses and complex production processes. Therefore, recent research is moving towards non-viral delivery bodies in order to overcome these disadvantages. Compared to viral delivery bodies, non-viral delivery bodies have the advantages of fewer side effects in terms of in vivo safety and lower production costs in terms of economics.

[0003] The most representative examples of nonviral delivery bodies used for nucleic acid delivery include complexes of cationic lipids and nucleic acids (lipoplexes) and complexes of polycationic polymers and nucleic acids (polyplexes). Such cationic lipids or polycationic polymers have been extensively studied for their ability to stabilize anionic drugs and facilitate their delivery to cells by forming complexes with anionic drugs through electrostatic interactions (Non-Patent Documents 1, 2).

[0004] However, the nanoparticles formed by these composites are susceptible to losing stability depending on the storage environment, making them vulnerable to long-term storage and potentially degrading in quality during transportation. Furthermore, nanoparticles are extremely difficult to manufacture because they require complex manufacturing processes to ensure sufficient stability.

[0005] Therefore, there is a need to develop a kit for manufacturing drug delivery nanoparticle compositions that is not significantly affected by storage conditions and 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 [Overview of the project] [Problems that the invention aims to solve]

[0007] The object of the present invention is to provide a kit for manufacturing a drug delivery nanoparticle composition in which drug-containing nanoparticles can be easily formed simply by mixing the components of the kit, making it easy for end consumers to use, and in which drug-containing nanoparticles can be easily formed immediately before use, thus enabling effective drug delivery into the body without being affected by storage or transportation environments. [Means for solving the problem]

[0008] To solve the aforementioned problems, the present invention provides a kit for producing nanoparticle compositions comprising: a first chamber containing an amphiphilic block copolymer and a cationic compound; and a second chamber containing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; wherein the kit does not contain polylactic acid or polylactic acid salts as components.

[0009] In one embodiment, the kit is for forming nanoparticles that deliver intracellular active ingredients.

[0010] In one embodiment, one or more chambers selected from the group consisting of the first chamber and the second chamber further contain an additional solvent.

[0011] In one embodiment, the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.

[0012] 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. [Effects of the Invention]

[0013] The nanoparticle composition production kit according to the present invention contains components for forming drug-containing nanoparticles separated in separate chambers, and therefore, unlike already formed nanoparticles, is not affected by storage or transport environments. 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 drawing]

[0014] [Figure 1] This graph shows the experimental results of the intracellular delivery efficiency of nanoparticles performed in Experimental Example 2 of the present invention. [Figure 2] This graph shows the experimental results of the intracellular delivery efficiency of nanoparticles performed in Experimental Example 3 of the present invention. [Modes for carrying out the invention]

[0015] The present invention will be described in more detail below. The kit for producing nanoparticle compositions according to the present invention comprises a first chamber containing an amphiphilic block copolymer and a cationic compound; and a second chamber containing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; and the kit does not contain polylactic acid or polylactic acid salts as components.

[0016] The kit of the present invention may be for producing nanoparticles by mixing immediately before administration. The kit of the present invention comprises two or more chambers, and the end user can easily form nanoparticles by simply mixing the chambers. The term “simply mixing” can include all acts of “mixing,” and means that no specific conditions are imposed on the act of mixing for the formation of nanoparticles. The mixing can be achieved by various methods such as dropping, vortexing, and decanting, but is not limited thereto. 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 rapidly, for example, within 1 minute, 30 seconds, or 15 seconds.

[0017] The kit of the present invention is characterized by not containing any polylactic acid and polylactic acid salts. Although polylactic acid or its salts can be used to induce the delivery effect of intracellular active ingredients, they have the drawback that precipitation can easily occur during the mixing process. Therefore, they are not included in the kit of the present invention in which the end user has to form nanoparticles simply by mixing.

[0018] The active ingredient of the nanoparticles formed simply by the end user mixing may form a complex by electrostatic interaction with a cationic compound, and the complex may be encapsulated inside the nanoparticle structure formed by an amphiphilic block copolymer.

[0019] Regarding the above-mentioned nanoparticles, in an aqueous environment, the hydrophilic part of the amphiphilic block copolymer forms the outer wall of the nanoparticles, the hydrophobic part of the amphiphilic block copolymer forms the inner wall of the nanoparticles, and inside the formed nanoparticles, a complex of the active ingredient and the cationic compound may be encapsulated. Such a nanoparticle structure improves the stability of the active ingredient in blood or body fluids.

[0020] The "nucleic acid" may be, for example, but not limited to, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof. The nucleic acid may express an antigen through a series of processes in the body and encode the antigen. For example, the nucleic acid may contain the base sequence of the antigen or may be DNA, RNA, or mRNA encoding such a polypeptide sequence.

[0021] The "polypeptide" may be recognizable as an antigen. The polypeptide means a protein that contains the polypeptide sequence of an antigen, its analog or precursor and can be recognized as an antigen through a series of processes in the body.

[0022] The aforementioned "virus" may be an oncolytic virus, and may be one or more selected from the group consisting of, for example, adenovirus, vaccinia virus, herpes simplex virus (HSV), and varicella stomatitis virus (VSV). In one embodiment, the oncolytic virus is an adenovirus. The adenovirus used in the embodiment of the present invention contains a luciferase gene that can be confirmed by imaging.

[0023] The aforementioned virus can express several types of therapeutic genes within an individual's body and is not limited to a specific molecular weight, protein, biological activity, or therapeutic field. The prophylactic virus can induce immunity against the target disease within the target body. Compositions containing the disease-preventive virus have 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 efficacy obtained through multiple inoculations.

[0024] In one embodiment, the particle size of the nanoparticles can be defined as a Z-average value. For example, it 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 it may be 10 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.

[0025] The aforementioned "Z-mean" may refer to the average hydrodynamic diameter of the particle distribution measured using dynamic light scattering (DSL). The nanoparticles have a simple dispersion particle distribution, and their polydispersity index may be, for example, 0.01 to 0.30, 0.05 to 0.25, or 0.1 to 0.2.

[0026] Furthermore, in one embodiment, the surface charge of the nanoparticles may be, for example, -40mV or more, -30mV or more, -20mV or more, or -10mV or more, or 40mV or less, 30mV or less, 20mV or less, or 10mV or less. In one embodiment, the surface charge of the nanoparticles may be, for example, -40 to 40mV, -30 to 30mV, -20 to 20mV, or -10 to 1mV. The surface charge can be measured in an environment similar to a biological environment, for example, in 10mM HEPES buffer (pH 7.2).

[0027] When the particle size and surface charge of the nanoparticles maintain the aforementioned levels, it is preferable in terms of the stability of the nanoparticle structure, the content of constituent components, the degree of absorption in the body, and the ease of sterilization as a pharmaceutical composition. For example, if 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.

[0028] The active ingredient may be, 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, relative to the total weight of the composition formed by the kit of the present invention, or it may be 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, relative to the total weight of the composition. In one embodiment, the active ingredient may be, for example, 0.05 to 30% by weight, 0.1 to 25% by weight, 0.25 to 20% by weight, 0.5 to 15% by weight, 1 to 10% by weight, or 1 to 5% by weight, relative to the total weight of the composition. If the content of the active ingredient is less than the above range relative to the weight of the total composition, the amount of delivery body used will be too large compared to the drug, which may lead to side effects from the delivery body. If it exceeds the above range, the nanoparticle size will be too large, which may reduce the stability of the nanoparticles and increase the loss rate during filter-sterilization.

[0029] In one embodiment, if the active ingredient is a virus, the nanoparticles are 1 × 10⁻⁶ viruses. 6 ~1 × 10 14 VP (virus particle), 1 x 10 7 ~1 × 10 13 VP, 1 x 10 8 ~1 × 10 12 VP, or 1 × 10 9 ~1 × 10 11 It can include VP.

[0030] In one embodiment, when the active ingredient is a nucleic acid or polypeptide, the amount of nucleic acid or polypeptide contained in the nanoparticles may be, for example, 5 ng to 150 μg, 10 ng to 100 μg, 10 ng to 50 μg, 10 ng to 10 μg, 10 ng to 500 ng, or 50 ng to 500 ng, but is not limited to these.

[0031] In certain embodiments, the cationic compound may be a cationic lipid or a cationic polymer, more specifically a cationic lipid.

[0032] 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'-trimethylaminoethanol It may be one or more combinations selected from the group consisting of [N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[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)carbamoylpropanoate tocopherol (AC-tocopherol), and N-(N'-methylaminoethane)carbamoylpropanoate tocopherol (MC-tocopherol).

[0033] When using such cationic lipids, it is preferable to reduce the amount of polycationic lipids with high intramolecular charge density in order to reduce the toxicity induced by the cationic lipids. More specifically, it is preferable to use cationic lipids that have one functional group per molecule capable of exhibiting a positive charge in aqueous solution.

[0034] Therefore, 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).

[0035] On the other hand, in one embodiment, the cationic polymer is selected from the group consisting of chitosan, glycochitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethyleneimine, dextran, hyaluronic acid, albumin, high molecular weight polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm), and more specifically, it may be one or more selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).

[0036] In one embodiment, the cationic lipid is given by the following formula (1) [ka] The cationic lipid may be represented by the formula (wherein n and m are independently 0 to 12 under the condition 2 ≤ n + m ≤ 12, a and b are independently 1 to 6, and R1 and R2 are independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms).

[0037] More specifically, in equation (1) above, n and m are independently between 1 and 9, and may also be 2 ≤ n + m ≤ 10.

[0038] More specifically, in formula (1) above, a and b may each be independently 2 to 4.

[0039] More specifically, in formula (1) above, R1 and R2 can each be independently selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, serotyl, myristrail, palmitrail, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and serotyl.

[0040] In one embodiment, the cationic lipid is 1,6-dioleoyltriethylenetetramid(N,N'-((ethane-1,2-diylbis(azandiyl))bis(ethane-2,1-diyl))oleamide), 1,8-dilinoleoyltetraethylenepentamide((9Z,9'Z,12Z,12'Z)-N,N'-(((azandiylbis(ethane-2,1-diyl))bis( It may also be one or more selected from the group consisting of azandiyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyristreoyldiethylenetriamide ((9Z,9'Z)-N,N'-(azandiylbis(ethane-2,1-diyl))bis(tetradecane-9-enamide)), 1,10-distearoylpentaethylenehexamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)distearamid) and 1,10-dioleoylpentaethylenehexamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl)oleamide).

[0041] The content of the cationic compound in the composition formed by the kit of the present invention may be, for example, 100 parts by weight or less, 90 parts by weight or less, 80 parts by weight or less, 70 parts by weight or less, 60 parts by weight or less, 50 parts by weight or less, 40 parts by weight or less, 30 parts by weight or less, 29 parts by weight or less, 28 parts by weight or less, 27 parts by weight or less, 26 parts by weight or less, 25 parts by weight or less, 24 parts by weight or less, 23 parts by weight or less, 22 parts by weight or less, 21 parts by weight or less, 20 parts by weight or less, 19 parts by weight or less, 18 parts by weight or less, 17 parts by weight or less, 16 parts by weight or less, 15 parts by weight or less, 14 parts by weight or less, 13 parts by weight or less, 12 parts by weight or less, 11 parts by weight or less, 10 parts by weight or less, 9 parts by weight or less, 8 parts by weight or less, 7 parts by weight or less, 6 parts by weight or less, or 5 parts by weight or less, per 1 part by weight of the active ingredient, or 0.1 parts by weight or more, 0.2 parts by weight or more, 0.3 parts by weight 0.4 parts by weight or more, 0.5 parts by weight or more, 0.6 parts by weight or more, 0.7 parts by weight or more, 0.8 parts by weight or more, 0.9 parts by weight or more, 1 part by weight or more, 1.1 parts by weight or more, 1.2 parts by weight or more, 1.3 parts by weight parts by weight or more, 1.4 parts by weight or more, 1.5 parts by weight or more, 1.6 parts by weight or more, 1.7 parts by weight or more, 1.8 parts by weight or more, 1.9 parts by weight or more, 2 parts by weight or more, 2.1 parts by weight or more, 2.2 parts by weight or more, 2.3 parts by weight The amount may be 100 parts or more, 2.4 parts by weight or more, 2.5 parts by weight or more, 2.6 parts by weight or more, 2.7 parts by weight or more, 2.8 parts by weight or more, 2.9 parts by weight or more, 3 parts by weight or more, 3.1 parts by weight or more, 3.2 parts by weight or more, 3.3 parts by weight or more, 3.4 parts by weight or more, 3.5 parts by weight or more, 3.6 parts by weight or more, 3.7 parts by weight or more, 3.8 parts by weight or more, 3.9 parts by weight or more, or 4 parts by weight or more, but is not limited to these.

[0042] In one embodiment, the content of the cationic compound in the composition may be 0.1 to 100 parts by weight, 0.5 to 50 parts by weight, 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 1 part by weight of the active ingredient, but is not limited thereto. If the content of the cationic compound in the composition is far less than the above levels, it may not be possible to form a stable complex with the active ingredient. Conversely, if it far exceeds the above levels, the nanoparticle size may become too large, reducing stability and potentially increasing the loss rate during filter sterilization.

[0043] When the active ingredient is a nucleic acid, the cationic compound and the nucleic acid bind together by electrostatic interaction to form a complex. In one embodiment, the ratio of the charge amounts of the nucleic acid (P) to 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, or 2 or more, and may also be 100 or less, 50 or less, or 20 or less, for example, 0.5 to 100, 1 to 50, or 2 to 20. 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, and if the ratio (N / P) exceeds 100, there is a risk of inducing toxicity. Furthermore, the N / P value may play an important role in the specific expression of the active ingredient in the spleen.

[0044] In one embodiment, the amphiphilic block copolymer may be an AB-type block copolymer containing hydrophilic A blocks and hydrophobic B blocks. In an aqueous solution, the AB-type block copolymer forms core-shell type polymer nanoparticles in which the hydrophobic B blocks form the core (inner wall) and the hydrophilic A blocks form the shell (outer wall).

[0045] 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.

[0046] More specifically, the hydrophilic A block may be one or more selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, copolymers of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0047] 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.

[0048] If necessary, a functional group or ligand that can reach a specific tissue or cell, or a functional group that can promote intracellular delivery, can be chemically attached to the end of the hydrophilic A block to regulate the in vivo distribution of the high molecular weight nanoparticle delivery body formed from the amphiphilic block copolymer and polylactic acid, or to increase the 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.

[0049] The hydrophobic B block is a biocompatible, biodegradable polymer, and in one embodiment, it may be one or more selected from the group consisting of polyester, polyanhydride, polyamino acid, polyorthoester, and polyphosphatidine.

[0050] More specifically, the hydrophobic B block may be one or more selected from the group consisting of polylactide, polyglycolide, polycaprolactone, polydioxan-2-one, copolymer of polylactide and glycoside, copolymer of polylactide and polydioxan-2-one, copolymer of polylactide and polycaprolactone, and copolymer of polyglycolide and polycaprolactone.

[0051] 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.

[0052] Furthermore, in one embodiment, the hydrophobic B block may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to the hydroxyl group at the terminal end of the hydrophobic B block in order to increase the hydrophobicity of the hydrophobic B block and improve the stability of the nanoparticles.

[0053] In one embodiment, the content of the amphiphilic block copolymer containing the hydrophilic block (A) and hydrophobic block (B) in the composition formed by the kit of the present invention may be, for example, 200 parts by weight or less, 190 parts by weight or less, 180 parts by weight or less, 170 parts by weight or less, 160 parts by weight or less, 150 parts by weight or less, 140 parts by weight or less, 130 parts by weight or less, 120 parts by weight or less, 110 parts by weight or less, 100 parts by weight or less, 90 parts by weight or less, 80 parts by weight or less, 70 parts by weight or less, 60 parts by weight or less, 50 parts by weight or less, 40 parts by weight or less, or 30 parts by weight or less, per 1 part by weight of the active ingredient, or 0.1 parts by weight or more, 0.5 parts by weight or more, 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, or 3 parts by weight or more. It may be 3.5 parts by weight or more, 4 parts by weight or more, 4.5 parts by weight or more, 5 parts by weight or more, 5.5 parts by weight or more, 5 parts by weight or more, 6.5 parts by weight or more, 7 parts by weight or more, 7.5 parts by weight or more, 8 parts by weight or more, 8.5 parts by weight or more, 9 parts by weight or more, 9.5 parts by weight or more, 10 parts by weight or more, 10.5 parts by weight or more, 11 parts by weight or more, 11.5 parts by weight or more, 12 parts by weight or more, 12.5 parts by weight or more, 13 parts by weight or more, 13.5 parts by weight or more, 14 parts by weight or more, 14.5 parts by weight or more, 15 parts by weight or more, 15.5 parts by weight or more, 16 parts by weight or more, 16.5 parts by weight or more, 17 parts by weight or more, 17.5 parts by weight or more, 18 parts by weight or more, 18.5 parts by weight or more, 19 parts by weight or more, 19.5 parts by weight or more, or 20 parts by weight or more, but is not limited to these.

[0054] For example, the content of the amphiphilic block copolymer in the composition may be, but is not limited to, 0.1 to 200 parts by weight, 0.5 to 180 parts by weight, 1 to 150 parts by weight, 10 to 100 parts by weight, 10 to 70 parts by weight, 15 to 50 parts by weight, or 15 to 30 parts by weight per 1 part by weight of the active ingredient. If the content of the amphiphilic block copolymer in the composition is much less than the above levels, the nanoparticle size may become too large, reducing the stability of the nanoparticles and potentially increasing the loss rate during filter sterilization. Conversely, if the content is much more than the above levels, the amount of the active ingredient that can be incorporated may be too small.

[0055] In one embodiment, the composition ratio of hydrophilic blocks (A) to hydrophobic blocks (B) in the amphiphilic block copolymer may be in the range of 40 to 70% by weight, more specifically 50 to 60% by weight, of the total weight of the copolymer. If the ratio of hydrophilic blocks (A) is less than 40% by weight of the total weight of the copolymer, the solubility of the polymer in water may be low, making it difficult to form nanoparticles. Therefore, the ratio of hydrophilic blocks (A) is preferably 40% by weight or more in order to give the copolymer sufficient water solubility to form nanoparticles. If the ratio of hydrophilic blocks (A) exceeds 70% by weight of the total weight of the copolymer, the hydrophilicity is too high, reducing the stability of the polymer nanoparticles, making it difficult to use as a solubilizing composition for active ingredient / cationic compound complexes. Therefore, considering nanoparticle stability, the ratio of hydrophilic blocks (A) is preferably 70% by weight or less.

[0056] In one embodiment, the amphiphilic block copolymer allows for the encapsulation of a complex of the active ingredient and the cationic compound within the nanoparticle structure in an aqueous solution. In this case, the weight ratio (a) of the active ingredient and cationic compound complex to the weight (b) of the amphiphilic block copolymer [a / b × 100; (weight of active ingredient + weight of cationic compound) / weight of amphiphilic block copolymer × 100] may be 1 to 60%, more specifically 1.5 to 50%, and even more specifically 2 to 40%. If the weight ratio (a / b × 100) is less than 1%, the content of the complex of the active ingredient and the cationic compound may be too low, making it difficult to achieve the effective content required for the active ingredient to function effectively. Conversely, if it exceeds 60%, a nanoparticle structure of appropriate size may not be formed when considering the molecular weight of the amphiphilic block copolymer and the amount of the complex of the active ingredient and the cationic compound.

[0057] In one embodiment, the weight of the cationic compound in the kit may be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, and may be 200 μg or less, 150 μg or less, 120 μg or less, 100 μg or less, 80 μg or less, 50 μg or less, 30 μg or less, or 25 μg or less. For example, it may be 1 μg to 200 μg, 5 μg to 100 μg, 5 μg to 80 μg, or 15 μg to 25 μg, but is not limited to these.

[0058] In one embodiment, the amphiphilic block copolymer in the kit may be 10 μg or more, 20 μg or more, 25 μg or more, 50 μg or more, 80 μg or more, 100 μg or more, or 120 μg or more, and may be 500 μg or less, 400 μg or less, 350 μg or less, 300 μg or less, or 250 μg or less, for example, 10 μg to 500 μg, 20 μg to 300 μg, 25 μg to 250 μg, or 120 μg to 250 μg, but is not limited to these.

[0059] Furthermore, in one embodiment, the amphiphilic block copolymer may be less than 40 parts by weight per 1 part by weight of the cationic compound, for example, 1 to 35 parts by weight, 1 to 30 parts by weight, 1 to 25 parts by weight, 1 to 20 parts by weight, 1 to 15 parts by weight, 1 to 10 parts by weight, 1 to 5 parts by weight, 5 to 40 parts by weight, 5 to 35 parts by weight, 5 to 30 parts by weight, 5 to 25 parts by weight, 5 to 20 parts by weight, or 5 to 15 parts by weight, but is not limited to these amounts.

[0060] In one embodiment, the first and / or second chambers may further contain an aqueous solution, a water-miscible organic solvent, or a combination thereof. The “aqueous solution” refers to, for example, water, sterile purified water, a buffer, an injection solution, etc., and may further contain an organic acid as a buffer. The aqueous solution may, but is not limited to, a citrate buffer, a PBS buffer, etc. The “water-miscible organic solvent” may, but is not limited to, a C1-C4 lower alcohol, acetone, acetonitrile, a water mixture thereof, or a mixture thereof.

[0061] In one embodiment, the amphiphilic block copolymer and the cationic compound may be contained in an emulsion state within the first chamber. In this case, the solvent is a mixture of a water-miscible organic solvent and an aqueous solution, and can be, for example, an acetate such as sodium acetate or ethanol mixed with 20 to 30 times the amount of water as ethanol.

[0062] In one embodiment, the second chamber may further contain a stabilizer suitable for improving the stability of the active ingredient. The stabilizer may further include, but is not limited to, pH adjusters, inorganic salts, sugars, surfactants, chelating agents, etc. The term "sugars" refers to monosaccharides, disaccharides, sugar alcohols which are reducing sugars thereof, polymers of monosaccharides or mixed polysaccharides, etc., and the term "polysaccharides" means sugars of three or more types. The monosaccharides include, for example, mannose, glucose, arabinose, fructose, and galactose; the disaccharides include sucrose, trehalose, maltose, lactose, cellobiose, genthiobiose, isomaltose, and melibiose; the sugar alcohols include mannitol, sorbitol, xylitol, erythritol, and maltitol; and the polysaccharides include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharides. The "pH adjusting agent" may be 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, sodium dioctyl sulfosuccinate, sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium cholate hydrate, sodium deoxycholate, sodium glycodeoxycholate, 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 aforementioned "chelating agent" may be, but is not limited to, citric acid, polyphenolic acid, EDTA, DTPA, EDDHA, or a combination thereof.The aforementioned "inorganic salt" refers to a salt of a 1 or 2 valent metal, and may be, but is not limited to, NaCl, KCl, MgCl2, CaCl2, MgSO4, CaSO4, CaCO3, MgCO3, etc.

[0063] For example, if the active ingredient is a virus, 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.

[0064] The "chamber" is suitable for containing nanoparticle material or a solvent containing it, and includes, but is not limited to, glass, plastic, paper, packs, etc.

[0065] The present invention will be described in detail below with reference to the following examples. However, these examples are intended solely to illustrate the present invention and its scope, and are not limited in any way. [Examples]

[0066] Manufacturing Example 1: Formation of virus-containing nanoparticles (1) Preparation of the first chamber composition 20 mg of 1,6-dioleoyltriethylenetetramid (dioTETA) and 50 mg of mPEG-PLA-tocopherol were each dissolved in 1 mL of 90% ethanol. The dioTETA and mPEG-PLA-tocopherol were mixed in the ratios shown in Table 1, and then mixed with 30 times the amount of PBS to prepare a composite emulsion. The prepared composition was filtered through a 0.22 μm hydrophilic filter. (See Table 1)

[0067] [Table 1]

[0068] (2) Preparation of the second chamber composition containing oncolytic virus VQAd CMV Luc virus (manufactured by ViraQuest, Lot#: 33088) in the form dispensed into A195 buffer (10 mM Tris, 10 mM histidine, 75 mM NaCl, 5% sucrose (w / v), 1 mM MgCl2, 0.02% (w / v) PS-80, 0.1 mM EDTA, 0.5% ethanol (v / v), pH 7.4) was counted to be 1×10 10 VP and manufactured.

[0069] (3) Preparation of nanoparticles Immediately before use, the first chamber composition and the second chamber composition were vortexed for 10 - 15 seconds and mixed to form nanoparticles.

[0070] Experimental Example 1: Confirmation of nanoparticle formation A test was conducted to confirm whether the nanoparticles of Examples 1 - 19 were successfully formed by mixing the first chamber composition and the second chamber composition immediately before use. As a result, in all of Examples 1 - 19, no precipitate was observed even with simple mixing, and it was confirmed that nanoparticles were successfully formed.

[0071] Experimental Example 2: Confirmation of intracellular delivery efficiency of nanoparticles containing oncolytic virus To evaluate the intracellular delivery efficiency of nanoparticles, MDA-MB435 cells with low CAR expression, which are suitable for evaluating virus delivery efficiency, were prepared. The nanoparticles of Examples 1 - 3, 6 - 8, and 11 - 13 were formed by mixing the first chamber composition and the second chamber composition immediately before intracellular injection, and were dispensed into cells at an amount corresponding to 500 moi based on the virus. After further incubation for 15 - 24 hours, luciferin was added to the cells and the amount of expressed luciferase was measured. As a control group, virus (naked Ad; Ad) instead of nanoparticles was used. The results are shown in Figure 1.

[0072] Manufacturing Example 2: Formation of mRNA-containing nanoparticles (1) Preparation of the first chamber composition 20 mg of dioTETA was dissolved in 20 mM sodium acetate, and 50 mg of mPEG-PLA-tocopherol was dissolved in purified water. When using polylactic acid, it was dissolved together with mPEG-PLA-tocopherol in purified water. After mixing dioTETA, mPEG-PLA-tocopherol, and polylactic acid in the ratios shown in Table 2 below, PBS was added to prepare a complex emulsion, which was then filtered through a 0.22 μm hydrophilic filter.

[0073] [Table 2]

[0074] (2) Preparation of the second chamber composition containing mRNA Luciferase mRNA was mixed with purified water. When preparing the nanoparticles, the mRNA was used at a concentration of 5 μg.

[0075] (3) Manufacturing of nanoparticles The first and second chamber compositions were mixed by vortexing for 10 to 15 seconds immediately before use to form nanoparticles.

[0076] Experimental Example 3: Confirmation of intracellular delivery efficiency of mRNA-containing nanoparticles To evaluate the intracellular delivery efficiency of nanoparticles compared to that in the presence of polylactic acid, HepG2 cells were prepared. Nanoparticles of Example 20 and Comparative Examples 1-3 were formed by mixing the first and second chamber compositions immediately before intracellular injection, and dispensed into cells in amounts corresponding to 250 ng of mRNA. After further incubation for 15-24 hours, luciferin was added to the cells, and the amount of luciferase expressed was measured. TransIT (Takara), a commonly used transduction reagent, was used as a control group. The results are shown in Figure 2.

Claims

1. A first chamber containing an amphiphilic block copolymer and a cationic compound; and A second chamber containing an active ingredient selected from nucleic acids, polypeptides, viruses, or combinations thereof; Includes, The kit does not contain polylactic acid or polylactic acid salts as components. This is for manufacturing nanoparticles immediately before administration. The compound comprises 1 part by weight of a cationic compound and less than 40 parts by weight of an amphiphilic block copolymer. The amphiphilic block copolymer is an A-B type block copolymer containing a hydrophilic A block and a hydrophobic B block. The hydrophilic A block is one or more selected from the group consisting of monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, and polyvinylpyrrolidone. The hydrophobic B block is one or more selected from the group consisting of polylactide, polyglycolide, polycaprolactone, polydioxan-2-one, copolymer of polylactide and glycoside, copolymer of polylactide and polydioxan-2-one, copolymer of polylactide and polycaprolactone, and copolymer of polyglycolide and polycaprolactone. The cationic compound is defined by the following formula (1): 【Chemistry 1】 (In the formula, n and m are independently 1 to 9 under the condition 2 ≤ n + m ≤ 12, and a and b are independently 2 to 4, R 1 and R 2 Each of these is independently selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, myristyl, palmityl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erusyl, docosahexaenyl, and serotyl. A kit for producing nanoparticle compositions which are cationic lipids as shown.

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

3. The kit for producing a nanoparticle composition according to claim 1, wherein one or more chambers selected from the group consisting of the first chamber and the second chamber further comprises 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 nanoparticle compositions according to claim 1, wherein the second chamber further comprises a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent, or a combination thereof.

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