Kit for preparing nanoparticle containing drug and nanoparticle composition for drug delivery

Abstract

The present invention relates to a kit for preparing nanoparticles containing a drug and a nanoparticle composition for delivering a drug, and more particularly, to a kit for preparing nanoparticles containing a drug and a nanoparticle composition for delivering a drug, the kit is designed to increase the cellular delivery efficiency of a drug by using nanoparticles comprising a cationic compound and an anionic polymer compound having at least one acidic functional group.

Description

Technical Field

[0001] The present invention relates to a kit for preparing nanoparticles containing a drug and a nanoparticle composition for delivering a drug, and more specifically, the present invention relates to a kit for preparing nanoparticles containing a drug and a nanoparticle composition for drug delivery, wherein the kit is designed to increase the cellular delivery efficiency of the drug by using nanoparticles containing a cationic compound and an anionic polymer compound having at least one acidic functional group. Background Art

[0002] For a long time, research has been conducted on safe and effective drug delivery technology in the treatment of anionic drugs including nucleic acids, and various delivery systems and delivery technologies have been developed. Delivery systems are mainly divided into viral delivery systems utilizing adenovirus or retrovirus, etc. and non-viral delivery systems utilizing cationic lipids and cationic polymers, etc. Known viral delivery systems face risks such as nonspecific immune responses, and the production process is complicated, so there are many problems in commercialization. Therefore, in recent years, the research direction is to improve these shortcomings using non-viral delivery systems. Compared with viral delivery systems, the advantages of non-viral delivery systems are that there are few side effects in terms of safety in vivo, and low production costs in terms of economy.

[0003] As the non-viral delivery system for delivering nucleic acid substances, representative is the complex (lipid complex (lipoplex)) of cationic lipid and nucleic acid and the complex (polymer complex (polyplex)) of polycation (polycation) polymer and nucleic acid.A lot of research has been carried out on these cationic lipids or polycationic polymers, and these cationic lipids or polycationic polymers form complex with anionic drugs by electrostatic interaction, thus stabilizing anionic drugs, and increasing intracellular delivery (De Paula D, Bentley MV, Mahato RI, Hydrophobization and bioconjugation for enhanced siRNA delivery and targeting, RNA 13 (2007) 431-56; Gary DJ, Puri N, Won YY, Polymer-based siRNA delivery: Perspectiveson the fundamental and phenomenological distinctions from polymer-based DNA delivery, J Control release 121 (2007) 64-73).

[0004] Nanoparticles containing drugs tend to lose stability easily according to the storage environment, so they are not easy to store for a long time, and the quality may be impaired during transportation. In addition, in order to ensure sufficient stability, a complex preparation process is required, so it has the characteristics of being very difficult to prepare. Therefore, it is necessary to develop a drug delivery composition that is not significantly affected by the storage environment, is easy for final consumers to use, and can further increase the cell delivery efficiency of the drug.

[0005] In addition, in recent years, it is necessary to develop a vaccine customized for a patient (personalized vaccine), and in the case of a pandemic, it is very important to quickly supply nanoparticles containing drugs to patients with the shortest process time. Therefore, a technology is needed to shorten the process time and provide prefabricated nanoparticles as a ready-made product regardless of the type of drug. Summary of the invention

[0006] Technical issues to be solved

[0007] The first aspect of the present invention provides a kit for preparing nanoparticles containing drugs, so that drugs can be easily contained in nanoparticles by simply mixing the components of the kit, so that the final consumer can use them easily. In addition, regardless of the type of drug (e.g., mRNA), the nanoparticles containing drugs can be easily and quickly prepared before use, so that the drugs can be effectively delivered to the body without being affected by the storage or transportation environment, and the cell delivery efficiency of the drugs can be further increased. In particular, the nanoparticles containing drugs formed by the kit according to the first aspect of the present invention can be specifically delivered to the spleen, thereby further improving the availability of anti-cancer vaccines.

[0008] In addition, the second aspect of the present invention provides a kind of nanoparticle composition for drug delivery, therefore, can be easily and quickly prepared before using the nanoparticle containing the drug regardless of the type of the drug (for example, mRNA), as long as the drug is determined, only simple mixing of the drug is required to easily contain the drug in the nanoparticle, so it is easy to prepare the nanoparticle containing the drug, and the final consumer is easy to use. In addition, even if the drug is changed into antigen mRNA with different base sequences such as OVA, mTrp2, hTrp2, etc., stable and effective nanoparticles containing the drug can be formed, so various drugs can be applied to the nanoparticles developed as ready-made products, and due to being less affected by the storage or transportation environment, the possibility of the denaturation of the drug can be minimized and applied to the patient, and the cell delivery efficiency of the drug can be further increased. In particular, the nanoparticle containing the drug formed by the mixed composition and the drug according to the second aspect of the present invention can be specifically delivered to the spleen, so that the availability of anticancer vaccines can be further improved.

[0009] Technical Solution

[0010] A first aspect of the present invention provides a kit for preparing drug-containing nanoparticles, the kit comprising: a first chamber, the first chamber comprising nanoparticles, the nanoparticles comprising a cationic compound and an anionic polymer compound; and a second chamber, the second chamber comprising a drug as an active ingredient selected from nucleic acids, polypeptides, viruses or a combination thereof, wherein the anionic polymer compound has at least one acid functional group.

[0011] In a specific embodiment of the first aspect, the drug-containing nanoparticles are used to deliver the drug into cells.

[0012] In a specific embodiment of the first aspect, the anionic polymer compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer or a combination thereof.

[0013] In a specific embodiment of the first aspect, the anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block.

[0014] In a particular embodiment of the first aspect, the anionic hydrophilic polymer comprises only hydrophilic blocks.

[0015] In a particular embodiment of the first aspect, the anionic hydrophobic polymer comprises only hydrophobic blocks.

[0016] In a specific embodiment of the first aspect, at least one selected from the first chamber and the second chamber further comprises an additional solvent.

[0017] In a specific embodiment of the first aspect, the solvent is an aqueous solvent, a water-miscible solvent or a mixture thereof.

[0018] In a specific embodiment of the first aspect, the second chamber further comprises one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

[0019] In a specific embodiment of the first aspect, the kit for preparing drug-containing nanoparticles can be composed of a drug as an active ingredient selected from nucleic acids, polypeptides, viruses or combinations thereof; a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from pH adjusters, inorganic salts, sugars, surfactants, chelating agents or combinations thereof.

[0020] In a specific embodiment of the first aspect, based on 1 part by weight of the cationic compound, the amount of the anionic polymer compound may be 0.01-15 parts by weight.

[0021] In a specific embodiment of the first aspect, the cationic compound and the anionic polymer compound can be used for the preparation of nanoparticles in the form of a solution filtered more than once.

[0022] The second aspect of the present invention provides a nanoparticle composition for drug delivery, which is a composition for drug delivery comprising nanoparticles, wherein the nanoparticles comprise a cationic compound and an anionic polymer compound, wherein the nanoparticles do not contain a drug, and wherein the anionic polymer compound has at least one acidic functional group.

[0023] In a specific embodiment of the second aspect, the nanoparticle composition for drug delivery is used to deliver the drug into cells.

[0024] In a specific embodiment of the second aspect, the drug is selected from nucleic acid, polypeptide, virus or a combination thereof.

[0025] In a specific embodiment of the second aspect, the anionic polymer compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer or a combination thereof.

[0026] In a specific embodiment of the second aspect, the anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block.

[0027] In a specific embodiment of the second aspect, the anionic hydrophilic polymer comprises only hydrophilic blocks.

[0028] In a particular embodiment of the second aspect, the anionic hydrophobic polymer comprises only hydrophobic blocks.

[0029] In a specific embodiment of the second aspect, the nanoparticle composition for drug delivery further comprises an additional solvent.

[0030] In a specific embodiment of the second aspect, the solvent is an aqueous solvent, a water-miscible solvent or a mixture thereof.

[0031] In a specific embodiment of the second aspect, the nanoparticle composition for drug delivery further comprises one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

[0032] In a specific embodiment of the second aspect, the nanoparticle composition for drug delivery may be composed of a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

[0033] In a specific embodiment of the second aspect, based on 1 part by weight of the cationic compound, the amount of the anionic polymer compound may be 0.01-15 parts by weight.

[0034] In a specific embodiment of the second aspect, the cationic compound and the anionic polymer compound can be used for the preparation of nanoparticles in the form of a solution filtered more than once.

[0035] Beneficial Effects

[0036] The kit for preparing drug-containing nanoparticles according to the first aspect of the present invention separates the drug and the nanoparticles and contains them in a separate chamber, so it is not affected by the storage or transportation environment. When using the kit, even if the type of drug (e.g., mRNA) changes, the end user can quickly prepare the drug-containing nanoparticles by simply mixing the ingredients of the chamber without going through a complicated process. Therefore, for example, in the case of a vaccine customized for a patient in a hospital (personalized vaccine) or a pandemic, once the therapeutic mRNA is produced, it can be simply mixed with the nanoparticles proposed in the present invention and immediately administered to the human body without even optimizing the production process for mRNA. In addition, compared with a kit containing an amphiphilic polymer, the kit for preparing drug-containing nanoparticles according to the present invention can further increase the cell delivery efficiency of the drug. In particular, the drug-containing nanoparticles formed by the kit for preparing drug-containing nanoparticles of the present invention can be specifically delivered to the spleen, thereby further improving the availability of anti-cancer vaccines.

[0037] In addition, the nanoparticle composition for drug delivery according to the second aspect of the present invention is not affected by the storage or transportation environment, when using the nanoparticle composition for drug delivery, even if the type of drug (for example, mRNA) changes, the end user does not go through a complicated process, as long as the composition and the drug are simply mixed, the nanoparticle containing the drug can be quickly prepared, so for example, in the case of a vaccine (personalized vaccine) or a pandemic customized for a patient in a hospital, because there is no need to optimize the production process for mRNA, once the final mRNA production is completed, it can be simply mixed with the composition of the present invention and can be immediately applied to the human body. In addition, the nanoparticle composition for drug delivery according to the present invention can further increase the cell delivery efficiency of the drug compared with the nanoparticle composition comprising an amphiphilic polymer. In particular, the nanoparticle containing the drug formed by mixing the composition of the present invention and the drug can be specifically delivered to the spleen, thus the availability of anticancer vaccines can be further improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 This is the agarose gel electrophoresis result of the experiment conducted in Example 1 of the present invention to confirm whether nanoparticles containing mRNA are prepared.

[0039] Figure 2 These are the agarose gel electrophoresis results of the experiments conducted in Examples 2 to 5 of the present invention and Comparative Examples 1 to 3 to confirm whether nanoparticles containing mRNA were prepared. DETAILED DESCRIPTION

[0040] Hereinafter, the present invention will be described in more detail.

[0041] [First aspect: Kit for preparing drug-containing nanoparticles]

[0042] According to the first aspect of the present invention, a kit for preparing drug-containing nanoparticles comprises: a first chamber, wherein the first chamber contains nanoparticles, wherein the nanoparticles contain cationic compounds and anionic polymer compounds; and a second chamber, wherein the second chamber contains a drug as an active ingredient selected from nucleic acids, polypeptides, viruses or a combination thereof, wherein the anionic polymer compound has at least one acidic functional group.

[0043] The kit of the present invention is composed of two or more chambers, and the end user can easily prepare drug-containing nanoparticles by simply mixing the contents in the chambers. The term "simply mixing" can include all "mixing" behaviors, and means that no specific conditions are given to the behavior of mixing. The mixing can be achieved in various ways such as dripping, stirring (vortexing), pouring, etc., but is not limited to this. According to a specific embodiment, when using the kit of the present invention, drug-containing nanoparticles can be quickly formed in an amount of more than 90%, more than 95% or more than 99% of the amount that can be formed theoretically, for example, within 1 minute, within 30 seconds or within 15 seconds.

[0044] In a specific embodiment, the cationic compound and the anionic polymer compound form nanoparticles through electrostatic interaction, and the end user only needs to simply mix the generated nanoparticles with the drug to form the drug-containing nanoparticles. Therefore, according to a specific embodiment, the drug-containing nanoparticles prepared by the kit of the present invention can be in a form in which at least a portion of the drug is combined with the outside of the nanoparticles. This drug-containing nanoparticle structure improves the stability of the drug in the blood or body fluids.

[0045] The “nucleic acid” may be, for example, DNA, RNA, siRNA, shRNA, miRNA, mRNA, aptamer, antisense oligonucleotide, or a combination thereof, but is not limited thereto.

[0046] The "polypeptide" may refer to an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a protein that is active in vivo, or a protein that contains a polypeptide sequence of an antigen, an analog thereof or a precursor thereof and can be recognized as an antigen through a series of processes in vivo.

[0047] The "virus" may be an oncolytic virus, for example, one or more selected from adenovirus, vaccinia virus, herpes simplex virus (HSV) and vesicular stomatitis virus (VSV). In a specific embodiment, the oncolytic virus is an adenovirus. The adenovirus used in a specific embodiment of the present invention comprises a luciferase gene, which can be confirmed by imaging.

[0048] The virus can express various therapeutic genes in the body of an individual and is not limited to a specific molecular weight, protein, biological activity or therapeutic field. The preventive virus can induce immunity against the target disease in the body of an individual. The advantage of nanoparticles containing viruses for disease prevention is that the immunity induced by the virus itself can be reduced, and target cells can be specified or amplified, and the hyperimmune response to the virus when it is re-administered can be reduced, so that effective effects can be obtained through multiple vaccinations.

[0049] In an example, the particle size of the nanoparticles can be defined by a Z-average value, for example, can be below 800 nm, below 600 nm, below 500 nm, below 400 nm, below 300 nm, below 200 nm, or below 180 nm, and can be above 10 nm, above 50 nm, or above 100 nm. In a specific embodiment, the particle size of the nanoparticles defined by a Z-average value can be, for example, 10-800 nm, 10-600 nm, 10-500 nm, or 10-400 nm.

[0050] The "Z-average" may refer to the average of the hydrodynamic diameters of the particle distribution measured using dynamic light scattering (DSL). The nanoparticles have a monodisperse particle distribution, and their polydispersity index may be, for example, 0.05 to 0.8, 0.1 to 0.7, or 0.2 to 0.6.

[0051] In addition, in an example, the surface potential of the nanoparticles can be, for example, more than -50mV, more than -45mV, more than -40mV or more than -35mV, and can be less than 40mV, less than 30mV, less than 20mV, less than 10mV or less than 0mV. In a specific embodiment, the surface potential of the nanoparticles can be, for example, -50 to 40mV, -45 to 30mV, -40 to 20mV, -40 to 10mV or -35 to 0mV. The surface potential can be measured in an environment close to a biological environment, for example, can be measured in a HEPES buffer (pH is 7.0 to 7.5) of 8-12mM.

[0052] When the particle size and surface potential of nanoparticles keep the above-mentioned level, it is preferred in terms of the stability of nanoparticle structure, the content of constituents, the absorbability in vivo and the convenience of sterilization. For example, when the medicine is a nucleic acid, more than one end of the nucleic acid can be selected from cholesterol, tocopherol and the fatty acid with carbon number of 10-24 and modified more than one. The cholesterol, tocopherol and the fatty acid with carbon number of 10-24 include each analog, derivative and metabolite of cholesterol, tocopherol and fatty acid.

[0053] Based on the total weight of the drug-containing nanoparticles prepared by the kit of the present invention, the content of the drug can be, for example, 30% by weight or less, 20% by weight or less, 10% by weight or less, 5% by weight or less, or 1% by weight or less, and can 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, or 0.15% by weight or more. In a specific embodiment, based on the total weight of the drug-containing nanoparticles, the content of the drug can be, for example, 0.001-30% by weight, 0.01-20% by weight, 0.05-10% by weight, 0.1-5% by weight, or 0.15-1% by weight. Based on the total weight of the drug-containing nanoparticles, when the content of the drug is less than the above range, the amount of the nanoparticles used as a delivery system is too much compared to the drug, and there may be side effects caused by the nanoparticles as a delivery system. When the content of the drug exceeds the above range, the size of the drug-containing nanoparticles becomes too large, the particle stability is reduced, and the loss rate during filtration sterilization may increase. When the drug is a virus, the drug-containing nanoparticles may contain 1×10 6 VP to 1×10 14 VP (Virus particle), 1×10 7 VP to 1×10 13 VP, 1×10 8 VP to 1×10 12 VP or 1×10 9 VP to 1×10 11 VP virus.

[0054] In a specific embodiment, the cationic compound can be a cationic lipid or a cationic polymer, more specifically a cationic lipid.

[0055] In a specific embodiment, the cationic lipid can be selected from 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'-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), cholesteryloxypropane-1-amine (COPA), N-(N'-aminoethane)carbamoyltocopheryl propionate (AC-tocopherol) and N-(N'-methylaminoethane)carbamoyltocopheryl propionate (MC-tocopherol), or a combination of two or more thereof.

[0056] When such a cationic lipid is used, in order to reduce the toxicity caused by the cationic lipid, it is preferred to use a small amount of a polycationic lipid having a high cation density in the molecule, more specifically, a cationic lipid having one functional group per molecule that can exhibit a positive charge in an aqueous solution.

[0057] Therefore, in a more preferred embodiment, the cationic lipid can be one or more selected from 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).

[0058] In addition, in a specific embodiment, the cationic polymer can be selected from chitosan, glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethylenimine, dextran, hyaluronic acid, albumin, high molecular weight polyethyleneimine (PEI), polyamine and polyvinylamine (PVAm), more specifically, it can be one or more selected from polyethyleneimine (PEI), polyamine and polyvinylamine (PVAm).

[0059] In a specific embodiment, the cationic lipid may be a cationic lipid of the following Chemical Formula 1:

[0060] [Chemical formula 1]

[0061]

[0062] In the formula,

[0063] n and m are each independently 0 to 12, and 2≤n+m≤12,

[0064] a and b are each independently 1 to 6,

[0065] R 1 and R 2 Each is independently selected from saturated hydrocarbon groups and unsaturated hydrocarbon groups having 11 to 25 carbon atoms.

[0066] More specifically, in the chemical formula 1, n and m can be independently 1 to 9, but must satisfy the condition: 2≤n+m≤10.

[0067] More specifically, in the chemical formula 1, a and b may be 2 to 4, respectively, independently.

[0068] More specifically, in the chemical formula 1, R 1 and R 2Each of the above groups may be independently selected from lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, cerotyl, myristoleyl, (9Z)-9-hexadecene-1-yl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl and cerotyl.

[0069] In a specific embodiment, the cationic lipid can be selected from 1,6-dioleoyl triethylenetetraamide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide; dioTETA), 1,8-dilinoleoyl tetraethylenepentaamide ((9Z,9'Z,12Z ,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamamide)((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamamide ide))), 1,4-dimyristoyl diethylene triamide ((9Z,9'Z)-N,N'-(azanediylbis(ethane-2,1-diyl))bis(tetradec-9-enamide))), 1,10-distearoyl pentaethylene hexamide (N,N'-(3,6,9,12-tetraazatetradec-1,14 At least one of N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl) distearamide and 1,10-dioleoyl pentaethylene hexaamide (N,N'-(3,6,9,12-tetraazatetradecane-1,14-diyl) dioleamide).

[0070] Based on 1 part by weight of the drug, the content of the cationic compound in the drug-containing nanoparticles prepared by the kit of the present invention can be, for example, less than 25 parts by weight, less than 20 parts by weight, less than 15 parts by weight, less than 10 parts by weight, or less than 5 parts by weight, and can be more than 0.5 parts by weight, more than 1 part by weight, more than 1.5 parts by weight, more than 2 parts by weight, or more than 2.5 parts by weight. In a specific embodiment, based on 1 part by weight of the drug, the content of the cationic compound in the drug-containing nanoparticles can be 0.5-25 parts by weight, 1-20 parts by weight, 1.5-15 parts by weight, 2-10 parts by weight, or 2.5-5 parts by weight. In addition, when the drug is a virus, more specifically an adenovirus, based on 1×10 10 Based on the virus of VP, the content of the cationic compound can be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, and can be less than 150 μg, less than 100 μg, less than 50 μg, or less than 30 μg, for example, 1-150 μg, 5-100 μg, 10-50 μg, or 15-30 μg. When the content of the cationic compound in the drug-containing nanoparticles is less than the above range, the drug may not be stably contained in the nanoparticles, and when the content of the cationic compound exceeds the above range, the size of the drug-containing nanoparticles becomes too large, the particle stability is reduced, and the loss rate may increase during filtration sterilization.

[0071] When the drug is a nucleic acid, the cationic compound binds to the nucleic acid through electrostatic interaction. In a specific embodiment, the ratio of the charge of the cationic compound (N) to the nucleic acid (P) (N / P: the ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) can be 0.5 or more, 0.7 or more, 0.9 or more, or 1 or more, and can be 100 or less, 50 or less, 20 or less, or 10 or less, for example, 0.5-100, 0.7-50, 0.9-20, or 1-10. When the above ratio (N / P) is less than the above range, a sufficient amount of nucleic acid cannot be included in the nanoparticles. On the other hand, when the above ratio (N / P) exceeds the above range, toxicity may be induced. In addition, the N / P ratio can play an important role in the specific expression of the active ingredient in the spleen.

[0072] In a specific embodiment, the anionic polymer compound may be an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof.

[0073] In a specific embodiment, the anionic amphiphilic block copolymer can be a block copolymer comprising a hydrophilic block and a hydrophobic block (e.g., an AB type block copolymer comprising a hydrophilic block (A) and a hydrophobic block (B)), wherein the hydrophilic block or the hydrophobic block may have at least one acidic functional group, and according to a specific embodiment, the hydrophobic block may have at least one acidic functional group. Such a block copolymer forms core-shell polymer nanoparticles in an aqueous solution, wherein the hydrophobic block forms the core (inner wall) and the hydrophilic block forms the shell (outer wall).

[0074] In a specific embodiment, the anionic hydrophilic polymer is a polymer containing only hydrophilic blocks, wherein the hydrophilic blocks have at least one acidic functional group. Such hydrophilic polymers form shell-type polymer nanoparticles, wherein the acidic functional groups interact electrostatically with cationic lipids in aqueous solution to form a hydrophilic block shell (exine).

[0075] In a specific embodiment, the anionic hydrophobic polymer is a polymer containing only a hydrophobic block, wherein the hydrophobic block has at least one acidic functional group. The acidic functional group of the hydrophobic polymer interacts electrostatically with the cationic lipid to form a shell-type polymer nanoparticle, in which the hydrophobic block forms the core (inner wall).

[0076] In a specific embodiment, the hydrophilic block may be one or more selected from polyalkylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyoxazoline, polyacrylamide and derivatives thereof.

[0077] More specifically, the hydrophilic block may be one or more selected from monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, polyethyloxazoline (PEOz), polymethyloxazoline (PMOz), a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0078] In a specific embodiment, the number average molecular weight (g / mol) of the hydrophilic block may be greater than 200, greater than 500, greater than 1000 or greater than 1500, and may be less than 50000, less than 20000, less than 10000 or less than 5000, but is not limited thereto.

[0079] In addition, as required, the functional group, ligand that can reach specific tissues or cells or the functional group that can promote intracellular delivery are chemically bonded to the end of the hydrophilic block, so that the in vivo distribution of the polymer nanoparticle delivery system can be adjusted, or the efficiency of the nanoparticle delivery system delivered into the cell can be improved. In a specific embodiment, the functional group or ligand can be selected from one or more of monosaccharides, polysaccharides, vitamins, peptides, proteins and antibodies against cell surface receptors. More specifically, the functional group or ligand can be selected from one or more of anisamide, vitamin B9 (folic acid), vitamin B12, vitamin A, galactose, lactose, mannose, hyaluronic acid, RGD peptide, NGR peptide, transferrin, antibodies against transferrin receptor, etc.

[0080] The hydrophobic block is a polymer with biocompatibility and biodegradability. In a specific embodiment, the hydrophobic block can be one or more selected from polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazene.

[0081] More specifically, the hydrophobic block may be one or more selected from polylactide (PLA), polyglycolide, polycaprolactone (PCL), polydioxane-2-one, a copolymer of polylactide and polyglycolide, a copolymer of polylactide and polydioxane-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

[0082] In a specific embodiment, the number average molecular weight (g / mol) of the hydrophobic block may be greater than 200, greater than 500, greater than 700 or greater than 1000, and may be less than 50000, less than 20000, less than 10000 or less than 6000, but is not limited thereto.

[0083] Furthermore, in a specific embodiment, the hydrophobic 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 end of the hydrophobic block.

[0084] The hydrophilic block may have no acidic functional group or may have at least one acidic functional group, and the hydrophobic block may have no acidic functional group or may have at least one acidic functional group, but at least one of the hydrophilic block and the hydrophobic block has at least one acidic functional group.

[0085] In a specific embodiment, the acidic functional group may be derived from an acid selected from the group consisting of an inorganic acid, a sulfonic acid, a carboxylic acid, and combinations thereof.

[0086] More specifically, the inorganic acid may be selected from phosphoric acid, nitric acid, chromic acid, and combinations thereof, the sulfonic acid may be selected from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and combinations thereof, and the carboxylic acid may be selected from acetic acid, citric acid, gluconic acid, lactic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, phthalic acid, and combinations thereof, but is not limited thereto.

[0087] In a preferred embodiment, the anionic amphiphilic block copolymer is an AB type copolymer composed of monomethoxy polyethylene glycol (number average molecular weight: 300-10000 g / mol) as a hydrophilic block and polylactide (number average molecular weight: 300-10000 g / mol) or polycaprolactone (number average molecular weight: 300-10000 g / mol) as a hydrophobic block, and the hydrophobic block may have a carboxylic acid group derived from succinic acid.

[0088] In a preferred embodiment, the anionic hydrophilic polymer is a polymer composed of a hydrophilic block, the hydrophilic block is monomethoxypolyethylene glycol (number average molecular weight: 300-10000 g / mol) or polymethyloxazoline (number average molecular weight: 300-10000 g / mol) or polyethyloxazoline (number average molecular weight: 300-10000 g / mol), and the anionic hydrophilic polymer may have a carboxylic acid group derived from succinic acid.

[0089] In a specific embodiment, the molar ratio of the anionic polymer compound contained in the first chamber to 1 mole of the cationic compound may be 0.01 or more, 0.015 or more, 0.02 or more, 0.025 or more, or 0.03 or more, and may be 1 or less, 0.95 or less, 0.9 or less, 0.85 or less, 0.8 or less, or 0.75 or less. More specifically, the molar ratio of the anionic polymer compound contained in the first chamber to 1 mole of the cationic compound may be 0.01-1 or 0.02-0.9 or 0.03-0.8, but is not limited thereto.

[0090] In a specific embodiment, in order to increase the intracellular delivery efficiency of the mRNA of the kit according to the present invention, the nanoparticles in the first chamber may further contain fusogenic lipids.

[0091] In a specific embodiment, based on the total weight of the drug-containing nanoparticles prepared by the kit of the present invention, the amount of the fusion lipid contained in the nanoparticles in the first chamber can be 0.01-50% by weight, more specifically 0.1-10% by weight.

[0092] In a specific embodiment, the fusogenic lipid is combined with the cationic compound through hydrophobic interaction to form a nanoparticle structure.

[0093] In a specific embodiment, the fusion lipid can be one or a combination of two or more selected from phospholipids, cholesterol and tocopherol.

[0094] More specifically, the phospholipid may be one or more selected from phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC) and phosphatidic acid may be in a form combined with one or two C10-24 fatty acids. The cholesterol and tocopherol include analogs, derivatives and metabolites of cholesterol and tocopherol.

[0095] More specifically, the fusion lipid can be selected from dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), dipalmitoleoylphosphatidylethanolamine (1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine, DPPE), dilinoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1- phosphatidylethanolamine), 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, dipalmitoleoylphosphocholine (1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine, DPPC), dioleoylphosphocholine (1,2-dioleoyl-sn-glycero-3-phosphocholine, DPPC),2-dioleoyl-sn-glycero-3-phosphocholine, DOPC), dilauroylphosphatidylcholine, dimyristoyl phosphatidylcholine, dipalmitoyl phosphatidylcholine, distearoyl phosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoyl phosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine phosphatidylcholine), 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroyl phosphatidic acid, dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoylphosphatidic acid, dioleoyl phosphatidic acid, dilinoleoyl phosphatidic acid, 1-palmitoyl-2-oleoylphosphatidic acid acid), 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol and tocopherol, or a combination of two or more thereof.

[0096] More specifically, the fusion lipid may be one or more selected from 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).

[0097] In a specific embodiment, among the components of the drug-containing nanoparticles prepared by the kit of the present invention, the content of the anionic polymer compound can be 0.01 parts by weight or more, 0.03 parts by weight or more, 0.05 parts by weight or more, 0.07 parts by weight or more, 0.1 parts by weight or more, 0.12 parts by weight or more, 0.15 parts by weight or more, 0.2 parts by weight or more, 0.3 parts by weight or more, 0.4 parts by weight or more, or 0.5 parts by weight or more, and can be 15 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, 5 parts by weight or less, 4 parts by weight or less, 3 parts by weight or less, or 1 part by weight or less. In addition, the content of the anionic polymer compound can also be adjusted within the above range according to the drug. For example, in one specific embodiment, when the drug is a virus, the content of the anionic polymer compound can be 3-15 parts by weight based on 1 part by weight of the cationic compound. In another specific embodiment, when the drug is a nucleic acid, the content of the anionic polymer compound can be 0.01-15 parts by weight, 0.05-15 parts by weight, 0.07-10 parts by weight, 0.1-5 parts by weight, 0.1-3 parts by weight or 0.15-1 parts by weight based on 1 part by weight of the cationic compound.

[0098] In a specific 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 "aqueous solution" may be used as the same meaning as an aqueous solution, for example, it may refer to water, sterile purified water, a buffer solution, an injection solution, etc., and may also be a buffer solution further comprising an organic acid. The aqueous solution may be, for example, a citric acid buffer solution, a PBS buffer solution, etc., but is not limited thereto. The "water-miscible organic solvent" may be a C1-C4 lower alcohol, acetone, acetonitrile, a water mixture thereof, or a mixture thereof, but is not limited thereto.

[0099] In a specific embodiment, the cationic compound and the anionic polymer compound can be used for the preparation of nanoparticles in the form of a solution filtered more than once. More specifically, the filtration can be performed using a hydrophilic filter. The material of the hydrophilic filter includes, for example, nylon, mixed cellulose ester (MCE), polyethylsulfone (PES), polyvinylidene difluoride (PVDF), cellulose acetate (CA), polytetrafluoroethylene (PTFE) and mixtures thereof, but is not limited thereto. In the case of a hydrophilic filter, the drug can be more successfully contained in the nanoparticles, and the stability of the nanoparticles containing the drug can be increased.

[0100] In a specific embodiment, the second chamber may further contain a stabilizer suitable for improving the stability of the drug. The stabilizer may also include a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent, etc., but is not limited thereto. The "sugar" may refer to monosaccharides, disaccharides, sugar alcohols as their reducing sugars, polymers of single or mixed polysaccharides, etc., and the polysaccharides may refer to sugars above trisaccharides. The monosaccharides include, for example, mannose, glucose, arabinose, fructose, galactose, etc.; the disaccharides include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, melibiose, etc.; the sugar alcohols include mannitol, sorbitol, xylitol, erythritol, maltitol, etc.; the polysaccharides include raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hydroxyethyl cellulose, oligosaccharides, but are not limited thereto. The "regulator" may be tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate or a combination thereof, the "surfactant" may be sodium dodecyl sulfate, sodium dioctyl sulfonate, sodium dioctyl sulfonate, chenodeoxycholic acid, sodium N-lauroyl sarcosine, lithium dodecyl sulfate, sodium 1-octane sulfonate, sodium cholate hydrate, sodium deoxycholate, sodium glycodeoxycholate, benzalkonium chloride, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyethylene 40 stearate, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 65 and polysorbate 80 or a combination thereof, but are not limited thereto. The "chelating agent" may be citric acid, polyphenolic carboxylic acid, EDTA, DTPA, EDDHA or a combination thereof, but are not limited thereto. The "inorganic salt" refers to a salt of a monovalent or divalent metal, which can be NaCl, KCl, MgCl 2 , CaCl 2 MgSO 4 、CaSO 4 、CaCO 3 MgCO 3 etc., but not limited to.

[0101] For example, when the drug is a virus, the second chamber may further contain 5-15 mM Tris, 5-15 mM histidine, 50-90 mM NaCl, 2-8% (w / v) sucrose, 0.5-1.5 mM MgCl 2, 0.005-0.05% (w / v) PS-80, 0.05-0.15 mM EDTA and 0.1-1.0% (v / v) ethanol, and the pH may be 7.0 to 8.0. In another specific embodiment, when the drug is a nucleic acid, the second chamber may further comprise a PBS buffer, for example, a PBS buffer having a pH of 7.0 to 8.0 and comprising 2.0-3.5 mM KCl, 1.0-2.5 mM KH 2 PO 4 , 125-145 mM NaCl and 7.5-9.5 mM Na 2 HPO 4 of solution.

[0102] In a specific embodiment, the kit for preparing drug-containing nanoparticles of the present invention can be composed of a drug as an active ingredient selected from nucleic acids, polypeptides, viruses or combinations thereof; a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from pH adjusters, inorganic salts, sugars, surfactants, chelating agents or combinations thereof, wherein the anionic polymer compound has at least one acidic functional group.

[0103] In one example, the particle size of the drug-containing nanoparticles can be defined by a Z-average value, for example, it can be 800 nm or less, 600 nm or less, 500 nm or less, or 400 nm or less, and can be 10 nm or more, 50 nm or more, 100 nm or more, 150 nm or more, or 200 nm or more. In a specific embodiment, the particle size of the drug-containing nanoparticles defined by a Z-average value can be, for example, 100-800 nm, 100-600 nm, 100-500 nm, 100-400 nm, or 200-400 nm.

[0104] The "chamber" is suitable for containing materials of nanoparticles or solvents containing nanoparticles, including glass, plastic, paper, packaging, etc., but not limited thereto.

[0105] [Second aspect: Nanoparticle composition for drug delivery]

[0106] According to the second aspect of the present invention, the nanoparticle composition for drug delivery comprises nanoparticles, wherein the nanoparticles comprise a cationic compound and an anionic polymer compound, wherein the nanoparticles do not comprise a drug, and wherein the anionic polymer compound has at least one acidic functional group.

[0107] In an example, the particle size of the nanoparticles can be defined by a Z-average value, for example, can be below 800 nm, below 600 nm, below 500 nm, below 400 nm, below 300 nm, below 200 nm, or below 180 nm, and can be above 10 nm, above 50 nm, or above 100 nm. In a specific embodiment, the particle size of the nanoparticles defined by a Z-average value can be, for example, 10-800 nm, 10-600 nm, 10-500 nm, or 10-400 nm.

[0108] The "Z-average" may refer to the average of the hydrodynamic diameters of the particle distribution measured using dynamic light scattering (DSL). The nanoparticles have a monodisperse particle distribution, and the polydispersity index thereof may be, for example, 0.05 to 0.8, 0.1 to 0.7, or 0.2 to 0.6.

[0109] In addition, in an example, the surface potential of the nanoparticle can be, for example, more than -50mV, more than -45mV, more than -40mV or more than -35mV, and can be less than 40mV, less than 30mV, less than 20mV, less than 10mV or less than 0mV. In a specific embodiment, the surface potential of the nanoparticle can be, for example, -50-40mV, -45-30mV, -40-20mV, -40-10mV or -35-0mV. The surface potential can be measured in an environment close to a biological environment, for example, can be measured in a HEPES buffer (pH is 7.0 to 7.5) of 8-12mM.

[0110] When the particle size and surface potential of nanoparticles keep the above-mentioned level, it is preferred in terms of the stability of nanoparticle structure, the content of constituents, the absorbability in vivo and the convenience of sterilization. For example, when the medicine is a nucleic acid, more than one end of the nucleic acid can be selected from cholesterol, tocopherol and the fatty acid with carbon number of 10-24 and modified more than one. The cholesterol, tocopherol and the fatty acid with carbon number of 10-24 include each analog, derivative and metabolite of cholesterol, tocopherol and fatty acid.

[0111] In a specific embodiment, the cationic compound can be a cationic lipid or a cationic polymer, more specifically a cationic lipid. This cationic compound is the same as that specifically described in the first aspect of the present invention.

[0112] In a specific embodiment, the anionic polymer compound can be an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer or a combination thereof. The anionic polymer compound is the same as that specifically described above in the first aspect of the present invention.

[0113] In one embodiment, the acidic functional group may be derived from an acid selected from an inorganic acid, a sulfonic acid, a carboxylic acid and a combination thereof, and the acid is the same as that specifically described above in the first aspect of the present invention.

[0114] In a specific embodiment, based on 1 part by weight of the cationic compound, the content of the anionic polymer compound in the nanoparticles contained in the nanoparticle composition for drug delivery of the present invention can be 0.01 parts by weight or more, 0.03 parts by weight or more, 0.05 parts by weight or more, 0.07 parts by weight or more, 0.1 parts by weight or more, 0.12 parts by weight or more, 0.15 parts by weight or more, 0.2 parts by weight or more, 0.3 parts by weight or more, 0.4 parts by weight or more, or 0.5 parts by weight or more, and can be 15 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, 5 parts by weight or less, 4 parts by weight or less, 3 parts by weight or less, or 1 part by weight or less.

[0115] More specifically, the amount of the anionic polymer compound may be 0.01-15 parts by weight, 0.05-15 parts by weight, 0.07-10 parts by weight, 0.1-5 parts by weight, 0.1-3 parts by weight, or 0.15-1 parts by weight based on 1 part by weight of the cationic compound.

[0116] The nanoparticle composition for drug delivery of the present invention can easily prepare nanoparticles containing drugs by simply mixing with drugs. The term "simply mixing" can include all "mixing" behaviors, and refers to the behavior of mixing without giving specific conditions. The mixing can be achieved in various ways such as dripping, stirring (vortex), pouring, etc., but is not limited to this. According to a specific embodiment, when using the nanoparticle composition for drug delivery of the present invention, nanoparticles containing drugs can be quickly formed in an amount of more than 90%, more than 95% or more than 99% of the amount that can be formed theoretically, for example, within 1 minute, within 30 seconds or within 15 seconds.

[0117] In one embodiment, the cationic compound and the anionic polymer compound form nanoparticles through electrostatic interaction, and the end user only needs to simply mix the formed nanoparticles with the drug to form the drug-containing nanoparticles. Therefore, according to one embodiment, the drug-containing nanoparticles prepared by mixing the nanoparticle composition for drug delivery of the present invention and the drug can be in a form in which at least a portion of the drug is combined with the outside of the nanoparticles. This drug-containing nanoparticle structure improves the stability of the drug in the blood or in body fluids.

[0118] In a specific embodiment, the drug can be selected from nucleic acids, polypeptides, viruses or a combination thereof, and the drug is the same as that specifically described in the first aspect of the present invention.

[0119] Based on the total weight of the drug-containing nanoparticles prepared by mixing with the drug delivery nanoparticle composition of the present invention, the content of the drug can be, for example, less than 30% by weight, less than 20% by weight, less than 10% by weight, less than 5% by weight, or less than 1% by weight, and can be more than 0.001% by weight, more than 0.01% by weight, more than 0.05% by weight, more than 0.1% by weight, or more than 0.15% by weight. Based on the total weight of the drug-containing nanoparticles, when the drug content is less than the above range, the amount of nanoparticles used as a delivery system is too much compared to the drug, and there may be side effects caused by the nanoparticles as a delivery system. When the drug content exceeds the above range, the size of the drug-containing nanoparticles becomes too large, the particle stability is reduced, and the loss rate during filtration sterilization may increase. When the drug is a virus, the drug-containing nanoparticles may contain 1×10 6 VP to 1×10 14 VP (viral particles), 1×10 7 VP to 1×10 13 VP, 1×10 8 VP to 1×10 12 VP or 1×10 9 VP to 1×10 11 VP virus.

[0120] In a specific embodiment, the content of anionic polymer compounds in the nanoparticles contained in the nanoparticle composition for drug delivery of the present invention can also be adjusted according to the drug. For example, in a specific embodiment, when the drug is a virus, the content of anionic polymer compounds can be 3-15 parts by weight based on 1 part by weight of the cationic compound, and in another specific embodiment, when the drug is a nucleic acid, the content of anionic polymer compounds can be 0.01-15 parts by weight, 0.05-15 parts by weight, 0.07-10 parts by weight, 0.1-5 parts by weight, 0.1-3 parts by weight or 0.15-1 parts by weight based on 1 part by weight of the cationic compound.

[0121] In a specific embodiment, based on 1 part by weight of the drug, the content of the cationic compound in the drug-containing nanoparticles prepared by mixing the nanoparticle composition for drug delivery of the present invention and the drug can be, for example, 25 parts by weight or less, 20 parts by weight or less, 15 parts by weight or less, 10 parts by weight or less, or 5 parts by weight or less, and can be 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, or 2.5 parts by weight or more. In a specific embodiment, based on 1 part by weight of the drug, the content of the cationic compound in the drug-containing nanoparticles can be 0.5-25 parts by weight, 1-20 parts by weight, 1.5-15 parts by weight, 2-10 parts by weight, or 2.5-5 parts by weight. In addition, when the drug is a virus, more specifically an adenovirus, the content of the cationic compound in the nanoparticles can be 1×10 10 Based on the virus of VP, the content of the cationic compound can be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, and can be 150 μg or less, 100 μg or less, 50 μg or less, or 30 μg or less, for example, 1-150 μg, 5-100 μg, 10-50 μg, or 15-30 μg. When the content of the cationic compound in the drug-containing nanoparticles is less than the above range, the drug may not be stably contained in the nanoparticles, and when the content of the cationic compound exceeds the above range, the size of the drug-containing nanoparticles becomes too large, the particle stability is reduced, and the loss rate may increase during filtration sterilization.

[0122] When the drug is a nucleic acid, the cationic compound binds to the nucleic acid through electrostatic interaction. In a specific embodiment, the ratio of the charge amount of the cationic compound (N) to the nucleic acid (P) (N / P: the ratio of the cationic charge of the cationic compound to the anionic charge of the nucleic acid) can be 0.5 or more, 0.7 or more, 0.9 or more, or 1 or more, and can be 100 or less, 50 or less, 20 or less, or 10 or less, for example, 0.5-100, 0.7-50, 0.9-20, or 1-10. When the above ratio (N / P) is less than the above range, a sufficient amount of nucleic acid cannot be included in the nanoparticles. On the other hand, when the above ratio (N / P) exceeds the above range, toxicity may be induced. In addition, the N / P ratio can play an important role in the specific expression of the active ingredient in the spleen.

[0123] In a specific embodiment, the nanoparticles contained in the nanoparticle composition for drug delivery of the present invention may further contain fusogenic lipids to increase their intracellular delivery efficiency when mixed with drugs (e.g., mRNA). This fusogenic lipid is the same as that specifically described in the first aspect of the present invention above.

[0124] In a specific embodiment, the amount of the fusogenic lipid contained in the nanoparticles may be 0.01-50 wt %, more specifically 0.1-10 wt %, based on the total weight of the drug-containing nanoparticles prepared by mixing with the drug (eg, mRNA).

[0125] In a specific embodiment, the nanoparticle composition for drug delivery of the present invention may further comprise an aqueous solution, a water-miscible organic solvent, or a combination thereof. The aqueous solution and the water-miscible organic solvent are the same as those specifically described above in the first aspect of the present invention.

[0126] In one embodiment, the cationic compound and the anionic polymer compound can be used in the preparation of nanoparticles in the form of a solution filtered more than once. More specifically, the filtration can be performed using a hydrophilic filter, which is the same as that specifically described in the first aspect of the present invention.

[0127] In a specific embodiment, the nanoparticle composition for drug delivery of the present invention may further comprise a stabilizer suitable for improving the stability of the drug, and such stabilizer is the same as that specifically described above in the first aspect of the present invention.

[0128] For example, when the drug to be mixed is a virus, the nanoparticle composition for drug delivery of the present invention may also contain 5-15 mM Tris, 5-15 mM histidine, 50-90 mM NaCl, 2-8% (w / v) sucrose, 0.5-1.5 mM MgCl 2 , 0.005-0.05% (w / v) PS-80, 0.05-0.15mM EDTA and 0.1-1.0% (v / v) ethanol, and the pH can be 7.0 to 8.0. In another specific embodiment, when the drug to be mixed is a nucleic acid, the nanoparticle composition for drug delivery of the present invention may also include a PBS buffer, for example, a buffer having a pH of 7.0 to 8.0 and containing 2.0-3.5mM KCl, 1.0-2.5mM KH 2 PO 4 , 125-145 mM NaCl and 7.5-9.5 mM Na 2 HPO 4 of solution.

[0129] In a specific embodiment, the nanoparticle composition for drug delivery of the present invention can be composed of a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof, wherein the anionic polymer compound has at least one acidic functional group.

[0130] In one example, the particle size of the drug-containing nanoparticles prepared by mixing the nanoparticle composition for drug delivery of the present invention and the drug can be defined by the Z-average value, for example, it can be 800nm ​​or less, 600nm or less, 500nm or less, or 400nm or less, and can be 10nm or more, 50nm or more, 100nm or more, 150nm or more, or 200nm or more. In a specific embodiment, the particle size of the drug-containing nanoparticles defined by the Z-average value can be, for example, 100-800nm, 100-600nm, 100-500nm, 100-400nm, or 200-400nm.

[0131] Hereinafter, the present invention will be described in more detail by the following examples, but these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention in any way.

[0132] [Example]

[0133] Example 1

[0134] (1) Preparation of Nanoparticle Compositions for Drug Delivery

[0135] 10 mL of a nanoparticle composition for drug delivery was prepared. The prepared nanoparticle composition for drug delivery corresponds to the first chamber of the first aspect of the present invention.

[0136] The nanoparticle composition for drug delivery is prepared by using 1,6-dioleoyltriethylenetetraamide (dioTETA) as a cationic compound, and an anionic polymer compound (mPEG-PLA-COOH), which is a block copolymer comprising monomethoxypolyethylene glycol (number average molecular weight: 2000 g / mol) as a hydrophilic block and polylactide (number average molecular weight: 1300 g / mol) as a hydrophobic block, wherein the hydrophobic block has a carboxylic acid group derived from succinic acid.

[0137] Specifically, dioTETA and mPEG-PLA-COOH were first mixed at a molar ratio of 1 / 0.03 (mole number of dioTETA / mPEG-PLA-COOH), and then RNase-free water and sucrose as a cryoprotectant were added thereto to make the final solution have a concentration of 4% (40 mg / mL). The ingredients and contents of the prepared drug delivery nanoparticle composition are shown in Table 1 below.

[0138] (2) Preparation of mRNA solution

[0139] The mRNA solution was prepared by diluting murine tyrosine-related protein-2 (mTrp2) mRNA with PBS. 5 μg of mRNA was used to prepare the mRNA-nanoparticles. The prepared mRNA solution corresponds to the second chamber of the first aspect of the present invention.

[0140] (3) Preparation of Nanoparticles Containing mRNA

[0141] The prepared nanoparticle composition for drug delivery and the mRNA solution (corresponding to the first chamber and the second chamber of the first aspect, respectively) are mixed to prepare a final drug (drug product, DP) (N / P ratio = 2.5) containing nanoparticles containing mRNA. The ingredients and contents of the prepared final drug (DP) are shown in Table 1 below.

[0142] [Table 1]

[0143]

[0144] After mixing the nanoparticle composition for drug delivery and the mRNA solution, the particle size (Size), particle size distribution (PDI) and surface charge (Zeta potential) were measured by a particle size analyzer (dynamic light scattering, DLS) to confirm whether nanoparticles were formed, and the results are shown in the following Table 2. According to Table 2, it was confirmed that particles were uniformly formed even after simple mixing.

[0145] [Table 2]

[0146]

[0147] (4) Experiments to confirm whether nanoparticles containing mRNA are prepared

[0148] When mixed drug delivery nanoparticle composition and mRNA solution, agarose gel electrophoresis is performed to confirm whether nanoparticle and mRNA are normally combined. After simple mixing of nanoparticle composition and mRNA solution, the volume corresponding to 400ng of mRNA is mixed with loading dye (loading dye). Afterwards, it is loaded on 1% agarose gel and electrophoresed under appropriate voltage and time, and then the image of mRNA band (band) is observed by UV transilluminator (UV transilluminator) device. At this time, Dyne Gel Safe Red Kit (Dyne Gel Safe Red Kit) (Dyne Bio Co., Ltd. (Dyne Bio)) is used as nucleic acid staining reagent.

[0149] The measurement results are shown in Figure 1In. According to Figure 1 In Example 1, due to the high binding force between the nanoparticles and mRNA, almost no exposed mRNA band appeared.

[0150] (5) Experiments to confirm the immune response induction effect of the preparation

[0151] During the screening of the preparation evaluation, the expression level of the reporter protein is detected using luciferase encoding mRNA. In order to evaluate whether the prepared drug delivery preparation (drug delivery nanoparticle composition-mRNA preparation) actually delivers the drug to the spleen (spleen) and causes an immune response and the immune response effect of the Trp2 (Tyrosine-related protein 2) antigen, which is an important factor of melanoma (melanoma), the murine (murine) Trp2 antigen of mouse origin is used to evaluate the efficacy of the preparation by IFN-enzyme linked immunospot (γenzyme linked immunospot) (ELISPOT).

[0152] Nanoparticle composition for drug delivery - mRNA preparation The nanoparticle composition for drug delivery prepared in Example 1 was used. As a control group, an LPX (lipoplex) preparation was used. Liposomes were prepared by mixing cationic lipid 1,2-di-O-octadecenyl-3-trimethylammonium propane (1,2-di-O-octadecenyl-3-trimethylammonium propane, DOTMA) and Hepler lipid dioleoylphosphatidylethanolamine (dioleoylphosphatidylethanolamine, DOPE), and then liposome-mRNA (lipoplex) preparations ( Nature , 2016, Vol.534, pages 396-401).

[0153] The nanoparticle composition-mRNA preparation for drug delivery and LPX as a control group (intravenous injection (iv) control group) were systemically administered to mice, and tissues and cells were extracted 7 days after administration, and the immune reactivity was analyzed and compared. The administration dose was changed to 10μg, 20μg, and 40μg, and the immune reactivity according to the dose was observed.

[0154] The results are shown in the following Table 3. According to Table 3, the nanoparticle composition-mRNA preparation for drug delivery showed a clear trend according to the dose, confirming that higher immunoreactivity was shown compared with the control group at all the administered doses.

[0155] [Table 3]

[0156]

[0157] Examples 2 to 5 and Comparative Examples 1 to 3

[0158] (1) Preparation of Nanoparticle Compositions for Drug Delivery

[0159] A nanoparticle composition for drug delivery was prepared according to the composition of the following Table 4. The prepared nanoparticle composition for drug delivery corresponds to the first chamber of the first aspect of the present invention.

[0160] Specifically, 1,6-dioleoyltriethylenetetraamide (dioTETA) was dissolved in 20 mM sodium acetate buffer to prepare a dioTETA solution with a concentration of 5 mg / mL, and an anionic polymer compound was dissolved in sterile distilled water at the concentrations described in Table 4 and prepared. Each solution was filtered and sterilized using a 0.22 μm hydrophilic filter, and then each solution was mixed in the ratio described in Table 5 below. After that, sterile distilled water and sucrose as a cryoprotectant were added to make the concentration in the final solution 4% (40 mg / mL), thereby preparing a nanoparticle composition for drug delivery. The content and final concentration of the prepared nanoparticle composition for drug delivery are shown in Table 5 below.

[0161] [Table 4]

[0162]

[0163] [Table 5]

[0164]

[0165] (2) Preparation of mRNA solution

[0166] 20 μg of luciferase mRNA (CleanCap® FireFly luciferase mRNA, 5-methoxyuridine, TriLink, catalog number L-7202) was diluted with phosphate buffer to prepare an mRNA solution. The prepared mRNA solution corresponds to the second chamber of the first aspect of the present invention.

[0167] (3) Preparation of Nanoparticles Containing mRNA

[0168] The prepared nanoparticle composition for drug delivery and the mRNA solution were simply mixed using a pipette to prepare a final drug (drug product, DP) containing nanoparticles containing mRNA. The ingredients and contents of the prepared final drug (DP) are shown in Table 5.

[0169] After mixing the nanoparticle composition for drug delivery and the mRNA solution, in order to confirm whether nanoparticles are formed, the particle size (Size), particle size distribution (PDI) and charge (Zeta potential) are measured by a particle size analyzer (dynamic light scattering, DLS), and the results are shown in the following Table 6. According to Table 6, it is confirmed that particles are stably formed even after simple mixing. As the nanoparticles of the comparative example, nanoparticles without adding a polymer and using only a cationic compound (Comparative Example 1), nanoparticles with an anionic carboxylic acid-free amphiphilic polymer (mPEG-PLA (2k-1.7k) added (Comparative Example 2) and nanoparticles with only a hydrophobic polylactide (PLA 1.7k) without a hydrophilic block added (Comparative Example 3).

[0170] [Table 6]

[0171]

[0172] (4) Experiments to confirm whether nanoparticles containing mRNA are prepared

[0173] When mixed drug delivery nanoparticle composition and mRNA solution, agarose gel electrophoresis is performed to confirm whether nanoparticle and mRNA are normally combined. After simple mixed drug delivery composition and mRNA solution, the volume corresponding to the mRNA of 500ng is taken and mixed with loading dye. Afterwards, it is loaded on 1% agarose gel and electrophoresed under appropriate voltage and time, and then the image of mRNA band is observed by ultraviolet transmission instrument device. At this time, dyne gel safety red test kit (dyne biological company) is used as nucleic acid staining reagent.

[0174] The measurement results are shown in Figure 2 In. According to Figure 2 , in Examples 2 to 5, due to the high binding force between the nanoparticles and the mRNA, the exposed mRNA band hardly appeared. On the other hand, in Comparative Examples 1 to 3, due to the relatively low binding force with the mRNA, the exposed mRNA band was observed.

Claims

1. A kit for preparing nanoparticles containing a drug, comprising: a first chamber comprising nanoparticles comprising a cationic compound and an anionic polymer compound; and a second chamber, wherein the second chamber contains a drug selected from nucleic acids, polypeptides, viruses or a combination thereof as an active ingredient, in, The anionic polymer compound has at least one acidic functional group.

2. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The drug-containing nanoparticles are used to deliver the drug into cells.

3. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The anionic polymer compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer or a combination thereof.

4. The kit for preparing drug-containing nanoparticles according to claim 3, wherein: The anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block, the anionic hydrophilic polymer comprises only a hydrophilic block, and the anionic hydrophobic polymer comprises only a hydrophobic block.

5. The kit for preparing drug-containing nanoparticles according to claim 4, wherein: The hydrophilic block is one or more selected from polyalkylene glycol, polyvinyl alcohol, polyvinyl pyrrolidone, polyoxazoline, polyacrylamide and their derivatives, and the hydrophobic block is one or more selected from polyester, polyanhydride, polyamino acid, polyorthoester and polyphosphazene.

6. The kit for preparing drug-containing nanoparticles according to claim 4, wherein: The hydrophilic block is one or more selected from monomethoxypolyethylene glycol, monoacetoxypolyethylene glycol, polyethylene glycol, polyethyloxazoline, polymethyloxazoline, a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone, and the hydrophobic block is one or more selected from polylactide, polyglycolide, polycaprolactone, polydioxane-2-one, a copolymer of polylactide and polyglycolide, a copolymer of polylactide and polydioxane-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone.

7. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The acidic functional group is derived from an acid selected from the group consisting of an inorganic acid, a sulfonic acid, a carboxylic acid, and combinations thereof.

8. The kit for preparing drug-containing nanoparticles according to claim 7, wherein: The inorganic acid is selected from phosphoric acid, nitric acid, chromic acid and combinations thereof, the sulfonic acid is selected from methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid and combinations thereof, and the carboxylic acid is selected from acetic acid, citric acid, gluconic acid, lactic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, phthalic acid and combinations thereof.

9. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: One or more selected from the first chamber and the second chamber further comprises an additional solvent.

10. The kit for preparing drug-containing nanoparticles according to claim 9, wherein: The solvent is an aqueous solvent, a water-miscible solvent or a mixture thereof.

11. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The second chamber further comprises one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

12. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The kit is composed of a drug as an active ingredient selected from nucleic acid, polypeptide, virus or a combination thereof; a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

13. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The amount of the anionic polymer compound is 0.01 to 15 parts by weight based on 1 part by weight of the cationic compound.

14. The kit for preparing drug-containing nanoparticles according to claim 1, wherein: The cationic compound and the anionic polymer compound are used in the preparation of nanoparticles in the form of a solution filtered once or more.

15. A nanoparticle composition for drug delivery, which is a composition for drug delivery comprising nanoparticles, wherein the nanoparticles comprise a cationic compound and an anionic polymer compound, wherein the nanoparticles do not contain a drug, and wherein, The anionic polymer compound has at least one acidic functional group.

16. The nanoparticle composition for drug delivery according to claim 15, wherein The anionic polymer compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof.

17. The nanoparticle composition for drug delivery according to claim 15, wherein The acidic functional group is derived from an acid selected from the group consisting of an inorganic acid, a sulfonic acid, a carboxylic acid, and combinations thereof.

18. The nanoparticle composition for drug delivery according to claim 15, wherein The nanoparticle composition for drug delivery consists of a cationic compound; an anionic polymer compound; a solvent; and one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent or a combination thereof.

19. The nanoparticle composition for drug delivery according to claim 15, wherein The amount of the anionic polymer compound is 0.01 to 15 parts by weight based on 1 part by weight of the cationic compound.

20. The nanoparticle composition for drug delivery according to claim 15, wherein The cationic compound and the anionic polymer compound are used in the preparation of nanoparticles in the form of a solution filtered once or more.

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