Kit for producing drug-containing nanoparticles and nanoparticle composition for drug delivery

Abstract

The present invention relates to a kit for producing drug-containing nanoparticles and a nanoparticle composition for drug delivery. More specifically, the present invention relates to a kit for producing drug-containing nanoparticles and a nanoparticle composition for drug delivery that are designed to enhance the efficiency of intracellular drug delivery by utilizing nanoparticles containing a cationic compound and an anionic polymeric compound having at least one acid functional group.

Description

[Technical Field]

[0001] The present invention relates to a kit for producing drug-containing nanoparticles and a nanoparticle composition for drug delivery, and more specifically to a kit for producing drug-containing nanoparticles and a nanoparticle composition for drug delivery that are designed to increase the efficiency of drug delivery into cells by utilizing nanoparticles containing a cationic compound and an anionic polymeric compound having at least one acid functional group. [Background technology]

[0002] Safe and efficient drug delivery technologies for treatment using anionic drugs, including nucleic acids, have long been studied, and various delivery vehicles and delivery technologies have been developed. Delivery vehicles are mainly divided into viral delivery vehicles using adenoviruses, retroviruses, etc., and non-viral delivery vehicles using cationic lipids, cationic polymers, etc. Viral delivery vehicles are known to pose many problems for commercialization due to risks such as non-specific immune responses and complex manufacturing processes. Therefore, recent research has been conducted toward improving these drawbacks by using non-viral delivery vehicles. Non-viral delivery vehicles have the advantages of fewer side effects in terms of safety in the body and lower manufacturing costs compared to viral delivery vehicles.

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

[0004] Drug-containing nanoparticles are susceptible to stability loss depending on the storage environment, making them vulnerable to long-term storage and potentially subject to quality loss during transportation. Furthermore, ensuring sufficient stability requires complex manufacturing processes, which require very strict manufacturing conditions. Therefore, there has been a need for the development of a drug delivery composition that is not significantly affected by the storage environment, is easy for end users to use, and can enhance the cellular delivery efficiency of drugs.

[0005] In addition, in recent years, there has been a demand for the development of personalized vaccines, and in pandemic situations, it is important to quickly provide drug-containing nanoparticles to patients with minimal processing time. Therefore, there is a need for a technology that can shorten the processing time and provide ready-made nanoparticles regardless of the type of drug. [Prior art documents] [Non-patent literature]

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

[0007] A first aspect of the present invention provides a kit for producing drug-containing nanoparticles, which allows end users to easily incorporate drugs into nanoparticles by simply mixing the kit components, making it easy to use. Furthermore, drug-containing nanoparticles can be easily and quickly produced immediately before use, regardless of the type of drug (e.g., mRNA). This allows drugs to be effectively delivered into the body without being affected by the storage or transportation environment, further improving the efficiency of drug cellular delivery. In particular, drug-containing nanoparticles formed by the kit according to the first aspect of the present invention can be delivered specifically to the spleen, thereby further enhancing the usefulness of anti-cancer vaccines.

[0008] The second aspect of the present invention provides a nanoparticle composition for drug delivery, which allows drug-containing nanoparticles to be easily and quickly prepared immediately before use, regardless of the type of drug (e.g., mRNA). Once a specific drug is selected, it can be easily incorporated into nanoparticles by simply mixing with the drug, simplifying the production of drug-containing nanoparticles and providing convenience for end users. Furthermore, stable and effective drug-containing nanoparticles can be formed even when drugs are changed between antigen mRNAs with different base sequences, such as OVA, mTrp2, and hTrp2. This allows various drugs to be applied to pre-made nanoparticles, and the risk of drug denaturation is minimized due to minimal impact from storage and transportation environments, allowing for administration to patients and further enhancing the efficiency of drug cellular delivery. In particular, drug-containing nanoparticles formed by mixing a drug with the composition according to the second aspect of the present invention can be specifically delivered to the spleen, thereby further enhancing the usefulness of anti-cancer vaccines. [Means for solving the problem]

[0009] A first aspect of the present invention provides a kit for producing drug-containing nanoparticles, comprising: a first chamber containing nanoparticles comprising a cationic compound and an anionic polymeric compound; and a second chamber containing a drug as an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof, wherein the anionic polymeric compound has at least one acid functional group.

[0010] In one embodiment of the first aspect, the drug-containing nanoparticles are for delivering the drug into a cell.

[0011] In one embodiment of the first aspect, the anionic polymeric compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof.

[0012] In one embodiment of the first aspect, the anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block.

[0013] In one embodiment of the first aspect, the anionic hydrophilic polymer comprises only hydrophilic blocks.

[0014] In one embodiment of the first aspect, the anionic hydrophobic polymer comprises only hydrophobic blocks.

[0015] In one embodiment of the first aspect, one or more selected from the group consisting of the first chamber and the second chamber further comprises an additional solvent.

[0016] In one embodiment of the first aspect, the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof. In one 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.

[0017] In one embodiment of the first aspect, the kit for producing drug-containing nanoparticles may consist of a drug as an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; a cationic compound; the 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.

[0018] In one embodiment of the first aspect, the amount of the anionic polymer compound may be 0.01 to 15 parts by weight relative to 1 part by weight of the cationic compound.

[0019] In one embodiment of the first aspect, the cationic compound and the anionic polymeric compound may be used in the production of nanoparticles in the form of a solution that has been filtered one or more times.

[0020] A second aspect of the present invention provides a drug delivery composition comprising nanoparticles, wherein the nanoparticles comprise a cationic compound and an anionic polymeric compound, and the nanoparticles do not comprise a drug, and wherein the anionic polymeric compound has at least one acid functional group.

[0021] In one embodiment of the second aspect, the drug delivery nanoparticle composition is for delivering a drug into a cell.

[0022] In one embodiment of the second aspect, the drug is selected from a nucleic acid, a polypeptide, a virus, or a combination thereof.

[0023] In one embodiment of the second aspect, the anionic polymeric compound is an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof.

[0024] In one embodiment of the second aspect, the anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block.

[0025] In one embodiment of the second aspect, the anionic hydrophilic polymer comprises only hydrophilic blocks.

[0026] In one embodiment of the second aspect, the anionic hydrophobic polymer comprises only hydrophobic blocks.

[0027] In one embodiment of the second aspect, the drug delivery nanoparticle composition further comprises an additional solvent. In one embodiment of the second aspect, the solvent is an aqueous solvent, a water-miscible solvent, or a mixture thereof.

[0028] In one embodiment of the second aspect, the drug delivery nanoparticle composition 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.

[0029] In one embodiment of the second aspect, the drug delivery nanoparticle composition may consist of a cationic compound; the anionic polymeric 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.

[0030] In one embodiment of the second aspect, the amount of the anionic polymer compound may be 0.01 to 15 parts by weight relative to 1 part by weight of the cationic compound.

[0031] In one embodiment of the second aspect, the cationic compound and the anionic polymeric compound can be used in the production of nanoparticles in the form of a solution that has been filtered one or more times. [Effects of the Invention]

[0032] In the kit for preparing drug-containing nanoparticles according to the first aspect of the present invention, the drug and nanoparticles are isolated from each other and contained in separate chambers, making them unaffected by storage or transportation environments. When using the kit, end users can quickly prepare drug-containing nanoparticles regardless of the type of drug (e.g., mRNA) by simply mixing the components of the kit without going through complicated processes. Therefore, in the case of personalized vaccines or pandemic situations, for example, once therapeutic mRNA is prepared, it can be quickly administered to the human body by simply mixing it with the nanoparticles presented in the present invention, without the need to optimize the mRNA production process. Furthermore, the kit for preparing drug-containing nanoparticles according to the present invention can further enhance the cellular drug delivery efficiency compared to kits containing amphiphilic polymers. In particular, the drug-containing nanoparticles formed by the kit for preparing a nanoparticle composition of the present invention can be specifically delivered to the spleen, further enhancing the usefulness of anti-cancer vaccines.

[0033] Furthermore, the nanoparticle composition for drug delivery according to the second aspect of the present invention is not affected by storage or transportation environments. When used, end users can quickly prepare drug-containing nanoparticles regardless of the type of drug (e.g., mRNA) by simply mixing the composition with the drug without going through complicated processes. Therefore, for example, in the case of personalized vaccines in hospitals or during pandemic situations, optimization of the manufacturing process based on mRNA is not required. Once the final mRNA is produced, it can be quickly administered to the human body by simply mixing it with the composition of the present invention. Furthermore, the nanoparticle composition for drug delivery according to the present invention can further enhance the cellular drug delivery efficiency compared to nanoparticle compositions containing amphiphilic polymers. In particular, drug-containing nanoparticles formed by mixing the composition according to the first aspect of the present invention with a drug can be delivered specifically to the spleen, thereby further enhancing the usefulness of anti-cancer vaccines. [Brief explanation of the drawings]

[0034] [Figure 1]1 is a photograph showing the results of agarose gel electrophoresis in an experiment to confirm the production of mRNA-containing nanoparticles carried out in Example 1 of the present invention. [Figure 2] 1 is a photograph showing the results of agarose gel electrophoresis in an experiment to confirm the production of mRNA-containing nanoparticles carried out in Examples 2 to 5 of the present invention and Comparative Examples 1 to 3. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention will now be described in further detail.

[0036] [First aspect: Kit for manufacturing drug-containing nanoparticles] A kit for producing drug-containing nanoparticles according to a first aspect of the present invention comprises: a first chamber containing nanoparticles comprising a cationic compound and an anionic polymeric compound; and a second chamber containing a drug as an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof, wherein the anionic polymeric compound has at least one acid functional group.

[0037] The kit of the present invention is composed of two or more chambers, and end users can easily produce drug-containing nanoparticles by simply mixing the contents of the chambers. The term "simply mixing" can include any act of "mixing" and means that the mixing act does not depend on specific conditions. The mixing can be performed by various methods, including, but not limited to, dropping, stirring, and decanting. According to one embodiment, when using the kit of the present invention, drug-containing nanoparticles can be rapidly formed in an amount of 90% or more, 95% or more, or 99% or more of the theoretically formable amount, for example, within 1 minute, 30 seconds, or 15 seconds.

[0038] In one embodiment, the cationic compound and the anionic polymer compound form nanoparticles through electrostatic interaction, and end users can form drug-containing nanoparticles simply by mixing the generated nanoparticles with a drug. Therefore, according to one embodiment, the drug-containing nanoparticles produced by the kit of the present invention can have at least a portion of the drug bound to the outside of the nanoparticles. Such a drug-containing nanoparticle structure improves the stability of the drug in blood or body fluids. The "nucleic acid" may be, for example, DNA, RNA, siRNA, shRNA, miRNA, mRNA, an aptamer, an antisense oligonucleotide, or a combination thereof, but is not limited to these.

[0039] The term "polypeptide" refers to a protein that has activity in the body, such as an antibody or a fragment thereof, a cytokine, a hormone or an analog thereof, or a protein that includes the polypeptide sequence of an antigen, an analog thereof or a precursor thereof, and that can be recognized as an antigen through a series of processes in the body.

[0040] The "virus" may be an oncolytic virus, such as one or more selected from the group consisting of adenovirus, vaccinia virus, herpes simplex virus (HSV), and vesicular stomatitis virus (VSV). In one embodiment, the oncolytic virus is an adenovirus. The adenovirus used in the present embodiment contains a luciferase gene, which can be confirmed by imaging.

[0041] The virus can express several types of therapeutic genes in the subject's body and is not limited to a specific molecular weight, protein, physiological activity, or therapeutic field. The prophylactic virus can induce immunity against the target disease in the subject's body. Nanoparticles containing disease-preventive viruses have the advantages of reducing immune induction by the virus itself, specifying or expanding target cells, reducing excessive immune responses to the virus upon re-administration, and achieving effective effects with multiple vaccinations.

[0042] In one embodiment, the particle size of the nanoparticles can be defined by a Z-average value, and may be, for example, 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 180 nm or less, or 10 nm or more, 50 nm or more, or 100 nm or more. In one embodiment, the particle size of the nanoparticles defined by a Z-average value may be, for example, 10 to 800 nm, 10 to 600 nm, 10 to 500 nm, or 10 to 400 nm.

[0043] The "Z-average" may refer to the average hydrodynamic diameter of a particle distribution measured using dynamic light scattering (DSL). The nanoparticles may have a monodisperse particle distribution, and the polydispersity index may be, for example, 0.05 to 0.8, 0.1 to 0.7, or 0.2 to 0.6.

[0044] In one embodiment, the surface potential of the nanoparticles may be, for example, -50 mV or more, -45 mV or more, -40 mV or more, or -35 mV or more, or may be 40 mV or less, 30 mV or less, 20 mV or less, 10 mV or less, or 0 mV or less. In one embodiment, the surface potential of the nanoparticles may be, for example, -50 to 40 mV, -45 to 30 mV, -40 to 20 mV, -40 to 10 mV, or -35 to 0 mV. The surface potential may be measured in an environment similar to a biological environment, for example, in 8 to 12 mM HEPES buffer (pH 7.0 to 7.5).

[0045] Maintaining the particle size and surface potential of the nanoparticles at these levels is preferable in terms of the stability of the nanoparticle structure, the amount of ingredients, bioavailability, and ease of sterilization. For example, when the drug is a nucleic acid, one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms include analogs, derivatives, and metabolites of cholesterol, tocopherol, and fatty acids, respectively.

[0046] The amount of the drug may 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, or 0.001% by weight or more, 0.01% by weight or more, 0.05% by weight or more, 0.1% by weight or more, or 0.15% by weight or more, based on the total weight of the drug-containing nanoparticles. In one embodiment, the drug may 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. If the amount of drug relative to the total weight of the drug-containing nanoparticles is less than the above range, the amount of nanoparticles used as delivery carriers may be too high compared to the drug, which may result in side effects attributable to the nanoparticle delivery carrier. If the amount of drug is greater than the above range, the size of the drug-containing nanoparticles may be too large, reducing particle stability and increasing the loss rate during filter sterilization. When the drug is a virus, the drug-containing nanoparticles may contain 1×10 6 ~1×10 14 VP (viral particle), 1 × 10 7 ~1×10 13 VP, 1×10 8 ~1×10 12 VP or 1 x 10 9 ~1×10 11 It can contain VPs.

[0047] In a specific embodiment, the cationic compound may be a cationic lipid or a cationic polymer, more specifically a cationic lipid.

[0048] 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′-trimethylaminoethane) [N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (TC-cholesterol), 3β-[N-(N',N'-dimethylaminoethane)carbamoyl]cholesterol (DC-cholesterol), 3β-[N-(N'-monomethylaminoethane)carbamoyl]cholesterol (MC-cholesterol), 3β-[N-(aminoethane)carbamoyl]cholesterol (AC-cholesterol), cholesteryloxypropan-1-amine (COPA), N-(N'-aminoethane)carbamoylpropanoic acid tocopherol (AC-tocopherol), and N-(N'-methylaminoethane)carbamoylpropanoic acid tocopherol (MC-tocopherol).

[0049] When using such cationic lipids, it is preferable to use as few polycationic lipids as possible that have a high intramolecular cation density in order to reduce the toxicity of the cationic lipids. More specifically, it is preferable to use polycationic lipids that have one functional group per molecule that can exhibit a positive charge in aqueous solution.

[0050] Therefore, in a more 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).

[0051] Meanwhile, in one embodiment, the cationic polymer may be selected from the group consisting of chitosan, glycol chitosan, protamine, polylysine, polyarginine, polyamidoamine (PAMAM), polyethyleneimine, dextran, hyaluronic acid, albumin, high-molecular-weight polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm), more specifically, one or more selected from the group consisting of polyethyleneimine (PEI), polyamine, and polyvinylamine (PVAm).

[0052] In a specific embodiment, the cationic lipid has the following formula (1): [ka] (In the formula, n and m each independently represent 0 to 12, provided that 2≦n+m≦12; a and b each independently represent 1 to 6; R1 and R2 are each independently selected from the group consisting of saturated and unsaturated hydrocarbon groups having 11 to 25 carbon atoms.

[0053] More specifically, in the formula (1), n ​​and m are each independently 1 to 9, but may be 2≦n+m≦10.

[0054] More specifically, in the formula (1), a and b may each independently be 2 to 4.

[0055] More specifically, in the formula (1), R1 and R2 may each independently be selected from the group consisting of lauryl, myristyl, palmityl, stearyl, arachidyl, behenyl, lignoceryl, celloyl, myristoleyl, palmitoleyl, sapienyl, oleyl, linoleyl, arachidonyl, eicosapentaenyl, erucyl, docosahexaenyl, and celloyl.

[0056] In one embodiment, the cationic lipid is 1,6-dioleoyltriethylenetetramide (N,N'-((ethane-1,2-diylbis(azanediyl))bis(ethane-2,1-diyl))dioleamide; dioTETA), 1,8-dilinoleoyltetraethylenepentamide ((9Z,9'Z,12Z,12'Z)-N,N'-(((azanediylbis(ethane-2,1-diyl))bis(azanediyl))bis(ethane-2,1-diyl))bis(octadeca-9,12-dienamide)), 1,4-dimyris The copolymer may be one or more selected from the group consisting of threaoyldiethylenetriamide ((9Z,9′Z)-N,N′-(azanediylbis(ethane-2,1-diyl))bis(tetradec-9-enamide)), 1,10-distearoylpentaethylenehexamide (N,N′-(3,6,9,12-tetraazatetradecane-1,14-diyl)distearamide), and 1,10-dioleoylpentaethylenehexamide (N,N′-(3,6,9,12-tetraazatetradecane-1,14-diyl)dioleamide).

[0057] The amount of the cationic compound in the drug-containing nanoparticles produced by the kit of the present invention may 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, relative to 1 part by weight of the drug, or may 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, relative to 1 part by weight of the drug. In one embodiment, the amount of the cationic compound in the drug-containing nanoparticles may be 0.5 to 25 parts by weight, 1 to 20 parts by weight, 1.5 to 15 parts by weight, 2 to 10 parts by weight, or 2.5 to 5 parts by weight, relative to 1 part by weight of the drug. On the other hand, when the drug is a virus, more specifically, an adenovirus ... 10 The amount of the cationic compound relative to the VP may be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, or 150 μg or less, 100 μg or less, 50 μg or less, or 30 μg or less, for example, 1 μg to 150 μg, 5 μg to 100 μg, 10 μg to 50 μg, or 15 μg to 30 μg. If the amount 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. If the amount of the cationic compound exceeds the above range, the particle size of the drug-containing nanoparticles may become too large, resulting in reduced particle stability and a high loss rate during filter sterilization.

[0058] When the drug is a nucleic acid, the cationic compound and the nucleic acid bind via electrostatic interaction. In one embodiment, the charge ratio between the nucleic acid (P) and the cationic compound (N) (N / P; the ratio of the positive charge of the cationic compound to the negative charge of the nucleic acid) may be 0.5 or more, 0.7 or more, 0.9 or more, or 1 or more, or may be 100 or less, 50 or less, 20 or less, or 10 or less, for example, 0.5 to 100, 0.7 to 50, 0.9 to 20, or 1 to 10. If the ratio (N / P) is below the above range, the nanoparticles may not contain a sufficient amount of nucleic acid. If the ratio (N / P) is above the above range, toxicity may be induced. Furthermore, the N / P ratio may play an important role in spleen-specific expression of an active ingredient.

[0059] In one embodiment, the anionic polymeric compound may be an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof.

[0060] In one embodiment, the anionic amphiphilic block copolymer is 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 has at least one acid functional group. According to one embodiment, the hydrophobic block may have at least one acid functional group. Such a block copolymer forms, in aqueous solution, core-shell polymeric nanoparticles in which the hydrophobic block forms the core (inner wall) and the hydrophilic block forms the shell (outer wall).

[0061] In one embodiment, the anionic hydrophilic polymer is a polymer containing only a hydrophilic block, where the hydrophilic block has at least one acid functional group. In an aqueous solution, such a hydrophilic polymer forms a shell-type polymeric nanoparticle in which the acid functional group electrostatically interacts with the cationic lipid and the hydrophilic block forms the shell (outer wall).

[0062] In one embodiment, the anionic hydrophobic polymer is a polymer containing only a hydrophobic block, where the hydrophobic block has at least one acid functional group, which electrostatically interacts with the cationic lipid to form a shell-type polymeric nanoparticle in which the hydrophobic block forms the core (inner wall).

[0063] In one embodiment, the hydrophilic block may be one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyoxazoline, polyacrylamide, and derivatives thereof.

[0064] More specifically, the hydrophilic block may be one or more selected from the group consisting of monomethoxypolyethylene glycol (mPEG), monoacetoxypolyethylene glycol, polyethylene glycol, polyethyloxazoline (PEOz), polymethyloxazoline (PMOz), a copolymer of polyethylene and propylene glycol, and polyvinylpyrrolidone.

[0065] In one embodiment, the number average molecular weight (g / mol) of the hydrophilic block may be, but is not limited to, 200 or more, 500 or more, 1,000 or more, or 1,500 or more, and 50,000 or less, 20,000 or less, 10,000 or less, or 5,000 or less.

[0066] Furthermore, if necessary, functional groups or ligands that can reach specific tissues or cells, or functional groups that can promote intracellular delivery, can be chemically bound to the ends of the hydrophilic block to regulate the biodistribution of the polymeric nanoparticle carrier or increase the efficiency of intracellular delivery of the nanoparticle carrier. 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, antibodies against transferrin receptors, etc. The hydrophobic block is a biocompatible, biodegradable polymer, which in one embodiment may be one or more selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazines.

[0067] More specifically, the hydrophobic block may be one or more selected from the group consisting of polylactide (PLA), polyglycolide, polycaprolactone (PCL), polydioxane-2-one, a copolymer of polylactide and glycolide, a copolymer of polylactide and polydioxane-2-one, a copolymer of polylactide and polycaprolactone, and a copolymer of polyglycolide and polycaprolactone. In one embodiment, the number average molecular weight (g / mol) of the hydrophobic block may be, but is not limited to, 200 or more, 500 or more, 700 or more, or 1,000 or more, and 50,000 or less, 20,000 or less, 10,000 or less, or 6,000 or less.

[0068] In one embodiment, the hydrophobic block may be modified by chemically bonding tocopherol, cholesterol, or a fatty acid having 10 to 24 carbon atoms to the terminal hydroxy group.

[0069] The hydrophilic block may have zero or at least one acid functional group, and the hydrophobic block may have zero or at least one acid functional group, but at least one of the hydrophilic block and the hydrophobic block has at least one acid functional group.

[0070] In one embodiment, the acid functionality may be derived from an acid selected from the group consisting of inorganic acids, sulfonic acids, carboxylic acids, and combinations thereof.

[0071] More specifically, the inorganic acid may be selected from the group consisting of phosphoric acid, nitric acid, chromic acid, and combinations thereof; the sulfonic acid may be selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and combinations thereof; and the carboxylic acid may be selected from the group consisting of 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 to these.

[0072] In a preferred embodiment, the anionic amphiphilic block copolymer is an AB type copolymer consisting of a hydrophilic block of monomethoxypolyethylene glycol (number average molecular weight: 300 to 10,000 g / mol) and a hydrophobic block of polylactide (number average molecular weight: 300 to 10,000 g / mol) or polycaprolactone (number average molecular weight: 300 to 10,000 g / mol), and the hydrophobic block may have a carboxylic acid group derived from succinic acid.

[0073] In a preferred embodiment, the anionic hydrophilic polymer is a polymer comprising a hydrophilic block of monomethoxypolyethylene glycol (number average molecular weight: 300 to 10,000 g / mol), polymethyloxazoline (number average molecular weight: 300 to 10,000 g / mol), or polyethyloxazoline (number average molecular weight: 300 to 10,000 g / mol), and may have a carboxylic acid group derived from succinic acid.

[0074] In one embodiment, the molar ratio of the anionic polymer compound to 1 mole of the cationic compound contained in the first chamber may be 0.01 or more, 0.015 or more, 0.02 or more, 0.025 or more, or 0.03 or more, or 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 to 1 mole of the cationic compound contained in the first chamber may be, but is not limited to, 0.01 to 1, 0.02 to 0.9, or 0.03 to 0.8.

[0075] In one embodiment, to increase the efficiency of intracellular delivery of mRNA by the kit of the present invention, the nanoparticles in the first chamber may further comprise a fusogenic lipid.

[0076] In one embodiment, the amount of fusogenic lipid contained in the nanoparticles in the first chamber may be 0.01 to 50% by weight, more specifically 0.1 to 10% by weight, relative to the total weight of the drug-containing nanoparticles produced by the kit of the present invention. In one embodiment, the fusogenic lipids bind to the cationic compound through hydrophobic interactions to form a nanoparticle structure.

[0077] In one embodiment, the fusogenic lipid may be one or a combination of two or more selected from the group consisting of phospholipids, cholesterol, and tocopherol.

[0078] More specifically, the phospholipid may be one or more selected from the group consisting of phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid. The phosphatidylethanolamine (PE), phosphatidylcholine (PC), and phosphatidic acid may be bound to one or two C10-24 fatty acids. The cholesterol and tocopherol include analogs, derivatives, and metabolites of cholesterol and tocopherol, respectively.

[0079] More specifically, the fusogenic lipid is selected from the group consisting of dilauroylphosphatidylethanolamine, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine, dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE), dilinoleoylphosphatidylethanolamine, 1-palmitoyl-2-oleoylphosphatidylethanolamine, 1,2-diphytanoyl-3-sn-phosphatidylethanolamine, 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), dilauroylphosphatidylethanolamine, The phosphatidylcholine may be one or a combination of two or more selected from the group consisting of dilauroylphosphatidylcholine, dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, dioleoylphosphatidylcholine, dilinoleoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, 1,2-diphytanoyl-3-sn-phosphatidylcholine, dilauroylphosphatidic acid, dimyristoylphosphatidic acid, dipalmitoylphosphatidic acid, distearoylphosphatidic acid, dioleoylphosphatidic acid, dilinoleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidic acid, 1,2-diphytanoyl-3-sn-phosphatidic acid, cholesterol, and tocopherol.

[0080] More specifically, the fusogenic lipid may be one or more selected from the group consisting of dioleoylphosphatidylethanolamine (DOPE), 1,2-dipalmitoleoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE).

[0081] In one embodiment, the amount of the anionic polymeric compound, among the components of the drug-containing nanoparticles produced by the kit of the present invention, relative to 1 part by weight of the cationic compound may 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, or may 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. The amount of the anionic polymeric compound can be adjusted within the above range depending on the drug. For example, when the drug is a virus, the amount of the anionic polymer compound may be 3 to 15 parts by weight relative to 1 part by weight of the cationic compound in one embodiment, and in another embodiment, when the drug is a nucleic acid, the amount of the anionic polymer compound may be 0.01 to 15 parts by weight, 0.05 to 15 parts by weight, 0.07 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 3 parts by weight, or 0.15 to 1 part by weight relative to 1 part by weight of the cationic compound.

[0082] In one embodiment, the first chamber and / or the second chamber may further contain an aqueous solution, a water-miscible organic solvent, or a combination thereof. The "aqueous solution" may be used interchangeably with "aqueous solution" and may refer to, for example, water, sterile water, a buffer solution, an injection solution, etc., or a buffer solution further containing an organic acid. The aqueous solution may be, for example, but is not limited to, a citrate buffer solution, a PBS buffer solution, etc. The "water-miscible organic solvent" may be, for example, but is not limited to, a C1-C4 lower alcohol, acetone, acetonitrile, an aqueous mixture thereof, or a mixture thereof.

[0083] In one embodiment, the cationic compound and the anionic polymer compound can be used to prepare nanoparticles in the form of a solution that has been filtered one or more times. More specifically, the filtration can be performed using a hydrophilic filter. The material of the hydrophilic filter can be, for example, but is not limited to, nylon, mixed cellulose ester (MCE), polyethylsulfone (PES), polyvinylidene difluoride (PVDF), cellulose acetate (CA), polytetrafluoroethylene (PTFE), and mixtures thereof. When hydrophilic filtration is performed, the drug can be more easily incorporated into the nanoparticles, potentially increasing the stability of the drug-containing nanoparticles.

[0084] In one embodiment, the second chamber may further contain a stabilizer suitable for improving the stability of the drug. Examples of stabilizers include, but are not limited to, pH adjusters, inorganic salts, sugars, surfactants, and chelating agents. The term "saccharides" refers to monosaccharides, disaccharides, their reducing sugars, sugar alcohols, and polymers of single or mixed polysaccharides. Polysaccharides may refer to trisaccharides or higher. Examples of monosaccharides include mannose, glucose, arabinose, fructose, and galactose. Examples of disaccharides include sucrose, trehalose, maltose, lactose, cellobiose, gentiobiose, isomaltose, and melibiose. Examples of sugar alcohols include mannitol, sorbitol, xylitol, erythritol, and maltitol. Examples of polysaccharides include, but are not limited to, raffinose, dextran, starch, hydroxyethyl starch, cyclodextrin, cellulose, hetastarch, and oligosaccharides. The "pH adjuster" may be, but is not limited to, Tris, glycine, histidine, glutamate, succinate, phosphate, acetate, aspartate, or a combination thereof. The "surfactant" may be, but is not limited to, sodium lauryl sulfate, dioctyl sodium sulfosuccinate, dioctyl sodium sulfonate, chenodeoxycholic acid, N-lauroylsarcosine sodium salt, lithium dodecyl sulfate, 1-octanesulfonic acid sodium salt, sodium cholate hydrate, sodium deoxycholate, glycodeoxycholic acid sodium salt, benzalkonium chloride, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polysorbate 20, 40, 60, 65, and 80, or a combination thereof. The "chelating agent" may be, but is not limited to, citric acid, polyphenolic acid, EDTA, DTPA, EDDHA, or a combination thereof. The "inorganic salt" refers to a salt of a monovalent or divalent metal, and includes, but is not limited to, NaCl, KCl, MgCl2, CaCl2, MgSO4, CaSO4, CaCO3, MgCO3, etc.

[0085] For example, when the drug is a virus, the second chamber may further contain 5-15 mM MTris, 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), with a pH of 7.0-8.0. In another embodiment, when the drug is a nucleic acid, the second chamber may further contain a PBS buffer solution, e.g., 2.0-3.5 mM KCl, 1.0-2.5 mM KH2PO4, 125-145 mM NaCl, and 7.5-9.5 mM Na2HPO4, with a pH of 7.0-8.0.

[0086] In one embodiment, the kit for producing drug-containing nanoparticles of the present invention comprises: a drug as an active ingredient selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; a cationic compound; an anionic polymeric 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 polymeric compound has at least one acid functional group.

[0087] In one embodiment, the particle size of the drug-containing nanoparticles can be defined by the Z-average value and may be, for example, 800 nm or less, 600 nm or less, 500 nm or less, or 400 nm or less, or 10 nm or more, 50 nm or more, 100 nm or more, 150 nm or more, or 200 nm or more. In one embodiment, the particle size of the drug-containing nanoparticles defined by the Z-average value may be, for example, 100 to 800 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, or 200 to 400 nm.

[0088] The "chamber" may be any suitable material for containing nanoparticle material or a solvent containing the same, including, but not limited to, glass, plastic, paper, or a pack.

[0089] [Second Aspect: Nanoparticle Compositions for Drug Delivery] A nanoparticle composition for drug delivery according to a second aspect of the present invention comprises nanoparticles, the nanoparticles comprising a cationic compound and an anionic polymeric compound, and the nanoparticles do not comprise a drug, wherein the anionic polymeric compound has at least one acid functional group.

[0090] In one embodiment, the particle size of the nanoparticles can be defined by a Z-average value, and may be, for example, 800 nm or less, 600 nm or less, 500 nm or less, 400 nm or less, 300 nm or less, 200 nm or less, or 180 nm or less, or 10 nm or more, 50 nm or more, or 100 nm or more. In one embodiment, the particle size of the nanoparticles defined by a Z-average value may be, for example, 10 to 800 nm, 10 to 600 nm, 10 to 500 nm, or 10 to 400 nm. The "Z-average" may refer to the average hydrodynamic diameter of a particle distribution measured using dynamic light scattering (DSL). The nanoparticles may have a monodisperse particle distribution, and the polydispersity index may be, for example, 0.05 to 0.8, 0.1 to 0.7, or 0.2 to 0.6.

[0091] In one embodiment, the surface potential of the nanoparticles may be, for example, -50 mV or more, -45 mV or more, -40 mV or more, or -35 mV or more, or may be 40 mV or less, 30 mV or less, 20 mV or less, 10 mV or less, or 0 mV or less. In one embodiment, the surface potential of the nanoparticles may be, for example, -50 to 40 mV, -45 to 30 mV, -40 to 20 mV, -40 to 10 mV, or -35 to 0 mV. The surface potential may be measured in an environment similar to a biological environment, for example, in an 8 to 12 mM HEPES buffer solution (pH 7.0 to 7.5).

[0092] When the particle size and surface potential of the nanoparticles are maintained at the above levels, it is preferable in terms of the stability of the nanoparticle structure, the amount of constituent components, absorbability in the body, and ease of sterilization. For example, when the drug is a nucleic acid, one or more ends of the nucleic acid may be modified with one or more selected from the group consisting of cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms. The cholesterol, tocopherol, and fatty acids having 10 to 24 carbon atoms include analogs, derivatives, and metabolites of cholesterol, tocopherol, and fatty acids, respectively.

[0093] In a specific embodiment, the cationic compound may be a type of cationic lipid or cationic polymer, more specifically, a cationic lipid, as specifically described in the first aspect of the present invention.

[0094] In one embodiment, the anionic polymer compound may be an anionic amphiphilic block copolymer, an anionic hydrophilic polymer, an anionic hydrophobic polymer, or a combination thereof, and such anionic polymer compounds are the same as those specifically described in the first aspect of the present invention.

[0095] In one embodiment, the acid functionality may be derived from an acid selected from the group consisting of inorganic acids, sulfonic acids, carboxylic acids, and combinations thereof, such acids being similar to those specifically described in the first aspect of the present invention.

[0096] In one embodiment, the amount of the anionic polymer compound in the nanoparticles contained in the drug delivery nanoparticle composition of the present invention may 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, relative to 1 part by weight of the cationic compound, or may 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.

[0097] More specifically, the amount of the anionic polymer compound may be 0.01 to 15 parts by weight, 0.05 to 15 parts by weight, 0.07 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 3 parts by weight, or 0.15 to 1 part by weight relative to 1 part by weight of the cationic compound.

[0098] The drug delivery nanoparticle composition of the present invention can easily produce drug-containing nanoparticles simply by mixing with a drug. The term "simply mixing" includes all acts of "mixing" and means that the mixing act does not depend on specific conditions. The mixing can be performed by various methods, including, but not limited to, dropping, stirring, and decanting. According to one embodiment, when using the drug delivery nanoparticle composition of the present invention, drug-containing nanoparticles can be rapidly formed in an amount of 90% or more, 95% or more, or 99% or more of the theoretically formable amount, for example, within 1 minute, 30 seconds, or 15 seconds.

[0099] In one embodiment, the cationic compound and the anionic polymer compound form nanoparticles through electrostatic interaction, and the end user can form drug-containing nanoparticles simply by mixing the formed nanoparticles with a drug. Therefore, according to one embodiment, the drug-containing nanoparticles produced by mixing the drug delivery nanoparticle composition of the present invention with a drug may have at least a portion of the drug bound to the outside of the nanoparticle. Such a drug-containing nanoparticle structure improves the stability of the drug in blood or body fluids.

[0100] In one embodiment, the drug may be selected from a nucleic acid, a polypeptide, a virus, or a combination thereof, and such drugs are as specifically described in the first aspect of the present invention.

[0101] The amount of the drug may be, for example, 30 wt% or less, 20 wt% or less, 10 wt% or less, 5 wt% or less, or 1 wt% or less, based on the total weight of the drug-containing nanoparticles prepared by mixing with the drug delivery nanoparticle composition of the present invention, or 0.001 wt% or more, 0.01 wt% or more, 0.05 wt% or more, 0.1 wt% or more, or 0.15 wt% or more. If the amount of drug relative to the total weight of the drug-containing nanoparticles is less than the above range, the amount of nanoparticles used as delivery carriers will be too high compared to the drug, which may result in side effects caused by the nanoparticle delivery carrier. If the amount of drug exceeds the above range, the particle size of the drug-containing nanoparticles will be too large, which may reduce particle stability and increase the loss rate during filter sterilization. When the drug is a virus, the drug-containing nanoparticles may contain 1×10 6 ~1×10 14 VP (viral particle), 1 × 10 7 ~1×10 13 VP, 1×10 8 ~1×10 12 VP or 1 x 10 9 ~1×10 11 It can contain VPs.

[0102] In one embodiment, the amount of the anionic polymer compound can be adjusted depending on the drug. For example, when the drug is a virus, the amount of the anionic polymer compound can be 3 to 15 parts by weight per part by weight of the cationic compound. In another embodiment, when the drug is a nucleic acid, the amount of the anionic polymer compound can be 0.01 to 15 parts by weight, 0.05 to 15 parts by weight, 0.07 to 10 parts by weight, 0.1 to 5 parts by weight, 0.1 to 3 parts by weight, or 0.15 to 1 part by weight per part by weight of the cationic compound.

[0103] In one embodiment, the amount of the cationic compound in the drug-containing nanoparticles produced by mixing the drug delivery nanoparticle composition of the present invention with a drug may 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, relative to 1 part by weight of the drug, or may 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, relative to 1 part by weight of the drug. In one embodiment, the amount of the cationic compound in the drug-containing nanoparticles may be 0.5 to 25 parts by weight, 1 to 20 parts by weight, 1.5 to 15 parts by weight, 2 to 10 parts by weight, or 2.5 to 5 parts by weight, relative to 1 part by weight of the drug. Furthermore, when the drug is a virus, more specifically, an adenovirus ... 10 The amount of the cationic compound relative to VP may be 1 μg or more, 5 μg or more, 10 μg or more, 15 μg or more, or 18 μg or more, or 150 μg or less, 100 μg or less, 50 μg or less, or 30 μg or less, for example, 1 μg to 150 μg, 5 μg to 100 μg, 10 μg to 50 μg, or 15 μg to 30 μg. If the amount 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. If the amount of the cationic compound exceeds the above range, the particle size of the drug-containing nanoparticles may become too large, resulting in reduced particle stability and a high loss rate during filter sterilization.

[0104] When the drug is a nucleic acid, the cationic compound and the nucleic acid bind via electrostatic interaction. In one embodiment, the charge ratio between the nucleic acid (P) and the cationic compound (N) (N / P; the ratio of the positive charge of the cationic compound to the negative charge of the nucleic acid) may be 0.5 or more, 0.7 or more, 0.9 or more, or 1 or more, or may be 100 or less, 50 or less, 20 or less, or 10 or less, for example, 0.5 to 100, 0.7 to 50, 0.9 to 20, or 1 to 10. If the ratio (N / P) is below the above range, the nanoparticles may not contain a sufficient amount of nucleic acid. If the ratio (N / P) is above the above range, toxicity may be induced. Furthermore, the N / P ratio may play an important role in spleen-specific expression of an active ingredient.

[0105] In one embodiment, the nanoparticles contained in the nanoparticle composition for drug delivery of the present invention further contain a fusogenic lipid to increase the intracellular delivery efficiency when mixed with a drug (e.g., mRNA), and such fusogenic lipids are the same as those specifically described in the first aspect of the present invention.

[0106] In one embodiment, when a drug (e.g., mRNA) is loaded into the nanoparticle composition for drug delivery of the present invention, the nanoparticles contained in the nanoparticle composition for drug delivery of the present invention may further contain a fusogenic lipid to enhance the efficiency of intracellular delivery of the drug (e.g., mRNA). Such fusogenic lipids are the same as those specifically described in the first aspect of the present invention.

[0107] In one embodiment, the amount of fusogenic lipid contained in the nanoparticles may be 0.01 to 50% by weight, more specifically 0.1 to 10% by weight, based on the total weight of the drug-containing nanoparticles produced by mixing the lipid with a drug (e.g., mRNA).

[0108] In one embodiment, the drug delivery nanoparticle composition of the present invention may further comprise an aqueous solution, a water-miscible organic solvent, or a combination thereof, as specifically described in the first aspect of the present invention.

[0109] In one embodiment, the cationic compound and the anionic polymer compound can be used to prepare nanoparticles in the form of a solution that has been filtered one or more times. More specifically, the filtration can be performed using a hydrophilic filter. Such a hydrophilic filter can be the same as those specifically described in the first aspect of the present invention.

[0110] In one embodiment, the drug delivery nanoparticle composition of the present invention may further comprise a stabilizer suitable for improving the stability of the drug, such as those specifically described in the first aspect of the present invention.

[0111] For example, when the drug to be mixed is a virus, the nanoparticle composition for drug delivery of the present invention 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 may have a pH of 7.0-8.0. In another embodiment, when the drug to be mixed is a nucleic acid, the nanoparticle composition for drug delivery of the present invention may further contain a PBS buffer solution, such as a solution containing 2.0-3.5 mM KCl, 1.0-2.5 mM KH2PO4, 125-145 mM NaCl, and 7.5-9.5 mM Na2HPO4, and having a pH of 7.0-8.0.

[0112] In one embodiment, the nanoparticle composition for drug delivery of the present invention is composed of a cationic compound; an anionic polymeric 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 polymeric compound has at least one acid functional group.

[0113] In one embodiment, the particle size of drug-containing nanoparticles produced by mixing the drug delivery nanoparticle composition of the present invention with a drug can be defined by the Z-average value and may be, for example, 800 nm or less, 600 nm or less, 500 nm or less, or 400 nm or less, or 10 nm or more, 50 nm or more, 100 nm or more, 150 nm or more, or 200 nm or more. In one embodiment, the particle size of the drug-containing nanoparticles defined by the Z-average value may be, for example, 100 to 800 nm, 100 to 600 nm, 100 to 500 nm, 100 to 400 nm, or 200 to 400 nm. The present invention will be described in more detail below with reference to the following examples, although the examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0114] Example Example 1 (1) Preparation of nanoparticle compositions for drug delivery 10 mL of the drug delivery nanoparticle composition was prepared, which corresponds to the first chamber in the first aspect of the present invention. The nanoparticle composition for drug delivery was prepared using 1,6-dioleoyltriethylenetetramine (dioTETA) as a cationic compound, a block copolymer containing 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, and an anionic polymer compound (mPEG-PLA-COOH) in which the hydrophobic block has a carboxylic acid group derived from succinic acid. Specifically, dioTETA and mPEG-PLA-COOH were mixed at a molar ratio of 1 / 0.03 (moles of dioTETA / moles of mPEG-PLA-COOH), and then RNase-free water and sucrose as a cryoprotectant were added to a final solution concentration of 4% (40 mg / mL). The components and amounts of the resulting nanoparticle composition for drug delivery are shown in Table 1 below.

[0115] (2) Preparation of mRNA solution An mRNA solution was prepared by diluting mTrp2 (mouse tyrosine-linked protein 2) mRNA with PBS. 5 μg of mRNA was used to prepare mRNA-nanoparticles. The prepared mRNA solution corresponds to the second chamber in the first aspect of the present invention.

[0116] (3) Production of mRNA-containing nanoparticles The prepared drug delivery nanoparticle composition and mRNA solution (corresponding to the first and second chambers in the first aspect, respectively) were mixed to prepare a final drug product (DP) containing mRNA-containing nanoparticles (N / P ratio = 2.5). The components and amounts of the final drug product (DP) are shown in Table 1 below.

[0117] [Table 1]

[0118] To confirm whether nanoparticles were formed after mixing the drug delivery nanoparticle composition with the mRNA solution, particle size (size), particle size distribution (PDI), and surface charge (zeta potential) were measured using a DLS (Digital Light Spectroscopy) analyzer, and the results are shown in Table 2. Table 2 confirms that particles were uniformly formed even after simple mixing.

[0119] [Table 2]

[0120] (4) Experiment to confirm the production of mRNA-containing nanoparticles Agarose gel electrophoresis was performed to confirm whether the nanoparticles and mRNA were properly bound when the drug delivery nanoparticle composition was mixed with the mRNA solution. After simply mixing the nanoparticle composition and the mRNA solution, an amount equivalent to 400 ng of mRNA was collected and mixed with a loading dye. The mixture was then loaded onto a 1% agarose gel and electrophoresed at the appropriate voltage and time. The mRNA band image was observed using a UV transilluminator. The Dyne GelSafe Red Kit (Dyne Bio) was used as the nucleic acid staining reagent.

[0121] The measurement results are shown in Figure 1. As can be seen from Figure 1, in Example 1, the binding strength between the nanoparticles and mRNA was high, so that almost no exposed mRNA band was observed.

[0122] (5) Experiment to confirm the immune response induction effect of the formulation In the formulation evaluation screening, we utilized the expression level of a reporter protein using mRNA encoding luciferase. To evaluate whether the manufactured drug delivery formulation (drug delivery nanoparticle composition-mRNA formulation) actually delivered the drug to the spleen and induced an immune response, we also evaluated the efficacy of the formulation by IFN-γ enzyme-linked immunospot (ELISPOT) using mouse Trp2 (tyrosine-linked protein 2) antigen, an important factor in melanoma.

[0123] The drug delivery nanoparticle composition-mRNA formulation used was the drug delivery nanoparticle composition prepared in Example 1. An LPX (lipoplex) formulation was used as a control. Liposomes were prepared by mixing the cationic lipid DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane) with the helper lipid DOPE (dioleoylphosphatidylethanolamine), and the liposome-mRNA (lipoplex) formulation was prepared (Nature, 2016, Vol. 534, pages 396-401).

[0124] The drug delivery nanoparticle composition-mRNA formulation and the control LPX (iv control group) were administered systemically to mice, and seven days after administration, tissues were excised and cells were extracted and analyzed to compare immunoreactivity. The doses were varied to 10, 20, and 40 μg, and the immunoreactivity was observed according to the dose.

[0125] The results are shown in Table 3. Table 3 shows that the nanoparticle composition for drug delivery - nanoparticle composition for mRNA formulation showed a clear trend depending on the dose, and it was confirmed that the immunoreactivity was higher than that of the control group at all doses.

[0126] [Table 3]

[0127] Examples 2 to 5 and Comparative Examples 1 to 3 (1) Preparation of nanoparticle compositions for drug delivery A nanoparticle composition for drug delivery was prepared according to the detailed composition shown in Table 4 below. The prepared nanoparticle composition for drug delivery corresponds to the first chamber in the first aspect of the present invention.

[0128] Specifically, 1,6-dioleoyltriethylenetetramine (dioTETA) was dissolved in 20 mM sodium acetate buffer to prepare a 5 mg / mL dioTETA solution, and the anionic polymer compound was dissolved in sterile distilled water to the concentrations listed in Table 4. Each solution was sterilized by filtration using a 0.22 μm hydrophilic filter and mixed in the ratios listed in Table 5 below. Then, sterile distilled water and sucrose as a cryoprotectant were added to the final solution to a concentration of 4% (40 mg / mL), producing a drug delivery nanoparticle composition. The amount and final concentration of the drug delivery nanoparticle composition produced are shown in Table 5 below.

[0129] [Table 4]

[0130] [Table 5]

[0131] (2) Preparation of mRNA solution An mRNA solution was prepared by diluting 20 μg of luciferase mRNA (CleanCap® FireFly luciferase, 5-methoxyuridine, TriLink, catalog L-7202) with phosphate buffer. The prepared mRNA solution corresponds to the second chamber in the first aspect of the present invention.

[0132] (3) Production of mRNA-containing nanoparticles The drug delivery nanoparticle composition and mRNA solution were mixed by simple pipetting to prepare the final drug product (DP) containing mRNA-containing nanoparticles. The components and amounts of the final drug product (DP) are shown in Table 5.

[0133] After mixing the drug delivery nanoparticle composition with the mRNA solution, the particle size (size), particle size distribution (PDI), and charge (zeta potential) were measured using a DLS (Digital Light Spectroscopy) to confirm whether nanoparticles were formed, and the results are shown in Table 6. Table 6 confirms that particles were stably formed even after simple mixing.

[0134] As comparative examples, nanoparticles using only a cationic compound without adding a polymer (Comparative Example 1), nanoparticles using an amphiphilic polymer (mPEG-PLA(2k-1.7k)) without adding an anionic carboxylic acid (Comparative Example 2), and nanoparticles using only a hydrophobic polylactide (PLA1.7k) without adding a hydrophilic block (Comparative Example 3) were used.

[0135] [Table 6]

[0136] (4) Experiment to confirm the production of mRNA-containing nanoparticles Agarose gel electrophoresis was performed to confirm whether the nanoparticles and mRNA were properly bound when the drug delivery nanoparticle composition was mixed with the mRNA solution. After simply mixing the drug delivery composition and the mRNA solution, an amount equivalent to 500 ng of mRNA was extracted and mixed with a loading dye. The gel was then loaded onto a 1% agarose gel and electrophoresed at the appropriate voltage and time. The mRNA bands were then observed using a UV transilluminator. The Dyne Gel Safe Red Kit (DyneBio) was used as the nucleic acid staining reagent.

[0137] The measurement results are shown in Figure 2. As can be seen from Figure 2, in Examples 2 to 5, the binding strength between the nanoparticles and mRNA was high, so almost no exposed mRNA bands were observed. On the other hand, in Comparative Examples 1 to 3, the binding strength with mRNA was relatively low, so exposed mRNA bands were observed.

Claims

1. a first chamber containing nanoparticles comprising a cationic compound and an anionic polymeric compound; and a second chamber containing a drug, the active ingredient of which is selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; Including, Here, the kit for producing drug-containing nanoparticles, wherein the anionic polymer compound has at least one acid functional group.

2. 2. The kit for producing drug-containing nanoparticles according to claim 1, wherein the drug-containing nanoparticles are for delivering the drug into cells.

3. 2. The kit for producing 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 anionic amphiphilic block copolymer comprises a hydrophilic block and a hydrophobic block; the anionic hydrophilic polymer comprises only hydrophilic blocks, The kit for producing drug-containing nanoparticles according to claim 3, wherein the anionic hydrophobic polymer contains only hydrophobic blocks.

5. the hydrophilic block is one or more selected from the group consisting of polyalkylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, polyoxazoline, polyacrylamide, and derivatives thereof; 5. The kit for producing drug-containing nanoparticles according to claim 4, wherein the hydrophobic block is one or more selected from the group consisting of polyesters, polyanhydrides, polyamino acids, polyorthoesters, and polyphosphazines.

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

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

8. the inorganic acid is selected from the group consisting of phosphoric acid, nitric acid, chromic acid, and combinations thereof; the sulfonic acid is selected from the group consisting of methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, and combinations thereof; The kit for manufacturing drug-containing nanoparticles described in claim 7, wherein the carboxylic acid is selected from the group consisting of 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 producing drug-containing nanoparticles according to claim 1, wherein one or more selected from the group consisting of the first chamber and the second chamber further contains an additional solvent.

10. 10. The kit for producing 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 manufacturing drug-containing nanoparticles described in claim 1, wherein the second chamber further contains one or more additives selected from a pH adjuster, an inorganic salt, a sugar, a surfactant, a chelating agent, or a combination thereof.

12. a drug whose active ingredient is selected from a nucleic acid, a polypeptide, a virus, or a combination thereof; cationic compounds; the anionic polymer compound; a solvent; and one or more additives selected from pH adjusters, inorganic salts, sugars, surfactants, chelating agents, or combinations thereof; The kit for producing drug-containing nanoparticles according to claim 1, comprising:

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

14. 2. The kit for producing drug-containing nanoparticles according to claim 1, wherein the cationic compound and the anionic polymer compound are used for producing nanoparticles in the form of a solution that has been filtered one or more times.

15. A drug delivery composition comprising nanoparticles, the nanoparticles contain a cationic compound and an anionic polymer compound; the nanoparticles are drug-free; The nanoparticle composition for drug delivery, wherein the anionic polymer compound has at least one acid functional group.

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

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

18. cationic compounds; the anionic polymer compound; a solvent; and one or more additives selected from pH adjusters, inorganic salts, sugars, surfactants, chelating agents, or combinations thereof; The nanoparticle composition for drug delivery according to claim 15, comprising:

19. 16. 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 per 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 polymeric compound are used in the preparation of the nanoparticles in the form of a solution that has been filtered one or more times.

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