Taylor reaction device and method for producing capsule particles

Abstract

The present invention addresses the problem of providing a method for producing capsule particles. Provided is a method for producing capsule particles by using a Taylor reaction device.

Description

Technical Field

[0001] The present disclosure relates to a Taylor reaction device and a method for manufacturing capsule particles. Background Art

[0002] As a means for delivering an encapsulant to a target, capsule particles can be cited. The capsule portion on the outside of the capsule particles usually encapsulates the content inside. Thereby, stability can be improved or the content can be delivered to the target. As an example of using capsule particles, cosmetics, pharmaceuticals, functional foods, printing technology, industrial chemicals, etc. can be cited. As methods for manufacturing capsule particles, various methods such as a solvent injection method, a handshake method, a reverse phase evaporation method, a transmembrane pH gradient method, a bubble method, a microfluidic method, a thin film hydration method, a heating method, a freeze-thaw method, a dehydration rehydration method, etc. are known.

[0003] Research and development of pharmaceuticals using various nucleic acids such as plasmid DNA, antisense oligonucleotides, siRNA (Small interfering RNA), miRNA (microRNA), mRNA (Messenger RNA) are underway. For example, an mRNA vaccine is a type of vaccine that causes an immune response by administering mRNA into a living body to express a target protein in the living body. As an antigen protein in the mRNA vaccine, a viral antigen or a cancer antigen can be cited. In recent years, an example is the spike protein of SARS-CoV-2 (novel coronavirus). In addition, attempts have also been made to treat genetic diseases by administering mRNA encoding a normal protein.

[0004] Nucleic acids are negatively charged polymers even when directly administered, and thus cannot penetrate cell membranes. In particular, as a problem, it can be cited that mRNA is rapidly decomposed in the blood and causes an inflammatory reaction in the living body. Therefore, various drug delivery systems are used for the delivery of nucleic acids.

[0005] As a drug delivery system for delivering nucleic acids, for example, lipid nanoparticles can be cited. Lipid nanoparticles are mainly spherical or spherical-like in shape. As lipids, fatty acids, acylglycerols, waxes, and mixtures thereof with surfactants can be used. Stabilizers can also be used. As stabilizers, biological membrane lipids such as phospholipids and sphingomyelins, bile salts, sterols (such as cholesterol), etc. can be used. For lipid nanoparticles, for example, refer to Lipid Nanoparticles: Production, Characterization and Stability by Shah et al., Springer, 2015.

[0006] As the lipid component of the lipid nanoparticle, for example, phospholipids, cholesterol, pH-responsive cationic lipids, and PEGylated (PEGylation: polyethylene glycolylation) lipids can be mentioned, and nucleic acids are encapsulated therein.

[0007] The manufacturing process of the lipid nanoparticle (LNP) encapsulating nucleic acids is generally as follows. First, a lipid ethanol solution is brought into contact with an aqueous nucleic acid solution (acidic) to mix the lipid solution and the nucleic acid solution (mixing step). Then, dilution is performed as needed (dilution step). Next, ultrafiltration or diafiltration is performed for concentration (purification step). At this stage, free nucleic acids are removed, the solvent (ethanol) is removed, replacement with the final medium is performed, and the lipid nanoparticles are concentrated to the target concentration. Then, filter filtration is performed.

[0008] As existing methods for mixing lipid components and nucleic acid components, a dropwise mixing method, an inline mixing method, and a microfluidic mixing method are known. The problems of the dropwise mixing method are that it is not suitable for continuous production because it is a batch process, and the quality of the obtained particles is non-uniform. Although inline mixing enables continuous production, since the flow path is in the millimeter range, the size of the obtained particles is large, the quality is non-uniform, and reproducibility is lacking. The microfluidic mixing method can control the particle size to be small and has high reproducibility. On the other hand, since the flow path is as small as about 0.1 mm, the yield is low.

[0009] A manufacturing method of capsule particles suitable for large-scale production is needed.

[0010] Prior Art Documents

[0011] Non-Patent Documents

[0012] Non-Patent Document 1: Shah et al., Lipid Nanoparticles: Production, Characterization and Stability, Springer, 2015. Summary of the Invention

[0013] Problems to be Solved by the Invention

[0014] The problem of the present inventors is to provide a manufacturing method of capsule particles that solves at least part of the problems of existing particle manufacturing.

[0015] Means for Solving the Problems

[0016] The inventors of the present invention have conducted extensive research to solve the above problems and have found that, as an example, capsule particles can be produced by using a Taylor reaction device, thus completing the present invention that includes this finding as an embodiment.

[0017] The present disclosure includes the following embodiments.

[0018] [1] A method for manufacturing capsule particles, wherein a Taylor reaction device is used.

[0019] [2] According to the manufacturing method described in Embodiment 1, wherein

[0020] The Taylor reaction device has an outer cylinder, an inner cylinder, and one or more inlets,

[0021] The inner cylinder is rotatably disposed within the outer cylinder and forms an annular reaction chamber therebetween,

[0022] At least one of the inlets is configured to allow a fluid to flow into the reaction chamber.

[0023] [3] According to the manufacturing method described in Embodiment 2, wherein

[0024] The one or more inlets include a first inlet and a second inlet, the first inlet and the second inlet are independent of each other, and are respectively connected to the reaction chamber,

[0025] A particle-forming component is injected into the reaction chamber from the first inlet,

[0026] An encapsulating component is injected into the reaction chamber from the second inlet,

[0027] The inner cylinder is rotated to mix the particle-forming component and the encapsulating component within the reaction chamber.

[0028] [4] According to the manufacturing method described in Embodiment 2, wherein

[0029] The one or more inlets include a first pipeline and a second pipeline, the first pipeline and the second pipeline are configured to merge, and the merged pipeline is connected to the reaction chamber via a common inlet,

[0030] A particle-forming component is injected from the first pipeline, an encapsulating component is injected from the second pipeline, the particle-forming component and the encapsulating component are mixed in front of the reaction chamber to generate a particle-forming component - encapsulating component mixed solution, and then the particle-forming component - encapsulating component mixed solution is injected into the reaction chamber via the common inlet, and the inner cylinder is rotated to further mix the particle-forming component - encapsulating component mixed solution within the reaction chamber.

[0031] [5] According to the manufacturing method described in Embodiment 3 or 4, wherein

[0032] The particle-forming component contains a polymer component or a lipid component,

[0033] The encapsulating component contains nucleic acid, protein, polypeptide, peptide or low-molecular compound.

[0034] [6] According to the manufacturing method described in Embodiment 5, wherein,

[0035] The particle-forming component contains a lipid component,

[0036] The encapsulating component contains nucleic acid.

[0037] [7] According to the manufacturing method described in Embodiment 3, wherein the particle-forming component is injected first, and then the encapsulating component is injected.

[0038] [8] According to the manufacturing method described in Embodiment 3, wherein the encapsulating component is injected first, and then the particle-forming component is injected.

[0039] [9] According to the manufacturing method described in Embodiment 3, wherein the particle-forming component and the encapsulating component are injected simultaneously.

[0040]

[10] According to the manufacturing method described in any one of Embodiments 2 to 9, wherein the circumferential speed of the inner cylinder is 0.5 to 47 m / s.

[0041]

[11] According to the manufacturing method described in any one of Embodiments 2 to 10, wherein the clearance width between the inner cylinder and the outer cylinder is 0.01 mm to 5 mm.

[0042]

[12] According to the manufacturing method described in any one of Embodiments 1 to 11, wherein the stirring time for carrying out the Taylor reaction is a stirring time of 1 minute or less.

[0043]

[13] According to the manufacturing method described in any one of Embodiments 3, 5 to 12, wherein the ratio of the injection speed of the particle-forming component to the injection speed of the encapsulating component is a ratio selected from the range of 1:1 to 1:9.

[0044]

[14] According to the manufacturing method described in any one of Embodiments 3 to 13, wherein the inner diameters of the first inlet and the second inlet are the same or substantially the same.

[0045]

[15] According to the manufacturing method described in any one of Embodiments 3 to 13, wherein the inner diameters of the first inlet and the second inlet are different.

[0046]

[16] A Taylor reaction device used in the manufacturing method of capsule particles, which has an outer cylinder, an inner cylinder and one or more inlets,

[0047] The inner cylinder is disposed inside the outer cylinder in a rotatable manner, and an annular reaction chamber is formed between the inner cylinder and the outer cylinder.

[0048] At least one of the inlets is configured to allow a fluid to flow into the reaction chamber.

[0049]

[17] According to the device described in Embodiment 16, wherein

[0050] One or more inlets include a first inlet and a second inlet, the first inlet and the second inlet are independent of each other, and are respectively connected to the reaction chamber.

[0051] The first inlet is for injecting a particle-forming component.

[0052] The second inlet is for injecting an encapsulating component.

[0053] The inner cylinder is for mixing the particle-forming component and the encapsulating component in the reaction chamber by rotation.

[0054]

[18] According to the device described in Embodiment 16, wherein

[0055] One or more inlets have a first pipeline and a second pipeline.

[0056] The first pipeline is for injecting a particle-forming component.

[0057] The second pipeline is for injecting an encapsulating component.

[0058] The first pipeline and the second pipeline merge in front of the reaction chamber.

[0059] A particle-forming component - encapsulating component mixed solution pipeline is formed on the downstream side of the merging part, and then is connected to the reaction chamber via a common inlet.

[0060] The inner cylinder is for further mixing the particle-forming component - encapsulating component mixed solution in the reaction chamber by rotation.

[0061]

[19] According to the device described in Embodiment 17 or 18, wherein

[0062] The particle-forming component contains a polymer component or a lipid component.

[0063] The encapsulating component contains nucleic acid, protein, polypeptide, peptide or low molecular compound.

[0064]

[20] According to the device described in Embodiment 19, wherein

[0065] The particle-forming component contains a lipid component.

[0066] The encapsulating component contains nucleic acid.

[0067]

[21] According to the device described in Embodiment 17 or Embodiment 19 which is subordinate to Embodiment 17, wherein, if the side for supplying liquid to the reaction chamber is set as the upstream side and the side for discharging liquid from the reaction chamber is set as the downstream side, then the first inlet is arranged on the upstream side and the second inlet is arranged on the downstream side.

[0068]

[22] According to the device described in Embodiment 17 or Embodiment 19 which is subordinate to Embodiment 17, wherein, if the side for supplying liquid to the reaction chamber is set as the upstream side and the side for discharging liquid from the reaction chamber is set as the downstream side, then the second inlet is arranged on the upstream side and the first inlet is arranged on the downstream side.

[0069]

[23] According to the device described in Embodiment 17 or Embodiment 19 which is subordinate to Embodiment 17, wherein the first inlet and the second inlet are arranged in the same cross-section perpendicular to the axial direction of the inner cylinder.

[0070]

[24] According to the device described in any one of Embodiments 16 to 23, wherein the gap width between the inner cylinder and the outer cylinder is 0.01 mm to 5 mm.

[0071]

[25] According to the device described in any one of Embodiments 17 and 19 to 24, wherein the ratio of the injection speed of the particle-forming component to the injection speed of the encapsulating component is a ratio selected from the range of 1:1 to 1:9.

[0072]

[26] According to the device described in any one of Embodiments 17 to 25, wherein the inner diameters of the first inlet and the second inlet are the same or substantially the same.

[0073]

[27] According to the device described in any one of Embodiments 17 to 25, wherein the inner diameters of the first inlet and the second inlet are different.

[0074] This specification includes the disclosure of Japanese Patent Application No. 2022-167048 which is the basis of the priority of this application.

[0075] Effects of the Invention

[0076] As an effect of the present disclosure, capsule particles can be manufactured. Description of the Drawings

[0077] Figure 1 It is a schematic diagram of a Taylor reaction device.

[0078] Figure 2 It shows the configuration of the inlets having the first pipeline and the second pipeline.

[0079] Figure 3 It shows the configuration of the first inlet and the second inlet.

[0080] Figure 4 Shows the configuration of the inlet with the first pipeline, the second pipeline, and the third pipeline.

[0081] Figure 5 Shows the configuration of the first inlet, the first inlet and the second inlet of the second pipeline. Figure 5 And Figure 1 Corresponds to the A-A' cross-section in

[0082] Figure 6 Shows the configuration of the first inlet, the second inlet, and the third inlet.

[0083] Figure 7 Is a graph showing the iron concentration in the plasma of a mouse before administration of nucleic acid lipid particles encapsulating TfR2 siRNA (Transferrin receptor 2 small interfering RNA).

[0084] Figure 8 Is a graph showing the iron concentration in the plasma of a mouse 1 day after administration of nucleic acid lipid particles encapsulating TfR2 siRNA.

[0085] Figure 9 Is a graph showing the iron concentration in the plasma of a mouse 7 days after administration of nucleic acid lipid particles encapsulating TfR2 siRNA.

[0086] Figure 10 Is a graph showing the concentration of HA (Heterophilic antibody) - specific IgG (immunoglobulin G) in the serum of a mouse after administration of nucleic acid lipid particles encapsulating A_Sin_GP1908_2015_H1 mRNA (Reference Example 2, Specimen 9, Specimen 10, and Specimen 11).

[0087] Figure 11 Is a graph showing the concentration of HA - specific IgG in the serum of a mouse after administration of nucleic acid lipid particles encapsulating A_Sin_GP1908_2015_H1 mRNA (Reference Example 3, Reference Example 4, Specimen 12, Specimen 13, and Specimen 14). Detailed implementation mode

[0088] In some embodiments, the present disclosure provides a method for manufacturing capsule particles using a Taylor reaction device.

[0089] A capsule particle refers to a particle whose content is encapsulated. In this specification, the content encapsulated in the capsule particle is sometimes referred to as the encapsulated component (the component to be encapsulated). Additionally, in this specification, the component surrounding the encapsulated content is sometimes referred to as the particle-forming component (particle-forming ingredient) or the capsule-forming component. Further, in the capsule particle, the encapsulated component and the particle-forming component can form a complex. Capsule particles include, but are not limited to, lipid particles and polymer particles. In some embodiments, the capsule particle can be a particle having an average particle diameter of 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 1 μm or more, 10 μm or more, 100 μm or more, such as 1 mm or more, 2 mm or less, 1 mm or less, 100 μm or less, 10 μm or less, 1 μm or less, such as 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, such as 10 nm or less, but is not limited thereto. Furthermore, the capsule particle can contain any content (encapsulated component). Additionally, the size or average particle diameter of the capsule particle can be appropriately adjusted according to the particle-forming component or the encapsulated component. For example, the particle diameter can vary according to the particle-forming component or the encapsulated component and their combination.

[0090] In some embodiments, the particle-forming component can include a polymer component, a lipid component, an auxiliary component, etc. As the polymer component, for example, biodegradable polymers can be cited. As such biodegradable polymers, poly(lactic acid), poly(glycolic acid), poly(lactic-co-glycolic acid) (PLGA), and poly(depsipeptide-lactic acid copolymer) can be cited, but are not limited thereto. As the lipid component, for example, zwitterionic lipids, neutral lipids, and cationic lipids can be cited. Specifically, phospholipids, sphingolipids, glycolipids, glycerolipids, sterol lipids, and polymer complex lipids can be cited, but are not limited thereto. In some embodiments, the particle-forming component can be used by dissolution, etc. As the encapsulated component, nucleic acids, proteins, polypeptides, peptides, low-molecular compounds, such as pharmaceutical compounds, cosmetics or cosmetic ingredients, pigments, skin care ingredients, nutritional ingredients, etc. can be cited, but are not limited thereto. In some embodiments, the encapsulated component can be used by dissolution, etc.

[0091] In this specification, unless otherwise specified, the average particle diameter refers to the volume average particle diameter obtained by measurement and calculation based on the principle of dynamic light scattering method. Here, in ascending order of particle diameter, there are n1, n2, …, ni, …, nk particles with particle diameters of d1, d2, …, di, …, dk, respectively. In addition, the volume of each particle is set as vi. At this time, the volume average diameter (mean volume diameter) can be obtained by the following formula.

[0092] [Formula 1]

[0093] MV = Σ(Vi·di) / Σ(di)

[0094] Polymer particles refer to particles containing a polymer material. In some embodiments, the polymer particles may be particles having an average particle diameter of 10 nm or more, 20 nm or more, 30 nm or more, 40 nm or more, 50 nm or more, 60 nm or more, 70 nm or more, 80 nm or more, 90 nm or more, 100 nm or more, 1 μm or more, 10 μm or more, 100 μm or more, for example, 1 mm or more, 2 mm or less, 1 mm or less, 500 μm or less, 400 μm or less, 300 μm or less, 200 μm or less, for example, 100 μm or less, 10 μm or less, 1 μm or less, for example, 100 nm or less, 90 nm or less, 80 nm or less, 70 nm or less, 60 nm or less, 50 nm or less, 40 nm or less, 30 nm or less, 20 nm or less, for example, 10 nm or less, but not limited thereto. In some embodiments, the polymer particles may be particles having an average particle diameter of 10 nm to 500 μm, for example, 20 nm to 400 μm, 30 nm to 300 μm, 20 nm to 200 μm, for example, 10 nm to 100 μm, but not limited thereto. Examples of the polymer particles include particles containing a biodegradable polymer as a particle-forming component. Examples of the biodegradable polymer include polymers containing polylactic acid. Examples of the polymers containing polylactic acid include poly(lactic-co-glycolic acid) (PLGA) and poly(polyphenolic acid peptide-lactic acid) copolymer, etc., but not limited thereto. The polymer particles may contain any content (encapsulated component).

[0095] Lipid particles refer to particles containing lipid components. The structure of lipid particles is not particularly limited and can be a lipid multilamellar, lipid bilayer, or lipid monolayer, or can partially have a lipid multilamellar, lipid bilayer, or lipid monolayer. As lipid particles, for example, particles having a lipid bilayer, such as liposomes or liposome-like particles having a lipid bilayer, particles having a lipid monolayer, such as lipid monolayer particles containing tetraether lipids, particles partially having a lipid monolayer and / or lipid bilayer, particles having a lipid multilamellar, lipid nanoparticles (LNP), etc. are mentioned, but not limited thereto. Lipid particles can contain any content (encapsulated component), but as the encapsulated component, for example, nucleic acids, proteins, peptides, low molecular weight compounds, pharmaceutical compounds are mentioned, preferably nucleic acids, and more preferably messenger RNA (mRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA) are mentioned, and further preferably mRNA (lipid particles having a nucleic acid as the encapsulated component are sometimes particularly referred to as nucleic acid lipid particles). In addition, liposomes refer to lipid particles having at least one lipid bilayer. As liposomes, small unilamellar vesicles (SUV), large unilamellar vesicles (LUV), multilamellar vesicles (MLV), oligolamellar vesicles (OLV), medium unilamellar vesicles (MUV), giant unilamellar vesicles (GUV), giant multilamellar vesicles (GMV), etc. are mentioned. The general particle size of LUV can be 100 nm or more, but there is also a classification of 50 nm or more. The general particle size of SUV can be 20 to 100 nm, but there is also a classification of less than 50 nm. Although any classification can be adopted, the same classification is used for both LUV and SUV. Each vesicle in MLV has two or more bilayers. Separate aqueous compartments can be formed within MLV. Liposomes can have a bilayer membrane composed of phospholipids. Liposome-like particles can have a bilayer membrane composed of nonionic surfactants. For example, refer to Japanese Patent Application Laid-Open No. 2013-536803. The description related to liposomes is incorporated into this specification by reference.

[0096] Lipid components are roughly classified into zwitterionic lipids, neutral lipids, anionic lipids, and cationic lipids. As lipid components, there is no particular limitation, and phospholipids, sphingolipids, glycolipids, glycerolipids, sterol lipids, polymer complex lipids, etc. are mentioned, but not limited thereto.

[0097] As cationic lipids, there can be mentioned lipids in which lipid molecules that are not affected by pH have a net positive charge, and lipids in which, at a selected pH such as physiological pH, a part of the lipid molecules have a net positive charge corresponding to the pKa (acid dissociation constant) of the lipid (sometimes referred to as pH-responsive cationic lipids). As lipids in which lipid molecules that are not affected by pH have a net positive charge, for example, N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium chloride (DOTAP), N,N-dioctadecylamidoglycylcarboxyspermine (DOGS), N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl-1-propylammonium trifluoroacetate (DOSPA), N-[1-(2,3-dimyristyloxy)propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE), 3β-N-(N',N'-dimethylaminoethane)-carbamoyl cholesterol (DC-Chol) can be mentioned, but not limited thereto. As pH-responsive cationic lipids, for example, 1,2-dioleoyl-3-dimethylammonium propane (DODAP), N,N-dimethyl-(2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-3-dimethylammonium propane (DLinDMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), heptatriaconta-6,9,28,31-tetraene-19-yl-4-(dimethylamino)butyrate (DLin-MC3-DMA) etc. can be mentioned, but not limited thereto. For example, refer to International Publication No. 2015 / 005253, International Publication No. 2021 / 060440. The descriptions related to cationic lipids are incorporated into this specification in the form of reference.

[0098] Amphiphilic lipids refer to lipids that have both hydrophilic groups and hydrophobic groups. Examples of amphiphilic lipids include, but are not limited to, ionic lipids, phospholipids, sphingolipids, glycolipids, glycerolipids, etc. Examples of phospholipids include glycerophospholipids and sphingophospholipids. Examples of glycerophospholipids include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidylcholine, etc. Examples of sphingophospholipids include sphingomyelin, sphingocholine, etc. Examples of glycolipids include glyceroglycolipids and glycosphingolipids. Examples of glyceroglycolipids include monogalactosyldiacylglycerol, digalactosyldiacylglycerol, sulfoquinovosyldiacylglycerol, etc. Examples of glycosphingolipids include ceramides, gangliosides, etc.

[0099] Examples of specific phospholipids include, but are not limited to, 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-diacyl-sn-glycero-3-phosphocholine (PC), 1,2-diacyl-sn-glycero-3-phosphatidylethanolamine (PE), 1,2-diacyl-sn-glycero-3-phosphatidylserine (PS), 1,2-diacyl-sn-glycero-3-phosphatidylglycerol (PG), 1,2-diacyl-sn-glycero-3-phosphoric acid (PA), 1,2-didecanoyl-sn-glycero-3-phosphocholine (DDPC), 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dilinoleoyl-sn-glycero-3-phosphocholine (DLoPC), 1,2-dierucoyl-sn-glycero-3-phosphocholine (DEPC), 1-myristoyl-2-palmitoyl-sn-glycero-3-phosphocholine (MPPC), 1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine (MSPC), 1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine (PMPC), 1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine (PSPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC), etc. For example, refer to International Publication No. 2021 / 060440. The description related to phospholipids is incorporated into this specification by reference.

[0100] As sterol esters, for example, sterols derived from animals, sterols derived from plants, sterols derived from microorganisms, etc. can be cited. As sterols derived from animals, for example, cholesterol, cholesterol succinate, dihydrocholesterol, 7-dehydrocholesterol, lanosterol, dihydrolanosterol, stigmastanol, etc. can be cited. As sterols derived from plants, for example, campesterol, brassicasterol, stigmasterol, sitosterol can be cited. As sterols derived from microorganisms, for example, ergosterol, mycosterol can be cited.

[0101] Polymer composite lipids refer to lipids added with non-ionic polyethers, non-ionic polyesters, non-ionic polyamino acids or non-ionic polypeptides, or polymers with their ends alkoxylated, etc. There are no particular restrictions on the polymers added to the polymer composite lipids, but non-ionic polyethers or non-ionic polyesters or polymers with their ends alkoxylated are preferred, non-ionic polyethers or non-ionic monoalkoxy polyethers are more preferred, polyalkylene glycols or monomethoxy polyalkylene glycols are further preferred, and polyethylene glycol or monomethoxy polyethylene glycol are even more preferred. Polyoxyalkylated lipids are lipids modified with polyalkylene glycols or their derivatives, and examples include polymethylene glycolated lipids, polyethylene glycolated lipids (PEG lipids), polypropylene glycolated lipids, polytetramethylene glycolated lipids, polyhexamethylene glycolated lipids (Polyhexamethyleneglycol lipid), etc., but are not limited thereto. The weight-average molecular weight of the polyalkylene glycol can be, for example, 200 to 10,000 Da. Examples of PEG lipids include PEG-cholesterol, PEG-phospholipid, PEG-ceramide, PEG-diglyceride, but are not limited thereto. The PEG moiety of the PEG lipid can have a molecular weight of, for example, 200 to 10,000 Da. The PEG moiety can be linear or branched. When modifying with PEG, stearylated polyethylene glycol, N-[carbonyl-methoxypolyethylene glycol-1000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, n-[carbonyl-methoxypolyethylene glycol-5000]-1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-750]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-1000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-2000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, N-[carbonyl-methoxypolyethylene glycol-5000]-1,2-distearoyl-sn-glycero-3-phosphoethanolamine, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (PEG-DMG) and other polyethylene glycol derivatives, etc., but are not limited thereto. For example, refer to International Publication No. WO 2020 / 262150. The description related to polyoxyalkylated lipids is incorporated into this specification by reference.

[0102] The lipid component(s) can be single or multiple. In some embodiments, the lipid component can be a lipid containing a cationic lipid. In other embodiments, the lipid component can include an amphiphilic lipid, a cationic lipid, a sterol lipid, and a polymeric complex lipid. In other specific embodiments, the content of each lipid in the overall lipid component used can be such that it contains 5 to 30 mol% phospholipid as the amphiphilic lipid, such as 12.5 mol%, such as 17.5 mol%, contains 10 to 75 mol% cationic lipid, such as 30 to 70 mol%, such as 40 to 65 mol%, such as 45 mol%, such as 60 mol%, contains 15 to 50 mol% cholesterol, such as 21 mol%, such as 41 mol% and contains 1 to 10 mol% polyoxyalkylated lipid, such as 1 to 5 mol%, such as 1.5 mol%. For example, refer to International Publication No. 2009 / 127060 and International Publication No. 2015 / 005253. The description related to the component ratio of the lipid component is incorporated into this specification.

[0103] Examples of the encapsulated component (contents) contained in the capsule particles include, but are not limited to, nucleic acids, proteins, polypeptides, peptides, low-molecular compounds, and pharmaceutical compounds. Examples of nucleic acids include DNA and RNA. Examples of RNA include messenger RNA (mRNA), small interfering RNA (siRNA), single-stranded RNA (antisense oligo), small nuclear RNA (snRNA), non-coding RNA (ncRNA), long non-coding RNA (lncRNA), microRNA (miRNA), double-stranded RNA (dsRNA), and circular RNA, but are not limited thereto. Examples of DNA include plasmids, single-stranded DNA, and double-stranded DNA, but are not limited thereto. Examples of low-molecular compounds include pharmaceutical compounds, anticancer agents, compounds acting on organisms, physiologically active substances, metabolites, etc., but are not limited thereto. Examples of proteins, polypeptides, or peptides include antigenic proteins, therapeutic proteins, physiologically active proteins, antibodies, etc., but are not limited thereto.

[0104] There is no particular limitation on the combination of the particle-forming component and the encapsulated component. In a specific embodiment, the combination of the particle-forming component and the encapsulated component can be selected in consideration of charge, polarity, etc. In some embodiments, when the encapsulated component is negatively charged, for example, a positively charged component (such as a lipid component) can be selected as the particle-forming component. In some embodiments, when the encapsulated component is positively charged, for example, a negatively charged component (such as a lipid component) can be selected as the particle-forming component. In some embodiments, when the encapsulated component is uncharged or almost uncharged, for example, an uncharged or almost uncharged component (such as a lipid component) can be selected as the particle-forming component. In some embodiments, when the encapsulated component is a highly hydrophobic component, the medium for dissolving the encapsulated component and the medium for dissolving the particle-forming component can be the same medium. In another embodiment, when the encapsulated component is a highly hydrophobic component, the medium for dissolving the encapsulated component and the medium for dissolving the particle-forming component can be different media.

[0105] In some embodiments of the present invention, the capsule particles are lipid particles (sometimes referred to as nucleic acid-lipid particles) using nucleic acid as the encapsulated component. Examples of the nucleic acid-lipid particles include lipid nanoparticles (LNPs) encapsulating nucleic acid and liposomes encapsulating nucleic acid. Examples of the lipid components for the particle-forming components include zwitterionic lipids, neutral lipids, cationic lipids, anionic lipids, amphiphilic lipids, sterol lipids, polymer complex lipids, etc., and they can be used alone or in combination of two or more. When multiple lipids are used as the lipid components in the nucleic acid-lipid particles, from the viewpoint of drug delivery in vivo, it is preferable to use cationic lipids, amphiphilic lipids, sterol lipids, and polymer complex lipids, and more preferably pH-responsive cationic lipids, phospholipids, cholesterol, and PEG lipids. The content of each lipid in the whole lipid components used can be appropriately adjusted according to the type of lipid used, the encapsulated nucleic acid, etc. For example, phospholipids can be set to 5 to 30 mol%, such as 12.5 mol%, such as 17.5 mol%, cationic lipids can be set to 10 to 75 mol%, such as 30 to 70 mol%, such as 40 to 65 mol%, such as 45 mol%, such as 60 mol%, cholesterol can be set to 15 to 50 mol%, such as 21 mol%, such as 41 mol%, and PEGylated lipids can be set to 1 to 10 mol%, such as 1 to 5 mol%, such as 1.5 mol%. The nucleic acid used as the encapsulated component is not particularly limited, but preferably includes messenger RNA (mRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), double-stranded DNA (dsDNA), single-stranded DNA (ssDNA), and more preferably mRNA. In addition, when a cationic lipid is used as the lipid component, the ratio (N / P ratio) of the number of molecules (N) of the cationic lipid in the nucleic acid-lipid particle to the number of phosphorus atoms (P) from the nucleic acid can be, for example, about 2.0 to 15.0, preferably about 2.0 to 12.0, more preferably about 2.0 to 9.0, and further preferably about 3.0 to 9.0. The lower limit of the N / P ratio can be, for example, 2.0, preferably 2.5, more preferably 3.0, and the upper limit can be, for example, 15.0, preferably 12.0, more preferably 9.0. The average particle size of the nucleic acid-lipid particles is not particularly limited, but can be, for example, 1 nm to 1 μm, preferably 10 nm to 500 nm, more preferably 30 nm to 300 nm, further preferably 50 nm to 200 nm, and most preferably 75 nm to 150 nm.

[0106] As Figure 1As shown, the Taylor reaction device 1 refers to a device for performing the Taylor reaction (also known as the Taylor vortex reaction). In some embodiments, the Taylor reaction device 1 has an inner cylinder 3 and an outer cylinder 2. A gap portion (d) is formed between the outer diameter (inner radius, IR) of the inner cylinder 3 and the inner diameter (outer radius, OR) of the outer cylinder 2, and this is used as the reaction chamber 5 (also known as the reaction tank) (OR = IR + d). Thus, the Taylor reaction device 1 has an outer cylinder 2 and an inner cylinder 3, and the inner cylinder 3 is rotatably disposed within the outer cylinder 2, with an annular reaction chamber 5 formed therebetween. The Taylor reaction device 1 may have one or more inlets 4 (including a first inlet 41 and a second inlet 42) for allowing liquid to flow into the reaction chamber 5. In this specification, the inlets 4, the first inlet 41, and the second inlet 42 are sometimes referred to as injection ports or injection nozzles. Additionally, the Taylor reaction device 1 may have at least one outlet 6 for allowing the liquid within the reaction chamber 5 to flow out to the outside. In this specification, the outlet 6 is sometimes referred to as the discharge port or the outflow port. Further, the reference numeral 7 is a control device that performs speed control of the motor 8 for rotating the inner cylinder 3 and supply amount control of the first pump 12, the second pump 14, and the third pump 16. The reference numeral 20 is a jacket for cooling the reaction chamber 5, and the reference numeral 21 is a cooler for jacket temperature regulation that circulates the cooling medium within the jacket 20 for temperature adjustment.

[0107] The encapsulating component and the particle-forming component can react in a solution state to form particles. Therefore, the respective solvents can be appropriately selected according to the properties of the encapsulating component and the particle-forming component. In the manufacture of nucleic acid-lipid particles, for example, the nucleic acid can be supplied to the device in a state dissolved in a citrate buffer solution, and the lipid can be supplied to the device in a state dissolved in ethanol. Thus, the encapsulating component and the particle-forming component can be prepared in a solution state through the respective solvents and can be supplied to the device separately.

[0108] In some embodiments, the method for manufacturing the particles of the present disclosure may have a step of mixing the particle-forming component and the encapsulating component. Inside the reaction chamber 5, when observed in a cross-sectional view in a direction perpendicular to the rotation axis of the inner cylinder 3, a group of vortex columns 5V are formed in multiple numbers axially in opposite directions to each other, and during the axial movement of these multiple vortex columns 5V, various solutions are mixed (Taylor vortex reaction). In a specific embodiment, first, it is supplied from the first tank 11 containing the solution of the particle-forming component via the first pump 12 from the first pipeline 4A, supplied from the second tank 13 containing the solution of the encapsulating component via the second pump 14 from the second pipeline 4B and mixed in the mixed solution pipeline 4D, and then it is supplied to the reaction chamber 5 of the Taylor reaction device 1 via the inlet 4 (which is also the common inlet described in the claims) (refer to Figure 1 , 2)。Since the mixing of the two liquids is carried out right before the injection into the reaction chamber 5, for convenience, in this specification, it is sometimes referred to as the mixing "in front of the reaction chamber (in front of the reaction tank)". In another embodiment, the particle-forming component and the encapsulating component do not come into contact with each other and are respectively supplied into the reaction chamber 5 of the Taylor reaction device 1 through independent injection ports (the first inlet 41 and the second inlet 42), and then the two liquids are first mixed inside the reaction chamber 5 (refer to Figure 3 )。Since the mixing of the two liquids is carried out inside the reaction chamber 5, for convenience, in this specification, it is sometimes referred to as the mixing "inside the reaction chamber (inside the reaction tank)". The order of injecting the solutions is not particularly limited. The particle-forming component and the encapsulating component can be injected simultaneously, or the particle-forming component can be injected first, or the encapsulating component can be injected first.

[0109] In another embodiment, the method for manufacturing the particles of the present disclosure can supply the pre-mixed liquid containing the particle-forming component and the encapsulating component from the first inlet 41 to the reaction chamber 5. In this case, the mixing of the particle-forming component and the encapsulating component can be carried out outside the Taylor reaction device 1. Additionally, a dilution solution can be injected from the second inlet 42 different from the first inlet 41 for injecting the mixed liquid (refer to Figure 3 )。For example, when both the particle-forming component and the encapsulating component are fat-soluble or hydrophobic, the particle-forming component and the encapsulating component can be dissolved in the same or different solvents and mixed. And such a pre-mixed solution can be used in the method for manufacturing the particles of the present disclosure.

[0110] In some embodiments, the method for manufacturing the particles of the present disclosure can have a step of further injecting a dilution solution. The dilution solution can be injected at any stage. In some embodiments, first, the particle-forming component is supplied from the first pipeline 4A, then the dilution solution is supplied from the third pipeline 4C and mixed to generate a particle-forming component-dilution solution mixed solution, and then the encapsulating component is supplied from the second pipeline 4B to the particle-forming component-dilution solution mixed solution and a particle-forming component-encapsulating component-dilution solution mixed solution is generated in the mixed solution pipeline 4D, and it can be supplied to the reaction chamber 5 through the inlet 4 (which is also the common inlet described in the claims) (refer to Figure 4)。In another embodiment, the encapsulating component and the diluting solution are first mixed to generate an encapsulating component-diluting solution mixed solution, and then the encapsulating component-diluting solution mixed solution and the particle-forming component are mixed to generate a particle-forming component-encapsulating component-diluting solution mixed solution, which can be supplied to the reaction chamber. In another embodiment, the particle-forming component and the encapsulating component are first mixed to generate a particle-forming component-encapsulating component mixed solution, and then the particle-forming component-encapsulating component mixed solution and the diluting solution are mixed to generate a first-second-third mixed solution, which can be supplied to the reaction chamber. In another embodiment, first, it is supplied from the first tank 11 containing the solution of the particle-forming component via the first pump 12 from the first pipeline 41A, and supplied from the second tank 13 containing the solution of the encapsulating component via the second pump 14 from the second pipeline 41B and mixed, and a particle-forming component-encapsulating component mixed solution is generated in the mixed solution pipeline 41D, which is supplied to the reaction chamber 5 via the first inlet 41. Then, it is supplied from the third tank 15 containing the diluting solution via the third pump 16 from the second inlet 42 into the reaction chamber 5, so that a particle-forming component-encapsulating component-diluting solution mixed solution can be generated (refer to Figure 1 , 5 ). The order of injecting the solutions into the reaction chamber is not particularly limited. The diluting solution can be first supplied to the reaction chamber, and then the particle-forming component-encapsulating component mixed solution can be supplied to the reaction chamber. In addition, the diluting solution and the particle-forming component-encapsulating component mixed solution can also be supplied to the reaction chamber simultaneously.

[0111] In some embodiments, the dilution solution can stabilize the encapsulated components. In another embodiment, the dilution solution can reduce the association of particles with each other by diluting the formed capsule particles. The type of the dilution solution can be any one of a neutral buffer solution, an acidic buffer solution, or a basic buffer solution, and can be appropriately selected according to the particle-forming components or the encapsulated components. As the dilution solution, for example, a citrate buffer solution, an acetate buffer solution, a phosphate buffer solution, a buffer solution containing Good's buffer (zwitterionic buffer), etc. can be mentioned, but it is not limited thereto. When the particle-forming components contain pH-responsive cationic lipids and the encapsulated component is a nucleic acid, the dilution solution can preferably be a neutral or weakly acidic buffer solution. For example, a phosphate buffer solution can be used as the neutral buffer solution, or a citrate buffer solution can be used as the weakly acidic buffer solution, etc. When the particle-forming components contain neutral amphiphilic lipids, the dilution solution can contain a buffer solution near neutrality, such as a phosphate buffer solution. When the particle-forming components contain anionic lipids, the dilution solution can contain, for example, a basic or weakly basic buffer solution. In some embodiments, in the manufacture of nucleic acid-lipid particles, in order to reduce the content of the solvent (such as ethanol) in which the lipid is dissolved and stabilize the particles, the same solvent as the solvent of the encapsulated component (for example, if the solvent of the encapsulated component uses a citrate buffer solution, then a citrate buffer solution is used) can be used as the dilution solution.

[0112] In another embodiment, first, the particle-forming components and the dilution solution are mixed to generate a particle-forming component-dilution solution mixed solution, and then the particle-forming component-dilution solution mixed solution is supplied to the reaction chamber. Then, the encapsulated component is supplied to the reaction chamber, so that a particle-forming component-encapsulated component-dilution solution mixed solution can be generated. The order of injecting the solutions into the reaction chamber is not particularly limited. The encapsulated component can also be supplied to the reaction chamber, and simultaneously or subsequently, the particle-forming component-dilution solution mixed solution can be supplied to the reaction chamber. In another embodiment, first, the encapsulated component and the dilution solution are mixed to generate an encapsulated component-dilution solution mixed solution, and then the encapsulated component-dilution solution mixed solution is supplied to the reaction chamber. Then, the particle-forming components are supplied to the reaction chamber, so that a particle-forming component-encapsulated component-dilution solution mixed solution can be generated. The order of injecting the solutions into the reaction chamber is not particularly limited. The particle-forming components can also be supplied to the reaction chamber, and simultaneously or subsequently, the encapsulated component-dilution solution mixed solution can be supplied to the reaction chamber. In another embodiment, the particle-forming components are supplied to the reaction chamber 5 through the first inlet 41, the encapsulated component is supplied to the reaction chamber 5 through the second inlet 42, and the dilution solution is supplied to the reaction chamber 5 through the third inlet 43, so that a particle-forming component-encapsulated component-dilution solution mixed solution can be generated in the reaction chamber 5 (refer to Figure 6)。There is no particular limitation on the order of injecting the solution into the reaction chamber. It is also possible to supply the particle-forming component to the reaction chamber, and simultaneously or subsequently supply the dilution solution to the reaction chamber, and simultaneously or subsequently supply the encapsulating component to the reaction chamber. It is also possible to supply the encapsulating component to the reaction chamber, and simultaneously or subsequently supply the particle-forming component to the reaction chamber, and simultaneously or subsequently supply the dilution solution to the reaction chamber. It is also possible to supply the encapsulating component to the reaction chamber, and simultaneously or subsequently supply the dilution solution to the reaction chamber, and simultaneously or subsequently supply the particle-forming component to the reaction chamber. It is also possible to supply the dilution solution to the reaction chamber, and simultaneously or subsequently supply the particle-forming component to the reaction chamber, and simultaneously or subsequently supply the encapsulating component to the reaction chamber. It is also possible to supply the dilution solution to the reaction chamber, and simultaneously or subsequently supply the encapsulating component to the reaction chamber, and simultaneously or subsequently supply the particle-forming component to the reaction chamber. It is also possible to supply the three liquids, i.e., the particle-forming component, the encapsulating component, and the dilution solution, to the reaction chamber simultaneously.

[0113] The same applies to solutions other than the particle-forming component, the encapsulating component, and / or the dilution solution. That is, in some embodiments, the method for manufacturing the particles of the present disclosure may further include a step of injecting one or more other solutions in addition to the particle-forming component, the encapsulating component, and / or the dilution solution. The other solutions may independently be a solution in which the particle-forming component is dissolved, a solution in which the encapsulating component is dissolved, or a dilution solution, or a liquid for mixing them.

[0114] In addition, regarding the method for manufacturing particles using the Taylor reaction device 1 of the present disclosure, the particles do not necessarily have to be manufactured only in the reaction chamber 5. For example, in a method of mixing the particle-forming component and the encapsulating component and then supplying them to the reaction chamber 5 of the Taylor reaction device 1, starting from the stage where the particle-forming component and the encapsulating component are mixed, a part of the target particles can be formed. The target particles formed through such a process also correspond to the target particles manufactured by the manufacturing method of the present disclosure or the target particles manufactured by the device of the present disclosure. In addition, when the mixing step is performed in front of the reaction tank, not only the target particles are formed in the reaction chamber 5, but also the homogenization of the particles can be achieved.

[0115] In some embodiments, the Taylor reaction device 1 has an inlet 4 (which is also the common inlet described in the claims) for injecting the particle-forming component-encapsulating component mixed solution in which the particle-forming component and the encapsulating component are mixed into the reaction chamber 5 (refer to Figure 2 ). In some embodiments, the particle manufacturing method includes a step of injecting the particle-forming component-encapsulating component mixed solution from the inlet 4 and rotating the inner cylinder 3 to further mix the particle-forming component-encapsulating component mixed solution. In the reaction chamber 5, the particle-forming component-encapsulating component mixed solution is supplied to the Taylor vortex reaction and can be further mixed.

[0116] In some embodiments, the Taylor reaction device 1 has a first inlet 41 for injecting particle-forming components into the reaction chamber 5 and a second inlet 42 for injecting encapsulating components into the reaction chamber 5 (see Figure 3 ). In some embodiments, the particle manufacturing method includes a step of injecting particle-forming components from the first inlet 41, injecting encapsulating components from the second inlet 42, and rotating the inner cylinder 3 to mix the particle-forming components and the encapsulating components. In the reaction chamber 5, the particle-forming components and the encapsulating components can be subjected to a Taylor vortex reaction.

[0117] In some embodiments, the Taylor reaction device 1 may further have a third inlet 43 for injecting a dilution solution into the reaction chamber 5 (see Figure 6 ). In some embodiments, the particle manufacturing method includes a step of injecting a dilution solution from the third inlet 43 and rotating the inner cylinder 3. In the reaction chamber 5, the dilution solution and the solution in the reaction chamber 5 can be subjected to a Taylor vortex reaction. The same applies to the fourth inlet for injecting a fourth solution, the fifth inlet for injecting a fifth solution, and the nth inlet for injecting an nth solution (n is a natural number of 6 or more). That is, in some embodiments, the Taylor reaction device 1 of the present disclosure may further have a fourth inlet, a fifth inlet... an nth inlet.

[0118] In some embodiments, the particles produced may be lipid particles. In some embodiments, the lipid particles may contain lipid components and encapsulated components such as nucleic acids. In some embodiments, the particle-forming components may contain lipid components, and the encapsulating components may contain encapsulated components such as nucleic acids. In another embodiment, the particle-forming components may contain encapsulated components such as nucleic acids, and the encapsulating components may contain lipid components.

[0119] In a specific embodiment, the dilution solution can reduce the content of an organic solvent (such as ethanol) in which the lipid is dissolved, thereby stabilizing the particles. The dilution solution can contact the solution containing lipid components at any time.

[0120] In some embodiments, the present disclosure provides a Taylor reaction device 1 for manufacturing particles. Examples of the particles produced include, but are not limited to, capsule particles. In some embodiments, the Taylor reaction device 1 has an inner cylinder 3 and an outer cylinder 2. A gap portion is formed between the outer diameter of the inner cylinder 3 and the inner diameter of the outer cylinder 2, and this is used as the reaction chamber 5. Therefore, the Taylor reaction device 1 has an outer cylinder 2 and an inner cylinder 3, and the inner cylinder 3 is disposed rotatably within the outer cylinder 2 and forms an annular reaction chamber 5 therebetween. The Taylor reaction device 1 may have one or more inlets 4 for allowing a liquid to flow into the reaction chamber 5. The Taylor reaction device 1 may have at least one outlet 6 for allowing the liquid in the reaction chamber 5 to flow out to the outside.

[0121] In some embodiments, the Taylor reaction apparatus 1 may be configured to mix the particle-forming component and the encapsulating component "in front of the reaction chamber". In this case, the first pipeline 4A for injecting the particle-forming component and the second pipeline 4B for injecting the encapsulating component merge ( Figure 2 ). For convenience, in the present specification, sometimes the part after the merging portion (i.e., the downstream side) is referred to as the particle-forming component-encapsulating component mixed solution pipeline 4D or the common pipeline. In the particle-forming component-encapsulating component mixed solution pipeline 4D, the particle-forming component and the encapsulating component are mixed. Then, the particle-forming component-encapsulating component mixed solution pipeline 4D supplies the particle-forming component-encapsulating component mixed solution into the reaction chamber 5 through the inlet 4 (which is also the common inlet described in the claims). In another embodiment, the Taylor reaction apparatus 1 may be configured to mix the particle-forming component and the encapsulating component "inside the reaction chamber". In this case, a first inlet 41 (also referred to as the first injection port) for injecting the particle-forming component and a second inlet 42 (also referred to as the second injection port) for injecting the encapsulating component are provided in the reaction chamber 5 ( Figure 3 ). Whether the particle-forming component and the encapsulating component are mixed "in front of the reaction chamber" or "inside the reaction chamber", the order of injecting the solutions is not particularly limited, and the particle-forming component and the encapsulating component may be injected simultaneously, or the particle-forming component may be injected first, or the encapsulating component may be injected first.

[0122] When the first inlet 41 for injecting the particle-forming component and the second inlet 42 for injecting the encapsulating component are provided in the reaction chamber 5, the first inlet 41 and the second inlet 42 may have any relative arrangement. In some embodiments, the first inlet 41 and the second inlet 42 may be arranged such that, when viewed from the direction of the rotation axis for rotating the inner cylinder 3 (viewed from a direction perpendicular to the circular cross-section of the inner cylinder 3), they form an angle of approximately 15 degrees, approximately 30 degrees, approximately 45 degrees, approximately 60 degrees, approximately 75 degrees, approximately 90 degrees, approximately 105 degrees, approximately 120 degrees, approximately 135 degrees, approximately 150 degrees, approximately 165 degrees, approximately 180 degrees, approximately 195 degrees, approximately 210 degrees, approximately 225 degrees, approximately 240 degrees, approximately 255 degrees, approximately 270 degrees, approximately 285 degrees, approximately 300 degrees, approximately 315 degrees, approximately 330 degrees, approximately 345 degrees, or approximately 360 degrees, but not limited thereto. Here, for the angle, "about" means a range of ±7.5 degrees of the indicated angle. In some embodiments, when the first inlet 41 and the second inlet 42 are viewed from a direction perpendicular to the rotation axis for rotating the inner cylinder 3 (viewed from a direction perpendicular to the rectangular cross-section of the inner cylinder), they may be adjacent or separated by a predetermined interval.

[0123] If the side where the liquid is supplied to the reaction chamber 5 is defined as the upstream side and the side where the liquid is discharged from the reaction chamber 5 is defined as the downstream side, then in some embodiments, the first inlet 41 can be arranged on the upstream side and the second inlet 42 can be arranged on the downstream side. In another embodiment, the second inlet 42 can be arranged on the upstream side and the first inlet 41 can be arranged on the downstream side. In another embodiment, the first inlet 41 and the second inlet 42 can be arranged on the same cross-section perpendicular to the axial direction of the inner cylinder 3( Figure 3 ). In this case, the first inlet 41 and the second inlet 42 are not one on the upstream and the other on the downstream, but both are on the same rotational cross-section. The third inlet 43 can also be arranged arbitrarily. That is, the third inlet 43 can be arranged on the upstream side of the first inlet 41, on the same rotational cross-section as the first inlet 41, on the downstream side of the first inlet 41 and on the upstream side of the second inlet 42, on the same rotational cross-section as the second inlet 42, or on the downstream side of the second inlet 42. Alternatively, the first inlet, the second inlet, and the third inlet 43 can be arranged on the same rotational cross-section( Figure 6 ). The same applies to the fourth inlet, the fifth inlet, ……, the nth inlet (n is a natural number of 6 or more).

[0124] In a specific embodiment, regarding the method for manufacturing particles and / or regarding the Taylor reaction device 1, the circumferential velocity of the inner cylinder 3 can be, for example, 0.5 to 47 m / s, for example, 0.4 m / s or more, 0.41 m / s or more, 0.42 m / s or more, 0.43 m / s or more, 0.44 m / s or more, 0.45 m / s or more, 0.46 m / s or more, 0.47 m / s or more, 0.48 m / s or more, 0.49 m / s or more, 0.5 m / s or more, 0.51 m / s or more, 0.52 m / s or more, 0.53 m / s or more, 0.54 m / s or more, 0.55 m / s or more, 0.6 m / s or more, 0.65 m / s or more, 0.7 m / s or more, 0.75 m / s or more, 0.8 m / s or more, 0.85 m / s or more, 0.9 m / s or more, 0.95 m / s or more, 1 m / s or more, 1.5 m / s or more, 2 m / s or more, 2.5 m / s or more, 3 m / s or more, 4 m / s or more, 5 m / s or more, 6 m / s or more, 7 m / s or more, 8 m / s or more, 9 m / s or more, 10 m / s or more, 11 m / s or more, 12 m / s or more, 13 m / s or more, 14 m / s or more, 15 m / s or more, 20 m / s or more, 25 m / s or more, 30 m / s or more, 35 m / s or more, 40 m / s or more, 41 m / s or more, 42 m / s or more, 43 m / s or more, 44 m / s or more, 45 m / s or more, 46 m / s or more, for example, 47 m / s or more, for example, 55 m / s or less, 54 m / s or less, 53 m / s or less, 52 m / s or less, 51 m / s or less, 50 m / s or less, 49 m / s or less, 48 m / s or less, 47 m / s or less, 46 m / s or less, 45 m / s or less, 44 m / s or less, 43 m / s or less, 42 m / s or less, 41 m / s or less, 40 m / s or less, 35 m / s or less, 30 m / s or less, 25 m / s or less, 20 m / s or less, 15 m / s or less, 14 m / s or less, 13 m / s or less, 12 m / s or less, 11 m / s or less, 10 m / s or less, 9 m / s or less, 8 m / s or less, 7 m / s or less, 6 m / s or less, 5 m / s or less, 4 m / s or less, 3 m / s or less, 2 m / s or less, 1 m / s or less, 0.95 m / s or less, 0.9 m / s or less, 0.85 m / s or less, 0.8 m / s or less, 0.75 m / s or less, 0.7 m / s or less, 0.65 m / s or less, 0.6 m / s or less, 0.55 m / s or less, 0.54 m / s or less, 0.53 m / s or less, 0.52 m / s or less, 0.51 m / s or less, 0.5 m / s or less, 0.49 m / s or less, 0.48 m / s or less, 0.47 m / s or less, 0.46 m / s or less, 0.45 m / s or less, 0.44 m / s or less, 0.Below 43 m / s, below 0.42 m / s, for example below 0.41 m / s, and can be any combination of their upper and lower limits, for example 0.4 - 55 m / s, 0.45 - 54 m / s, 0.5 - 53 m / s, 0.5 - 52 m / s, 0.5 - 51 m / s, 0.5 - 50 m / s, 0.5 - 49 m / s, 0.5 - 48 m / s, 0.5 - 47 m / s, 0.51 - 46 m / s, 0.52 - 45 m / s, 0.53 - 44 m / s, 0.54 - 43 m / s, 0.55 - 42 m / s, 0.6 - 41 m / s, 0.7 - 40 m / s, 0.8 - 35 m / s, 0.9 - 30 m / s, 1 - 25 m / s, 1.5 - 20 m / s, 2 - 15 m / s, 3 - 14 m / s, 4 - 13 m / s, 5 - 12 m / s, 6 - 11 m / s, 7 - 10 m / s, for example 8 - 9 m / s, but not limited to this. In addition, in this specification, unless otherwise specified, the circumferential speed is defined by the following formula.

[0125] [Formula 2]

[0126] Circumferential speed = Outer diameter of the inner cylinder × π × Rotational speed

[0127] (In the formula, π represents pi)

[0128] In a specific embodiment, when the inner diameter of the outer cylinder is the rotational speed of the inner cylinder 3 can be set to, for example, 500 - 30000 rpm, 550 - 25000 rpm, 600 - 20000 rpm, 650 - 15000 rpm, 700 - 10000 rpm, 750 - 9000 rpm, 800 - 8000 rpm, 850 - 7000 rpm, 900 - 6000 rpm, for example 1000 - 6000 rpm, but not limited to this. In a specific embodiment, when the inner diameter of the outer cylinder is the outer diameter of the inner cylinder can be set to or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, or more, Above Above Above Above Above Above Above Above Above Above, but not limited to this.

[0129] In a specific embodiment, regarding the method for manufacturing particles and / or regarding the Taylor reaction device 1, the clearance width d between the inner cylinder 3 and the outer cylinder 2 can be set to 0.01 mm or more, 0.02 mm or more, 0.03 mm or more, 0.04 mm or more, 0.05 mm or more, 0.06 mm or more, 0.07 mm or more, 0.08 mm or more, 0.09 mm or more, 0.1 mm or more, 0.11 mm or more, 0.12 mm or more, 0.13 mm or more, 0.14 mm or more, 0.15 mm or more, for example 0.21 mm or more, 0.75 mm or more, 1.5 mm or more, for example 5 mm or less, 4.9 mm or less, 4.8 mm or less, 4.7 mm or less, 4.6 mm or less, 4.5 mm or less, 4.4 mm or less, 4.3 mm or less, 4.2 mm or less, 4.1 mm or less, 4 mm or less, 3.5 mm or less, 3 mm or less, 2.5 mm or less, 2 mm, 1.5 mm or less, 1.0 mm or less, for example 0.01 mm to 5 mm, 0.02 mm to 4.9 mm, 0.03 mm to 4.8 mm, 0.04 mm to 4.7 mm, 0.05 mm to 4.6 mm, 0.06 mm to 4.5 mm, 0.07 mm to 4.4 mm, 0.08 mm to 4.3 mm, 0.09 mm to 4.2 mm, 0.1 mm to 4.1 mm, 0.11 mm to 4 mm, 0.12 mm to 3.5 mm, 0.13 mm to 3 mm, 0.14 mm to 2 mm, for example 0.15 mm to 1.5 mm, but not limited to this.

[0130] Regarding the method for manufacturing particles and / or regarding the Taylor reaction device 1, the circumferential velocity and the clearance width d can be set to the stirring force R which is a calculation formula for simplifying the shear force. The stirring force R is calculated by the following formula.

[0131] [Formula 3]

[0132] Stirring force R = circumferential velocity ÷ clearance width d

[0133] In some embodiments, the rotational speed of the inner cylinder 3 and the clearance width d between the inner cylinder 3 and the outer cylinder 2 can be set so that the stirring force R is 0.5 / 5 < R < 47 / 0.01, 100 - 5,000,000 s-1 , 104 to 4,900,000 s -1 , 105 to 4,900,000 s -1 , 110 to 4,800,000 s -1 , 120 to 4,710,000 s -1 , 130 to 4,700,900 s -1 , 140 to 4,700,000 s -1 , 150 to 4,600,000 s -1 , 160 to 4,500,000 s -1 , 170 to 4,400,000 s -1 , 180 to 4,300,000 s -1 , 190 to 4,200,000 s -1 , 200 to 4,100,000 s -1 , 250 to 4,000,000 s -1 , 300 to 3,900,000 s -1 , 400 to 3,800,000 s -1 , 500 to 3,700,000 s -1 , 600 to 3,600,000 s -1 , 700 to 3,500,000 s -1 , 800 to 3,400,000 s -1 , 900 to 3,300,000 s -1 , 1000 to 3,200,000 s -1 , 1500 to 3,100,000 s -1 , 2000 to 3,000,000 s -1 , 3000 to 2,500,000 s -1 , 4000 to 2,000,000 s -1 , 5000 to 1,500,000 s -1 , 10,000 to 1,000,000 s -1 or a range of any combination of their upper and lower limits.

[0134] Each solution can be mixed with each other at an appropriate injection rate and can be injected into the reaction chamber 5. A person skilled in the art can appropriately set the injection rate of the solution. In addition, a person skilled in the art can appropriately set the ratio of the injection rates of the respective solutions. In some embodiments, the ratio of the injection rate of the particle-forming component to the injection rate of the encapsulating component can be set to a ratio selected from the range of 1:1 to 1:9, such as 1:2 to 1:9, 1:3 to 1:9, such as 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, such as 1:9, but not limited thereto. The injection rate can be determined according to the combination of the particle-forming component and the encapsulating component. The injection rate of the particle-forming component or the encapsulating component can be, for example, 1 mL / min or more, 1.1 mL / min or more, 1.2 mL / min or more, 1.3 mL / min or more, 1.4 mL / min or more, 1.5 mL / min or more, 1.6 mL / min or more, 1.7 mL / min or more, 1.8 mL / min or more, 1.9 mL / min or more, 2 mL / min or more, 2.1 mL / min or more, 2.2 mL / min or more, 2.3 mL / min or more, 2.4 mL / min or more, 2.5 mL / min or more, 2.6 mL / min or more, 2.7 mL / min or more, 2.8 mL / min or more, 2.9 mL / min or more, 3 mL / min or more, 3.5 mL / min or more, 4 mL / min or more, 4.5 mL / min or more, 5 mL / min or more, 6 mL / min or more, 7 mL / min or more, 8 mL / min or more, 9 mL / min or more, 10 mL / min or more, 20 mL / min or more, 30 mL / min or more, 40 mL / min or more, 50 mL / min or more, 60 mL / min or more, 70 mL / min or more, 80 mL / min or more, 90 mL / min or more, 100 mL / min or more, 110 mL / min or more, 120 mL / min or more, 130 mL / min or more, 140 mL / min or more, for example 150 mL / min or more, 150 mL / min or less, 140 mL / min or less, 130 mL / min or less, 120 mL / min or less, 110 mL / min or less, 100 mL / min or less, 90 mL / min or less, 80 mL / min or less, 70 mL / min or less, 60 mL / min or less, 50 mL / min or less, 40 mL / min or less, 30 mL / min or less, 20 mL / min or less, 10 mL / min or less, 9 mL / min or less, 8 mL / min or less, 7 mL / min or less, 6 mL / min or less, 5 mL / min or less, 4.5 mL / min or less, 4 mL / min or less, 3.5 mL / min or less, 3 mL / min or less, 2.9 mL / min or less, 2.8 mL / min or less, 2.7 mL / min or less, 2.6 mL / min or less, 2.5 mL / min or less, 2.4 mL / min or less, 2.3 mL / min or less, 2.2 mL / min or less, 2.1 mL / min or less, 2 mL / min or less, 1.9 mL / min or less, 1.8 mL / min or less, 1.7 mL / min or less, 1.6 mL / min or less, 1.5 mL / min or less, 1.4 mL / min or less, 1.3 mL / min or less, 1.2 mL / min or less, 1.1 mL / min or less, for example 1 mL / min or less.

[0135] In some embodiments, the stirring time for carrying out the Taylor reaction can be set to 5 minutes or less, 4 minutes or less, 3 minutes or less, 2 minutes or less, 90 seconds or less, 60 seconds or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 15 seconds or less, for example 10 seconds or less, but not limited thereto. In some embodiments, the stirring time for carrying out the Taylor reaction can be set to 1 second or more, 2 seconds or more, 3 seconds or more, 4 seconds or more, for example 5 seconds or more, but not limited thereto. In addition, in this specification, unless otherwise specified, the "stirring time" refers to the time from when the injected liquid flows into the reaction tank until it passes through the outlet port. Sometimes the time from when the injected liquid flows into the reaction tank until it passes through the outlet port is referred to as the one-pass time. Additionally, the stirring time is the time from when the latest injected liquid flows into the reaction tank until it passes through the outlet port in the case where there are multiple inlet ports.

[0136] In some embodiments, the inner diameters of the first inlet 41 and the second inlet 42 can be the same or substantially the same. Substantially the same means including values within ±5%. In another embodiment, the inner diameters of the first inlet 41 and the second inlet 42 can be different.

[0137] Examples

[0138] Hereinafter, the present invention will be specifically described by way of examples. In addition, these examples are only for illustrating the present invention and do not limit the scope of the present invention.

[0139] [Example 1]

[0140] Preparation of nucleic acid lipid particles encapsulating siRNA

[0141] As specimens 1 to 3, nucleic acid-lipid particles encapsulating siRNA were prepared as follows. 1,2-Distearoyl-sn-glycero-3-phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma-Aldrich, Inc.), cationic lipid 1 (the compound described in WO2015 / 005253) (hereinafter referred to as LP1), and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethyleneglycol with a molecular weight of about 2000 for polyethylene glycol (hereinafter referred to as PEG2000-DMG, NOF CORPORATION) were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000-DMG = 10:48:40:2 so that the total lipid concentration was 1.96 mg / mL.

[0142] On the other hand, siRNA (AD-47882) against Transferin Receptor2 was dissolved in citrate buffer (20 mM Citrate Buffer, pH 4.0) to be 51.4 μg / mL. In addition, AD-47882 is the siRNA against mouse TfR2 described in WO2012 / 177921.

[0143] The above lipid solution and siRNA solution were mixed by pumping them into a Taylor reaction device in which the inner cylinder rotated at the "circumferential speed" described in Table 1 and had the "gap width" between the inner cylinder and the outer cylinder as described in Table 1 from their respective "injection ports" described in Table 1 at the "injection speed (the sum of the injection speeds of the lipid solution and the siRNA solution)" described in Table 1, and a dispersion of nucleic acid-lipid particles was obtained from the outlet port. The injection speed ratio of the lipid solution to the siRNA solution was set to 1:3. In the device of this example, the first inlet was on the upstream side and the second inlet was on the downstream side. The encapsulating component, i.e., the siRNA solution, was injected from the first inlet, and the particle-forming component, i.e., the lipid solution, was injected from the second inlet. In addition, regarding the "injection port" shown in Table 1, "inside the reaction tank (inside the reaction chamber)" and "in front of the reaction tank (in front of the reaction chamber)" are conditions related to the mixing position of the two liquids. If the injection port is "inside the reaction tank", the two liquids can be mixed in the reaction tank. If the injection port is "in front of the reaction tank", the two liquids can be mixed in front of the reaction tank, and then the mixed solution is injected into the reaction part of the Taylor reaction device, i.e., the gap between the inner cylinder and the outer cylinder. The same applies to Tables 2 to 5. In addition, regarding the "with or without dilution" shown in Table 1, in the case of diluting the nucleic acid-lipid particles, a citrate buffer (20 mM Citrate Buffer, pH 4.0) was pumped as a dilution solution from the injection port in front of the outlet port at a speed equal to the "injection speed" described in Table 1. The obtained dispersion of nucleic acid-lipid particles was dialyzed with about 25 to 50 times the amount of PBS (polybutylene succinate, pH 7.4) for 12 to 18 hours (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) to remove ethanol, and a purified dispersion of nucleic acid-lipid particles encapsulating siRNA was obtained.

[0144] In addition, LP1 was synthesized according to the method described in WO2015 / 005253. For reference, the description of WO2015 / 005253 related to the synthesis of LP1 is incorporated into this specification.

[0145] [Example 2]

[0146] Preparation of Nucleic Acid-Lipid Particles Encapsulating dsDNA

[0147] As samples 4 to 8, nucleic acid-lipid particles encapsulating dsDNA were prepared as follows. 1,2-Distearoyl-sn-glycero-3-phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma-Aldrich, Inc.), LP1, and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethylene glycol with a polyethylene glycol molecular weight of approximately 2000 (hereinafter referred to as PEG2000-DMG, NOF CORPORATION) were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000-DMG = 12.5:41:45:1.5 so that the total lipid concentration was 3.16 mg / mL.

[0148] On the other hand, β-catenin dsDNA was diluted and prepared with a citrate buffer (20 mM Citrate Buffer, pH 4.0) to be 52.7 μg / mL.

[0149] The above lipid solution and dsDNA solution were mixed by pumping them into a Taylor reaction device with an inner cylinder rotating at the "circumferential speed" described in Table 2 and having a "gap width" between the inner cylinder and the outer cylinder described in Table 2 from the "injection port" described in Table 2 at the "injection speed (the sum of the injection speeds of the lipid solution and the dsDNA solution)" described in Table 2, and a dispersion of nucleic acid-lipid particles was obtained from the outlet port. The injection speed ratio of the lipid solution to the dsDNA solution was set to 1:3. In addition, regarding the "with or without dilution" shown in Table 2, in the case of diluting the nucleic acid-lipid particles, a citrate buffer (20 mM Citrate Buffer, pH 4.0) was pumped as a dilution solution from the injection port in front of the outlet port at a speed equal to the "injection speed" described in Table 2. The dispersion of the nucleic acid-lipid particles was dialyzed with approximately 25 to 50 times the amount of PBS (pH 7.4) for 12 to 18 hours (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) to remove ethanol, and a dispersion of purified nucleic acid-lipid particles encapsulating dsDNA was obtained.

[0150] In addition, LP1 was synthesized according to the method described in WO2015 / 005253.

[0151] [Example 3]

[0152] Preparation of Nucleic Acid Lipid Particles Encapsulating mRNA

[0153] As Specimens 9 - 11, nucleic acid lipid particles encapsulating mRNA were prepared in the following manner. 1,2 - Distearoyl - sn - glycero - 3 - phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma - Aldrich, Inc.), LP1, and 1,2 - Dimyristoyl - sn - Glycero - 3 - Methoxypolyethyleneglycol with a polyethylene glycol molecular weight of approximately 2000 (hereinafter referred to as PEG2000 - DMG, NOF CORPORATION) were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000 - DMG = 12.5:41:45:1.5 to make the total lipid concentration 3.16 mg / mL.

[0154] On the other hand, A_Sin_GP1908_2015_H1 mRNA was diluted and prepared with a citrate buffer (20 mM Citrate Buffer, pH 4.0) to make it 52.7 μg / mL.

[0155] The above - mentioned lipid solution and mRNA solution were mixed by pumping them from the "injection port" described in Table 3 at the "injection speed (the sum of the injection speeds of the lipid solution and the mRNA solution)" described in Table 3 into an inner cylinder rotating at the "circumferential speed" described in Table 3 and having the "gap width" between the inner cylinder and the outer cylinder of the Taylor reaction device, and a dispersion of nucleic acid lipid particles was obtained from the outlet port. The injection speed ratio of the lipid solution to the mRNA solution was set to 1:3. In addition, regarding the "with or without dilution" shown in Table 3, in the case of diluting the nucleic acid lipid particles, a citrate buffer (20 mM Citrate Buffer, pH 4.0) was pumped as a dilution solution from the injection port in front of the outlet port at a speed equal to the "injection speed" described in Table 3. The dispersion of nucleic acid lipid particles was dialyzed (Float - A - Lyzer G2, MWCO: 1,000 kD, Spectra / Por) with approximately 25 - 50 times the volume of PBS (pH 7.4) for 12 - 18 hours to remove ethanol, and a dispersion of purified nucleic acid lipid particles encapsulating mRNA was obtained.

[0156] In addition, LP1 was synthesized according to the method described in WO2015 / 005253.

[0157] [Example 4]

[0158] Preparation of Nucleic Acid Lipid Particles Encapsulating mRNA

[0159] As Specimens 12 and 13, nucleic acid lipid particles encapsulating mRNA were prepared in the following manner. 1,2-Distearoyl-sn-glycero-3-phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma-Aldrich, Inc.), LP1, and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethyleneglycol with a polyethylene glycol molecular weight of approximately 2000 (hereinafter referred to as PEG2000-DMG, NOF CORPORATION) were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000-DMG = 12.5:41:45:1.5 to make the total lipid concentration 3.16 mg / mL.

[0160] On the other hand, A_Sin_GP1908_2015_H1 mRNA was diluted and prepared with a citrate buffer (20 mM Citrate Buffer, pH 4.0) to make it 52.7 μg / mL.

[0161] The above lipid solution and mRNA solution were mixed by pumping them into a Taylor reaction device with an inner cylinder rotating at the "circumferential speed" described in Table 4 and having a "gap width" between the inner cylinder and the outer cylinder as described in Table 4 from the "injection port" described in Table 4 at the "injection speed (the sum of the injection speeds of the lipid solution and the mRNA solution)" described in Table 4, and a dispersion of nucleic acid lipid particles was obtained from the outlet port. The injection speed ratio of the lipid solution to the mRNA solution was set to 1:3. In addition, a citrate buffer (20 mM Citrate Buffer, pH 4.0) was pumped as a dilution solution from the injection port in front of the outlet port at a speed equal to the "injection speed" described in Table 4. The dispersion of nucleic acid lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) with approximately 25 to 50 times the amount of 10 mM histidine and 300 mM sucrose (pH 7.0) for 12 to 18 hours to remove ethanol, and a dispersion of refined nucleic acid lipid particles encapsulating mRNA was obtained.

[0162] In addition, LP1 was synthesized according to the method described in WO2015 / 005253.

[0163] [Example 5]

[0164] Preparation of Nucleic Acid Lipid Particles Encapsulating mRNA

[0165] As Specimen 14, nucleic acid lipid particles encapsulating mRNA were prepared as follows. 1,2-Distearoyl-sn-glycero-3-phosphocholine (hereinafter referred to as DSPC, NOF CORPORATION), cholesterol (hereinafter referred to as Chol, Sigma-Aldrich, Inc.), cationic lipid 2 (the compound described in WO2015 / 005253) (hereinafter referred to as LP2), and 1,2-dimyristoyl-sn-glycero-3-methoxypolyethyleneglycol with a molecular weight of about 2000 (hereinafter referred to as PEG2000-DMG, NOF CORPORATION) were dissolved in ethanol at a molar ratio of DSPC:Chol:LP2:PEG2000-DMG = 17.5:21:60:1.5 so that the total lipid concentration was 3.08 mg / mL.

[0166] On the other hand, A_Sin_GP1908_2015_H1 mRNA was diluted and prepared with a citrate buffer (20 mM Citrate Buffer, pH 4.0) to make it 51.3 μg / mL.

[0167] The above lipid solution and mRNA solution were mixed by pumping them into a Taylor reaction device with an inner cylinder rotating at the "circumferential speed" described in Table 5 and having a "gap width" between the inner cylinder and the outer cylinder as described in Table 5 from the "injection port" described in Table 5 at the "injection speed (the sum of the injection speeds of the lipid solution and the mRNA solution)" described in Table 5, and a dispersion of nucleic acid-lipid particles was obtained from the outlet port. The injection speed ratio of the lipid solution to the mRNA solution was set to 1:3. In addition, a citrate buffer (20 mM Citrate Buffer, pH 4.0) was pumped as a dilution solution from the injection port in front of the outlet port at a speed equal to the "injection speed" described in Table 5. The dispersion of nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) with about 25 to 50 times the volume of 10 mM histidine and 300 mM sucrose (pH 7.0) for 12 to 18 hours to remove ethanol, and a purified dispersion of nucleic acid-lipid particles encapsulating mRNA was obtained.

[0168] In addition, LP2 was synthesized according to the method described in WO2015 / 005253. For reference, the description of WO2015 / 005253 related to the synthesis of LP2 is incorporated into this specification.

[0169] [Example 6]

[0170] Characterization of nucleic acid-lipid particles encapsulating nucleic acids

[0171] For Samples 1 to 14, characterization of the dispersion containing nucleic acid-lipid particles was performed. The methods for each characterization are described.

[0172] (1) Encapsulation efficiency of nucleic acid

[0173] The encapsulation efficiency of nucleic acid was measured using the Quant-iT RiboGreen RNA Quantitation Kit (Invitrogen) according to the attached instructions.

[0174] That is, in the presence and absence of 0.015% Triton X-100 surfactant, the nucleic acid in the dispersion of nucleic acid-lipid particles was quantified, and the encapsulation efficiency was calculated by the following formula.

[0175] [Equation 4]

[0176] {([Amount of nucleic acid in the presence of surfactant] - [Amount of nucleic acid in the absence of surfactant]) / [Amount of nucleic acid in the presence of surfactant]} x 100 (%)

[0177] (2) Average particle size, polydispersity index (PDI)

[0178] The average particle size and polydispersity index of the nucleic acid-lipid particles were measured using a Zeta Potential / Particle Sizer (NICOMP (trademark) 380ZLS (PARTICLE SIZING SYSTEMS)) from Particle Sizing Systems. The average particle size shown in the table represents the volume-average particle size. In addition, PDI is a polydispersity index calculated as the square of (standard deviation / average particle size), and is sometimes simply referred to as the dispersity.

[0179] The results of the property evaluations are shown in Tables 1 to 5. As shown in the tables, nucleic acid-lipid particles with an average particle size of 80 to 200 nm and a nucleic acid encapsulation rate of 85% or more were obtained. This demonstrated that lipid particles encapsulating nucleic acids can be produced by the Taylor reaction device. In addition, it was also demonstrated that particles that can be produced by a microchannel can also be produced by the Taylor reaction device.

[0180] (Reference Example) Preparation of Nucleic Acid-Lipid Particles Based on Microchannel Mixing

[0181] (Reference Example 1) Preparation and Property Evaluation of Nucleic Acid-Lipid Particles Encapsulating siRNA

[0182] Reference Example 1 was prepared as follows. DSPC, Chol, LP1, and PEG2000-DMG were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000-DMG = 10:48:40:2 to give a total lipid concentration of 1.96 mg / mL. On the other hand, siRNA (AD-47882) against Transferin Receptor2 was dissolved in citrate buffer (20 mM Citrate Buffer, pH 4.0) to give a concentration of 51.4 μg / mL. The above lipid solution and mRNA solution were mixed in a microchannel using a nanoparticle synthesis system (NanoAssemblr BenchTop, Precision Nanosystems Inc.) at a volume ratio of 1:3 to obtain a crude dispersion of nucleic acid-lipid particles. The dispersion of the nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) against about 25 to 50 volumes of buffer for 12 to 18 hours to remove ethanol, and a purified dispersion of nucleic acid-lipid particles encapsulating siRNA was obtained.

[0183] For the nucleic acid-lipid particles of Reference Example 1 obtained, property evaluation was carried out in the same manner as in Example 6, and the results are shown in Table 6.

[0184] (Reference Examples 2 and 3) Preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA

[0185] Reference Examples 2 and 3 were prepared as follows. DSPC, Chol, LP1, and PEG2000-DMG were dissolved in ethanol at a molar ratio of DSPC:Chol:LP1:PEG2000-DMG = 12.5:41:45:1.5 so that the total lipid concentration was 3.16 mg / mL. On the other hand, A_Sin_GP1908_2015_H1 mRNA (sequence information) was diluted and prepared with a citrate buffer (20 mM Citrate Buffer, pH 4.0) to be 52.7 μg / mL. Using a nanoparticle synthesis system (NanoAssemblr BenchTop, PrecisionNanosystems Inc.), the above lipid solution and mRNA solution were mixed in a microchannel at a volume ratio of 1:3 to obtain a crude dispersion of nucleic acid-lipid particles. The dispersion of nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) with about 25 to 50 times the volume of buffer for 12 to 18 hours to remove ethanol, and a refined dispersion of nucleic acid-lipid particles encapsulating mRNA was obtained.

[0186] For the nucleic acid-lipid particles of Reference Examples 2 and 3 obtained, property evaluation was carried out in the same manner as in Example 6, and the results are shown in Table 6.

[0187] (Reference Example 4) Preparation and property evaluation of nucleic acid-lipid particles encapsulating mRNA

[0188] Reference Example 4 was prepared as follows. DSPC, Chol, LP2, and PEG2000-DMG were dissolved in ethanol at a molar ratio of DSPC:Chol:LP2:PEG2000-DMG = 17.5:21:60:1.5 to give a total lipid concentration of 3.08 mg / mL. On the other hand, A_Sin_GP1908_2015_H1mRNA (sequence information) was diluted with citrate buffer (20 mM Citrate Buffer, pH 4.0) to a concentration of 51.3 μg / mL. The above lipid solution and mRNA solution were mixed in a microchannel using a nanoparticle synthesis system (NanoAssemblr BenchTop, PrecisionNanosystems Inc.) at a volume ratio of 1:3 to obtain a crude dispersion of nucleic acid-lipid particles. The dispersion of nucleic acid-lipid particles was dialyzed (Float-A-Lyzer G2, MWCO: 1,000 kD, Spectra / Por) with about 25 - 50 volumes of buffer for 12 - 18 hours to remove ethanol, and a refined dispersion of nucleic acid-lipid particles encapsulating mRNA was obtained.

[0189] For the obtained nucleic acid-lipid particles of Reference Example 4, property evaluation was carried out in the same manner as in Example 6, and the results are shown in Table 6.

[0190] As shown in Table 6, for Reference Examples 1 - 4, nucleic acid-lipid particles with an average particle size of 90 - 140 nm and a nucleic acid encapsulation rate of 95% or more were obtained.

[0191] [Table 1]

[0192]

[0193] TR2 siRNA

[0194] DSPC∶Chol∶LP1∶PEG2000-DMG = 10∶48∶40∶2

[0195] [Table 2]

[0196]

[0197] βcatenin dsDNA

[0198] DSPC∶Chol∶LP1∶PEG2000-DMG = 12.5∶41∶45∶1.5

[0199] [Table 3]

[0200]

[0201] A_Sin_GP1908_2015_H1 mRNA

[0202] DSPC∶Chol∶LP1∶PEG2000-DMG = 12.5∶41∶45∶1.5

[0203] [Table 4]

[0204]

[0205] ＊ Syringe pump

[0206] A_Sin_GP1908_2015_H1 mRNA

[0207] DSPC∶Chol∶LP2∶PEG2000-DMG = 12.5∶41∶45∶1.5

[0208] [Table 5]

[0209]

[0210] ＊ Syringe pump

[0211] A_Sin_GP1908_2015_H1 mRNA

[0212] DSPC∶Chol∶LP2∶PEG2000-DMG = 17.5∶21∶60∶1.5

[0213] [Table 6]

[0214]

[0215] [Test Example 1] Pharmacodynamic evaluation test of nucleic acid lipid particles encapsulating TfR2 siRNA in normal mice

[0216] It has been reported that in the above-mentioned Example 1 and Reference Example 1, TfR2 siRNA encapsulated in lipid particles increased the plasma iron concentration in normal mice (WO2012 / 177921). Therefore, the following tests were conducted using nucleic acid lipid particles encapsulating TfR2 siRNA (Specimens 1 to 3) manufactured using a Taylor reaction device and nucleic acid lipid particles encapsulating TfR2 siRNA (Reference Example 1) manufactured by microchannel mixing.

[0217] Male C57BL / 6NJcl mice (manufactured by CLEA Japan, Inc.) were intravenously administered (i.v.) with TfR2 siRNA encapsulated in lipid particles at a dose of 0.3 mg / kg (0.3 mpk) (n = 4 / group). As a vehicle group, PBS was intravenously administered (n = 4 / group). Blood was collected from the tail vein before (Pre) treatment with siRNA or PBS, 1 day after (Day1) treatment, and 7 days after (Day7) treatment, and the plasma iron concentration (plasma Fe (ug / mL)) was measured using a Metallo assay iron measurement kit (manufactured by Metallogenics). The results of each plasma iron concentration are shown separately in Figures 7 - 9 (mean ± standard error).

[0218] As Figures 7 - 9 shown, a significant increase in plasma iron concentration was observed 1 day and 7 days after administration of nucleic acid lipid particles encapsulating TfR2 siRNA compared to the vehicle group. These results indicate that the nucleic acid lipid particles encapsulating TfR2 siRNA manufactured using the Taylor reaction device function in vivo.

[0219] [Test Example 2] Immunogenicity of monovalent LNP-mRNA: Ability to induce the production of HA-specific IgG by nucleic acid lipid particles encapsulating A Singapore GP19082015H1 mRNA

[0220] To investigate the in vivo immune induction ability of nucleic acid lipid particles encapsulating mRNA manufactured using the Taylor reaction device, the following tests were conducted using nucleic acid lipid particles encapsulating A_Sin_GP1908_2015_H1 mRNA (Specimens 9 - 14) and nucleic acid lipid particles encapsulating A_Sin_GP1908_2015_H1 mRNA manufactured by microfluidic mixing (Reference Examples 2 - 4).

[0221] In the gastrocnemius muscle of 7-week-old BALB / c mice, 0.2 μg of Reference Example 2, Specimen 9, Specimen 10, and Specimen 11 were administered twice at two-week intervals. Blood was collected two weeks after the last administration to prepare serum. Similarly, Reference Example 3, Reference Example 4, Specimen 12, Specimen 13, and Specimen 14 were administered to 6-week-old BALB / c mice to prepare serum. Serum-specific IgG against HA was detected by Enzyme-linked immunosorbent assay (ELISA).

[0222] The solid-phase treatment of the ELISA method was carried out as follows: 0.5 μg / mL of recombinant A / Michigan / 45 / 2015 (influenza antigen A / Michigan / 45 / 2015) HA was added to a 96-well plate at 25 μL / well and left standing overnight at 4°C. At the same time, in order to prepare a standard curve, mouse IgG of known concentration was serially diluted and solid-phase treated in the same manner. After removing the solid-phase liquid, it was washed 3 times with DPBS (DPBST) containing 0.05% Tween 20 (polysorbate-20), and blocked with Dulbecco's phosphate-buffered saline (DPBS) (DPBST / BSA) containing 1% bovine serum albumin (BSA) and 0.05% Tween 20. The serum was serially diluted with DPBST / BSA. After the blocking treatment, it was washed 3 times with DPBST, and the diluted serum was added at 25 μL / well. DPBST / BSA was added to the wells for the standard curve instead of the diluted serum. After reacting at room temperature for more than 1 hour, it was washed 3 times with DPBST, and horseradish peroxidase (HRP)-labeled anti-mouse IgG diluted with DPBST / BSA was added. After reacting at room temperature for 1 hour, it was washed 3 times with DPBST, and 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate was added at 30 μL / well. After leaving it to develop color at room temperature for about 10 minutes, a color development stop solution containing hydrochloric acid was added at 30 μL / well, and the absorbance at 450 nm was measured. The specific IgG concentration in the serum was calculated based on the standard curve.

[0223] The results of administering Reference Example 2, Specimen 9, Specimen 10, and Specimen 11 are shown in Figure 10 . Specimen 10 showed a specific IgG induction level similar to that of Reference Example 2, and Specimen 9 and Specimen 11 showed a specific IgG induction level higher than that of Reference Example 2.

[0224] Similarly, the results of administering Reference Example 3, Reference Example 4, Specimen 12, Specimen 13, and Specimen 14 are shown in Figure 11 . Reference Example 3 and Reference Example 4 showed similar specific IgG induction levels. In addition, Specimen 12, Specimen 13, and Specimen 14 showed specific IgG induction levels similar to those of the two reference examples.

[0225] These results indicate that the nucleic acid lipid particles encapsulating A_Sin_GP1908_2015_H1mRNA produced using the Taylor reaction device induce the production of HA-specific IgG in vivo.

[0226] Industrial Applicability

[0227] According to the present disclosure, capsule particles can be manufactured. This can be used, for example, in the manufacture of RNA vaccines or siRNA pharmaceuticals. In addition, the capsule particles can be continuously manufactured. In addition, the capsule particles can be mass-produced, but the present invention is not limited thereto.

[0228] In this specification, a plurality of documents including patent applications and manufacturer manuals are cited. The disclosures of these documents are not regarded as relevant to the patentability of the present invention, but are incorporated herein by reference in their entirety. More specifically, all the cited documents are incorporated herein by reference in the same manner as if each document was specifically and individually shown to be incorporated by reference. In the case of inconsistencies or contradictions between the cited documents and the present specification, the present specification prevails.

[0229] Description of Reference Numerals

[0230] 1: Taylor reaction device,

[0231] 2: Outer cylinder,

[0232] 3: Inner cylinder,

[0233] 4: Inlet,

[0234] 4A: First pipeline,

[0235] 4B: Second pipeline,

[0236] 4D: Mixed solution pipeline,

[0237] 5: Reaction chamber,

[0238] 5V: Vortex row,

[0239] 6: Outlet,

[0240] 7: Control device,

[0241] 8: Motor,

[0242] 11: First tank,

[0243] 12: First pump,

[0244] 13: Second tank,

[0245] 14: Second pump,

[0246] 15: Third tank,

[0247] 16: Third pump,

[0248] 20: Outer cover,

[0249] 21: Cooler for outer cover temperature adjustment,

[0250] 41: First inlet

[0251] 41A: The first pipeline,

[0252] 41B: The second pipeline,

[0253] 41D: The mixed solution pipeline,

[0254] 42: The second fluid inlet,

[0255] 43: The third fluid inlet,

[0256] IR: Inner cylinder outer diameter,

[0257] d: Clearance width.

Claims

1. A method for manufacturing capsule particles, wherein, a Taylor reaction device is used.

2. The manufacturing method according to claim 1, wherein, the Taylor reaction device has an outer cylinder, an inner cylinder, and one or more inlets, the inner cylinder is rotatably disposed within the outer cylinder and forms an annular reaction chamber therebetween, at least one of the inlets is configured to allow a fluid to flow into the reaction chamber.

3. The manufacturing method according to claim 2, wherein, the one or more inlets include a first inlet and a second inlet, the first inlet and the second inlet are independent of each other and are respectively connected to the reaction chamber, a particle-forming component is injected into the reaction chamber from the first inlet, an encapsulating component is injected into the reaction chamber from the second inlet, the inner cylinder is rotated to mix the particle-forming component and the encapsulating component within the reaction chamber.

4. The manufacturing method according to claim 2, wherein, the one or more inlets include a first pipeline and a second pipeline, the first pipeline and the second pipeline are configured to merge, and the merged pipeline is connected to the reaction chamber via a common inlet, a particle-forming component is injected from the first pipeline, an encapsulating component is injected from the second pipeline, the particle-forming component and the encapsulating component are mixed in front of the reaction chamber to generate a particle-forming component - encapsulating component mixed solution, and then the particle-forming component - encapsulating component mixed solution is injected into the reaction chamber via the common inlet, and the inner cylinder is rotated to further mix the particle-forming component - encapsulating component mixed solution within the reaction chamber.

5. The manufacturing method according to claim 3 or 4, wherein, the particle-forming component contains a polymer component or a lipid component, the encapsulating component contains a nucleic acid, a protein, a polypeptide, a peptide, or a low-molecular compound.

6. The manufacturing method according to claim 5, wherein, the particle-forming component contains a lipid component, the encapsulating component contains a nucleic acid.

7. The manufacturing method according to claim 3, wherein, the particle-forming component is injected first, and then the encapsulating component is injected.

8. The manufacturing method according to claim 3, wherein, the encapsulating component is injected first, and then the particle-forming component is injected.

9. The manufacturing method according to claim 3, wherein, the particle-forming component and the encapsulating component are injected simultaneously.

10. The manufacturing method according to any one of claims 2 to 9, wherein, the circumferential speed of the inner cylinder is 0.5 to 47 m / s.

11. The manufacturing method according to any one of claims 2 to 10, wherein, the clearance width between the inner cylinder and the outer cylinder is 0.01 mm to 5 mm.

12. The manufacturing method according to any one of claims 1 to 11, wherein, the stirring time for performing the Taylor reaction is a stirring time of 1 minute or less.

13. The manufacturing method according to any one of claims 3, 5 to 12, wherein, the ratio of the injection speed of the particle-forming component to the injection speed of the encapsulating component is a ratio selected from the range of 1:1 to 1:

9.

14. The manufacturing method according to any one of claims 3 to 13, wherein, the inner diameters of the first inlet and the second inlet are the same or substantially the same.

15. The manufacturing method according to any one of claims 3 to 13, wherein, The inner diameters of the first fluid inlet and the second fluid inlet are different.

16. A Taylor reaction device, which is used in a method for manufacturing capsule particles, wherein, it includes an outer cylinder, an inner cylinder, and one or more fluid inlets, the inner cylinder is rotatably disposed inside the outer cylinder, and a ring-shaped reaction chamber is formed between the inner cylinder and the outer cylinder, at least one of the fluid inlets is configured to allow a fluid to flow into the reaction chamber.

17. The device according to claim 16, wherein, the one or more fluid inlets include a first fluid inlet and a second fluid inlet, the first fluid inlet and the second fluid inlet are independent of each other and are respectively connected to the reaction chamber, the first fluid inlet is used for injecting a particle-forming component, the second fluid inlet is used for injecting an encapsulating component, the inner cylinder is used for mixing the particle-forming component and the encapsulating component in the reaction chamber by rotation.

18. The device according to claim 16, wherein, the one or more fluid inlets have a first pipeline and a second pipeline, the first pipeline is used for injecting a particle-forming component, the second pipeline is used for injecting an encapsulating component, the first pipeline and the second pipeline merge in front of the reaction chamber, a particle-forming component - encapsulating component mixed solution pipeline is formed on the downstream side of the merging part, and then it is connected to the reaction chamber via a common fluid inlet, the inner cylinder is used for further mixing the particle-forming component - encapsulating component mixed solution in the reaction chamber by rotation.

19. The device according to claim 17 or 18, wherein, the particle-forming component contains a polymer component or a lipid component, the encapsulating component contains a nucleic acid, a protein, a polypeptide, a peptide, or a low molecular compound.

20. The device according to claim 19, wherein, the particle-forming component contains a lipid component, the encapsulating component contains a nucleic acid.

21. The device according to claim 17 or claim 19 which depends on claim 17, wherein, if the side for supplying liquid to the reaction chamber is set as the upstream side and the side for discharging liquid from the reaction chamber is set as the downstream side, then the first fluid inlet is disposed on the upstream side and the second fluid inlet is disposed on the downstream side.

22. The device according to claim 17 or claim 19 which depends on claim 17, wherein, if the side for supplying liquid to the reaction chamber is set as the upstream side and the side for discharging liquid from the reaction chamber is set as the downstream side, then the second fluid inlet is disposed on the upstream side and the first fluid inlet is disposed on the downstream side.

23. The device according to claim 17 or claim 19 which depends on claim 17, wherein, the first fluid inlet and the second fluid inlet are disposed in the same cross-section perpendicular to the axial direction of the inner cylinder.

24. The device according to any one of claims 16 to 23, wherein, the clearance width between the inner cylinder and the outer cylinder is 0.01 mm to 5 mm.

25. The device according to any one of claims 17, 19 to 24, wherein, the ratio of the injection speed of the particle-forming component to the injection speed of the encapsulating component is a ratio selected from the range of 1:1 to 1:

9.

26. The device according to any one of claims 17 to 25, wherein, the inner diameters of the first fluid inlet and the second fluid inlet are the same or substantially the same.

27. The device according to any one of claims 17 to 25, wherein, The inner diameters of the first fluid inlet and the second fluid inlet are different.

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