Method for producing lipid particles and apparatus for producing lipid particles

Abstract

To produce lipid particles with uniform particle size. [Solution] The method for producing lipid particles according to the present disclosure includes the steps of: A) mixing a lipid solution containing lipid particle components and a solvent with a mixed fluid containing a diluent to obtain a mixed fluid containing lipid particle components before lipid particle formation; and B) supplying a diluent to the mixed fluid, and then further mixing the mixed fluid to which the diluent has been supplied to form lipid particles.

Description

[Technical Field]

[0001] The present disclosure relates to a method for producing lipid particles and an apparatus for producing lipid particles. [Background Art]

[0002] As a method for producing lipid particles such as liposomes, the method described in Patent Document 1 has been conventionally known. In this method, lipid particles are continuously produced by mixing a first solution and a second solution using a microfluidic chip. [Prior Art Document] [Patent Document]

[0003] [Patent Document 1] Japanese National Publication of International Patent Application No. 2018-515324 [Summary of the Invention] [Problem to be Solved by the Invention]

[0004] Here, there is a demand for producing lipid particles having a uniform particle diameter.

[0005] An object of one aspect of the present disclosure is to produce lipid particles having a uniform particle diameter. [Means for Solving the Problem]

[0006] The gist of the present disclosure is the following [1] to

[10] . [1] A) a step of mixing a lipid solution containing a lipid particle component and a solvent with a mixed fluid containing a diluent to obtain a mixed fluid containing the lipid particle component before lipid particle formation; B) a step of, after supplying a diluent to the mixed fluid, further mixing the mixed fluid to which the diluent has been supplied to form lipid particles; a method for producing lipid particles comprising the steps. [2] The method for producing lipid particles according to [1], wherein in step A), the concentration of the solvent in the mixed fluid is 50% or more and 90% or less. [3] A) The method for producing lipid particles according to [1] or [2], wherein in step A, the standard deviation of the mass fraction of the solvent in a cross-section perpendicular to the flow direction of the mixed fluid after mixing is 0.01 or less. [4] A method for producing lipid particles according to any one of [1] to [3], wherein step B) is repeated two or more times. A lipid particle manufacturing apparatus used in any one of the lipid particle manufacturing methods described in [5] [1] to [4], A) An upstream mixer that mixes the mixed fluid in the process, A lipid particle manufacturing apparatus comprising: a downstream mixer provided downstream of the upstream mixer, which, in step B), supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, and then further mixes the mixed fluid to which the diluent has been supplied. [6] The lipid particle manufacturing apparatus according to [5], wherein the downstream mixer is provided downstream of the upstream mixer and spaced apart from the upstream mixer. [7] The lipid particle manufacturing apparatus according to [5], wherein the downstream mixer is provided downstream of the upstream mixer and integrally with the upstream mixer. [8] The upstream mixer comprises an upstream supply unit for supplying the diluent to the lipid solution, an upstream mixing tube extending downstream from the upstream supply unit, and an upstream mixing element provided inside the upstream mixing tube for mixing a mixed fluid containing the lipid solution and the diluent to obtain a mixed fluid containing lipid particle components before lipid particle formation, The downstream mixer comprises a downstream supply unit that supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, a downstream mixing pipe extending downstream from the downstream supply unit, and a downstream mixing element provided inside the downstream mixing pipe for mixing the mixed fluid to which the diluent has been supplied. A lipid particle manufacturing apparatus as described in any one of [5] to [7]. [9] The lipid particle manufacturing apparatus according to [8], wherein the flow velocity of the mixed fluid flowing through the downstream mixing tube is slower than the flow velocity of the mixed fluid flowing through the upstream mixing tube.

[10] A lipid particle manufacturing apparatus according to any one of [5] to [9], wherein a plurality of downstream mixers are provided in series downstream of the upstream mixer. [Effects of the Invention]

[0007] According to one aspect of this disclosure, lipid particles with uniform particle size can be produced. [Brief explanation of the drawing]

[0008] [Figure 1] This is an explanatory diagram of a lipid particle manufacturing apparatus according to the embodiments and Example 1 of the present disclosure, in which the downstream mixer is provided spaced apart from the upstream mixer. [Figure 2] This is a perspective view of the upstream mixing element (or downstream mixing element) according to the embodiments and Example 1 of this disclosure. [Figure 3] This is an explanatory diagram of a lipid particle manufacturing apparatus according to an embodiment of the present disclosure, in which a downstream mixer is integrated with an upstream mixer. [Figure 4] This is an explanatory diagram of the lipid particle manufacturing apparatus according to Comparative Example 1. [Figure 5] This figure shows the average particle size and the majority dispersion index (pdi) of liposomes produced using the lipid particle production apparatus of Example 1 and Comparative Example 1 in Experimental Example 1. [Figure 6] This figure shows the relationship between the average particle size and the majority dispersion index (pdi) of liposomes produced in Experimental Example 2 by changing the production conditions using the lipid particle production apparatus of Example 1 and Comparative Example 1. [Figure 7] This figure shows the trends in the average particle size and the majority dispersion index (pdi) of liposomes when the ethanol concentration of the mixed fluid flowing through the upstream mixing tube is changed in Experimental Example 3. [Figure 8] In Experimental Example 4, the standard deviation of the mass fraction of ethanol in the mixed fluid is shown when the mixed fluid flowing through the upstream mixing tube of the lipid particle manufacturing apparatus of Example 1 is simulated. [Figure 9]In Experimental Example 4, shows the standard deviation of the mass fraction of ethanol in the mixed fluid when simulation analysis is performed on the mixed fluid flowing through the inside of the downstream mixing tube of the lipid particle production apparatus of Example 1. Description of Embodiments

[0009] Method for Producing Lipid Particles In order to solve the above problem, a method for producing lipid particles according to one aspect of the present disclosure includes: A) a step of mixing a lipid solution containing a lipid particle component and a solvent with a diluent to obtain a mixed fluid containing the lipid particle component before lipid particle formation; B) after supplying a diluent to the mixed fluid, further mixing the mixed fluid to which the diluent has been supplied to form lipid particles.

[0010] Lipid particles are spherical vesicles composed of lipid particle components. Lipid particles are formed through several self-assembly steps, for example, aggregation of a plurality of lipid particle components, formation of a lipid membrane, and spheroidization of the lipid membrane. The term "before lipid particle formation" refers to a state in which many lipid particle components in the mixed fluid are in the stage of aggregation or lipid membrane formation, and have not yet formed spherical vesicles which are the final form of self-assembly.

[0011] The method for producing lipid particles according to one aspect of the present disclosure is a method for continuously producing lipid particles using a dilution method. In the dilution method, a lipid solution is diluted by mixing a mixed fluid containing the lipid solution and a diluent, thereby promoting self-assembly of the lipid particle components to form lipid particles. Self-assembly of lipid particle components tends to proceed easily when the solvent concentration is within a predetermined range.

[0012] In the present disclosure, in step A), the above mixed fluid is mixed to obtain a mixed fluid containing a lipid particle component before lipid particle formation. This makes it possible to equalize the solvent concentration in the mixed fluid, and increase the area within the predetermined range in which lipid particle components easily undergo self-assembly in the mixed fluid. Thereby, self-assembly of the lipid particle components can be allowed to proceed uniformly.

[0013] In step B), a diluent is supplied to the mixed fluid containing the lipid particle components before lipid particle formation. After supplying the diluent, the mixed fluid is further mixed to form lipid particles. The solvent concentration in the mixed fluid to which the diluent has been supplied is lower than the solvent concentration in the mixed fluid before the diluent is supplied. In such a mixed fluid with reduced solvent concentration, the self-assembly of lipid particle components proceeds further, and lipid particles with uniform particle size are formed.

[0014] The lipid particle components contained in the lipid solution have the property of being prone to self-assembly in a mixed fluid containing a solvent at a predetermined concentration, for example, 50% or more and 90% or less. In step A), the concentration of the solvent in the mixed fluid is preferably 50% or more and 90% or less, more preferably 55 to 85%, and still more preferably 60 to 80%. In a mixed fluid with a solvent concentration within this range, the lipid particle components are prone to self-assembly, and lipid particles with a relatively large average particle size can be obtained.

[0015] In step A), the standard deviation of the mass fraction of the solvent in the cross-section perpendicular to the flow direction of the mixed fluid after mixing is preferably 0.01 or less. In this case, the mixed fluid after mixing can be uniformly mixed, and the self-assembly of the lipid particle components can proceed uniformly.

[0016] Step B) is preferably repeated two or more times, and for example, step B) may be repeated three or more times. When step B) is repeated two or more times, it is preferable that the solvent concentration in the mixed fluid after the diluent is supplied in any step except the final step is, for example, 50% or more and 90% or less, and the solvent concentration in the mixed fluid after the diluent is supplied in the final step is, for example, less than 50%. In this case, the solvent concentration at which the self-assembly of lipid particle components easily proceeds can be precisely controlled in multiple stages, and lipid particles with more uniform particle size can be formed.

[0017] A) The mixing ratio of the lipid solution and the diluent in step A is not particularly limited, but for example, the mixing ratio of the lipid solution and the diluent is preferably 1:0.18 to 1:0.8 by volume, and more preferably 1:0.25 to 1:0.65. In this case, the lipid particle components begin to self-assemble in the mixed fluid, and the morphology of the lipid particle components can be changed to a pre-lipid particle form such as an aggregate or a lipid membrane.

[0018] In step B), it is preferable that the solvent concentration in the mixed fluid after supplying the diluent is lower than the solvent concentration in the mixed fluid before lipid particle formation in step A. When step B) is performed only once, or at least in the final step when it is performed multiple times, the solvent concentration in the mixed fluid after supplying the diluent is preferably a solvent concentration that facilitates lipid particle formation, for example, less than 50%. [Lipid solution] A lipid solution refers to a solution containing lipid particulate matter and a solvent. Examples of lipid particulate matter include amphiphilic substances and lipids. A lipid solution should preferably contain at least amphiphilic substances.

[0019] Amphiphilic substances are compounds that possess both a lipophilic group (hydrophobic group) and a hydrophilic group (polar group) in a single molecule, and have affinity for both water and oil. Examples of amphiphilic substances include phospholipids and surfactants.

[0020] Phospholipids are substances that have a central skeleton of glycerol or sphingosine, with fatty acids and phosphate groups bonded to it, and further ester-bonded alcohols to the phosphate groups. Phospholipids may be one or more selected from, for example, phosphatidylcholine and phosphatidylglycerol. Examples of phosphatidylcholine include 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine (DOPC), lecithin, or combinations thereof. Examples of lecithin include soy-derived lecithin and egg-derived lecithin.

[0021] Lipids are organic components that dissolve in a solvent. Examples of lipids include cholesterol and other cholesterol-based substances. Lipid solutions may contain phospholipids, or phospholipids and cholesterol-based substances.

[0022] The solvent should be a liquid capable of dissolving the lipid particle components. Examples of solvents include alcohols such as ethanol. Of these, ethanol is preferred as the solvent.

[0023] The concentration of lipid particle components in the lipid solution is preferably such that the lipid particle components are soluble, for example, 0.1 to 120 mg / ml, and more preferably 1 to 90 mg / ml.

[0024] The lipid solution may consist of lipid particle components and a solvent, or it may contain other components. The solvent concentration in the lipid solution is preferably greater than 90% and less than or equal to 100%, and more preferably between 99% and 100%. In this case, the initiation of self-assembly of the lipid particle components in the lipid solution can be delayed.

[0025] In addition to lipid particle components and solvents, lipid solutions may also contain drugs, nutrients, and cosmetic ingredients. By adding drugs to the lipid solution, the lipid particles can be used as carriers for drug delivery. [Diluent] The diluent is a solution used to dilute the lipid solution. The diluent may be, for example, pure water or a solution mainly composed of water. A solution mainly composed of water may be, for example, physiological saline, phosphate buffer, or acetate buffer. [Lipid particle manufacturing equipment] A lipid particle manufacturing apparatus in one aspect of the present disclosure, a lipid particle manufacturing apparatus used in the above-described method for manufacturing lipid particles, A) An upstream mixer that mixes the mixed fluid in the process, A lipid particle manufacturing apparatus comprising a downstream mixer located downstream of an upstream mixer, which, in step B), supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, and then further mixes the mixed fluid to which the diluent has been supplied.

[0026] According to one embodiment of the lipid particle manufacturing apparatus of this disclosure, lipid particles with uniform particle size can be manufactured.

[0027] As shown in Figure 1, in a lipid particle manufacturing apparatus 10 according to one embodiment of the present disclosure, the downstream mixer 2 may be provided downstream of the upstream mixer 1 and spaced apart from the upstream mixer 1. In this case, a longer time can be secured between the time the lipid particle components self-assemble to some extent in the upstream mixer 1 and when they are mixed in the downstream mixer 2, making it easier for more lipid particle components to aggregate with each other. As a result, relatively large lipid particles can be formed.

[0028] As shown in Figure 3, in one embodiment of the lipid particle manufacturing apparatus 10 of this disclosure, the downstream mixer 2 may be provided integrally with the upstream mixer 1. In this case, the number of parts of the lipid particle manufacturing apparatus 10 can be reduced, and the overall structure of the apparatus can be simplified. [Upstream mixer] The upstream mixer 1 may include an upstream supply unit 11 that supplies a diluent to the lipid solution, an upstream mixing pipe 13 that extends downstream from the upstream supply unit 11, and an upstream mixing element 12 provided inside the upstream mixing pipe 13 that mixes a mixed fluid containing the lipid solution and the diluent to obtain a mixed fluid containing lipid particle components before lipid particle formation.

[0029] The upstream supply section 11 is, for example, the point where the flow pipe 3 through which the lipid solution containing lipid particle components and solvent flows and the upstream inlet pipe 4 for introducing the diluent merge. In the upstream supply section 11, the diluent is supplied to the lipid solution to obtain a mixed fluid containing the lipid solution and the diluent.

[0030] The upstream mixing element 12 may be provided over a predetermined length from a proximal position of the upstream supply unit 11 to the downstream side. The lipid solution is supplied with a diluent from the upstream supply unit 11 and immediately stirred by the upstream mixing element 12. This allows the length of the upstream mixer 1 to be shortened. "Proximal position of the upstream supply unit 11" refers to the position of the upstream supply unit 11, or a proximal position upstream or downstream of the upstream supply unit 11. [Downstream Mixer] The downstream mixer 2 is located downstream of the upstream mixer 1. The downstream mixer 2 may include a downstream supply unit 21 that supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, a downstream mixing pipe 23 extending downstream from the downstream supply unit 21, and a downstream mixing element 22 provided inside the downstream mixing pipe 23 that mixes the mixed fluid to which the diluent has been supplied.

[0031] The downstream supply section 21 is the part to which a diluent is supplied to the mixed fluid containing lipid particle components before lipid particle formation. When a diluent is supplied to the mixed fluid in the downstream supply section 21, the solvent concentration in the mixed fluid after the supply of the diluent becomes lower than the solvent concentration in the mixed fluid before the supply of the diluent.

[0032] As shown in Figure 1, if the downstream mixer 2 is installed at a distance from the upstream mixer 1, the downstream supply unit 21 only needs to be connected to a connecting pipe 17 connected to the upstream mixing pipe 13 and a downstream inlet pipe 6 for introducing the diluent.

[0033] As shown in Figure 3, if the downstream mixer 2 is provided integrally with the upstream mixer 1, the downstream supply unit 21 only needs to have the upstream mixing pipe 13 of the upstream mixer 1 integrally connected to it, and the downstream inlet pipe 6 for introducing the diluent connected to it.

[0034] The downstream mixing element 22 may be provided over a predetermined length extending downstream from a position proximal to the downstream supply unit 21. The mixed fluid is immediately agitated in the downstream mixing element 22 after the diluent is supplied in the downstream supply unit 21. This allows the length of the downstream mixer 2 to be shortened. "Proximal position of the downstream supply unit 21" refers to the position of the downstream supply unit 21, or a proximal position upstream or downstream of the downstream supply unit 21.

[0035] In the upstream mixer 1, it is preferable that the solvent concentration in the mixed fluid within the upstream mixing tube 13 is within a predetermined range. The solvent concentration in the mixed fluid flowing through the upstream mixing tube 13 is preferably 50% to 90%, more preferably 55 to 85%, and most desirablely 60 to 80%. In this case, the lipid particle components contained in the lipid solution are more likely to self-assemble.

[0036] In the downstream mixer 2, it is preferable that the mixed fluid in the downstream mixing tube 23 has a lower solvent concentration than the mixed fluid in the upstream mixing tube 13 of the upstream mixer 1.

[0037] The flow velocity of the mixed fluid in the upstream mixing pipe 13 is not particularly limited, but for example, 2.0 to 0.1 m / sec is good, more preferably 1.5 to 0.2 m / sec, and 1.0 to 0.5 m / sec is desirable.

[0038] The flow velocity of the mixed fluid flowing through the downstream mixing pipe 23 should be slower than the flow velocity of the mixed fluid flowing through the upstream mixing pipe 13. The slower the flow velocity of the mixed fluid in the downstream mixing pipe 23, the larger the average particle size of the resulting lipid particles can be, and the particle size can also be made more uniform.

[0039] The area of ​​the cross-section of the downstream mixing pipe 23 perpendicular to the flow direction within the pipe may be larger, the same as, or smaller than the area of ​​the cross-section of the upstream mixing pipe 13 perpendicular to the flow direction within the pipe. As one embodiment to slow the flow velocity of the mixed fluid in the downstream mixing pipe 23 down to that of the mixed fluid in the upstream mixing pipe 13, it is preferable that the area of ​​the cross-section of the downstream mixing pipe 23 perpendicular to the flow direction within the pipe is larger than the area of ​​the cross-section of the upstream mixing pipe 13 perpendicular to the flow direction within the pipe.

[0040] It is preferable that multiple downstream mixers 2 are provided in series downstream of the upstream mixer 1. For example, the configuration is not limited to having only one downstream mixer 2; two or more downstream mixers 2 may be provided in series. The solvent concentration in the mixed fluid can be diluted in multiple stages by multiple downstream mixers 2, making the particle size of lipid particles more uniform. [Upstream mixing pipe and downstream mixing pipe] The upstream mixing pipe 13 and the downstream mixing pipe 23 may be composed of cylindrical members such as cylinders or rectangular tubes. The cylindrical members may be composed of high-rigidity members. The cylindrical members may be transparent or opaque, and may be composed of resin, metal, or inorganic material. Transparent cylindrical members may be made of acrylic resin or polytetrafluoroethylene (PTFE), and opaque cylindrical members may be made of stainless steel (SUS) or titanium.

[0041] The cylindrical member may be configured to open and close along the flow direction. By unfolding the cylindrical member along the flow direction, it becomes easier to rearrange, adjust the position of, and adjust the number of structural members constituting each mixing element located within the cylindrical member. [Upstream mixing elements and downstream mixing elements] As shown in Figure 1, when the upstream mixer 1 and the downstream mixer 2 are spaced apart, the upstream mixing element 12 and the downstream mixing element 22 are also spaced apart. In this case, the upstream mixing element 12 and the downstream mixing element 22 may be separate components, or they may be connected by a connecting member. When the upstream mixing element 12 and the downstream mixing element 22 are connected by a connecting member, for example, the upstream mixing element 12 and the downstream mixing element 22 may be connected by a connecting member that extends along the flow direction. This makes the upstream mixing element 12 and the downstream mixing element 22 a single integrated unit, making them easier to handle.

[0042] As shown in Figure 3, if the upstream mixer 1 and the downstream mixer 2 are provided as a single unit, the upstream mixing element 12 and the downstream mixing element 22 may be a single integrated unit. Alternatively, the upstream mixing element 12 and the downstream mixing element 22 may be separate units.

[0043] The upstream mixing element 12 and the downstream mixing element 22 can be configured in various ways to agitate the mixed fluid. The upstream mixing element 12 and the downstream mixing element 22 may have the same structure as each other, or they may have different structures. The upstream mixing element 12 and the downstream mixing element 22 may be structural materials such as wool. As the wool, quartz wool is often used, for example, quartz wool for gas chromatography is often used.

[0044] The structural member may be one or multiple. The structural member may be, for example, a twisted member 18 having a shape twisted around an axis S, as shown in Figure 2. Alternatively, the structural member may be a veneer projecting radially inward from the pipe wall of the upstream mixing pipe 13 or the downstream mixing pipe 23, or a vane radiating radially outward from the radial center of the upstream mixing pipe 13 or the downstream mixing pipe 23.

[0045] [Lipid particles] Lipid particles obtained by the manufacturing method or apparatus of the present disclosure may be, for example, liposomes, micelles, or LNPs (lipid nanoparticles). Liposomes are spherical vesicles formed from a lipid bilayer. Micelles are formed when lipid particle components aggregate to form a colloid. Micelles have a structure in which the hydrophilic groups of the lipid particle components (e.g., amphiphilic substances) are located on the outside and the hydrophobic groups are located on the inside.

[0046] The average particle size of lipid particles may be 200 nm or larger, even if there is a particle size suitable for the application.

[0047] The average particle size of lipid particles may be measured, for example, by dynamic light scattering (Zetasizer Nano ZS (Malvern)).

[0048] Lipid particles produced according to one embodiment of this disclosure can be used, for example, in the fields of food, cosmetics, or pharmaceuticals. Lipid particles used in food, cosmetics, or pharmaceuticals have a variety of average particle sizes. The average particle size of lipid particles used in food, cosmetics, or pharmaceuticals may be, for example, in the range of 50 to 200 nm, or outside of this range. [Examples]

[0049] (Example 1) Figure 1 is an explanatory diagram of a lipid particle manufacturing apparatus according to Example 1 of this disclosure. The lipid particle manufacturing apparatus 10 of Example 1 comprises an upstream mixer 1 and a downstream mixer 2.

[0050] The upstream mixer 1 comprises an upstream supply section 11, an upstream mixing pipe 13 extending downstream from the upstream supply section 11, and an upstream mixing element 12 provided inside the upstream mixing pipe 13. The upstream supply section 11 is the point where a flow pipe 3 through which a lipid solution containing lipid particle components and a solvent flows, and an upstream inlet pipe 4 for introducing a diluent, merge. The upstream end of the flow pipe 3 is inserted into a lipid solution container 30. The flow pipe 3 is equipped with a pump 31 for sending the lipid solution in the lipid solution container 30 toward the upstream supply section 11. The upstream end of the upstream inlet pipe 4 is inserted into a diluent container 40. The upstream inlet pipe 4 is equipped with a pump 41 for sending the diluent in the diluent container 40 toward the upstream supply section 11. An upstream mixing element 12 is provided inside the upstream mixing pipe 13. The upstream mixing element 12 is located within the upstream mixing pipe 13, extending from a proximal position downstream of the upstream supply section 11 to the downstream end of the upstream mixing pipe 13. The downstream end of the upstream mixing pipe 13 is connected to a connecting pipe 17. The upstream mixing element 12 is not located within the connecting pipe 17.

[0051] The downstream mixer 2 comprises a downstream supply section 21, a downstream mixing pipe 23 extending downstream from the downstream supply section 21, and a downstream mixing element 22 provided inside the downstream mixing pipe 23. The downstream supply section 21 is the point where the connecting pipe 17 and the downstream introduction pipe 6 for introducing the diluent are joined. The upstream end of the downstream introduction pipe 6 is inserted into the diluent container 40. The downstream introduction pipe 6 is equipped with a pump 61 for sending the diluent from the diluent container 40 to the downstream supply section 21. The downstream mixing element 22 is positioned inside the downstream mixing pipe 23, extending from a proximal position downstream of the downstream supply section 21 to the downstream end of the downstream mixing pipe 23. The downstream end of the downstream mixing pipe 23 has an extension pipe 27 in which the downstream mixing element 22 does not exist. The downstream end of the extension pipe 27 is inserted into the mixed fluid container 70.

[0052] The upstream mixing pipe 13 and the downstream mixing pipe 23 are cylindrical bodies made of acrylic resin. The internal diameter of the upstream mixing pipe 13 is 0.6 mm, and the internal diameter of the downstream mixing pipe 23 is 2 mm. The length of the connecting pipe 17 between the upstream mixing pipe 13 and the downstream mixing pipe 23 in the flow direction is 100 mm. The upstream mixing element 12 and the downstream mixing element 22 are separate entities, and the distance between them is 100 mm. The length of the upstream mixing element 12 in the flow direction is 12 mm, and the length of the downstream mixing element 22 in the flow direction is 80 mm.

[0053] Figure 2 is a perspective view of the upstream mixing element 12 and the downstream mixing element 22. Both the upstream mixing element 12 and the downstream mixing element 22 consist of a twisted member 18. The twisted member 18 has a left-handed twisted portion 15 in which a strip-shaped member is twisted 180 degrees to the left around the axis S in the direction from upstream to downstream, and a right-handed twisted portion 16 in which it is twisted 180 degrees to the right around the axis S. The twisted member 18 is arranged so that the left-handed twisted portion 15 and the right-handed twisted portion 16 are alternately repeated in the flow direction. The twisted member 18 is fixed inside the upstream mixing pipe 13 or the downstream mixing pipe 23 by a stopper or the like. The diameter of the twisted member 18 as the upstream mixing element 12 (length in the direction perpendicular to the axis S) is approximately equal to the inner diameter of the upstream mixing pipe 13. The diameter (length in the direction perpendicular to the axis S) of the torsion member 18 as the downstream mixing element 22 is approximately equal to the inner diameter of the downstream mixing pipe 23. The torsion member 18 is fabricated from metal materials such as stainless steel, inorganic materials such as ceramics, or synthetic resin materials by laser beam processing, electron beam processing, electrical discharge machining, 3D printing, etc.

[0054] (Comparative Example 1) Figure 4 is an explanatory diagram of the lipid particle manufacturing apparatus of Comparative Example 1. The lipid particle manufacturing apparatus 9 of Comparative Example 1 does not have an upstream mixer. In the downstream supply section 21 of the downstream mixer 2 of the lipid particle manufacturing apparatus 9, the upstream inlet pipe 4 and the downstream inlet pipe 6 for introducing the diluent are joined. The diameter of the downstream mixing pipe 23 of the downstream mixer 2 is 2 mm. Other aspects of the lipid particle manufacturing apparatus 9 are the same as those of the lipid particle manufacturing apparatus 10 of Example 1. [Experimental Example 1] [1. Raw materials] <Lipid solution> Lipid particle component: Egg-derived lecithin (egg phosphatidylcholine (EggPC)) Solvent: 99.5% ethanol Concentration of lipid particle component in the solvent: 30 mg / ml <Diluent> pure water <Experimental conditions> (1) Example 1 The flow velocity of the mixed fluid in the upstream mixing tube of the upstream mixer was set to 0.5 m / sec, the mixing ratio of the lipid solution and diluent in the upstream mixer was set to lipid solution:diluent = 1:0.4 (volume ratio), and the ethanol concentration in the mixed fluid in the upstream mixing tube was set to 70%.

[0055] The flow velocity of the mixed fluid in the downstream mixing tube was set to 0.09 m / sec or 0.14 m / sec. When the flow velocity of the mixed fluid in the downstream mixing tube was 0.09 m / sec, the mixing ratio of the mixed fluid to the diluent in the downstream mixer was set to mixed fluid:diluent = 1:1 (volume ratio), and the ethanol concentration in the mixed fluid in the downstream mixing tube was set to 35%. When the flow velocity of the mixed fluid in the downstream mixing tube was 0.14 m / sec, the mixing ratio of the mixed fluid to the diluent in the downstream mixer was set to mixed fluid:diluent = 1:2 (volume ratio), and the ethanol concentration in the mixed fluid in the downstream mixing tube was set to 23%. To stabilize the manufactured lipid particles, the mixed fluid contained in the mixed fluid container was mixed with the diluent so that the ethanol concentration was 5% or less.

[0056] (2) Comparative Example 1 The flow velocity of the mixed fluid in the downstream mixing tube of the downstream mixer was set to 0.14 m / sec or 0.09 m / sec. When the flow velocity of the mixed fluid in the downstream mixing tube was 0.09 m / sec, the mixing ratio of the mixed fluid to the diluent in the downstream mixer was set to mixed fluid:diluent = 1:1.8 (volume ratio), and the ethanol concentration in the mixed fluid in the downstream mixing tube was set to 35%. When the flow velocity of the mixed fluid in the downstream mixing tube was 0.14 m / sec, the mixing ratio of the mixed fluid to the diluent in the downstream mixer was set to mixed fluid:diluent = 1:3.3 (volume ratio), and the ethanol concentration in the mixed fluid in the downstream mixing tube was set to 23%. To stabilize the manufactured lipid particles, the mixed fluid contained in the mixed fluid container was mixed with diluent so that the ethanol concentration was 5% or less. <Measurement of average liposome particle size and polydispersity index> The mixed fluid contained in the mixed fluid container was removed, and the particle size distribution of liposomes in the mixed fluid was measured using dynamic light scattering. Specifically, using an analytical instrument (Zetasizer Nano ZS (Malvern)), a laser was irradiated onto Brownian-motion liposomes in the mixed fluid, and the scattering intensity was measured. The particle size of the liposomes was calculated from the temporal variation of the measured laser intensity. Scattering intensity distributions for each particle size were created, and the average value was calculated from these results. The calculated average value was defined as the average particle size of the liposomes.

[0057] The polydispersity index of liposome particle size was determined by photon correlation spectroscopy using an analytical instrument (Zetasizer Nano ZS (Malvern))). <Mixed fluid in the upstream and downstream mixing tubes> Liposomes were produced under the above conditions using the lipid particle production apparatus of Example 1 and Comparative Example 1. The average particle size and the number of dispersibility index (pdi) of the liposomes are shown in Figure 5.

[0058] When liposomes were produced using the lipid particle production apparatus of Example 1, the average particle size of the liposomes was larger, and the polydispersity index (pdi) of the liposome particle size was smaller, compared to when liposomes were produced using the lipid particle production apparatus of Comparative Example 1. Furthermore, when the flow velocity of the mixed fluid flowing through the downstream mixing tube was 0.09 m / sec, the average particle size of the liposomes was larger, and the polydispersity index (pdi) of the liposome particle size was smaller, compared to when the flow velocity was 0.14 m / sec.

[0059] In Example 1, the solvent concentration in the mixed fluid was homogenized to 70% by mixing in the upstream mixer. As a result, the ethanol concentration in the mixed fluid, which is conducive to the self-assembly of lipid particle components, became more abundant in the 70% range. Therefore, the lipid particle components self-assemble in the mixed fluid, and the self-assembly of lipid particle components can proceed uniformly. It is presumed that by further reducing the ethanol concentration and mixing in the downstream mixer, lipid particles with a relatively large average particle size and uniform particle size are formed.

[0060] In Comparative Example 1, the ethanol concentration in the mixed fluid drops sharply from 99.5% to 50% or less after dilution with the diluent, resulting in fewer opportunities for the lipid particle components to self-assemble in the downstream mixer. Therefore, it is presumed that the average particle size of the lipid particles is small, and uniform particle size is difficult to achieve. [Experimental Example 2] Liposomes were produced using the lipid particle production apparatus of Example 1 and Comparative Example 1. In Test Examples A, B, C, D, and E of Example 1, the production conditions were adjusted so that the average particle size of the liposomes was approximately the same as that of Test Examples a, b, c, d, and e of Comparative Example 1, respectively. To stabilize the produced lipid particles, a diluent was added to the mixed fluid contained in the mixed fluid container so that the ethanol concentration was 5% or less. Tables 1 and 2 show the production conditions for Example 1 and Comparative Example 1.

[0061] [Table 1]

[0062] [Table 2]

[0063] Figure 6 shows the average particle size and majority dispersion index (pdi) of liposomes produced using the lipid particle production apparatus of Example 1 and Comparative Example 1. When lipid particles with similar average particle size were produced using the respective lipid particle production apparatuses of Example 1 and Comparative Example 1, the majority dispersion index of the lipid particle size was higher for Comparative Example 1 than for Example 1. This indicates that when the diluent is mixed in two stages, as in Example 1, the particle size becomes more uniform than when the diluent is mixed in one stage, as in Comparative Example 1. [Experimental Example 3] In Experimental Example 3, when producing liposomes using the lipid particle production apparatus of Example 1, the ethanol concentration in the mixed fluid flowing through the upstream mixing tube was varied. By adjusting the mixing ratio of the lipid solution and the diluent, the ethanol concentration in the mixed fluid flowing through the upstream mixing tube was set to 90%, 80%, 70%, 60%, and 50%. The flow velocity of the mixed fluid flowing through the downstream mixing tube was kept constant at 0.14 m / sec. To stabilize the produced lipid particles, the mixed fluid contained in the mixed fluid container was mixed with the diluent so that the ethanol concentration was 5% or less. Other conditions in Experimental Example 3 were the same as those in Experimental Example 1. The average particle size and the number of dispersibility index (pdi) of the liposomes are shown in Figure 7.

[0064] When the ethanol concentration in the mixed fluid flowing through the upstream mixing tube was 50% to 90%, liposomes with uniform particle size could be produced. When the ethanol concentration was 60% to 80%, the average particle size of the liposomes was larger compared to the 50% or 90% cases. When the ethanol concentration was 90% or 70% to 50%, the particle size of the liposomes was more uniform compared to the 80% case.

[0065] [Experimental Example 4] In Experimental Example 4, a simulation analysis was performed assuming the production of liposomes using the lipid particle manufacturing apparatus of Example 1. In the simulation analysis, the standard deviation of the mass fraction of ethanol in the mixed fluid flowing through the upstream and downstream mixing tubes was measured. The simulation analysis was performed using the finite volume method for fluid simulation. The conditions for the fluid simulation were as follows: <Upstream mixing pipe> • Solvent: 99.5% ethanol • Diluent: Pure water • Ratio of solvent to diluent (by volume): 3:1 • Flow velocity of the mixed fluid consisting of solvent and diluent: 0.5 m / sec • Solvent concentration in the mixed fluid: 70% • Diameter of the upstream mixing tube: 0.6mm • Length of the upstream mixing element inside the upstream mixing tube: 10.8 mm <Downstream mixing pipe> • Diluent: Pure water • Ratio of mixed fluid to diluent (by volume): 1:1 • Fluid mixture velocity: 0.09 m / sec • Solvent concentration in the mixed fluid after supplying the diluent: 35% • Diameter of the downstream mixing tube: 2mm • Length of the downstream mixing element inside the downstream mixing pipe: 36 mm • Spacing between upstream and downstream mixing elements: 100 mm Figure 8 shows the standard deviation of the mass fraction of ethanol in the mixed fluid when the mixed fluid flowing through the upstream mixing tube of the lipid particle manufacturing apparatus in Example 1 was simulated. Figure 9 shows the standard deviation of the mass fraction of ethanol in the mixed fluid when the mixed fluid flowing through the downstream mixing tube of the lipid particle manufacturing apparatus in Example 1 was simulated.

[0066] In the upstream mixing tube of the lipid particle manufacturing apparatus of Example 1, the standard deviation of the lipid fraction of ethanol in the mixed fluid was 0.01 or less at a point 0.005 m downstream from the upstream supply section. It was confirmed that the mixed fluid was uniformly mixed by the upstream mixing element in the upstream mixing tube.

[0067] Furthermore, in the downstream mixing tube, the standard deviation of the lipid fraction of ethanol in the mixed fluid was 0.01 or less at a point 0.02 m downstream from the downstream supply section. This confirmed that the mixed fluid was uniformly mixed by the downstream mixing element in the downstream mixing tube.

[0068] This disclosure is not limited to the embodiments described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in each embodiment are also included in the technical scope of this disclosure. [Industrial applicability]

[0069] This disclosure can be used, for example, in the fields of food, cosmetics, or pharmaceuticals. [Explanation of Symbols]

[0070] 1: Upstream mixer 10: Lipid particle manufacturing device 11: Upstream supply section 12: Upstream mixing element 13: Upstream mixing pipe 15: Left-hand twist 16: Right-hand twist 17: Connecting pipe 18: Torsional member 2: Downstream mixer 21: Downstream supply section 22: Downstream mixing element 23: Downstream mixing pipe 27: Extension tube 3: Distribution pipe 30: Lipid solution container 31, 41, 61: Pumps 4: Upstream inlet pipe 40: Diluent container 6: Downstream inlet pipe 70: Mixed fluid container

Claims

1. A) A step of mixing a lipid solution containing lipid particle components and a solvent with a diluent to obtain a mixed fluid containing lipid particle components before lipid particle formation, B) A method for producing lipid particles, comprising the steps of supplying a diluent to the mixed fluid, and then further mixing the mixed fluid to which the diluent has been supplied to form lipid particles.

2. A) The method for producing lipid particles according to claim 1, wherein in step A, the concentration of the solvent in the mixed fluid is 50% or more and 90% or less.

3. A) The method for producing lipid particles according to claim 1, wherein in step A, the standard deviation of the mass fraction of the solvent in a cross-section perpendicular to the flow direction of the mixed fluid after mixing is 0.01 or less.

4. B) The method for producing lipid particles according to claim 1, wherein the process is repeated two or more times.

5. A lipid particle manufacturing apparatus used in the method for manufacturing lipid particles according to any one of claims 1 to 4, A) An upstream mixer that mixes the mixed fluid in the process, A lipid particle manufacturing apparatus comprising: a downstream mixer provided downstream of the upstream mixer, which in step B) supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, and then further mixes the mixed fluid to which the diluent has been supplied.

6. The lipid particle manufacturing apparatus according to claim 5, wherein the downstream mixer is provided at a distance from the upstream mixer.

7. The lipid particle manufacturing apparatus according to claim 5, wherein the downstream mixer is provided integrally with the upstream mixer.

8. The upstream mixer comprises an upstream supply unit that supplies the diluent to the lipid solution, an upstream mixing tube extending downstream from the upstream supply unit, and an upstream mixing element provided inside the upstream mixing tube that mixes the lipid solution and the diluent to obtain a mixed fluid containing lipid particle components before lipid particle formation. The downstream mixer comprises a downstream supply unit that supplies a diluent to a mixed fluid containing lipid particle components before lipid particle formation, a downstream mixing pipe extending downstream from the downstream supply unit, and a downstream mixing element provided inside the downstream mixing pipe for mixing the mixed fluid to which the diluent has been supplied. The lipid particle manufacturing apparatus according to claim 5.

9. The lipid particle manufacturing apparatus according to claim 8, wherein the flow velocity of the mixed fluid flowing through the downstream mixing tube is slower than the flow velocity of the mixed fluid flowing through the upstream mixing tube.

10. The lipid particle manufacturing apparatus according to claim 5, wherein a plurality of downstream mixers are provided in series downstream of the upstream mixer.

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