Flow channel structure and method for producing self-assembled material particles using the same

Abstract

The present invention relates to a flow path structure that includes a flow path A10 having an inlet port 11 and a flow path B20 having an inlet port 21. The flow paths A10, B20 intersect three-dimensionally at one or more locations downstream of the inlet ports 11, 21, and include, in the intersection regions, a mutual hole 32 in respective flow path walls through which the flow paths A10, B20 communicate. The flow paths A10, B20 merge at a merging section 30 positioned downstream of the furthest-downstream intersection region and have at least one outlet port 31 downstream of the merging section 30. Alternatively, the flow paths A10, B20 do not merge, and independently have an outlet port 12 and an outlet port 22. The present invention relates to a method for manufacturing self-organizing material particles, the method using said flow path structure and including a step for obtaining a liquid that contains self-organizing material particles by diluting, in a dilution medium, a solution containing a self-organizing material. The present invention provides: a fluid structure with which it is possible to produce self-organizing material particles having an average particle diameter of 100 nm or more even under conditions in which the flow rate is relatively high; and a method for manufacturing self-organizing material particles, the method using said fluid structure.

Description

Technical Field

[0001] The present invention relates to a flow path structure and a method for manufacturing self-organized substance particles using the same. Cross-reference to related applications This application claims the priority of Japanese Patent Application No. 2022-17619 filed on February 8, 2022, and the entire description thereof is incorporated herein by specific reference.

Background Art

[0002] Amphiphilic molecules such as lipids are known to self-organize multiple lipid molecules in a polar solvent such as water to form particles (lipid nanoparticles) in which the polar groups of each lipid molecule are exposed to the solvent side (non-polar groups are isolated from the solvent). Such self-organized substance particles have been increasingly put into practical use as nucleic acid delivery carrier particles in recent years. For example, lipid nanoparticles are also used as mRNA delivery carriers in the mRNA vaccine for the novel coronavirus (SARS-CoV-2) that was granted emergency use authorization in 2020. Recently, it has been reported that the delivery efficiency and activity of nucleic acids differ depending on the particle size of lipid nanoparticles. Therefore, microfluidic devices have attracted attention as a manufacturing method for precisely controlling the particle size of lipid nanoparticles (Patent Documents 1 and 2). By using a microfluidic device, the particle size of lipid nanoparticles can be controlled in a flow rate-dependent manner, and it has also been used in the development of Onpattro (registered trademark), the world's first siRNA formulation.

[0003] Patent Document 1: WO2018 / 190423 Patent Document 2: WO2021 / 064998 Patent Document 3: Japanese Patent Application Laid-Open No. 2019-503271 (WO2017 / 117647)

Summary of the Invention

Problems to be Solved by the Invention

[0004] On the other hand, microfluidic devices reported to date have faced the challenge of reduced particle size when particles are fabricated under high-flow conditions for mass production. For example, larger lipid nanoparticles with an average particle size of 100 nm or more have shown higher drug efficacy in some cases, and larger average particle sizes also allow for selective drug delivery to different tissues than smaller particles. Therefore, there is a great demand for lipid nanoparticles with relatively large average particle sizes and high size uniformity. Similar demand is also expected for self-assembled material particles made from materials other than lipids in the future.

[0005] For example, when using the microfluidic devices described in Patent Documents 1 and 2, under the conditions used by the present inventors in their studies, lipid nanoparticles of approximately 100 nm could be prepared at a total flow rate of 100 μL / min. However, when the total flow rate was increased to 500 μL / min, the average particle size of the obtained lipid nanoparticles was approximately 30 nm.

[0006] Furthermore, when using the Dean vortex branching microfluidic device described in Patent Document 3, the particle size of the lipid nanoparticles obtained at a total flow rate of 2 to 10 mL / min was approximately 80 nm or less in all cases.

[0007] Furthermore, the microfluidic devices described in Patent Documents 1 and 2 have a structure in which the channel width (cross-sectional area) changes intermittently and is less than half of the original width along the way. Therefore, under high flow rate conditions, the pressure loss becomes large, and there is a risk of device damage, making them unsuitable for the mass production of lipid nanoparticles, which requires high flow rates.

[0008] The problems to be solved and the objectives of the present invention are to provide a fluid structure capable of producing relatively large self-assembled material particles, such as lipid nanoparticles, with an average particle size of 100 nm or more, even under relatively high flow rate conditions, for example, a total flow rate of 2 mL / min, and a method for producing self-assembled material particles such as lipid nanoparticles using this fluid structure. In this specification, the average particle size refers to the Z-average particle diameter as defined in ISO 13321. [Means for solving the problem]

[0009] This invention is based on the principle of forming self-assembled material particles, and, unlike, for example, Patent Document 3 which aims to minimize the time required to completely mix the two liquids, it was arrived at by considering the dilution time of the self-assembled material-containing solution necessary for increasing particle size, and by investigating a flow path shape that can achieve relatively high flow rates. Specifically, it is as follows.

[0010] [1] It has a flow path A (10) with an inlet (11) and a flow path B (20) with an inlet (21), Flow channels A and B (10, 20) intersect three-dimensionally at one or more locations downstream of the inlet (11, 21), and each has a common hole (32) in the intersecting region that connects flow channels A and B (10, 20) in the flow channel wall. Flow paths A and B (10, 20) merge at a confluence (30) located in the downstream intersection region, and have at least one outlet (31) downstream of the confluence (30) (however, part or all of the section from the upstream intersection region to the outlet (31) is a section for diluting the fluid flowing through the flow path), or Flow paths A and B (10, 20) do not merge and have independent outlets (12) and outlet (22) (however, part or all of the section from the intersection region to outlet (12) and outlet (22) is a section for diluting the fluid flowing through the flow path). A channel structure. [2] A flow channel structure for producing self-assembled material particles, wherein in a portion for diluting the fluid, a self-assembled material-containing solution introduced from an inlet (11) or an inlet (21) is diluted with a diluent introduced from the other inlet to form self-assembled material particles. [3] The channel structure according to [1] or [2], wherein channel A(10) is regularly or irregularly bent or straight in two or three dimensions, and channel B(20) is regularly or irregularly bent or straight in two or three dimensions. [4] A channel structure according to any one of items [1] to [3], having two or more intersection regions. [5] A channel structure according to any one of items [1] to [4], wherein the opening surface of the common hole of channel A(10) and the opening surface of the common hole of channel B(20) form substantially the same surface. [6] Flow channels A and B (10, 20) periodically intersect, and the flow channel structure is as described in any one of [1] to [5]. [7] The flow channel structure according to any one of [1] to [6], wherein the cross-sectional area of ​​the flow channel A(10) is constant or periodically varies at least in part between the inlet (11) and the confluence (30) or outlet (12). [8] The channel structure according to any one of [1] to [7], wherein the cross-sectional area of ​​the straight section of channel B(20) is constant or periodically varies between the inlet (11) and the confluence (30) or outlet (22). [9] The channel structure described in any one of [1] to [8], wherein the intersection angle of channels A and B (10, 20) is such that the angle θ formed by the center line of channel A in the direction of flow and the center line of channel B in the direction of flow is in the range of 30° to 150°.

[10] The channel structure described in any one of [1] to [9], wherein the widths of channels A and B (10, 20) are independently in the range of 10 to 2000 μm, and the heights of channels A and B (10, 20) are independently in the range of 10 to 1000 μm.

[11] The shape of the planes of the outer and inner edges of the bends in the two- or three-dimensionally curved channel A(10) is polygonal or approximately circular. The flow channel structure according to any one of [1] to

[10] , wherein the shape of the planes of the outer and inner edges of the bends in the flow channel B(20) which bends in two or three dimensions is polygonal or substantially circular.

[12] A substrate A having a continuous groove A for a flow channel A on at least one main surface of a flat substrate A substrate B having continuous grooves B for a flow channel B on at least one main surface of a flat substrate It has a structure in which continuous groove A and continuous groove B are bonded together so that they face each other. Channel A is regularly or irregularly bent in two dimensions or is straight, and channel B is regularly or irregularly bent in two dimensions or is straight. A flow channel structure as described in any one of items [1] to

[11] .

[13] A channel structure according to any one of [1] to

[12] , further comprising a second channel structure downstream of the outlet of the first channel structure.

[14] A method for producing self-assembled material particles, comprising the step of diluting a self-assembling material-containing solution with a diluent to obtain a liquid containing self-assembling material particles, The manufacturing method, wherein the above step is carried out using a flow channel structure described in any one of items [1] to

[13] .

[15] The manufacturing method according to

[14] , wherein a self-assembling material-containing solution is introduced from the inlet of channel A and a diluent is introduced from the inlet of channel B, or a self-assembling material-containing solution is introduced from the inlet of channel B and a diluent is introduced from the inlet of channel A.

[16] The manufacturing method according to claim 14 or 15, wherein in a common hole in the intersection region of the flow channel structure, at least a portion of the self-organizing material-containing solution flow branches off and merges with a diluent medium flow, and at least a portion of the diluent medium flow branches off and merges with the self-organizing material-containing solution flow, and in the dilution portion of the flow channel structure, a dilution layer of the self-organizing material-containing solution is formed at the interface between the merged self-organizing material-containing solution flow and the diluent medium flow, and self-organizing material particles are formed in the dilution layer.

[17] The manufacturing method according to any one of

[14] to

[16] , wherein the total flow rate of the self-assembling material-containing solution and diluent to channel A and channel B is in the range of 1 μl / min to 1000 ml / min.

[18] The method for production according to any one of

[14] to

[17] , wherein the ratio (V2 / V1) of the flow rate V2 of the diluent medium to the flow rate V1 of the self-assembling substance-containing solution is in the range of 1:1 to 1:20.

[19] The method for production according to any one of

[14] to

[18] , wherein the self-assembling substance is a lipid or an amphiphilic substance.

[20] The method for production according to any one of

[14] to

[19] , wherein the diluent medium is selected from an aqueous solution, a buffer solution, an aqueous solution containing nucleic acid, an aqueous solution containing protein, an aqueous solution containing peptide, an aqueous solution containing adjuvant, and a mixture thereof.

[21] The method for production according to any one of

[14] to

[20] , wherein the self-assembling substance particles contained in the liquid containing the self-assembling substance particles are nanosized.

[22] The method for production according to any one of

[14] to

[21] , wherein the Z-average particle diameter of the nanosized self-assembling substance particles is in the range of 10 to 1000 nm.

[23] The method for production according to any one of

[14] to

[22] , wherein the Z-average particle diameter of the nanosized self-assembling substance particles is in the range of 20 to 200 nm. [Effect of the Invention]

[0011] According to the present invention, it is possible to provide a fluid structure capable of producing self-assembling substance particles such as relatively large lipid nanoparticles having an average particle diameter of 100 nm or more even under conditions where the flow rate is relatively large. Further, according to the present invention, by using the fluid structure of the present invention, it is possible to produce self-assembling substance particles such as relatively large lipid nanoparticles having an average particle diameter of 100 nm or more even under conditions where the flow rate is relatively large.

[0012] In one aspect of the present invention, it is also possible to produce self-assembling substance particles having high size uniformity with an average particle diameter of less than 100 nm. [Brief Description of the Drawings]

[0013] [Figure 1-1] Figure 1-1 is a perspective view showing only the flow channels of a flow channel structure, illustrating an example of a flow channel structure according to the present invention. [Figure 1-2] Figure 1-2 shows only the flow channels of the flow channel structure in Figure 1-1, as well as a plan view, front view, and side view. [Figure 1-3] Figure 1-3 is a plan view showing only the flow channels of the flow channel structure in Figure 1-1, with dimensions W1 to W8 indicated. [Figure 2] Figure 2 shows a perspective view, plan view, front view, and side view of the flow channel structure of the present invention, illustrating only the flow channel of an example of the flow channel structure. [Figure 3] Figure 3 shows a perspective view, plan view, front view, and side view of an example of a flow channel structure of the present invention, illustrating only the flow channel. [Figure 4] Figure 4 shows a perspective view, plan view, front view, and side view of the flow channel structure of the present invention, illustrating only the flow channel of an example of the flow channel structure. [Figure 5-1] Figure 5-1 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 5-2] Figure 5-2 is a schematic diagram illustrating an example of the flow channel structure of the present invention. [Figure 5-3] Figure 5-3 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 5-4] Figure 5-4 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 5-5] Figure 5-5 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 5-6] Figure 5-6 is a schematic diagram illustrating an example of the flow channel structure of the present invention. [Figure 5-7] Figure 5-7 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 5-8] Figure 5-8 is a schematic diagram illustrating an example of a flow channel structure according to the present invention. [Figure 6] Figure 6 is a plan view of an example of a flow channel structure according to the present invention. [Figure 7]Figure 7 is an explanatory diagram of the flow channel structure used in the embodiment. [Figure 8] Figure 8 shows the experimental results for Example 1. [Figure 9] Figure 9 shows the experimental results for Example 1. [Figure 10] Figure 10 shows the experimental results of Example 1. [Figure 11] Figure 11 shows the experimental results for Example 2. [Figure 12] Figure 12 shows the experimental results for Example 3. [Figure 13] Figure 13 shows the simulation results for Reference Example 2-1. [Figure 14] Figure 14 shows the simulation results for Reference Example 2-2. [Figure 15] Figure 15 shows the simulation results for Reference Example 2-3. [Figure 16] Figure 16 shows the simulation results for Reference Example 2-4. [Figure 17] Figure 17 shows the simulation results for Reference Example 2-5. [Figure 18] Figure 18 shows the particle size measurement results for Example 5. [Figure 19] Figure 19 shows the relative luminescence intensity measurement results for Example 5. [Figure 20] Figure 20 shows the relative luminescence intensity measurement results for Example 6. [Modes for carrying out the invention]

[0014] <Flow channel structure> The flow channel structure of the present invention is It has a flow path A (10) with an inlet (11) and a flow path B (20) with an inlet (21), Flow channels A and B (10, 20) intersect three-dimensionally at one or more locations downstream of the inlet (11, 21), and each has a common hole (32) in the intersecting region that connects flow channels A and B (10, 20) in the flow channel wall. Flow paths A and B (10, 20) merge at a confluence (30) located in the downstream intersection region, and have at least one outlet (31) downstream of the confluence (30) (however, part or all of the section from the upstream intersection region to the outlet (31) is a section for diluting the fluid flowing through the flow path) (hereinafter sometimes referred to as dilution section 1), or Flow paths A and B (10, 20) do not merge and each has its own outlet (12) and outlet (22) independently (however, part or all of the section from the intersection region to outlet (12) and outlet (22) is a section for diluting the fluid flowing through the flow path) (hereinafter sometimes referred to as dilution section 2).

[0015] The flow channel structure of the present invention is preferably for the production of self-assembled material particles, wherein, in a portion for diluting the fluid, a self-assembled material-containing solution introduced from an inlet (11) or an inlet (21) is diluted by a diluent introduced from the other inlet to form self-assembled material particles.

[0016] The flow path A(10) may be regularly or irregularly bent or straight in two or three dimensions, and the flow path B(20) may be regularly or irregularly bent or straight in two or three dimensions.

[0017] The flow channel structure of the present invention has one or more intersection regions, and may also have two or more intersection regions. When there is one intersection region, one or both of flow channels A(10) and B(20) may be linear, and if they are not linear, they may be regularly or irregularly curved in two or three dimensions. Figures 5-7 and 5-8 show embodiments in which both flow channels A(10) and B(20) are linear.

[0018] The flow channel structure of the present invention will be described below based on Figure 1, which shows an example of a flow channel structure. Figure 1-1 is a perspective view showing only the flow channel of the flow channel structure, and the flow channel is incorporated into the main body of the structure which is not shown. Figure 1-2 is a plan view, front view, and side view showing only the flow channel of the flow channel structure of Figure 1-1. Figure 1-3 is a plan view showing only the flow channel of the flow channel structure of Figure 1-1, and the dimensions W1 to W8 are shown.

[0019] The flow channel structure having a dilution section 1 has a flow channel A, indicated by 10, which has an inlet 11, and a flow channel B, indicated by 20, which has an inlet 21. Flow channels A and B merge at a confluence section 30 located downstream of the inlets 11 and 21, and there may be at least one outlet 31 downstream of the confluence section 30. The confluence section 30 is located at the downstreammost intersection of flow channels A and B. Part or all of the section from the upstreammost intersection section to the outlet 31 is for diluting the fluid flowing through the flow channel. Depending on the fluid velocity, the ratio of the fluid to be diluted to the fluid of the diluent, and the length of the dilution section, dilution may be completed in part of the section from the upstreammost intersection section to the outlet 31, or dilution may continue throughout the entire section from the upstreammost intersection section to the outlet 31. When dilution is completed in part of the section from the upstreammost intersection section to the outlet 31, that section is for diluting the fluid flowing through the flow channel. If dilution continues throughout the entire section from the uppermost intersection to the outlet 31, then the entire section from the uppermost intersection to the outlet 31 is a part that dilutes the fluid flowing through the channel.

[0020] In a flow channel structure having a dilution section 2, flow channels A and B do not merge, and each has an outlet. Part or all of the section from the intersection region to outlets 12 and 22 is a section for diluting the fluid flowing through the flow channel. Similar to the case of dilution section 1, depending on the fluid velocity, the ratio of the fluid to be diluted to the fluid of the diluting medium, and the length of the dilution section, dilution may be completed in part of the section from the intersection region to outlets 12 and 22, dilution may continue in the entire section from the intersection region to outlets 12 and 22, dilution may be completed in part of the section from the intersection region to outlet 12 and dilution may be completed in the entire section from the intersection region to outlet 22, or dilution may be completed in the entire section from the intersection region to outlet 12 and dilution may be completed in part of the section from the intersection region to outlet 22. If dilution is completed between the intersection region and outlet 12, and in part between the intersection region and outlet 22, then the area between the intersection region and outlet 12, and the area between the intersection region and outlet 22, respectively, are areas for diluting the fluid flowing through the channel. If dilution continues throughout the entire area between the intersection region and outlet 12, and the entire area between the intersection region and outlet 22, then the entire area between the intersection region and outlet 12, and the area between the intersection region and outlet 22, respectively, are areas for diluting the fluid flowing through the channel. As shown in the embodiments in Figures 5-7 and 5-8, channel A and channel B have outlets 12 and 22 independently without merging, and each has a dilution area between the intersection region and outlets 12 and 22.

[0021] There are no particular restrictions on the number of times channels A and B intersect. This can be determined as appropriate, taking into account the intended use of the channel structure and the dimensions of channels A and B. For example, it can range from 1 or 2 to 100 times. The minimum number of intersections can be, for example, 1, 2, 3, 4, or 5 times, and the maximum number can be, for example, 30, 25, 20, 15, or 10 times.

[0022] Flow channels A and B intersect three-dimensionally at two or more locations between the inlets 11 and 21 and the confluence 30, or at one location between the inlets 11 and 21 and the outlets 12 and 22, and in each intersection region, the side wall of the flow channel has a common hole 32 that connects flow channels A and B.

[0023] The opening surface of the common hole in channel A and the opening surface of the common hole in channel B can be substantially the same plane. In this case, the fluid flowing through channel A and the fluid flowing through channel B flow while forming a contact surface at the common hole. Alternatively, the common hole can be a passage (connecting passage) that connects the opening of the common hole in channel A and the opening of the common hole in channel B. In this case, a portion of the fluid flowing through channel A and a portion of the fluid flowing through channel B flow into the connecting passage and form a contact surface between the two fluids. Alternatively, channels A and B can intersect at the common hole such that they have a common flow path. In this case, since they have a common flow path, the fluids flowing through the two channels will mix at that point.

[0024] The cross-sectional area of ​​flow path A is constant between the inlet 11 and the confluence 30 or outlet 12, or can vary periodically in at least part of it. From the viewpoint of maintaining laminar flow, it is preferable that it be constant, and from the viewpoint of promoting diffusion within the fluid, it is preferable that it vary periodically.

[0025] The cross-sectional area of ​​flow path B is constant between the inlet 21 and the confluence 20 or outlet 22, or can vary periodically in at least part of it. From the viewpoint of maintaining laminar flow, it is preferable that it be constant, and from the viewpoint of promoting diffusion within the fluid, it is preferable that it vary periodically.

[0026] The dimensions of channels A and B can be determined as appropriate, taking into consideration the intended use of the channel structure, and are not intended to be limited. The width and height of channel A can be, for example, in the range of 1 μm to 10 mm and 1 μm to 10 mm, respectively, and the width and height of channel B can be, for example, in the range of 1 μm to 10 mm and 1 μm to 10 mm, respectively. Preferably, the width and height of channel A can be in the range of 10 μm to 2 mm (2000 μm) and 10 μm to 1 mm (1000 μm), respectively, and the width and height of channel B can be in the range of 10 μm to 2 mm (2000 μm) and 10 μm to 1 mm (1000 μm), respectively. In the channel structure shown in Figure 1-3, the width of channels A and B is 200 μm, and the height of channels A and B is 100 μm. However, while larger dimensions are more suitable for mass production at high flow rates, the dimensions can be appropriately selected within the range in which particles with the desired particle size (average particle diameter) can be obtained.

[0027] Figures 2-4 show examples of channel structures with channel dimensions different from those in Figure 1. The channel structures shown in Figures 3 and 4 are examples of channel structures in which the cross-sectional area of ​​the straight section of channel A fluctuates periodically.

[0028] Various variations are possible in the shape and structure of channel A and channel B in the channel structure of the present invention. These are illustrated in Figures 5-1 to 5-8.

[0029] Figure 5-1 shows that channel B is straight, while channel A is regularly bent in two dimensions, with the bent sections having a curved planar shape. Figure 5-2 shows an example where channel B is straight, channel A is regularly bent in two dimensions, the bent sections have a refracted shape in plan, and the intersection angle of channels A and B is not 90°. Figure 5-3 shows that channel B is straight, and channel A is regularly bent in two dimensions. The bent sections have a planar shape similar to that in Figure 1, with some sections of channel A being interrupted, and channel A and channel B merging in the intersection region. Figure 5-4 shows an example where channel B is straight, channel B is regularly bent in two dimensions, the bent sections have a curved planar shape, and the intersection angle of channels A and B is not 90°.

[0030] Figure 5-5 shows that channel B is linear, while channel A is regularly bent in three dimensions, and the bent sections have a planar shape similar to that in Figure 1. Figure 5-6 shows that channel B is linear, and channel A consists of two channels, both of which are regularly bent in two dimensions, with the bent sections having a refracted planar shape, similar to Figure 1. The two channels A sequentially intersect channel B in the longitudinal direction. Each of the two channels A has an inlet and an outlet. Figure 5-7 shows that channel B is linear, and channel A consists of three linear channels. The three channels A sequentially intersect channel B in the longitudinal direction, each having one intersection point. Each of the three channels A has an inlet and an outlet. Figure 5-8 shows that channel B is straight, and channel A is also straight. Channels A and B intersect at one point. Channels A and B each have an inlet and an outlet.

[0031] There are no particular restrictions on the intersection angle of channels A and B, but the angle θ formed by the center line in the direction of flow of channel A and the center line in the direction of flow of channel B can be in the range of, for example, 30° to 150°. Typically it is 90°, but preferably it is in the range of 45° to 120°. Typically it is 90° as shown in Figure 1, but as shown in Figures 5-2 and 5-4, there may also be configurations in which the angle θ alternates between, for example, about 60° and about 120°, other than 90°.

[0032] Channel A is regularly or irregularly bent in two or three dimensions, and channel B is regularly or irregularly bent in two or three dimensions or is linear. In the example in Figure 1, channel A is regularly bent in two dimensions, channel B is linear, and channels A and B intersect periodically. Channels A and B can intersect irregularly. Figure 5-5 shows an example where channel A is linear and channel B is regularly bent in three dimensions.

[0033] The shape of the planes at the outer and inner edges of the bends in a two- or three-dimensional channel A can be polygonal or approximately circular, and the shape of the planes at the outer and inner edges of the bends in a two- or three-dimensional channel B can also be polygonal or approximately circular. Figure 5-1 shows the case of approximately circular shape, and Figure 5-2 shows the case of polygonal shape.

[0034] The flow channel structure of the present invention may be a flow channel structure having, for example, a substrate A having a continuous groove A for a flow channel A on at least one main surface of a substrate with a flat surface, and a substrate B having a continuous groove B for a flow channel B on at least one main surface of a substrate with a flat surface, bonded together so that the continuous groove A and the continuous groove B face each other, wherein the flow channel A is regularly or irregularly bent in two dimensions, and the flow channel B is regularly or irregularly bent in two dimensions or is linear.

[0035] Figure 6 is a plan view showing the main body and channels of four different channel structures (1) to (4) with different widths W. In each channel structure, channel A is regularly curved in two dimensions, and channel B is straight. The right side of the figure shows the inlets (two locations) for channel A and channel B, and one outlet of the channel where channels A and B merge. (1), (2), (3), and (4) are examples of channels A that are regularly curved in two dimensions with widths W of 200 μm, 400 μm, 600 μm, and 800 μm, respectively.

[0036] The flow channel structure of the present invention may further have a second flow channel structure downstream of the outlet of the first flow channel structure, and the first and second flow channel structures may be the same or different. The outlet of the first flow channel structure is connected to the inlet of flow channel A of the second flow channel structure, or to the inlet of flow channel B of the second flow channel structure. More specifically, the flow channel structure may be configured such that the outlet of the flow channel structure having dilution portion 1 is connected to the inlet of flow channel A or flow channel B of the flow channel structure having dilution portion 2, or such that the outlet of flow channel A or flow channel B of the flow channel structure having dilution portion 2 is connected to the inlet of flow channel A or flow channel B of the flow channel structure having dilution portion 1, or a combination thereof. For example, Figure 5-6 shows a configuration in which the outlet of flow channel B of the flow channel structure having dilution portion 2 is connected to the inlet of flow channel B of the flow channel structure having dilution portion 1. Figure 5-7 shows an example in which three flow channel structures, each having dilution sections 1 and 2, are connected. The upstream flow channel structure has dilution section 1, and the outlet of its flow channel B is connected to the inlet of the middle flow channel B. The middle flow channel structure also has dilution section 1, and the outlet of its flow channel B is connected to the inlet of the flow channel B of the downstream flow channel structure, which has dilution section 2.

[0037] <Method for manufacturing self-assembled material particles> The present invention relates to a method for producing self-assembled material particles, comprising the step of diluting a self-assembled material-containing solution with a diluent to obtain a liquid containing self-assembled material particles, wherein the step is carried out using the flow channel structure of the present invention.

[0038] A solution containing a self-assembling material is introduced through the inlet of channel A, and a diluent is introduced through the inlet of channel B; or, a solution containing a self-assembling material is introduced through the inlet of channel B, and a diluent is introduced through the inlet of channel A.

[0039] The self-organizing substance-containing solution fluid and diluent fluid introduced into the flow channel from each inlet come into contact with a common hole in the flow channel structure, and the self-organizing substance-containing solution is diluted with the diluent to form self-organizing substance particles. More specifically, at the common hole in the intersection region of the flow channel structure, at least a portion of the self-organizing substance-containing solution flow branches off into the diluent fluid flow and merges with the diluent fluid flow, and at least a portion of the diluent fluid flow branches off into the self-organizing substance-containing solution flow and merges with the self-organizing substance-containing solution flow. In each flow after the merger, a diluted layer of the self-organizing substance-containing solution is formed at the interface between the merged self-organizing substance-containing solution flow and the diluent fluid flow, either up to the next common hole or in the dilution section up to the outlet, and self-organizing substance particles are formed in the diluted layer. By intersecting the flow channels three-dimensionally and branching off only a portion of the fluid at the common hole, a continuous diluted layer in the direction of flow, suitable for forming relatively large-particle-sized particles, can be formed. The flow channel structure of the present invention is a structure that can realize the above-described fluid behavior and, consequently, particle formation. The mechanism by which a diluted layer of the self-assembling material-containing solution is formed, and how self-assembling material particles are formed within this diluted layer, is similar to the mechanism described in Figure 7 of PLoS ONE 12(11):e0187962.(2017) (hereinafter abbreviated as PLoS document).

[0040] The formation of a dilution layer in a self-assembling material-containing solution is shown as a simulation result in Reference Examples 2-1 to 2-5. The simulation assumes that the ethanol (EtOH) flow is the self-assembling material-containing solution flow and the water flow is the dilution medium flow. For example, Reference Example 1 is an image of the dilution layer formation in a flow channel structure shown in Figure 3, where the upper channel is 200 μm wide (W) and 100 μm deep (D) for water flow, and the lower channel is 200 μm wide (W) and 50 μm deep (D) for EtOH flow, with six intersection regions where they merge and lead to the inlet, with a total flow rate of 500 μL / min and a water / EtOH flow rate ratio of 5 (5 / 1). The results are shown in Figure 13. The EtOH flow comes into contact with the water flow at the first common hole, partially branches off into the intersecting water flow, and merges into the water flow channel. Meanwhile, the water flow also comes into contact with the EtOH flow at the first common hole, branches off into the intersecting EtOH flow, and merges into the EtOH flow channel. The top view shows how the EtOH flow, drawn into the intersecting water flow at the common hole, flows into the water flow channel. The cross-sectional view shows how the water flow, drawn into the intersecting EtOH flow at the common hole, flows into the EtOH flow channel. However, since the water / EtOH flow rate ratio is 5, and the water flow rate is high, it can be seen that a large amount of water flow merges with the EtOH flow from the common hole even in the EtOH flow channel. The EtOH concentration of the fluid in the channel is shown in color, and it can be seen that a dilution layer of EtOH is formed at the contact surface between the EtOH flow and the water flow after the merger. Furthermore, in the second and subsequent common pores, if there is undiluted EtOH flow or EtOH flow in the process of dilution, a similar merging of EtOH flow and water flow occurs due to the same draw-in, and the formation of a diluted layer is repeated.

[0041] According to the mechanism by which self-assembled material particles are formed in a dilution layer as described in the PLoS literature, self-assembled material particles are formed when the solvent that dissolves the self-assembled material is within a certain concentration range in the dilution layer (in the case of POPC lipid nanoparticles in the literature, this is generally in the range of 60-80% EtOH concentration). The longer the residence time in the dilution layer within this solvent concentration range, the larger the particle size tends to be, and the shorter the residence time, the smaller the particle size tends to be. From the results in Figure 13, a dilution layer is formed at the interface between the EtOH flow and the water flow in the two channels after the common pore, and the formed dilution layer is maintained as the flow moves in each flow direction. The longer the time for which the dilution layer is maintained without significant disturbance and the EtOH concentration can be maintained within a certain range, the larger the particle size becomes. Therefore, it can be seen that the channel structure and manufacturing method of the present invention are rational for obtaining large-particle size particles.

[0042] Reference Example 2-2 is a simulation result using the same flow channel structure as Reference Example 1, with a total flow rate of 1000 μL / min. The results are shown in Figure 14. It is inferred that the dilution of the EtOH flow proceeds rapidly, the time during which a solvent concentration capable of particle formation can be maintained is shorter compared to Reference Example 1, and the particle size tends to be smaller.

[0043] Reference Examples 2-3 are simulation results using the same flow channel structure as Reference Examples 1 and 2, with a total flow rate of 2000 μL / min. The results are shown in Figure 15. It is inferred that the dilution of the EtOH flow proceeds even more rapidly, the time during which a solvent concentration capable of particle formation can be maintained is shorter compared to Reference Examples 1 and 2, and the particle size tends to become even smaller.

[0044] Reference Example 2-4 is an image of the dilution layer formation in a channel structure shown in Figure 4, where the upper channel is for water flow, with a width (W) of 400 μm and a depth (D) of 100 μm, and the lower channel is for EtOH flow, with a width (W) of 200 μm and a depth (D) of 50 μm, and has seven intersection regions where they merge to reach the inlet. The result is shown in Figure 16. Compared to Reference Example 1, it can be seen that much of the EtOH flow branches into the water flow and forms a dilution layer in the water flow, and the dilution proceeds gradually. In other words, when the width of the channel for water flow is widened, even at the same flow rate, much of the EtOH flow branches into the water flow and forms a dilution layer in the water flow, and a state of high EtOH concentration is maintained for a relatively long time, and particles with uniform and large particle size tend to be formed.

[0045] Reference Example 2-5 is a simulation result using the same flow channel structure as Reference Example 2-4, with a total flow rate of 2000 μL / min. The results are shown in Figure 17. It is inferred that the dilution of the EtOH flow proceeds rapidly, the time during which a solvent concentration capable of particle formation can be maintained is shorter compared to Reference Example 2-4, and the particle size tends to be smaller.

[0046] The results of these simulations show that in the flow channel structure of the present invention, particle size and particle size uniformity can be controlled by controlling the width and depth of the two flow channels, the flow rate ratio of the self-assembled material-containing solution flow to the diluent flow, and the total flow rate. Furthermore, the examples demonstrate that by controlling the above conditions when using actual self-assembled material-containing solution flows and diluent flows, self-assembled material particles with high particle size uniformity and a desired particle size can be produced.

[0047] In the present invention, the self-assembled material particles formed are particles that contain the self-assembled material as a particle component. These particles are obtained when self-assembled materials associate with each other and form particles, and the encapsulated material coexisting in the system during particle formation can also be incorporated into the particles. The components of particles formed under conditions in which the encapsulated material coexists are at least the self-assembled material and the encapsulated material.

[0048] The self-assembling material-containing solution and diluent can be introduced into the channel structure into channels A and B, for example, so that the total flow rate is between 1 μl / min and 1000 ml / min. However, the total flow rate is not intended to be limited to this range and can be appropriately determined considering the structure and dimensions of the channel structure, the type of self-assembling material-containing solution and diluent, the particle size of the desired self-assembling material particles, and the encapsulation efficiency of the encapsulated material. In the present invention, from the viewpoint of mass production of self-assembling material particles, the total flow rate of the self-assembling material-containing solution and diluent can be in the range of 100 μl / min to 1000 ml / min, and from the viewpoint of obtaining homogeneous self-assembling material particles, it can be in the range of 100 μl / min to 100 ml / min.

[0049] The ratio (V1:V2) of the flow rate V1 of the self-assembling material-containing solution to the flow rate V2 of the diluent can be, for example, in the range of 1:1 to 1:20. However, it is not intended to be limited to this range, and can be appropriately selected within the range in which the desired particles are obtained.

[0050] Self-assembling materials can be, for example, lipids or amphiphilic substances.

[0051] The self-assembling material-containing solution can be, for example, any solution selected from the group consisting of neutral lipid-containing solutions, anionic lipid-containing solutions, cationic lipid-containing solutions, and polymer-containing solutions, but is not intended to be limited to these. The self-assembling material in the present invention is any material that has a self-assembly function and, as described above, can associate with other self-assembling materials and form particles. Lipids, which are examples of self-assembling materials, are not particularly limited, but include, for example, naturally derived lipids such as soy lecithin, hydrogenated soy lecithin, egg yolk lecithin, phosphatidylcholines (e.g., egg-derived eggPC), phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, phosphasphingomyelins, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, sphingolipids, sterols, lysophospholipids, and other naturally derived lipids, as well as non-naturally derived lipids, including cationic non-naturally derived lipids that are considered suitable as components of nucleic acid delivery liposomes, such as N,N-dioleyl-N,N-dimethylammonium chloride (DODAC); N-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); and N,N-distearyl-N,N-dimethylammonium propyl Mido(DDAB); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1,2-dimyristyloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), Lipofectin®, Lipofectamine ( (Registered Trademark), Transfectum (Registered Trademark), DODAP, DODMA, DMDMA, 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleyl-3-dimethylaminopropane (DLinDAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleyl-2-Linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 2,2-Dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-Dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-Heptatriaconta-6,9,28,31-Tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride (DLin-TMA-Cl), 1,2-Dilinoleyl-3-trimethylaminopropane Chloride salt (DLin-TAP-Cl), 1,2-dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), 3-(N,N-dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-dioleylamino)-1,2-propanediol (dio) (DOAP), 1,2-dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA) and 2,2-dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA), 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), etc. can be used. The above-mentioned 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl-4-(dimethylamino)butanoate (DLin-MC3-DMA) and its analogues are described in Japanese Patent Publication No. 2013-245190, Japanese Patent Publication No. 2016-84297, and Japanese Patent Publication No. 2019-151589, and the entirety of those descriptions is incorporated herein by reference as a particular disclosure.

[0052] Other examples of self-assembling materials, such as amphiphilic materials, include amphiphilic polymer compounds, such as polystyrene-polyethylene oxide block copolymers, polyethylene oxide-polypropylene oxide block copolymers, polylactic acid polyethylene glycol copolymers, and polycaprolactone-polyethylene glycol copolymers, among others.

[0053] The encapsulated substances are not particularly limited, but examples include biomacromolecules such as nucleic acids, peptides, proteins, and glycans, metal ions, low- or medium-molecular-weight organic compounds, organometallic complexes, and metal particles. From the perspective of application, examples include drugs such as anticancer agents, antioxidants, antibacterial agents, anti-inflammatory agents, vitamins, artificial blood (hemoglobin), vaccines, hair growth agents, moisturizers, dyes, whitening agents, and pigments, as well as physiologically active substances and cosmetics. If these encapsulated substances are water-soluble, they can be contained within the aqueous phase of the formed particles. In the case of water-soluble charged substances, they can form aggregates with self-assembling substances having the opposite charge, and at the same time, these aggregates can be used as a core to form self-assembling substance particles which can then be contained within the particles. If the substances are poorly soluble in water, they can be contained within the hydrophobic portion of the self-assembling membrane formed by the self-assembling substance, or they can be contained within the particles as aggregates formed by bonding with the hydrophobic portion of the self-assembling substance. Furthermore, the substance to be encapsulated can be made water-soluble or sparingly soluble in advance through a pretreatment process, or formed into an aggregate, and this pretreatment process can be carried out in a flow path suitable for that process. When the pretreatment process is carried out in a flow path, the discharge port of the flow path in which the pretreatment process is carried out can be connected to the first or second inlet passage of the flow path structure of the present invention.

[0054] The water-miscible organic solvent used to dissolve the self-assembling material and prepare the particle solution is not particularly limited, but examples include water-miscible organic solvents such as alcohols, ethers, esters, ketones, and acetals. In particular, alcohols such as methanol, ethanol, t-butanol, butanediols, 1-propanol, 2-propanol, and 2-butoxyethanol, especially alkanols having 1 to 6 carbon atoms, are preferred. Other examples include ethers such as tetrahydrofuran, acetonitrile, and acetone.

[0055] As a diluent, water or aqueous solutions that are basically water-based, such as physiological saline, phosphate buffer solution, acetate buffer solution, citrate buffer solution, malate buffer solution, etc., are used as appropriate depending on the intended use of the particles to be formed. In addition to aqueous solutions and buffer solutions, the diluent may also be a solution that further contains water-soluble substances. Water-soluble substances can be low molecular weight, medium molecular weight, high molecular weight, nucleic acids, proteins, peptides, and physiologically active substances containing them, pharmaceuticals, cosmetic materials, vaccines, adjuvants, etc. Diluents that further contain water-soluble substances can be, for example, aqueous solutions containing low molecular weight, aqueous solutions containing medium molecular weight, aqueous solutions containing high molecular weight, aqueous solutions containing nucleic acids, aqueous solutions containing proteins, aqueous solutions containing peptides, aqueous solutions containing physiologically active substances, aqueous solutions containing pharmaceuticals, aqueous solutions containing cosmetic materials, aqueous solutions containing vaccines, aqueous solutions containing adjuvants, and mixtures thereof.

[0056] The self-assembled material particles containing the encapsulated substance obtained by the method of the present invention can be nano-sized, and the Z-mean particle diameter can be, for example, in the range of 10 to 1000 nm or in the range of 20 to 200 nm. However, it is not intended to be limited to this range. The Z-mean particle diameter is also called the cumulative average (harmonic intensity averaged over the particle diameters) and is defined in ISO 13321. [Examples]

[0057] The present invention will be described in more detail below based on examples. However, the examples are illustrative of the present invention and are not intended to limit the present invention to these examples.

[0058] Example 1 Lipid particles were prepared using the six different channel structures shown in Tables 1-1 and 1-2 below. In Table 1-2, W5, W7, W2, and W4 are the dimensions of the parts shown in Figure 1-3, and the confluence-outlet (mm) indicates the distance (mm) from the confluence of channels A and B to the outlet of the channel structure shown in Figure 6.

[0059] • Upper channel: Buffer solution flow [25mM acetate buffer (pH 4.0)] ·Lower flow path: Lipid EtOH liquid flow • Lipid solution: 10mM DOTAP / DSPC / Cholesterol / DMG-PEG 2K (40 / 11.5 / 47.5 / 1%) • Dialysis: D-PBS (-) ·Particle size measurement: DLS

[0060] [Table 1-1]

[0061] [Table 1-2]

[0062] The results are shown in Figures 8-10. In Figures 8-10, the particle size on the vertical axis represents the Z-average particle diameter, and the particle diameter in the following explanation also refers to the average particle diameter. The results shown in Figure 8, obtained using four different channel structures (No. 1, 3, 5, and 6) with different upper channel widths, show that in FRR3, as the upper channel width increases from 200 nm to 800 nm, particles with a diameter of 100 nm or more are formed even at high flow rates of 1000 μL / min or more. Similar trends are observed in FRR5 and FRR7. In particular, in channel No. 6 and FRR3, particles with a diameter of 100 nm or more can be obtained even at 2000 μL / min. A smaller FRR tends to make it easier to obtain particles with a larger diameter at high flow rates. As the upper channel width increases, the formation of particles with a diameter of 100 nm or more tends to become easier.

[0063] The results shown in Figure 9, obtained using two different channel structures (No. 1 and 4) with different lower channel depths, indicate that for FRR3, with a lower channel depth of 50 μm, particles with a diameter of 100 nm or larger are formed at flow rates of 1000 μL / min or less. A similar trend is observed for FRR5. For FRR7, particles with a diameter of 100 nm or larger are formed at flow rates of 500 μL / min or less. In all FRRs, channel structure No. 4, with a lower channel depth of 100 μm, does not produce large particles with a diameter of 100 nm or larger, even at low flow rates. This result suggests that for lipid EtOH liquid flow, a shallower lower channel depth is preferable to obtain large particles with a diameter of 100 nm or larger. Although a comparative diagram is not shown, a comparison of the results for channel structures No. 2 and No. 3, which have a larger upper channel width than channel structures No. 1 and No. 4, shows that the channel with the deeper lower channel (No. 2) has a slower flow velocity even at the same flow rate, and as a result, the particle size of the generated particles tends to be larger.

[0064] Figure 10 shows the results obtained using two different channel structures (No. 2 and 4) with varying upper channel widths and a lower channel depth of 100 μm. With an upper channel width of 200 μm, no large particles with a diameter of 100 nm or more can be obtained in any FRR, even at low flow rates, within the experimental range. However, it is possible to obtain particles with a diameter of 100 nm or more by further reducing the flow rate. When the upper channel width is doubled to 400 μm, particles with a diameter of 100 nm or more are formed in FRR3 and FRR5 at flow rates of 1000 μL / min or less. In FRR7, particles with a diameter of 80 μm are formed at a flow rate of 1000 μL / min.

[0065] To obtain large particles at high flow rates, it is advantageous to have a sufficiently wide upper channel.

[0066] Example 2 Lipid particles were prepared under the same conditions as in Example 1 (FRR=3) in a channel structure with a chamfered upper channel in channel No. 1. Chamfering of the channel is a structure in which the corners of the inner surface of the side wall of the channel are curved, intended to smooth the flow of fluid. The results are shown in Figure 11. The particle size on the vertical axis of Figure 11 represents the Z-average particle diameter. There was no significant change in the particle size of the obtained lipid particles with or without chamfering of the upper channel of the channel structure, but the particle size was larger when the chamfered structure was present.

[0067] Example 3 Lipid particles were prepared using the single-cross channel structure shown in Figure 5-8 under the same conditions as in Example 1 (however, FRR=5). The results are shown in Figure 12. However, Figure 12(1) shows the results for a channel structure with a channel width of 200 μm and intersecting channels being orthogonal, Figure 12(2) shows the results for a channel structure with a channel width of 800 μm and an intersection angle of 90 degrees, Figure 12(3) shows the results for a channel structure with a channel width of 800 μm and an intersection angle of 35 degrees, and Figure 12(4) shows the results for a channel structure with a channel width of 800 μm and an intersection angle of 60 degrees. Even with a single-intersecting channel structure, lipid particles with an average particle size in the range of 60 to 150 μm were obtained at flow rates in the range of 500 μL / min to 2000 μL / min.

[0068] Example 4 Lipid particles encapsulating nucleic acids were prepared using the three channel structures shown in Table 2 below. In the table, Z-mean represents the Z-mean particle diameter, Number represents the number-mean particle diameter, and PDI represents the polydispersity. These values ​​were calculated using the software attached to the Zetasizer Nano (Malvern Panalytical) from the scattering intensity of the particle suspension measured by the same instrument. Note that the Z-mean particle diameter is the average particle diameter determined based on the measured scattering intensity (light intensity), and the number-mean particle diameter is the average particle diameter weighted by the number of particles.

[0069] • Nucleic acid solution: 60 (FRR7) or 70 (FRR3) μg / mL siRNA in 25 mM acetate buffer (pH 4.0) • Lipid solution: 10mM DOTAP / DSPC / Cholesterol / DMG-PEG 2K (40 / 11.5 / 47.5 / 1%) • Dialysis: D-PBS (-) • Particle size measurement: Dynamic light scattering (DLS) was used. • Measurement of nucleic acid encapsulation rate: Ribogreen® assay was used.

[0070] [Table 2]

[0071] The results shown in Table 2 indicate that the channel structure used in the examples of the present invention can produce lipid particles encapsulating nucleic acids with a Z-average particle size in the range of 100 to 200 nm. Nucleic acid encapsulation was high, at over 96%. In particular, in channel No. 4 (flow rates of 300 μL / min and 2000 μL / min) and channel No. 6 (flow rate of 2000 μL / min), particles with a PDI of 0.1 or less were produced. These results demonstrate that even with a DOTAP particle system equipped with siRNA, it is possible to produce particles with a particle size of 100 nm or larger and a PDI of 0.1 or less.

[0072] Example 5 mRNA-LNPs were prepared using the channel (W800) shown in Figure 6(4). The lipid composition was the same as that of Spike-Vax: SM-102 / DSPC / cholesterol / DMG-PEG2k = 50 / 10 / 38.5 / 1.5 mol%. Lipid concentration: 8 mM in ethanol Aqueous solution: 48.9 μg / mL mRNA (luciferase expression) in 10 mM citrate buffer (pH 3.0)

[0073] After mixing in the flow path (flow rate: 500, 1000, or 2000 μL / min, FRR3), the mixture was dialyzed overnight with PBS, and the average particle size (Z-average nm) was measured after dialyzing. The results are shown in Figure 18. There was a tendency for the average particle size to decrease as the flow rate increased, but in all cases it was greater than 100 nm.

[0074] Three types of dialyzed mRNA-LNPs were diluted in culture medium to a mRNA concentration of 200 ng / mL and dosed into HeLa cells. After 24 hours of incubation, the luminescence intensity and total protein concentration of luciferase were measured using ONE-Glo and BCA assays kit. Relative luminescence intensity (RLU / mg protein) was then determined. The results are shown in Figure 19. In all three cases, the Z-mean particle size was greater than 100 nm (Figure 18), and the relative luminescence intensity did not vary significantly (Figure 19).

[0075] Example 6 mRNA-LNPs were prepared using the channel (W800) shown in Figure 6(4) and the microfluidic device described in WO2018 / 190423 (Patent Document 1) (hereinafter referred to as "iLiNP" or "iLiNP device"). The lipid composition was the same as that of Spike-Vax: SM-102 / DSPC / cholesterol / DMG-PEG2k = 50 / 10 / 38.5 / 1.5 mol%. Lipid concentration: 8 mM in ethanol Aqueous solution: 48.9 μg / mL mRNA (luciferase expression) in 10 mM citrate buffer (pH 3.0)

[0076] After mixing in a channel (flow rate: 1000 μL / min (channel in Figure 6(4)), 500 μL / min (iLiNP) FRR 3), the mixture was dialyzed overnight with PBS, and the average particle size (Z-average nm) was measured after dialyzing. The dialyzed mRNA-LNP was diluted in culture medium to a concentration of 200 ng / mL mRNA and dosed into HeLa cells. After 24-hour incubation, the luminescence intensity of luciferase and the total protein concentration were measured using ONE-Glo and the BCA assays kit. The relative luminescence intensity (RLU / mg protein) was then determined. The results are shown in Figure 20. mRNA-LNP with an average particle size of 155 nm prepared using the channel in Figure 6(4) (W800) showed approximately 7 times greater activity than mRNA-LNP with an average particle size of 80 nm prepared using the iLiNP device.

[0077] Reference example 2-1 Figure 3 shows a channel structure with an upper channel measuring 200 μm in width (W) and 100 μm in depth (D) for water flow, and a lower channel measuring 200 μm in width (W) and 50 μm in depth (D) for EtOH flow. The channels have six intersection regions and merge before reaching the inlet. Figure 12 visualizes the formation of a dilution layer when the total flow rate is 500 μL / min and the water / EtOH flow rate ratio is 5. The results are shown in Figure 12.

[0078] Reference example 2-2 This simulation uses the same flow path structure as in Reference Example 2-1, with a total flow rate of 1000 μL / min. The results are shown in Figure 13.

[0079] Reference example 2-3 This simulation uses the same flow path structure as in Reference Example 2-1, with a total flow rate of 2000 μL / min. The results are shown in Figure 14.

[0080] Reference example 2-4 Figure 5 shows a channel structure with an upper channel measuring 400 μm wide (W) and 100 μm deep (D) for water flow, and a lower channel measuring 200 μm wide (W) and 50 μm deep (D) for EtOH flow. The channels have seven intersection regions and merge before reaching the inlet. Figure 15 visualizes the formation of a dilution layer in this channel structure when the total flow rate is 500 μL / min and the water / EtOH flow rate ratio is 5. The results are shown in Figure 15.

[0081] Reference example 2-5 This simulation uses a flow channel structure similar to that in Reference Example 2-4, with a total flow rate of 2000 μL / min. The results are shown in Figure 16. [Industrial applicability]

[0082] The present invention is useful in the field of flow channel structures and the production of self-assembled material nanoparticles using flow channel structures.

Claims

1. It has a flow path A (10) having an inlet (11) and a flow path B (20) having an inlet (21), Flow channels A and B (10, 20) intersect three-dimensionally at one or more locations downstream of the inlet (11, 21), and each has a common hole (32) in the intersecting region that connects flow channels A and B (10, 20) in the flow channel wall. Flow paths A and B (10, 20) merge at a confluence (30) located in the downstream intersection region, and have at least one outlet (31) downstream of the confluence (30) (however, part or all of the section from the upstream intersection region to the outlet (31) is a section for diluting the fluid flowing through the flow path), or Flow paths A and B (10, 20) do not merge and have independent outlets (12) and outlets (22) (however, part or all of the section from the intersection region to outlets (12) and outlets (22) is a section for diluting the fluid flowing through the flow path). A channel structure for the production of self-assembled material particles.

2. The flow channel structure according to claim 1, wherein in the portion for diluting the fluid, a self-assembling substance-containing solution introduced from an inlet (11) or an inlet (21) is diluted by a diluent introduced from the other inlet to form self-assembling substance particles.

3. The channel structure according to claim 1 or 2, wherein channel A (10) is regularly or irregularly bent or straight in two or three dimensions, and channel B (20) is regularly or irregularly bent or straight in two or three dimensions.

4. A flow channel structure according to claim 1 or 2, having two or more intersection regions.

5. The flow channel structure according to claim 1 or 2, wherein the opening surface of the common hole in flow channel A (10) and the opening surface of the common hole in flow channel B (20) form substantially the same surface.

6. The flow channels A and B (10, 20) intersect periodically, as described in claim 1 or 2.

7. The flow channel structure according to claim 1 or 2, wherein the cross-sectional area of ​​the flow channel A (10) is constant between the inlet (11) and the confluence (30) or outlet (12), or fluctuates periodically in at least a portion thereof.

8. The flow channel structure according to claim 1 or 2, wherein the cross-sectional area of ​​the straight section of the flow channel B (20) is constant or periodically fluctuates between the inlet (11) and the confluence (30) or outlet (22).

9. The channel structure according to claim 1 or 2, wherein the intersection angle of channels A and B (10, 20) is such that the angle θ formed by the center line of channel A in the direction of flow and the center line of channel B in the direction of flow is in the range of 30° to 150°.

10. The channel structure according to claim 1 or 2, wherein the widths of channels A and B (10, 20) are independently in the range of 10 to 2000 μm, and the heights of channels A and B (10, 20) are independently in the range of 10 to 1000 μm.

11. The plane shapes of the outer and inner edges of the bends in the two- or three-dimensionally curved channel A (10) are polygonal or substantially circular. The flow channel structure according to claim 1 or 2, wherein the shape of the planes of the outer and inner edges of the bends in the flow channel B (20) that bends in two or three dimensions is polygonal or substantially circular.

12. A substrate A having a continuous groove A for a flow channel A on at least one main surface of a flat substrate A substrate B having continuous grooves B for a flow channel B on at least one main surface of a flat substrate It has a structure in which continuous groove A and continuous groove B are bonded together so that they face each other. Channel A is regularly or irregularly bent in two dimensions or is straight, and channel B is regularly or irregularly bent in two dimensions or is straight. The flow channel structure according to claim 1 or 2.

13. The flow channel structure according to claim 1 or 2, further comprising a second flow channel structure downstream of the outlet of the first flow channel structure.

14. A method for producing self-assembled material particles, comprising the step of diluting a self-assembling material-containing solution with a diluent to obtain a liquid containing self-assembling material particles, The manufacturing method comprising carrying out the above step using the flow channel structure described in claim 1.

15. The manufacturing method according to claim 14, wherein a self-assembling material-containing solution is introduced from the inlet of channel A and a diluent is introduced from the inlet of channel B, or a self-assembling material-containing solution is introduced from the inlet of channel B and a diluent is introduced from the inlet of channel A.

16. The manufacturing method according to claim 14 or 15, wherein the total flow rate of the self-assembling material-containing solution and diluent to flow channels A and B is in the range of 1 μl / min to 1000 ml / min.

17. The manufacturing method according to claim 14 or 15, wherein the ratio of the flow rate V2 of the diluent to the flow rate V1 of the self-assembling material-containing solution (V2 / V1) is in the range of 1:1 to 1:

20.

18. The manufacturing method according to claim 14 or 15, wherein the self-assembling material is a lipid or an amphiphilic material.

19. The manufacturing method according to claim 14 or 15, wherein the diluent is selected from aqueous solutions, buffer solutions, nucleic acid-containing aqueous solutions, protein-containing aqueous solutions, peptide-containing aqueous solutions, adjuvant-containing aqueous solutions, and mixtures thereof.

20. The manufacturing method according to claim 14 or 15, wherein the self-assembled material particles contained in the liquid containing the self-assembled material particles are nano-sized.

21. The manufacturing method according to claim 14 or 15, wherein the Z-average particle diameter of the nano-sized self-assembled material particles is in the range of 10 to 1000 nm.

22. The manufacturing method according to claim 14 or 15, wherein the Z-average particle diameter of the nano-sized self-assembled material particles is in the range of 20 to 200 nm.

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