Flow channel structure, fluid stirring method, and method for producing lipid particles

Abstract

To provide a channel structure, a fluid agitation method, and a lipid particle manufacturing method in which a fluid can be more uniformly mixed by generating a lateral vortex.SOLUTION: A channel structure according to an embodiment comprises a first channel, and a second channel confluent with the first channel. An end of a first channel side of the second channel has a region with a depth shallower than the first channel. The direction of the first channel is bent when viewed from the direction of the second channel, and in a mixed region of the second channel and the first channel, the first channel is closed relative to a longer direction of the second channel.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Embodiments of the present invention relate to a flow channel structure, a fluid stirring method, and a method for producing lipid particles.

Background Art

[0002] In order to quickly and uniformly mix two liquids, it is appropriate to stir them. In recent years, microchannels have been used for handling fluids. However, when the amount of the two liquids is small, it is difficult to generate turbulent flow in a microchannel with a small Reynolds number, and it is difficult to stir and mix the two liquids. Therefore, attempts have been made to promote mixing by generating a steady swirling flow (vortex, swirl) in the microchannel. Generating a swirling flow in a microchannel requires three-dimensional fluid control, and for this purpose, a delicate mold, channel processing, or high-precision lamination molding of a plurality of channels is required. ​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​

[0006] The problem that this invention aims to solve is to provide a flow channel structure, a fluid stirring method, and a method for producing lipid particles that can mix fluids more uniformly by generating transverse vortices. [Means for solving the problem]

[0007] A flow channel structure according to the embodiment comprises a first flow channel and a second flow channel that merges with the first flow channel, wherein the end of the second flow channel on the first flow channel side has a region that is shallower in depth than the first flow channel, the direction of the first flow channel is bent when viewed from the direction of the second flow channel, and in the mixing region of the second flow channel and the first flow channel, the first flow channel is closed with respect to the longitudinal direction of the second flow channel. [Brief explanation of the drawing]

[0008] [Figure 1] Figure 1 is a plan view and a cross-sectional view showing an example of a flow channel structure according to the first embodiment. [Figure 2] Figure 2 is a perspective view showing an example of a flow channel structure according to the first embodiment. [Figure 3] Figure 3 is a cross-sectional view showing an example of a cross-sectional view of a flow channel structure according to the embodiment. [Figure 4] Figure 4 is a plan view showing an example of a flow channel structure according to the second embodiment. [Figure 5] Figure 5 is a plan view showing an example of a flow channel structure according to the third embodiment. [Figure 6] Figure 6 is a plan view showing an example of a flow channel structure according to the fourth embodiment. [Figure 7] Figure 7 is a plan view showing an example of a flow channel structure according to the fourth embodiment. [Figure 8] Figure 8 is a plan view showing an example of a flow channel structure according to the fourth embodiment. [Figure 9] Figure 9 is a plan view showing an example of a flow channel structure according to the fifth embodiment. [Figure 10] Figure 10 is a cross-sectional view showing an example of a flow channel structure according to an embodiment. [Figure 11] Figure 11 is a diagram showing an example of lipid particles of an embodiment. [Figure 12] Figure 12 is a flowchart showing an example of a method for producing lipid particles of an embodiment. [Figure 13] Figure 13 is a diagram showing an example of a flow path structure used in the method for producing lipid particles of an embodiment. [Figure 14] Figure 14 is an image showing the experimental results of Example 1. [Figure 15] Figure 15 is an image showing the experimental results of Example 2. [[ID=!15]] [Figure 16] Figure 16 is an image showing the simulation results of Example 2. [Figure 17] Figure 17 is a photograph showing the experimental results of Example 3. [Figure 18] Figure 18 is a photograph showing the experimental results of Example 4. [Figure 19] Figure 19 is a photograph showing the experimental results of Example 4. [Figure 20] Figure 20 is a graph showing the experimental results of Example 4. [Figure 21] Figure 21 is a graph showing the experimental results of Example 4. [Figure 22] Figure 22 is an image showing the simulation results of Example 5. [Figure 23] Figure 23 is a plan view showing the flow path structure used in Example 6. [Figure 24] Figure 24 is a graph showing the experimental results of Example 6. [Figure 25] Figure 25 is a plan view showing the flow path structure used in Example 7. [Figure 26] Figure 26 is an image showing the simulation results of Example 10.

Mode for Carrying Out the Invention

[0009] The embodiments will be described below with reference to the attached drawings. In each embodiment, substantially identical components will be denoted by the same reference numerals, and their descriptions may be partially omitted. The drawings are schematic, and the relationship between the thickness of each part and its planar dimensions, the ratio of the thicknesses of each part, etc., may differ from those in reality.

[0010] (First Embodiment) As shown in the plan view of Figure 1(a), the flow channel structure 1 of the first embodiment includes a first flow channel 2 and a second flow channel 3 that merges with the first flow channel 2. The first flow channel 2 and the second flow channel 3 are cavities formed inside the flow channel structure 1, that is, their top surfaces have lids and are configured to be liquid-tight. The third flow channel and the fourth flow channel, which will be described later, are also similarly shaped cavities formed inside the flow channel structure.

[0011] The end of the second channel 3 on the side of the first channel 2 has a first region that is shallower than the first channel 2. The first region will also be referred to as the first shallow region 4. As shown in Figure 1(b), a cross-sectional view taken along B-B' in Figure 1(a), the first shallow region 4, for example, has a bottom surface that protrudes further upstream than the first shallow region 4 (hereinafter referred to as the "deep region 5") and further than the first channel 2, narrowing the lumen of the channel. The depth of the deep region 5 and the first channel 2 may be the same. In this figure, the direction of fluid flow is indicated by arrows. As shown in the figure, the direction of fluid flow in the first channel 2 is different from that of the second channel 3. The region of the first channel 2 where fluid from the second channel 3 merges is referred to as the "mixing region 6".

[0012] For example, the first channel 2 and the second channel 3 are microchannels.

[0013] Figure 2 shows what happens when fluid flows through the second channel 3. The arrows indicate the direction of fluid flow. When the fluid passes through the first shallow section 4 and flows into the first channel 2, transverse vortices are generated. These transverse vortices are swirling flows whose axis of rotation coincides with the long axis of the first channel 2. In this example, the right end of the first channel 2 on the right side of the second channel 3 is closed and forms a wall, so the fluid flows along the long axis of the first channel 2 to the left (to the right in Figure 2, while generating transverse vortices). The generation of transverse vortices allows the fluid to be thoroughly mixed and stirred.

[0014] As shown in Figure 1(b), it is preferable that the depth d1 of the first shallow section 4 is less than half the depth d2 of the first flow channel 2. It is even more preferable that d1 / d2 is 1 / 3 or less. By setting the depth to this extent, the flow velocity increases when the fluid flows from the first shallow section 4 into the first flow channel 2, making it easier for transverse vortices to be generated.

[0015] While a shallower depth d1 in the first shallow section 4 allows for a flow velocity that generates larger and stronger transverse vortices, making it too shallow can lead to excessive pressure loss and potentially cause blockage if foreign matter is present. Therefore, when designing the d1 / d2 to be shallower, such as 1 / 3, 1 / 4, or 1 / 5, through experiments or simulations, a reasonable depth d1 should be determined based on the manufacturing precision of the flow path, pressure loss, and robustness against foreign matter.

[0016] In practice, since the accuracy of the preferred methods used to manufacture this channel structure, such as mold forming or machining, is generally 5 μm, it is desirable that the depth of the shallow section 4 be at least 10 μm in order to avoid blockage of the channel due to errors.

[0017] Furthermore, in order to form a steady and stable transverse vortex and to prevent unexpected reactions due to cavitation, it is best to avoid reducing the depth of the shallow section 4 to a level where the flow velocity reaches the turbulent region.

[0018] The length of the first shallow section 4 is preferably the same as or greater than the width of the second flow path 3. This allows the flow turbulence generated when the fluid flows from the deep section 5 to the first shallow section 4 to be adequately contained within the first shallow section 4. By suppressing the flow turbulence, transverse vortices can be generated more efficiently. However, making the first shallow section 4 unnecessarily long is undesirable as it may excessively increase the pressure (fluid resistance), and the length of the first shallow section 4 is usually preferably about three times the width of the flow path at most. However, if the discharge performance of the pump allows, it may be made longer than three times the width if necessary due to the arrangement of the flow path, etc.

[0019] The second flow channel 3 merges with the first flow channel 2 at a right angle, for example. The angle θ1 between the second flow channel 3 and the first flow channel 2 does not necessarily have to be a right angle, but the larger the angle θ1, the more likely laminar flow confluence is to occur. Therefore, it is preferable that the angle θ1 be as close to a right angle as possible. Also, in the example in Figure 1, the flow in the first flow channel 2 is configured to bend to the left when viewed from the second flow channel 3, but it may also be configured to bend to the right.

[0020] The cross-section of the first channel 2 is preferably a square with equal width and depth, as shown in Figure 3(a). However, it does not need to be a perfect square; it may be an approximate square with one side slightly longer than the other. Also, if possible, a shape with rounded corners at the bottom of the cross-section, as shown in Figure 3(b), is also preferable, or the bottom of the cross-section may be rounded with a radius equal to half the distance of the sides of the square, as shown in Figure 3(c). Such a cross-sectional shape results in transverse vortices that are closer to a perfect circle and are maintained for a longer period. As a result, the fluid can be mixed and stirred well. It is not necessary for the entire first channel 2 to have such a cross-sectional shape; it is sufficient if at least the mixing region 6 has such a cross-sectional shape.

[0021] The width and depth of the deep section 5 of the second channel 3 and the first channel 2, the depth of the first shallow section 4, and the amount of fluid supplied are not limited and are determined according to the type of fluid. For example, in order to generate transverse vortices and prevent laminar flow, it is preferable to adjust the Reynolds number in the channel sections with normal depths other than the first shallow section 4 to be 10 or more. Also, in order to avoid turbulence in order to create uniform transverse vortices, it is preferable to keep the Reynolds number below 2300 at least. More preferably, taking into account the performance of commonly available pumps and the effective strength of the transverse vortices, the Reynolds number is preferably 50 to about 1000.

[0022] For example, if the cross-section of the deep section 5 and the first channel 2 is 0.3 mm square, it is preferable to have a flow velocity of approximately 0.5 m / s or more. Assuming the fluid is similar to water, the Reynolds number in this case is around 100 at room temperature.

[0023] For example, shortening the length of one side of the flow channel cross-section while maintaining a constant Reynolds number affects the pressure by a factor of two. Therefore, in a flow channel with sides of 0.3 mm, setting the depth d1 of the first shallow section 4 to 0.1 mm increases the pressure loss in the first shallow section 4 tenfold. Furthermore, a pressure increase of around tenfold in the first shallow section 4 increases the need to change the specifications of the pump applicable to the flow rate range, and in that case, the types of pumps available are also limited. For this reason, it is desirable to design the depth of the deep section 5 and the first flow channel 2 so that the depth d1 of the first shallow section 4 is 0.1 mm or more. In addition, it is preferable to limit the upper limit of the pressure increase to around tenfold in order to reduce the load on the pump.

[0024] On the other hand, when using a pump in this flow channel structure, it is preferable to use a pump that does not cause pulsation. Such pumps with a delivery rate of about 1 ml / sec are readily available. Considering this, the width and depth of the cross-section of the deep section 5 and the first flow channel 2 may be reasonably limited to about 3 mm.

[0025] As described above, the flow channel structure 1 of the embodiment allows for better mixing and stirring of the fluid by generating transverse vortices. As will be explained in more detail later, this flow channel structure does not require the formation of a tunnel structure in the base material during manufacturing, and can be configured by covering the groove-shaped flow channels with flat covers (i.e., a configuration that does not involve stacking of flow channels). Therefore, it does not require high machining precision during manufacturing and can be manufactured simply and at low cost.

[0026] (Second Embodiment) The flow channel structure of the second embodiment further includes a third flow channel 7 connected in series immediately upstream of the point where the first flow channel merges with the second flow channel. For example, as shown in the flow channel structure 10 in Figure 4(a), the third flow channel 7 and the first flow channel 2 form a single linear flow channel, and the second flow channel 3 merges with the first flow channel 2 at a right angle. In this case, both the angle θ1 between the second flow channel 3 and the first flow channel 2 and the angle θ2 between the second flow channel 3 and the third flow channel 7 are right angles.

[0027] In a further embodiment, as shown in Figure 4(b), the second channel 3 and the third channel 7 merge into the first channel 2 at, for example, the same angle, forming a Y-shape overall. Preferably, the angle θ2 between the second channel 3 and the third channel 7 is a right angle. Furthermore, these two channels merge into the first channel 2 at the same angle; in other words, the second channel 3 and the third channel 7 are connected symmetrically to each other with respect to the major axis of the first channel 2 as the axis of symmetry. For example, when angle θ2 is a right angle, the angle θ1 between the second channel 3 and the first channel 2 is, for example, 135°.

[0028] In the flow channel structures 10 and 11, fluid flows not only from the second flow channel 3 but also from the third flow channel 7. As a result, the two fluids merge in the mixing region 6. Furthermore, the first shallow section 4 generates transverse vortices in the mixing region 6, causing the two fluids to mix and agitate.

[0029] The flow channel structure 11 may result in less turbulence in the flow immediately after the confluence with the second flow channel 3 compared to the flow channel structure 10. While flow turbulence increases the stirring effect, reducing turbulence allows for more uniform mixing, and in this case, the lifetime (energy) of the transverse vortices is not wasted, allowing the transverse vortices to be sustained longer. Therefore, when uniform mixing is desired over the stirring effect, it is preferable to use the flow channel structure 11 rather than the flow channel structure 10. Conversely, when more rapid mixing is desired, it is preferable to use the flow channel structure 10.

[0030] In the flow channel structures 10 and 11 of the second embodiment, it is preferable that the depth d1 of the first shallow section 4 is less than half the depth d2 of the first flow channel 2. For example, if the first shallow section 4 is not provided, and fluids flow from the second flow channel 3 and the third flow channel 7 at nearly the same flow rate, after the confluence, the fluids will each occupy half of the cross-sectional area, thus strengthening the tendency for laminar flow after the confluence. Therefore, if d1 / d2 is set to half, a similar situation will be realized, making it difficult for transverse vortices to be generated. Consequently, setting d1 / d2 to less than half may make it easier for transverse vortices to be generated.

[0031] The flow path structures 10 and 11 of the second embodiment can be used, for example, to mix two fluids, enabling more efficient and uniform mixing of the two fluids.

[0032] (Third embodiment) The flow channel structure of the third embodiment includes a group of flow channels (mixing unit) for further mixing the fluid at the downstream end of the first flow channel of the flow channel structure of the first or second embodiment. Figure 5 shows an example of the flow channel structure 20 of the third embodiment. The flow channel structure 20 includes a merging unit 21 and a mixing unit 22. In this figure, for convenience, shallow sections (the first shallow section 4a to the third shallow section 4c of the third embodiment) are shown with a diagonal line pattern. The direction of fluid flow is indicated by arrows.

[0033] The confluence unit 21 is equipped with a group of flow channels for the confluence of two fluids. The confluence unit 21 has the same structure as, for example, the flow channel structure 10 or 11 of the second embodiment. Here, the same structure as the flow channel structure 11 is shown in the diagram. In the confluence unit 21, as described in the second embodiment, transverse vortices are generated in the mixing region 6a as the fluid passes through the first shallow section 4a, and the two fluids flowing from the second flow channel 3 and the third flow channel 7, respectively, are mixed in the first flow channel 2. The fluid then flows to the downstream mixing unit 22.

[0034] The mixing unit 22 is connected downstream of the confluence unit 21 and includes a group of channels for further mixing and stirring the fluids that have been combined in the confluence unit 21. This group of channels includes, for example, a first branch-combining channel 23 and a second branch-combining channel 24 that split the fluid flowing in from the first channel 2 into two separate flows and merge these two flows into the fourth channel.

[0035] For example, the second branched confluence channel 24 has a second region (second shallow section 4b) in its middle that is shallower than its upstream and downstream sides, and its downstream section bends to merge with the fourth channel. The second shallow section 4b and the bend generate transverse vortices, allowing the fluid to be mixed and stirred. Also, the end of the first branched confluence channel 23 on the fourth channel side has a third region (third shallow section 4c) that is shallower than the fourth channel. The third shallow section 4c generates transverse vortices when the fluid merges with the fourth channel, allowing the fluid to be mixed and stirred.

[0036] The structures of the first branching / merging channel 23 and the second branching / merging channel 24 will be described in more detail below. The first branching / merging channel 23 includes, for example, a branching section 23a, an intermediate section 23b, and a merging section 23c, extending from upstream to downstream. Similarly, the second branching / merging channel 24 includes, for example, a branching section 24a, an intermediate section 24b, and a merging section 24c.

[0037] Branch sections 23a and 24a are connected to the downstream end of the first flow path 2 and are parts that branch the fluid. Preferably, branch sections 23a and 24a are connected at the same angle, i.e., symmetrically with respect to the first flow path 2 as an axis, in order to make the flow rates equal, and they also have the same flow path width and depth. The angle between branch section 23a and branch section 24a is not limited, but is, for example, a right angle.

[0038] However, it is possible to generate transverse vortices in the second shallow section 4b and 4c downstream even if the flow rates at the two branching points are not necessarily equal, so it is also possible to make the size or angle of the flow paths different. However, in that case, the flow rate of one of them will be reduced, and the difficulty of pressure adjustment (required precision) may increase in the flow path with the lower flow rate. Therefore, branching to approximately equal amounts is preferable for the robustness of the product.

[0039] At intermediate sections 23b and 24b downstream of branch sections 23a and 24a, respectively, the flow path bends at an angle parallel to the long axis of the first flow path 2. Subsequently, at the confluence sections 23c and 24c downstream, it bends further inward and connects to the fourth flow path 25.

[0040] The intermediate section 24b is a second shallow section 4b having a depth of less than half the depth of the confluence section 24c, for example. In the mixing unit 22, one of the flow paths has a third shallow section 4c, so when branching occurs, the pressure balance may be uneven and the branching may not be even. Therefore, by arranging the second shallow section 4b, for example, the pressures of the two branches can be made equal. The second shallow section 4b can also be provided in the branching section 24a, but it is preferable to place it in the intermediate section 24b because the branching is simpler. Because the flow path is bent at the confluence section 24c downstream of the second shallow section 4b, transverse vortices are generated near the upstream of the confluence section 24c (mixing region 6b). This allows for further agitation of the fluid there. The cross-sectional shape of the flow path in the mixing region 6b is preferably one of the shapes shown in Figure 3.

[0041] The confluence sections 23c and 24c are connected to the fourth flow path 25 at the same angle to each other, that is, symmetrically with respect to the fourth flow path 25 as the axis. Preferably, the angle between the confluence section 23c and the confluence section 24c is a right angle.

[0042] For example, the confluence section 23c is a third shallow section 4c having a depth of less than half the depth of the fourth flow path 25. The third shallow section 4c generates transverse vortices near the entrance of the fourth flow path 25 (mixing region 6c). This allows for further mixing and stirring of the fluid. The cross-sectional shape of the flow path in the mixing region 6c is preferably one of the shapes shown in Figure 3.

[0043] If the goal is simply to generate transverse vortices, it is not necessarily required to provide a second shallow section 4b; it is possible to adjust the pressure and make the entire depth of the second branched confluence channel 24 the same. However, the configuration shown in Figure 5, in which shallow sections are provided in both the first branched confluence channel 23 and the second branched confluence channel 24, is preferable because, even if one channel becomes blocked due to the ingress of foreign matter, the fluid can still be mixed and stirred by passing through the shallow section in the other channel.

[0044] In Figure 5, the channel (here, the second branched merging channel 24) located diagonally opposite the channel (here, the second channel 3) having a shallow section 4a in the merging unit 21 has a second shallow section 4b in its intermediate section 24b. However, as shown in Figure 6, which will be described later, the first branched merging channel 23 and the second branched merging channel 24 may be arranged in reverse.

[0045] (Fourth Embodiment) The flow channel structure according to the fourth embodiment comprises a plurality of mixing units 22. For example, as shown in Figure 6(a), the flow channel structure 30 comprises three mixing units arranged in series, namely a first mixing unit 22a, a second mixing unit 22b, and a third mixing unit 22c.

[0046] In the flow channel structure 31 shown in Figure 6(b), the first branched confluence channel 23 (with a third shallow section 4c at the confluence) and the second branched confluence channel 24 (with a second shallow section 4b in the middle) are arranged inverted around the fourth flow channel 25 as the axis in the second mixing unit 22b. By alternately arranging mixing units in which the arrangement of the first branched confluence channel 23 and the second branched confluence channel 24 is reversed, as in this example, the fluid can be mixed more evenly.

[0047] The number of mixing units 22 is not limited to three; it may be two, four, five, six, or more.

[0048] In a further embodiment, a flow path structure may be used in which multiple mixing units 22a to 2c are arranged in parallel, as shown in the flow path structure 40 in Figure 7. For example, the fluid is branched upstream, passes through multiple mixing units 22a to 2c, and then rejoins into a single flow path downstream. This arrangement can reduce the resistance to fluid transfer even when the flow rate is high compared to the case where the fluid is arranged in series. When a fluid transfer pump is used, the load on the pump is reduced.

[0049] Furthermore, a structure combining series and parallel arrangements may be used. In this case, the resistance of the fluid delivery can be adjusted, and the effect of agitation and mixing can be enhanced. For example, the flow path structure 50 shown in Figure 8 has four sets of flow path structures, each consisting of two mixing units 22 arranged in series, and these four sets of flow path structures are arranged in parallel. Also, at the point where the fluids merge downstream of the parallel-arranged mixing units 22, it is preferable to place a shallow section in one of the merging flow paths to promote mixing and agitation. The flow path structure combining series and parallel arrangements is not limited to the example shown in Figure 8, and can be modified according to the type of fluid or application.

[0050] According to the flow path structure of the fourth embodiment, the fluid can be mixed and stirred even more effectively than when there is only one mixing unit 22.

[0051] (Fifth embodiment) In the fifth embodiment, as shown in Figure 9, the flow path structure 60 is such that the third flow path 7 and the first flow path 2 of the confluence unit 21 form a single, linear flow path. The second flow path 3 merges with the first flow path 2 at a right angle (i.e., a flow path structure similar to that in Figure 4(a)).

[0052] Furthermore, the junction 23c of the first mixing unit 22a is connected in series with the fourth flow path 25, forming a straight flow path that is integrated with the fourth flow path 25. The junction 24c merges with the fourth flow path 25 at a right angle. In the second mixing unit 22b, the first branched merging flow path 23 and the second branched merging flow path 24 are arranged in reverse, but similarly, the junction 23c and the fourth flow path 25 form a straight flow path, to which the junction 24c merges at a right angle.

[0053] The first channel 2 and the fourth channel 25 are bent along the axis of symmetry of the two branched channels just before the next branch. Alternatively, the first channel 2 and the fourth channel 25 may be connected in series to the next branch 23a without being bent.

[0054] In this example, any number of mixing units 22 may be connected, for example, one, three, four, five, six, or more.

[0055] By merging two shallow channels at a right angle, the fluid can be mixed and stirred more quickly. This is thought to be partly due to increased flow turbulence at the point of merging. While flow turbulence has little effect on the uniformity of mixing, it can enhance the stirring effect. Therefore, when speed of stirring is more important than uniformity, this type of channel structure is preferable.

[0056] (Method of manufacturing a channel structure) The manufacturing method for the flow channel structure described above (hereinafter collectively referred to as "flow channel structure 100") will be explained below with reference to Figure 10. As shown in Figure 10(a), the flow channel structure 100 comprises, for example, a substrate 102 on which a groove 101 that functions as a flow channel is formed, and a plate-shaped lid portion 103 joined to the substrate 102 so as to close the top surface of the groove 101.

[0057] The material of the substrate 102 can be selected appropriately from resins such as acrylic, polyethylene, and polypropylene, glass, ceramics, or metal, depending on the application. For example, if the channel structure is for medical use, cycloolefin polymer is a preferred example. If it is to be reused many times, glass or ceramics such as quartz are preferred due to their stability, and if temperature control is required, metal with a corrosion-resistant treatment on the surface may be used. The grooves 101 can be formed, for example, by press working using a mold or by cutting. In areas corresponding to shallow areas, the grooves 101 should be formed or cut shallower than in other areas.

[0058] The material of the lid portion 103 can be the same as that described for the substrate 102. The lid portion 103 may be, for example, in the form of a plate. Alternatively, a thin film-like lid portion 104 may be used, as shown in Figure 10(b).

[0059] The film-like lid portion 104 can also be fitted with sensor terminals 105 for monitoring the fluid state. Alternatively, the lid portion 104 can be given various functions or characteristics, such as high thermal conductivity or a function (not shown) for performing specific processing on specific substances.

[0060] If there is concern that the lid portion 104 may bulge due to internal pressure, the bulging may be suppressed by pressing the retaining plate 106 onto the lid portion 104 from above, as shown in Figure 10(c). The retaining plate 106 may be equipped with a heat exchange fluid channel 107 located inside it, or an electrical terminal (not shown) having a sensor function.

[0061] In this way, the flow channel structure 100 can be manufactured by a simple procedure of forming grooves 101 in the substrate 102 and joining the lid portion 103 or 104. Therefore, since it is not necessary to form grooves in both the substrate 102 and the lid portion 103, and precise alignment of the two is not required, it is highly suitable for mass production.

[0062] In a further embodiment, the shallow groove 101 may be made to the same depth as the other parts, and a lid 104 having a protrusion at the corresponding location may be attached. That is, the flow path lumen of the shallow part 4 formed in this way is narrowed by a recess from above. Although this structure involves more manufacturing steps such as forming and positioning the lid compared to the structure with a protruding bottom as described above, it is still possible to generate transverse vortices.

[0063] (Fluid stirring method) According to the embodiment, a fluid stirring method is provided. The fluid stirring method includes flowing a fluid to be stirred through the flow channel structure of the embodiment. According to the fluid stirring method, by using the flow channel structure of the embodiment, the fluid can be mixed and stirred more effectively.

[0064] This method, when using the flow channel structure of the first embodiment, includes flowing the first fluid from the second flow channel 3 to the first flow channel 2. Furthermore, when using the flow channel structures of the second to fifth embodiments, this method further includes flowing the second fluid through the third flow channel 7. The first fluid and the second fluid may be different types of fluids, and according to the flow channel structures of the second to fifth embodiments, the first fluid and the second fluid can be mixed and stirred more effectively. In addition, more uniform mixing is possible.

[0065] (Method for producing lipid particles containing a drug) The following describes a method for producing lipid particles containing a drug using the flow channel structure of the embodiment.

[0066] First, the lipid particles produced by this method will be described. As shown in Figure 11, the lipid particles 200 consist of a lipid membrane formed by the arrangement of lipid molecules and are hollow and approximately spherical in shape. The drug 202 is encapsulated in the lumen 201 of the lipid particle 200. The lipid particles 200 can be used, for example, to deliver the drug 202 into cells.

[0067] The manufacturing method includes the following steps, as shown in Figure 12, for example: Concentrating the drug (in the case of nucleic acids) (concentration step S1), Using the flow channel structure of the embodiment, a first solution containing lipids for lipid particle material is flowed through either the second flow channel 3 or the third flow channel 7 in an organic solvent, and a second solution containing a drug is flowed through the other in an aqueous solvent to mix the first solution and the second solution to obtain a mixed solution (mixing step S2). By reducing the concentration of the organic solvent in the mixture, the lipids are atomized to produce lipid particles containing the drug (particleization step S3), and The lipid particle solution is concentrated (concentration step S4).

[0068] This manufacturing method can be carried out using, for example, the flow channel structure shown in Figure 13. Figure 13(a) shows a flow channel structure 301 for flocculation having a configuration for performing a condensation step S1, (b) shows an embodiment of a flow channel structure 302 for performing a mixing step S2, (c) shows a flow channel structure 303 for particle formation having a configuration for performing a particle formation step S3, and (d) shows a flow channel structure 304 for concentration having a configuration for performing a concentration step S4.

[0069] The following describes an example of the procedure for this manufacturing method.

[0070] First, prepare the first solution and the second solution. The first solution contains lipids in an organic solvent. These lipids are the materials that make up the lipid particles 200. The second solution contains drug 202 in an aqueous solvent.

[0071] • Condensation process S1 Drug 202 is, but is not limited to, a nucleic acid. A nucleic acid drug 202 may be, for example, a nucleic acid containing DNA, RNA and / or other nucleotides, and may be, for example, mRNA of a specific gene, DNA encoding a gene, DNA containing a gene expression cassette with a gene and other sequences for gene expression such as a promoter, a vector, etc. If drug 202 is a nucleic acid, an aggregation step S1 in which the nucleic acid (drug 202) is aggregated may be performed first.

[0072] Nucleic acid condensation is carried out, for example, using nucleic acid condensation peptides. By condensing nucleic acids into smaller particles, nucleic acid condensation peptides can reduce the particle size of lipid particles 200 and allow more nucleic acids to be contained within the lipid particles 200. As a result, less nucleic acid may remain outside the lipid particles 200, which could cause aggregation of the lipid particles 200.

[0073] A preferred nucleic acid condensed peptide is, for example, a peptide containing 45% or more cationic amino acids. A more preferred nucleic acid condensed peptide has RRRRRR (first amino acid sequence) at one end and the sequence RQRQR (second amino acid sequence) at the other end. Between the first amino acid sequence and the second amino acid sequence, there are zero or one or more intermediate sequences consisting of RRRRRR or RQRQR. In addition, there are two or more neutral amino acids between two adjacent sequences among the first amino acid sequence, the second amino acid sequence, and the intermediate sequence. The neutral amino acids are, for example, G or Y. The other end may have RRRRRR (first amino acid sequence) instead of the second amino acid sequence.

[0074] The above nucleic acid condensed peptide preferably has the following amino acid sequence: RQRQRYYRQRQRGGRRRRRR (Sequence ID 1) RQRQRGGRRRRRR (Sequence ID 2) RRRRRRYYRQRQRGGRRRRRR (Sequence code 3).

[0075] Furthermore, a nucleic acid condensate peptide having the following amino acid sequence can be used in combination with any of the above nucleic acid condensate peptides. This peptide can further condense the nucleic acid condensate condensed with the above nucleic acid condensate peptide.

[0076] GNQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (M9)(Sequence ID 4) As shown in Figure 13(a), the flocculation channel structure 301 for performing the flocculation process S1 is, for example, a Y-shaped channel. At the upstream end of one of the branched channels 311 of the Y-shape, for example, a flocculant inlet 312 is provided, and a flocculant containing nucleic acid condensate peptide is flowed through it. At the upstream end of the other channel 313, a drug inlet 314 is provided, and a solution containing nucleic acid (drug 202) in an aqueous solvent is flowed through it. The aqueous solvent is, for example, water, saline solution such as physiological saline, glycine aqueous solution, or buffer solution. As a result, the flocculant and the solution containing drug 202 are mixed in channel 315 where channels 311 and 313 merge. Mixing yields a second solution containing condensed drug 202.

[0077] The condensation step S1 does not necessarily need to be performed using a flow channel; it can be done by mixing and stirring a solution containing a flocculant and nucleic acid (drug 202) in an aqueous solvent.

[0078] Since the above effects are achieved, it is preferable to perform the condensation step S1 when drug 202 is a nucleic acid. However, if drug 202 is not a nucleic acid, or if it is a nucleic acid but does not need to be condensed, it is not necessary to perform the condensation step S1.

[0079] ·Mixing process S2 Next, the first solution and the second solution are mixed together.

[0080] The second solution may be prepared as described above if drug 202 is a nucleic acid. Alternatively, if a non-condensable nucleic acid or non-nucleic acid drug 202 is used, the second solution can be prepared by mixing drug 202 with any of the above aqueous solvents selected according to its type. Non-nucleic acid drugs 202 include, for example, proteins, peptides, amino acids, other organic compounds, or inorganic compounds as active ingredients. Drug 202 may be, for example, a drug for treating a disease or a diagnostic agent. However, drug 202 is not limited to these, and may be any substance that can be encapsulated in lipid particles 200.

[0081] Drug 202 may further contain reagents as needed, such as pH adjusters, osmotic pressure adjusters, and / or drug activators. Examples of pH adjusters include organic acids such as citric acid and their salts. Examples of osmotic pressure adjusters include sugars or amino acids. Drug activators are reagents that assist the activity of the active ingredient. These may be added after the condensation step S1 is performed.

[0082] Drug 202 may be a single substance or may contain multiple substances. The concentration of drug 202 in the second solution is preferably, for example, 0.01% to 1.0% (by weight).

[0083] The first solution can be prepared by mixing a lipid with an organic solvent. The lipid may be, for example, a lipid that is a major component of biological membranes. Alternatively, the lipid may be artificially synthesized. The lipid may include, for example, phospholipids or sphingolipids, such as base lipids like diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, kephalin or cerebroside, or combinations thereof.

[0084] For example, as a base lipid, 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1,2-Stearoyl-sn-glycero-3-phosphoethanolamine (DSPE), 1,2-Dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC), 1-Palmitoyl-2-Oleoyl-sn-Glycerol-3-Phosphatidylcholine (POPC), 1,2-di-O-octadecyl-3-trimethylammonium propane (DOTMA), 1,2-Dioleoyl-3-dimethylammonium propane (DODAP), 1,2-Dimyristoyl-3-dimethylammonium propane (14:0 DAP), 1,2-Dipalmitoyl-3-dimethylammonium propane (16:0 DAP), 1,2-Distearoyl-3-dimethylammonium propane (18:0 DAP), N-(4-carboxybenzyl)-N,N-dimethyl-2,3-bis(oleoyloxy)propane (DOBAQ), 1,2-Dioleoyl-3-trimethylammonium propane (DOTAP), 1,2-Dioleoyl-sn-glycero-3-phosphochlorin (DOPC), 1,2-Dilinoleoyl-sn-glycero-3-phosphochlorin (DLPC), 1,2-Dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), or cholesterol, Alternatively, it is preferable to use any combination of these. In particular, it is preferable to use DOTAP and / or DOPE.

[0085] The lipids preferably further comprise a first lipid compound and / or a second lipid compound, which are biodegradable lipids. The first lipid compound can be represented by the formula Q-CHR2. (In the formula, Q is a nitrogen-containing aliphatic group that contains two or more tertiary nitrogen atoms and does not contain oxygen. R is independent of C 12 ~C 24 It is an aliphatic group, At least one R contains a linking group LR selected from the group consisting of -C(=O)-O-, -OC(=O)-, -OC(=O)-O-, -SC(=O)-, -C(=O)-S-, -C(=O)-NH-, and -NHC(=O)- in its main chain or side chain.

[0086] The first lipid compound is a lipid having a structure represented by the following formula, for example. [ka] [ka] [ka] [ka]

[0087] In particular, it is preferable to use the lipid compound of formula (1-01) and / or the lipid compound of formula (1-02).

[0088] The second lipid compound can be represented by the formula P-[XWY-W'-Z]2. (In the formula, P is an alkylene oxy containing one or more ether bonds in its main chain. Each of X is independently a divalent linking group containing a tertiary amine structure. W is independently a C1-C6 alkylene. Each Y is independently a divalent linking group selected from the group consisting of single bonds, ether bonds, carboxylic acid ester bonds, thiocarboxylic acid ester bonds, thioester bonds, amide bonds, carbamate bonds, and urea bonds. Each W' is independently either a single bond or a C1-C6 alkylene. Z is independently a fat-soluble vitamin residue, a sterol residue, or C 12 ~C 22 (It is an aliphatic hydrocarbon group.)

[0089] The second lipid compound is a lipid having a structure represented by the following formula, for example. [ka] [ka] [ka]

[0090] In particular, it is preferable to use the compound of formula (2-01).

[0091] When the first and second lipid compounds are included, it is possible to increase the amount of drug 202 encapsulated in the lipid particles 200, thereby improving the efficiency of drug 202 introduction into cells. Furthermore, it is possible to reduce cell death in the introduced cells. The base lipid is preferably present in an amount of 30% to about 80% (molar ratio) of the total lipid material. Alternatively, it may be composed of nearly 100% base lipid. The first and second lipid compounds are preferably present in an amount of about 20% to about 70% (molar ratio) of the total lipid material.

[0092] The lipids may also preferably include lipids that prevent aggregation of the lipid particles 200. For example, the lipids that prevent aggregation may further preferably include PEG-modified lipids, such as polyethylene glycol (PEG) dimyristoyl glycerol (DMG-PEG), polyamide oligomers derived from omega-amino (oligoethylene glycol) alkanoic acid monomers (U.S. Patent No. 6,320,017), or monosialogangliosides. Such lipids are preferably present in an amount of about 1% to about 10% (molar ratio) of the total lipid material of the lipid particles 200.

[0093] The lipids may further include lipids that are relatively low in toxicity to adjust toxicity; lipids that have functional groups to bind ligands to the lipid particles 200; and lipids that suppress the leakage of encapsulated substances such as sterols and cholesterol. In particular, the inclusion of cholesterol is preferred.

[0094] For example, the lipid particles 200 preferably contain a compound of formula (1-01) or formula (1-02) and / or a compound of formula (2-01), DOPE and / or DOTAP, cholesterol, and DMG-PEG.

[0095] The type and composition of lipids are appropriately selected considering the acid dissociation constant (pKa) of the target lipid particles 200, the size of the lipid particles 200, the type of encapsulated material, and their stability in the cells into which they are introduced. For example, in order to obtain the desired composition of lipids constituting the lipid particles 200, the composition of lipids contained in the first solution should be set to the same ratio.

[0096] The organic solvent in the first solution is, for example, ethanol, methanol, isopropyl alcohol, ether, chloroform, benzene, or acetone. The concentration of lipids in the organic solvent is preferably, for example, 0.1% to 0.5% (by weight).

[0097] The mixing of the first solution and the second solution is performed using the flow channel structure 302 of the embodiment, as shown in Figure 13(b). Here, the flow channel structure 302 is shown as the flow channel structure of the fourth embodiment, but the flow channel structure 302 is not limited to this, and it is also possible to use the flow channel structures of the second, third, or fifth embodiment, for example.

[0098] When the condensation process S1 is performed, the downstream end of the channel 315 of the condensation channel structure 301 is connected to the upstream end of the second channel 3 of the channel structure 302 of the embodiment, and the second solution is supplied to the second channel 3. When the condensation process S1 is not performed, a second solution inlet (not shown) is provided at the upstream end of the second channel 3, and the second solution is supplied from there. The third channel 7 is equipped with, for example, a first solution inlet 321 at its upstream end, from which the first solution is supplied. As a result, the first solution and the second solution are mixed to obtain a mixed liquid 8. If a mixing unit 22 is provided, the mixed liquid 8 is further mixed and stirred there. For example, when the condensation process S1 is not performed, the first solution may be flowed through the second channel 3 and the second solution through the third channel 7.

[0099] ·Particleization process S3 Next, in the particle formation step S3, the concentration of the organic solvent in the mixture 8 is reduced. For example, it is preferable to relatively reduce the organic solvent concentration by adding a large amount of aqueous solution to the mixture 8. For example, three times the amount of aqueous solution as the mixture 8 is added to the mixture 8. The same aqueous solvent used in the first solution can be used as the aqueous solution. By reducing the organic solvent concentration, the lipids are atomized, and lipid particles 200 containing the drug 202 can be produced. As a result, a lipid particle solution 9 containing lipid particles 200 is obtained.

[0100] As shown in Figure 13(c), the particle formation channel structure 303 that performs the particle formation process S3 is, for example, a Y-shaped channel. The upstream end of one of the branched Y-shaped channels 331 is connected to, for example, the downstream end of the channel structure 302 (the fourth channel 25 in this example), from which the mixed liquid 8 is supplied. The upstream end of the other channel 332 is equipped with, for example, an aqueous solution inlet 333, from which the aqueous solution flows. As a result, the aqueous solution is mixed with the mixed liquid 8 in the channel 334 where channels 331 and 332 merge. As a result, the lipids are formed into particles, and lipid particles 200 containing the drug 202 are produced, and a lipid particle solution 9 containing the lipid particles 200 is obtained.

[0101] The particle formation step S3 does not necessarily have to be carried out using a flow channel; for example, an aqueous solution may be added to the mixed liquid 8 collected in the container.

[0102] In this way, lipid particles 200 can be manufactured.

[0103] ·Concentration process S4 The lipid particle production method of the embodiment may further include concentrating the lipid particle solution 9 as needed (concentration step S4). Concentration is performed, for example, by removing some of the solvent and / or excess lipids and drug 202 from the lipid particle solution 9. Concentration can be performed, for example, by ultrafiltration. For ultrafiltration, it is preferable to use an ultrafiltration filter with a pore size of 2 nm to 100 nm. For example, Amicon® Ultra-15 (Merck) can be used as a filter. By performing the concentration step S4, a lipid particle solution 9 with high purity and concentration can be obtained. The concentration of lipid particles 200 in the concentrated lipid particle solution 9 is 1 × 10⁻⁶. 13 pieces / mL~5×10 13 It is preferable that the concentration is approximately 1 / mL. However, the concentration step S4 is not necessarily required.

[0104] As shown in Figure 13(d), the concentration channel structure 304 that performs the concentration process S4 comprises a channel 341 and a filter 342 provided on the wall surface of the channel 341. The upstream end of the channel 341 is connected, for example, to the channel 335 of the particle formation channel structure 303.

[0105] The filter 342 is provided, for example, in place of a portion of the wall surface of the flow path 341. Any of the ultrafiltration filters described above can be used as the filter 342.

[0106] By flowing the lipid particle solution 9 through the channel 341, residual material and excess solvent are discharged outside the channel 341 through the filter 342, while the lipid particles 200 remain in the channel 341 and flow downstream, concentrating the lipid particle solution 9. The downstream end of the channel 341 may be equipped with an outlet 343 for recovering the concentrated lipid particle solution 9, or it may be connected to a tank for recovering the lipid particle solution 9.

[0107] The concentration step S4 does not necessarily have to be performed using a flow path; for example, the lipid particle solution 9 collected in the container may be filtered using a filter.

[0108] Furthermore, the lipid particle manufacturing method of the embodiment may include further processing to improve the quality of the lipid particles 200 as needed. Quality improvement may include, for example, preventing leakage of the drug 202 from the lipid particles 200, improving the amount of drug 202 encapsulated in the lipid particles 200, improving the proportion of lipid particles 200 encapsulating the drug 202 (encapsulation rate), reducing and preventing aggregation of lipid particles 200, and / or reducing variations in the size of the lipid particles. For example, the lipid particle solution 9 may be cooled. Such processing may also be carried out using a flow path.

[0109] Each of the above-described channels is, for example, a microchannel. The flow of fluid within the channels, the injection of fluid into the channels, the removal of fluid from the tank and / or the storage of the lipid particle solution 9 into the container can be carried out, for example, by a pump or extrusion mechanism configured and controlled to perform these operations automatically.

[0110] The method for producing lipid particles in the embodiment does not necessarily require the condensation step S1 and the concentration step S4 as described above, but may include at least the mixing step S2 and the particle formation step S3.

[0111] According to the lipid particle manufacturing method of the embodiment, since the mixing step S2 is performed using the flow channel structure of the embodiment, the first solution and the second solution can be uniformly and thoroughly mixed and stirred, making it possible to produce higher quality lipid particles 200. For example, effects such as an improvement in the amount of drug 202 encapsulated, a reduction in the average particle size of the lipid particles 200, and an improvement in the proportion of lipid particles containing drug 202 can be obtained.

[0112] [example] Example 1 A channel structure similar to that shown in Figure 4(a) was manufactured. The cross-sectional dimensions of the first channel 2 were set to width × depth 0.3 mm × 0.3 mm, with the depth of the shallow section 4 being 1 / 3 (0.1 mm). Water was flowed from the upstream side (right side in the figure) of the third channel 7 towards the left side in the figure, and water containing a fluorescent dye was flowed from the second channel 3. The channel structure was then photographed with a fluorescence microscope. The flow rate in each channel was the same, and the linear velocity of the first channel 2 was set so that the Reynolds number was 50 or higher.

[0113] The captured images are shown in Figure 14. It became clear that transverse vortices were generated in the first channel 2 over a distance of several millimeters from the mixing region 6.

[0114] Example 2 A Y-shaped channel structure similar to that shown in Figure 4(b) was manufactured. The third channel 7 and the first channel 2 had sides of 0.3 mm square. The second channel 3 had a depth of 1 / 3 (0.1 mm) immediately before the mixing region 6.

[0115] Water was flowed through the third channel 7, and water containing a fluorescent dye was flowed through the second channel 3, and images were taken with a light microscope. The flow rate in each channel was the same. The same experiment was also performed with the channel structure manufactured in Example 1, and the two were compared.

[0116] Figure 15(a) shows an image of the channel structure for Example 1, and Figure 15(b) shows an image of the channel structure for Example 2. It was found that the channel structure for Example 2 showed less turbulence in the flow immediately after the confluence compared to the channel structure for Example 1, which is preferable for uniform mixing.

[0117] Furthermore, the generation of transverse vortices in the flow channel structure shown in Figure 4(b) was simulated. The simulation was performed using the fluid analysis software ANSYS® Fluent®. The simulation image is shown in Figure 16. From the image, it became clear that transverse vortices were generated in the mixing region 6.

[0118] Example 3 A mixing unit 22 was added downstream of the flow channel structure (confluence unit 21) of Example 2 to produce a flow channel structure similar to that shown in Figure 5. The flow channels other than the shallow section were 0.3 mm square on each side, and the depth of the shallow section was 1 / 3 (0.1 mm). A fluid containing a fluorescent dye was flowed from the second flow channel 3 of the confluence unit 21, and water was flowed from the third flow channel 7.

[0119] Figure 17 shows a photograph of the mixing unit 22 from the first channel 2 of the confluence unit 21. Transverse vortices were observed over a distance of approximately 1 mm at the confluence section 24c immediately after the second shallow section 4b and at the fourth channel 25 immediately after the third shallow section 4c, indicating that mixing was promoted there. This result indicates that further mixing can be achieved by providing the mixing unit 22.

[0120] Example 4 A channel structure similar to that shown in Figure 6(a) was manufactured by arranging three mixing units 22 in series downstream of the channel (confluence unit 21) of Example 2. The normal channels, excluding the shallow section, had sides of 0.3 mm square, while the shallow section had a depth of 1 / 3 (0.1 mm). Ethanol was flowed through the second channel 3 of the confluence unit, and water was flowed through the third channel 7. Schlieren images were taken of the confluence unit, the confluence of the first mixing unit, the confluence of the second mixing unit, and the confluence of the third mixing unit.

[0121] The captured images are shown in Figure 18. Transverse vortices were observed downstream of the shallow areas of each unit. Furthermore, as the mixture flowed from the confluence unit to the third mixing unit, the unevenness (cloudy areas) observed due to the difference in refractive index between water and ethanol disappeared, indicating that the mixture was well combined.

[0122] Furthermore, using a similarly configured flow channel structure, water containing a fluorescent dye flowed through one channel of the confluence unit, and water flowed through the other channel, and images were taken with a fluorescence microscope. The captured images are shown in Figure 19. The images show that as the mixture flows from the first mixing unit to the third mixing unit, the streaky shadows that form when the fluorescent dye and water mix disappear, indicating that they are mixed uniformly.

[0123] Furthermore, Figure 20 shows graphs illustrating the fluorescence intensity (normalized value) at the branching point of the first mixing unit (Figure 19(a)), the channel connecting the first and second mixing units (Figure 19(b)), the branching point of the second mixing unit (Figure 19(c)), the channel connecting the second and third mixing units (Figure 19(d)), the branching point of the third mixing unit (Figure 19(e)), and the downstream channel of the third mixing unit (Figure 19(f)). Figure 21 shows a graph illustrating the luminance dispersion (normalized value), i.e., the squared difference from the average value of the fluorescence intensity.

[0124] Figure 20 clearly shows that the variation in fluorescence intensity decreases as the data flows from (a) to (f).

[0125] Furthermore, as shown in Figure 21, the luminance dispersion was approximately 0.46 at point (a), approximately 0.05 at points (b) and (c), approximately 0.02 at points (d) and (e), and approximately 0.05 at point (f). These results clearly show that the fluorescence intensity approaches the average value as the data progresses from point (a) to point (f).

[0126] Therefore, it became clear that the two liquids were uniformly mixed as they flowed from the first mixing unit to the third mixing unit.

[0127] Example 5 In Examples 1 to 4, the shallow section was set to 1 / 3 the depth of the other flow channels. However, in Example 5, for a flow channel structure with the same shape as in Example 1, the depth of the shallow section was set to 1 / 1, 1 / 2, 1 / 3, or 1 / 6, and the dependence of transverse vortex generation on the depth of the shallow section was simulated.

[0128] The simulation images are shown in Figure 22. At 1 / 1, almost no transverse vortices were generated. At 1 / 2, the generation of transverse vortices was very small. Significant transverse vortices were generated from 1 / 3, and even stronger transverse vortices were generated at the thinner 1 / 6.

[0129] Therefore, it became clear that the depth of the shallow portion is preferably less than 1 / 2, and more preferably 1 / 3 or less.

[0130] Example 6 Flow channel structures A (Example 1) and B (Example 2), shown in Figure 23, were manufactured, and two-liquid mixing was simulated and compared using these flow channel structures.

[0131] In the flow channel structure A, two mixing units 22 are arranged in series downstream of a merging unit 21. Let α1 be the first flow channel of the merging unit in flow channel structure A, α2 be the flow channel connecting the first mixing unit and the second mixing unit, and α3 be the flow channel after the merging of the second mixing unit (the fourth flow channel).

[0132] The flow channel structure B is configured such that two mixing units 22 are located downstream of the confluence unit 21, and the two flow channels intersect at a right angle at the confluence of the confluence unit and the mixing units (similar to the flow channel structure shown in Figure 9). The first flow channel of the confluence unit in the flow channel structure B is denoted as β1, the flow channel after the confluence of the first mixing unit is denoted as β2, and the flow channel after the confluence of the second mixing unit is denoted as β3. Furthermore, the flow channel after the bend downstream of flow channel β1 (immediately before the branching point of the first mixing unit) is denoted as γ1, the flow channel after the bend downstream of flow channel β2 (immediately before the branching point of the second mixing unit) is denoted as γ2, and the flow channel after the bend downstream of flow channel β3 is denoted as γ3.

[0133] Under conditions where the Reynolds number is at least 50, equal amounts of ethanol and water were introduced from each confluence unit, and the enotal concentrations at each position in the channels α1-3, β1-5, and γ1-3 were simulated. The results are shown in Figure 24. The maximum ethanol concentration in each of α, β, and γ converged to approximately 44%, the concentration of complete mixing, as the mixing progressed from 1 to 3. However, in channel structure B (β, γ), the convergence was clearly faster than in channel structure A (α).

[0134] This is thought to be partly due to the increased flow turbulence that occurs when a shallow channel is joined perpendicularly to a normal channel. Since flow turbulence can be expected to have a stirring effect, if the speed of stirring is more important than uniformity, a structure like channel structure B, in which a shallow channel is joined perpendicularly to a normal channel, is desirable.

[0135] Example 7 Example 7 describes an experiment in which DNA-encapsulated lipid particles were produced using the flow channel structure of the embodiment, and the amount of DNA encapsulated in the lipid particles was measured.

[0136] As shown in Figure 25, a flow channel structure C (Comparative Example 1) having a Y-shaped structure without shallow sections, a flow channel structure D (Example 3) in which three mixing units are arranged in series in a confluence unit, and a flow channel structure E (Example 4) in which six mixing units are arranged in series in a confluence unit were manufactured.

[0137] 180 μl of 0.1 mg / ml nLuc plasmid DNA was dissolved in 1620 μl of 10 mM HEPES (pH 7.3) to obtain a DNA solution (second solution). Six types of lipids were mixed as lipid particle material in the ratio FFT10:FFT20:DOPE:DOTAP:Cholesterol:DNG-PEG2000 = 35:70:21:9.4:88.5:9.4 (molar ratio), and dissolved in 1800 μl of ethanol to obtain a lipid solution (first solution).

[0138] DNA solution (second solution) and lipid solution (first solution) were each filled into syringes and connected to a syringe pump. A liquid delivery tube was connected to each syringe connected to the syringe pump, and these liquid delivery tubes were connected to the two input ports of the confluence unit of the flow path structure C to E, respectively. A liquid delivery tube was also connected to the output port and connected to a tube for collecting the mixed solution. Then, the solution was delivered using the syringe pump and mixed in the flow path. Of the fluid collected from the output port, the first 800 μl was discarded, and finally 2400 μl of DNA-lipid mixture was collected. 7.2 ml of 10 mM HEPES (pH 7.3) was added to 2.4 ml of this DNA-lipid mixture to form particles and obtain a dilute lipid particle solution. 9.6 ml of the dilute lipid particle solution was centrifuged and concentrated to 240 μl using an ultrafiltration filter (Amicon® Ultra15, Merck) to obtain the lipid particle solution.

[0139] 890 μl of purified water (water for injection, manufactured by Otsuka Pharmaceutical Co., Ltd.) and 10 μl of lipid particle solution were mixed. The resulting mixture was placed in a cuvette for particle size measurement, and the particle size and polydispersity index (pdi) were measured using the particle size measurement mode of Zetasaizer® Nano ZSP (Malvern). Next, the zeta potential of the same diluted solution was measured using a cuvette for zeta potential measurement in zeta potential measurement mode.

[0140] Also, Quant-iT TM The concentration of DNA encapsulated within lipid particles in a lipid particle solution was measured using the PicoGreen® ds DNA Assay Kit (Thermo Fisher Scientific). Solution A was prepared by mixing 0.5 μl of lipid particle solution with 99.5 μl of 10 mM HEPES (pH 7.3). Additionally, a solution containing 0.5 μl of lipid particle solution, 84.5 μl of 10 mM HEPES (pH 7.3), and 1% Triton was also prepared. TM -X 100 10 μl and heparin 5 μl were mixed to prepare a solution (Solution B) in which DNA was eluted from lipid particles.

[0141] Each sample was allowed to stand at room temperature for 30 minutes, then 100 μl of PicoGreen solution was added, and the fluorescence intensity was measured using QuantiFlour® (Promega). Calibration curve samples were measured simultaneously, and the amount of DNA in each was calculated. The difference between the amount of DNA in solution B and the amount of DNA in solution A was defined as the amount of DNA contained within the lipid particles. The measurement results are shown in Table 1.

[0142] [Table 1]

[0143] Compared to the Y-shaped channel structure C without a shallow section, the amount of DNA encapsulated was improved by approximately 190% by using the channel structures D and E of the embodiment. Furthermore, when using the channel structures D and E of the embodiment, the average particle size could be reduced further, and channel structure E, which has six mixing units, yielded lipid particles with an even smaller average particle size.

[0144] Example 8 Example 8 describes an experiment in which mRNA-encapsulated lipid particles were produced using the channel structure of the embodiment, and the amount of mRNA encapsulated in the lipid particles was measured.

[0145] 180 μl of mRNA encoding NanoLuc® at a concentration of 0.1 mg / ml was dissolved in 1620 μl of 10 mM HEPES (pH 7.3) to obtain the mRNA solution (second solution). Six types of lipids for the preparation of lipid particles were mixed in the ratio FFT10:FFT20:DOPE:DOTAP:Cholesterol:DNG-PEG2000 = 35:70:21:9.4:88.5:9.4 (molar ratio), dissolved in 1800 μl of ethanol to obtain the lipid solution (first solution).

[0146] The mRNA solution (second solution) and the lipid solution (first solution) were each filled into syringes and connected to a syringe pump. A fluid delivery tube was connected to each syringe connected to the syringe pump, and these tubes were connected to the two input ports of the flow channel structures D and E prepared in Example 7, respectively. A fluid delivery tube was also connected to the output port and connected to a tube for collecting the mixed solution. The solutions were then delivered using the syringe pump and mixed in the flow channel. Of the fluid collected from the output port, the first 800 μl was discarded, and finally 2400 μl of mRNA-lipid mixture was collected. 7.2 ml of 10 mM HEPES (pH 7.3) was added to 2.4 ml of this mRNA-lipid mixture to form particles and obtain a dilute lipid particle solution. 9.6 ml of the dilute lipid particle solution was centrifuged and concentrated to 240 μl using an ultrafiltration filter (AmiconUltra15) to obtain a lipid particle solution.

[0147] 890 μl of purified water (water for injection, manufactured by Otsuka Pharmaceutical Co., Ltd.) and 10 μl of lipid particle solution were mixed and placed in a cuvette for particle size measurement. The particle size and polydispersity index (pdi) were measured using the particle size measurement mode of the Zetasaizer Nano ZSP. Next, the zeta potential of the same diluted solution was measured using the zeta potential measurement mode with a cuvette for zeta potential measurement. The results are shown in Table 2.

[0148] [Table 2]

[0149] Compared to channel structure D, which has three mixing units, lipid particles produced by channel structure E, which has six mixing units, were found to have a smaller average particle size and to have undergone more advanced mixing.

[0150] Example 9 Example 9 describes an experiment in which the abundance of lipid particles containing mRNA was measured in lipid particles prepared using the channel structure of the embodiment.

[0151] 180 μl of mRNA encoding NanoLuc® at a concentration of 0.1 mg / ml was dissolved in 1620 μl of 10 mM HEPES (pH 7.3) to obtain the mRNA solution (second solution). Six types of lipids for the preparation of lipid particles were mixed in the ratio FFT10:FFT20:DOPE:DOTAP:Cholesterol:DNG-PEG2000 = 35:70:21:9.4:88.5:9.4 (molar ratio), dissolved in 1800 μl of ethanol to obtain the lipid solution (first solution).

[0152] The mRNA solution (second solution) and the lipid solution (first solution) were each filled into syringes and connected to a syringe pump. A fluid delivery tube was connected to each syringe connected to the syringe pump, and these tubes were connected to the two input ports of the flow channel structure C-E prepared in Example 7. A fluid delivery tube was also connected to the output port and connected to a tube for collecting the mixed solution. The solutions were then delivered using the syringe pump and mixed in the flow channel. Of the fluid collected from the output port, the first 800 μl was discarded, and finally 2400 μl of mRNA-lipid mixture was collected. 7.2 ml of 10 mM HEPES (pH 7.3) was added to 2.4 ml of this mRNA-lipid mixture to form particles and obtain a dilute lipid particle solution. 9.6 ml of the dilute lipid particle solution was concentrated by centrifugation using an ultrafiltration filter (AmiconUltra15) until it was 240 μl to obtain the lipid particle solution.

[0153] The abundance of mRNA-encapsulating lipid particles was measured using NanoSight® NS300 (Malvern). 10 μl of lipid particle solution was mixed and diluted with 990 μl of 10 mM HEPES (pH 7.3). 5 μl of QantiFlour (RNAdye) and 985 μl of HEPES (pH 7.3) were mixed with 10 μl of the dilute lipid particle solution. After vortexing, the mixture was left to stand at room temperature for 30 minutes in the dark. Then, the lipid particle staining solution was irradiated with a laser using NanoSight NS300, and the number of particles that achieved a certain level of lateral scattered light intensity was defined as the total number of lipid particles (C). Furthermore, the same sample was fluorescently excited by laser irradiation, and the number of particles that achieved a certain level of fluorescence intensity was defined as the number of mRNA-encapsulating lipid particles (D). The ratio of D to C was calculated to determine the abundance of nucleic acid-encapsulating lipid particles. The results are shown in Table 3.

[0154] [Table 3]

[0155] Compared to channel structure C, which lacks a shallow section, it was found that the abundance of lipid particles containing mRNA was significantly improved in channel structures D and E of the embodiment. This result indicates that the mRNA solution and lipid solution are uniformly mixed according to channel structures D and E of the embodiment.

[0156] Example 10 We simulated vortex generation in a flow channel structure in which multiple mixing units 22 were connected in series after a confluence unit 21. The simulation image is shown in Figure 26. From this image, it became clear that transverse vortices were generated downstream of the shallow parts of each mixing unit.

[0157] While several embodiments of the present invention have been described, these embodiments are presented as examples only and are not intended to limit the scope of the invention. These novel embodiments can be carried out in a variety of other forms, and various omissions, substitutions, and modifications can be made without departing from the spirit of the invention. These embodiments and their variations are included in the scope and spirit of the invention, as well as in the claims of the invention and its equivalents. The invention described in the original claims of the parent application is listed below. [1] It comprises a first flow path and a second flow path that merges with the first flow path, A channel structure wherein the end of the second channel on the side of the first channel has a first region that is shallower in depth than the first channel. [2] The first and second flow channels are cavities formed inside the flow channel structure [1], the flow channel structure described above. [3] The channel structure according to [1] or [2], wherein the depth of the first region is less than half the depth of the first channel. [4] The second flow channel merges perpendicularly with the first flow channel, and is a flow channel structure according to any one of [1] to [3]. [5] A channel structure according to any one of [1] to [4], further comprising a third channel connected in series immediately upstream of the confluence point of the first channel with the second channel. [6] The channel structure described in [5], wherein the third channel is a cavity formed inside the channel structure. [7] The channel structure according to [5] or [6], wherein the third channel and the first channel form an integrated straight channel, and the second channel merges with the first channel at a right angle. [8] The channel structure according to [5] or [6], wherein the second channel and the third channel are connected to the first channel symmetrically with respect to the long axis of the first channel, and the angle between the second channel and the third channel is a right angle. [9] The system further comprises a mixing unit connected to the downstream end of the first flow path, The mixing unit includes a first branch-to-combine channel and a second branch-to-combine channel that split the fluid flowing in from the first channel into two to form two separate flows, and merge the two separate flows into a fourth channel. The second branching channel has a second region in its middle that is shallower than its upstream and downstream sides, and its downstream section bends and merges with the fourth channel. The end of the first branching channel on the fourth channel side has a third region that is shallower in depth than the fourth channel. A flow channel structure as described in any one of [1] to [8].

[10] The channel structure described in [9], wherein the fourth channel is a cavity formed inside the channel structure.

[11] The depth of the second region is less than half the depth of the upstream and downstream channels. The depth of the third region is less than half the depth of the fourth channel. The flow channel structure described in [9] or

[10] .

[12] The confluence portions of the first branched confluence channel and the second branched confluence channel into the fourth channel are connected to the fourth channel at angles symmetrical with respect to the long axis of the fourth channel, as described in any one of [9] to

[11] .

[13] The channel structure according to any one of [9] to

[11] , wherein the confluence portion of the second branching channel to the fourth channel is connected in series with the fourth channel and forms a straight channel integral with the fourth channel, and the confluence portion of the first branching channel merges perpendicularly with the fourth channel.

[14] A flow channel structure according to any one of [9] to

[13] , comprising a plurality of the mixing units connected in series.

[15] A flow channel structure according to any one of [9] to

[13] , comprising a plurality of the mixing units connected in parallel.

[16] A method for stirring a fluid using a flow channel structure, The flow channel structure comprises a first flow channel and a second flow channel that merges with the first flow channel, and the end of the second flow channel on the first flow channel side has a first region that is shallower in depth than the first flow channel. This includes flowing a first fluid from the second flow path to the first flow path, A method for stirring fluids.

[17] The method according to

[16] , wherein the depth of the first region is less than half the depth of the first channel.

[18] The flow channel structure further comprises a third flow channel connected in series immediately upstream of the confluence point of the first flow channel with the second flow channel. The present invention further includes further flowing a second fluid from the third flow path into the first flow path, The method described in

[16] or

[17] .

[19] The aforementioned flow channel structure further comprises a mixing unit downstream of the first flow channel, The mixing unit includes a first branch-to-combine channel and a second branch-to-combine channel that split the fluid flowing in from the first channel into two to form two separate flows, and merge the two separate flows into a fourth channel. The end of the first branching channel on the fourth channel side has a second region that is shallower in depth than the fourth channel. The second branching channel has a third region in its middle section that is shallower than its upstream and downstream sections, and its downstream section bends and merges with the fourth channel. The method described in any one of

[16] to

[18] .

[20] The depth of the second region is less than half the depth of the upstream and downstream channels. The depth of the third region is less than half the depth of the fourth channel. The method described in

[19] . [twenty one] The method according to

[19] or

[20] , wherein the flow channel structure comprises a plurality of the mixing units connected in series. [twenty two] The method according to

[19] or

[20] , wherein the flow channel structure comprises a plurality of the mixing units connected in parallel. [twenty three] A method for producing lipid particles containing a drug using a channel structure described in any one of [5] to

[15] , From either the second or third channel, a first solution containing the lipid material of the lipid particles is flowed through an organic solvent, and from the other, a second solution containing the drug is flowed through an aqueous solvent to mix the first solution and the second solution and obtain a mixed solution, and By reducing the concentration of the organic solvent in the mixture, the lipids are atomized to produce lipid particles containing the drug. including, A method for producing lipid particles. [twenty four] The method according to

[23] , wherein the agent is a nucleic acid, and further comprises agglutinating the nucleic acid before mixing the first solution and the second solution. [twenty five] The method according to

[23] or

[24] , further comprising the step of concentrating the lipid particle solution containing the lipid particles after the particleization. [Explanation of Symbols]

[0158] 1, 10, 11, 20, 30, 31, 40, 50, 60, 100... flow channel structures, 2...First channel, 3...Second channel, 4, 4a...First shallow area, 4b...Second shallow area, 4c...Third shallow area, 7...Third channel, 8...Mixed solution, 9...Lipid particle solution, 21... Merging unit, 22... Mixing unit, 22a...First mixing unit, 22b... Second mixing unit, 22c... Third mixed unit, 23...First branching / merging channel, 24...Second branching / merging channel, 25...The fourth channel, 200...Lipid particles, 202...Medication.

Claims

1. It comprises a first flow path and a second flow path that merges with the first flow path, The end of the second flow channel on the side of the first flow channel has a first region that is shallower in depth than the first flow channel, and the first region does not include a tapered portion. The direction of the first flow path is curved when viewed from the direction of the second flow path. A channel structure in which, in the mixing region of the second channel and the first channel, the first channel is closed with respect to the longitudinal direction of the second channel.

2. The flow channel structure according to claim 1, wherein the direction of the first flow channel intersects the plane formed by the depth direction of the first region and the direction of the second flow channel.

3. The channel structure according to claim 1 or 2, wherein each of the first channel and the second channel is a cavity formed inside the channel structure, and the top surfaces of each of the cavities are on the same plane.

4. The channel structure according to any one of claims 1 to 3, wherein the depth of the first region is less than half the depth of the first channel.

5. The flow channel structure according to any one of claims 1 to 4, wherein the second flow channel merges with the first flow channel at a right angle.

6. comprising a first flow path and a second flow path that merges with the first flow path, The end of the second channel on the side of the first channel has a first region that is shallower in depth than the first channel. The direction of the first flow path is curved when viewed from the direction of the second flow path. In the mixing region of the second channel and the first channel, the first channel is closed with respect to the longitudinal direction of the second channel. A flow channel structure further comprising a third flow channel connected in series immediately upstream of the confluence point of the first flow channel with the second flow channel.

7. The channel structure according to claim 6, wherein the third channel is a cavity formed inside the channel structure.

8. The flow channel structure according to claim 6 or 7, wherein the third flow channel and the first flow channel form an integrated linear flow channel, and the second flow channel merges with the first flow channel at a right angle.

9. The flow channel structure according to claim 6 or 7, wherein the second flow channel and the third flow channel are connected to the first flow channel symmetrically with respect to the long axis of the first flow channel, and the angle between the second flow channel and the third flow channel is a right angle.

10. The system further comprises a mixing unit connected to the downstream end of the first flow path, The mixing unit includes a first branch-to-combine channel and a second branch-to-combine channel that split the fluid flowing in from the first channel into two to form two separate flows, and merge the two separate flows into a fourth channel. The second branching channel has a second region in its middle section that is shallower than its upstream and downstream sections, and its downstream section bends and merges with the fourth channel. The end of the first branching channel on the fourth channel side has a third region that is shallower in depth than the fourth channel. A flow channel structure according to any one of claims 1 to 9.

11. The channel structure according to claim 10, wherein the fourth channel is a cavity formed inside the channel structure.

12. The depth of the second region is less than half the depth of the upstream and downstream channels. The depth of the third region is less than half the depth of the fourth channel. The flow channel structure according to claim 10 or 11.

13. The channel structure according to any one of claims 10 to 12, wherein the confluence portions of the first branching channel and the second branching channel into the fourth channel are connected to the fourth channel at angles symmetrical with respect to the long axis of the fourth channel.

14. The channel structure according to any one of claims 10 to 12, wherein the confluence portion of the second branching channel to the fourth channel is connected in series with the fourth channel and forms a straight channel integral with the fourth channel, and the confluence portion of the first branching channel merges perpendicularly with the fourth channel.

15. A flow path structure according to any one of claims 10 to 14, comprising a plurality of the mixing units connected in series.

16. A flow channel structure according to any one of claims 10 to 14, comprising a plurality of the mixing units connected in parallel.

17. A method for stirring a fluid using a flow channel structure, The flow channel structure comprises a first flow channel and a second flow channel that merges with the first flow channel, the end of the second flow channel on the first flow channel side having a first region that is shallower in depth than the first flow channel, and the first region does not include a tapered portion. The direction of the first flow path is curved when viewed from the direction of the second flow path. In the mixing region of the second channel and the first channel, the first channel is closed with respect to the longitudinal direction of the second channel. This includes flowing a first fluid from the second flow path to the first flow path, A method for stirring fluids.

18. The fluid stirring method according to claim 17, wherein the direction of the first flow path intersects the plane formed by the depth direction of the first region and the direction of the second flow path.

19. The method according to claim 17, wherein each of the first flow path and the second flow path is a cavity formed inside the flow path structure, and the top surfaces of each of the cavities are on the same plane.

20. The method according to any one of claims 17 to 19, wherein the depth of the first region is less than half the depth of the first flow channel.

21. A method for stirring a fluid using a flow channel structure, The flow channel structure comprises a first flow channel, a second flow channel that merges with the first flow channel, and a third flow channel connected in series to the first flow channel immediately upstream of the point where it merges with the second flow channel, wherein the end of the second flow channel on the side of the first flow channel has a first region that is shallower in depth than the first flow channel. The direction of the first flow path is curved when viewed from the direction of the second flow path. In the mixing region of the second channel and the first channel, the first channel is closed with respect to the longitudinal direction of the second channel. The first fluid is to flow from the second flow path to the first flow path, This includes further flowing a second fluid from the third flow path into the first flow path, A method for stirring fluids.

22. The flow channel structure further comprises a mixing unit downstream of the first flow channel, The mixing unit includes a first branch-to-combine channel and a second branch-to-combine channel that split the fluid flowing in from the first channel into two to form two separate flows, and merge the two separate flows into a fourth channel. The end of the first branching channel on the fourth channel side has a second region that is shallower in depth than the fourth channel. The second branching channel has a third region in its middle section that is shallower than its upstream and downstream sections, and its downstream section bends and merges with the fourth channel. The method according to any one of claims 17 to 21.

23. The depth of the second region is less than half the depth of the upstream and downstream channels. The depth of the third region is less than half the depth of the fourth channel. The method according to claim 22.

24. The method according to claim 22 or 23, wherein the flow channel structure comprises a plurality of mixing units connected in series.

25. The method according to claim 22 or 23, wherein the flow channel structure comprises a plurality of mixing units connected in parallel.

26. A method for producing lipid particles containing a drug using a flow channel structure according to any one of claims 6 to 9, From either the second or third channel, a first solution containing the lipid material of the lipid particles is flowed into an organic solvent, and from the other, a second solution containing the drug is flowed into an aqueous solvent to mix the first solution and the second solution and obtain a mixed solution, and By reducing the concentration of the organic solvent in the mixture, the lipids are atomized to produce lipid particles containing the drug. including, A method for producing lipid particles.

27. The method according to claim 26, wherein the agent is a nucleic acid, and further comprises agglutinating the nucleic acid before mixing the first solution and the second solution.

28. The method according to claim 26 or 27, further comprising the step of concentrating the lipid particle solution containing the lipid particles after the particleization.

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