Nanoparticle production method

Abstract

[Problem] To provide a nanoparticle production method whereby desired nanoparticles can be easily produced using a microfluidic device. [Solution] A method for manufacturing nanoparticles according to the present invention comprises a step for introducing a first liquid including at least one material through a first fluid inlet flow path 10a of a microfluidic device 1a into a mixing flow path 20, and introducing a second liquid into the mixing flow path 20 through a second fluid inlet flow path 10b and a third fluid inlet flow path 10c on the sides of the first fluid inlet flow path 10a. The Reynolds number in the mixing flow path 20 is set to a predetermined value or more, and the flow of the first liquid in the mixing flow path 20 is made asymmetric so that the flow of the first liquid immediately after entering the mixing flow path 20 through the first fluid inlet flow path 10a is biased toward one of both side walls of the mixing flow path 20.

Description

Nanoparticle manufacturing method

[0001] The present invention relates to a method for producing nanoparticles using a microfluidic device.

[0002] A manufacturing method using a microfluidic device is known as a method for manufacturing nanoparticles that can be used as carriers in drug delivery systems (DDS) or in pharmaceuticals. For example, Patent Document 1 describes a technology for chemically synthesizing reconstituted HDL (rHDL) with a size (approximately 10 nm) similar to that of natural high-density lipoprotein (HDL) by mixing a first liquid containing a phospholipid (DMPC: dimyristoyl phosphatidylcholine) with a second liquid containing apolipoprotein AI (apoA-I) using a microfluidic device.

[0003] The nanoparticle manufacturing method of Patent Document 1 uses a microfluidic device having three fluid inlet channels (a central fluid inlet channel and fluid inlet channels on either side of it) and a mixing channel where these fluid inlet channels converge to produce rHDL. In this microfluidic device, a first liquid is introduced into the central fluid inlet channel, and a second liquid is introduced into the fluid inlet channels on either side of it.

[0004] The first liquid flows into the mixing channel through a central fluid inlet channel, and the second liquid flows into the mixing channel through fluid inlet channels on either side of the central fluid inlet channel. It is believed that the first and second liquids form a microvortex in the mixing channel that is symmetrical (mirror symmetrical) with respect to the central fluid inlet channel, thereby rapidly mixing the DMPC contained in the first liquid and the apolipoprotein AI contained in the second liquid, thereby reconstituting rHDL.

[0005] In recent years, rHDL (DSPC-rHDL) using distearoyl phosphatidylcholine (DSPC) as the phospholipid has been shown to be a promising carrier in drug delivery systems or as a pharmaceutical. Cholic acid dialysis has been used as a technique for reconstituting DSPC-rHDL. However, this method requires time and effort to remove cholic acid by dialysis, and residual cholic acid can be toxic.

[0006] Therefore, the present inventors attempted to reconstitute DSPC-rHDL using the nanoparticle manufacturing method described in Patent Document 1. However, the nanoparticle manufacturing method described in Patent Document 1 was unable to reconstitute DSPC-rHDL, and it was suggested that this was due to insufficient mixing efficiency of the first and second liquids in the mixing channel.

[0007] Furthermore, phospholipids with high phase transition temperatures, such as DSPC and HSPC (hydrogenated soy phosphatidylcholine), are thought to rapidly self-assemble when the first and second liquids are mixed, making it difficult or impossible to reconstitute rHDL using the nanoparticle production method described in Patent Document 1. Therefore, there is a need for a technology that uses microfluidic devices to produce various nanoparticles (e.g., rHDL, liposomes, lipid nanoparticles, polymer nanoparticles, polymer-lipid hybrids) that can be used as carriers in drug delivery systems, pharmaceuticals, cosmetics, or quasi-drugs.

[0008] U.S. Pat. No. 10,864,162

[0009] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for producing nanoparticles that can easily produce desired nanoparticles using a microfluidic device.

[0010] In order to achieve the above-mentioned object, the method for producing nanoparticles according to the present invention comprises the steps of introducing a first liquid containing at least one material into a mixing channel through a first fluid inlet channel of a microfluidic device, and introducing a second liquid into the mixing channel through a second fluid inlet channel and a third fluid inlet channel on either side of the first fluid inlet channel, wherein the Reynolds number in the mixing channel is set to a predetermined value or greater, and the flow of the first liquid in the mixing channel is made asymmetric so that the flow of the first liquid immediately after entering the mixing channel through the first fluid inlet channel is biased toward one of the side walls of the mixing channel.

[0011] In the method for producing nanoparticles according to the present invention, the Reynolds number in the mixing channel is set to a predetermined value or higher, and the flow of the first liquid in the mixing channel is made asymmetric so that the flow of the first liquid immediately after entering the mixing channel through the first fluid inlet channel is biased toward one of the side walls of the mixing channel. This is thought to make it easier for the first liquid and the second liquid to mix in the early stage of their flow into the mixing channel, improving the mixing efficiency of the first liquid and the second liquid.

[0012] This promotes rapid and uniform mixing of the first and second liquids, allowing for convenient production of desired nanoparticles based on at least one material contained in the first liquid. In particular, the nanoparticle production method according to the present invention is suitable for producing minute nanoparticles with a particle size of, for example, about 10 nm, and it has been confirmed that it is possible to reconstitute rHDL using DSPC as a phospholipid, which has been difficult to produce using conventional nanoparticle production methods using microfluidic devices.

[0013] The Reynolds number may be equal to or greater than 151. In this case, the larger the Reynolds number, the more likely the flow of the first liquid will be biased toward one of the side walls of the mixing channel at the initial stage of the first liquid flowing into the mixing channel (upstream of the mixing channel). This improves the mixing efficiency of the first liquid and the second liquid, making it possible to easily produce minute nanoparticles.

[0014] The Reynolds number may be 281 or greater. In this case, the formation of a microvortex (a minute whirlpool) tends to eliminate the boundaries between the region where the first liquid and the second liquid mix and the regions on either side of it (regions mainly containing the second liquid). This makes it easier for the first liquid and the second liquid to mix between these regions, further improving the mixing efficiency of the first liquid and the second liquid. This promotes rapid and uniform mixing of the first liquid and the second liquid, making it possible to easily produce minute nanoparticles.

[0015] The distance between the first fluid inlet channel and the second fluid inlet channel may be greater than the widths of the first fluid inlet channel, the second fluid inlet channel, and the third fluid inlet channel, and the distance between the first fluid inlet channel and the third fluid inlet channel may be greater than the widths of the first fluid inlet channel, the second fluid inlet channel, and the third fluid inlet channel. In this case, the formation of a microvortex tends to eliminate the boundaries between the region where the first liquid and the second liquid mix and the regions on both sides of the region (regions mainly containing the second liquid). This promotes rapid and uniform mixing of the first liquid and the second liquid, making it possible to easily produce minute nanoparticles.

[0016] The method may further include a step of discharging the first liquid and the second liquid from the mixing channel through a fluid outlet channel located downstream of the mixing channel. The fluid outlet channel may be configured as a single fluid outlet channel having a width narrower than a width of the mixing channel.

[0017] In this case, the first liquid and the second liquid are concentrated in a single fluid outlet channel, which increases the Reynolds number in the fluid outlet channel and promotes mixing of the first liquid and the second liquid in the fluid outlet channel, thereby allowing the first liquid and the second liquid to mix not only in the mixing channel but also in the fluid outlet channel, further improving the mixing efficiency of the first liquid and the second liquid.

[0018] The temperature of the mixing channel may be 45° C. or higher. In this case, agglomerates of at least one material contained in the first liquid are less likely to occur in the mixing channel, and fine nanoparticles are more likely to be formed than in conventional nanoparticle manufacturing methods.

[0019] The temperature of the mixing channel may be room temperature or higher (for example, 10° C. or higher, preferably 15° C. or higher, or 20° C. or higher). Even in this case, experiments by the present inventors have revealed that agglomerates of at least one material contained in the first liquid are less likely to occur in the mixing channel, and that fine nanoparticles are more likely to be formed than in conventional nanoparticle manufacturing methods.

[0020] The at least one material may include a lipid. In the method for producing nanoparticles according to the present invention, the at least one material contained in the first liquid may be, for example, a phospholipid (DMPC, DPPC, DSPC, DAPC, HSPC, etc.).

[0021] The second liquid may be an aqueous solvent and may contain at least one water-soluble material, such as apolipoprotein AI (apoA-I).

[0022] The at least one water-soluble material may include at least one of a protein and a peptide. For example, by mixing a first liquid containing phospholipid (DSPC) with a second liquid containing apolipoprotein AI (apoA-I), it is possible to reconstitute DSPC-rHDL having a small particle size that can be used as a carrier in a drug delivery system or as a pharmaceutical.

[0023] FIG. 1 is an exploded perspective view of a microfluidic device and the like used in a method for producing nanoparticles according to one embodiment of the present invention. FIG. 2 is a plan view of the microfluidic device shown in FIG. 1. FIG. 3 is a view of the microfluidic device and the like shown in FIG. 1 immersed in a constant-temperature water bath. FIG. 4A is a conceptual diagram showing the flow state of the first liquid and the second liquid flowing through the mixing channel in the microfluidic device shown in FIG. 2 when the Reynolds number in the mixing channel is equal to or greater than a first predetermined value. FIG. 4B is a conceptual diagram showing the flow state of the first liquid and the second liquid flowing through the mixing channel in the microfluidic device shown in FIG. 2 when the Reynolds number in the mixing channel is equal to or greater than a second predetermined value. FIG. 4C is a conceptual diagram showing the flow state of the first liquid and the second liquid flowing through the mixing channel in the microfluidic device shown in FIG. 2 when the Reynolds number in the mixing channel is less than the first predetermined value. FIG. 5A is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 151. FIG. 5B is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 173. FIG. 5C is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 281. FIG. 5D is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 306. FIG. 5E is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 345. FIG. 5F is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 383. FIG. 5G is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 479. FIG. 5H is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 575. FIG. 5I is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is about 108.FIG. 5J is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 130. FIG. 6A is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the flow rate ratio of the second liquid introduced into the second fluid inlet channel to the second liquid introduced into the third fluid inlet channel is 10:7 and the Reynolds number in the mixing channel is approximately 175. FIG. 6B is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 197. FIG. 6C is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 316. FIG. 6D is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 395. FIG. 6E is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 474. FIG. 7 is a photograph showing the state of the first and second liquids flowing through the mixing channel when the total flow rate of the first and second liquids is 5.5 mL / min and the temperature of the mixing channel is approximately 55°C. FIG. 8 is a diagram showing the results of size exclusion chromatography when the total flow rate of the first and second liquids is set to 5.5 mL / min, 7.7 mL / min, 8.8 mL / min, 11 mL / min, and 13.2 mL / min, respectively. FIG. 9 is a diagram showing the results of size exclusion chromatography when the temperature of the mixing channel is set to approximately 45°C and approximately 55°C. FIG. 10 is a photograph showing the state of the first and second liquids flowing through the mixing channel when the fluid outlet channel is narrowed so that the fluids are discharged from the mixing channel through a single main fluid outlet channel. FIG. 11 is a diagram showing the results of size exclusion chromatography when the fluid outlet channel is narrowed so that the fluids are discharged from the mixing channel through a single main fluid outlet channel. FIG. 12 is an exploded perspective view of a microfluidic device, etc., used in a method for producing nanoparticles according to another embodiment of the present invention. Fig. 13 is a plan view of the microfluidic device shown in Fig. 12. Fig. 14A is a photograph showing the states of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is about 1437 using the microfluidic device shown in Fig. 13.FIG. 14B is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 2395, using the microfluidic device shown in FIG. 13 . FIG. 14C is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 1081, using the microfluidic device shown in FIG. 13 . FIG. 15 is a plan view of another pattern of the microfluidic device shown in FIG. 12 . FIG. 16 is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 1081, using the microfluidic device shown in FIG. 15 . FIG. 17 is a plan view of another pattern of the microfluidic device shown in FIG. 12 . FIG. 18 is a photograph showing the state of the first liquid and the second liquid flowing through the mixing channel when the Reynolds number in the mixing channel is approximately 1081, using the microfluidic device shown in FIG. 17 . FIG. 19 shows the results of size exclusion chromatography using the apparatus shown in FIGS. 12 and 13, with the temperature of the mixing channel set to about 20° C. and the Reynolds number set to 1081.

[0024] Hereinafter, embodiments of the present invention will be described with reference to the drawings. Note that the contents shown in the drawings are merely shown schematically and exemplarily to facilitate understanding of the present invention, and the appearance and dimensional ratios may differ from the actual product. Furthermore, the present invention is not limited to the following embodiments.

[0025] First Embodiment The microfluidic device 1 shown in FIG. 1 is an apparatus used in a method for producing nanoparticles according to one embodiment of the present invention, and is used to produce nanoparticles that can be used, for example, as carriers in drug delivery systems, pharmaceuticals, cosmetics, or quasi-drugs.

[0026] The microfluidic device 1 has a base chip 2a with a groove 9 formed therein, and a cover chip 2b that covers the base chip 2a. A clamping plate 4a and a clamping plate 4b are fixed to the microfluidic device 1 with a plurality of screws 8a and a plurality of nuts 8b. Tubes 7a to 7e are fixed to the clamping plate 4a via connectors 70a to 70f. These components will be described in detail below.

[0027] 1 and 2, the X-axis is an axis parallel to the longitudinal direction of the base chip 2a, the Y-axis is an axis parallel to the lateral direction of the base chip 2a, and the Z-axis is an axis perpendicular to the X-axis and Y-axis. The X-axis, Y-axis, and Z-axis are perpendicular to each other. Hereinafter, the positive side of the Z-axis will be referred to as the upper side, and the negative side of the Z-axis will be referred to as the lower side.

[0028] The base chip 2a is composed of a flat plate and has a groove 9. The groove 9 is formed on one main surface (top surface) of the base chip 2a. The base chip 2a is composed of any material suitable for the flow of fluid through the groove 9. The material that constitutes the base chip 2a is not particularly limited, but may be a polymer, plastic (polypropylene, polydimethylsiloxane, polycarbonate, polyethylene terephthalate, acrylic resin, etc.), glass, silicon, ceramic, metal, etc.

[0029] The cover chip 2b is made of a plate similar to the base chip 2a, and is placed on top of the base chip 2a so as to cover the surface of the base chip 2a on which the groove 9 is formed. By placing the cover chip 2b on the base chip 2a, a closed space is formed between the base chip 2a and the cover chip 2b along the groove 9. This allows the fluid to flow inside the groove 9 without leaking from the groove 9.

[0030] The cover chip 2b is made of the same material as the base chip 2a, but may be made of a different material from the base chip 2a. At one end of the cover chip 2b in the X-axis direction, through-holes 3a to 3c are formed at regular intervals along the Y-axis. At the other end of the cover chip 2b in the X-axis direction, through-holes 3d to 3f are formed at regular intervals along the Y-axis.

[0031] The clamping plates 4a and 4b are each composed of a flat plate and sandwich the base chip 2a and the cover chip 2b from above and below. As a result, the base chip 2a and the cover chip 2b are fixed (pressed) from above and below by the clamping plates 4a and 4b. The material constituting the clamping plates 4a and 4b is not particularly limited, but may be a polymer, plastic (polypropylene, polydimethylsiloxane, polycarbonate, polyethylene terephthalate, acrylic resin, etc.), glass, silicon, ceramic, metal, etc.

[0032] At one end of the clamping plate 4a in the X-axis direction, through-holes 5a to 5c are formed at regular intervals along the Y-axis, and at the other end of the clamping plate 4a in the X-axis direction, through-holes 5d to 5f are formed at regular intervals along the Y-axis.

[0033] Connectors 70a to 70c attached to one ends of tubes 7a to 7c fit into the through holes 5a to 5c of clamping plate 4a. At least a portion of connectors 70a to 70c pass through the through holes 5a to 5c and fit into the through holes 3a to 3c of cover chip 2b. Connectors 70d to 70f attached to one ends of tubes 7d (7d1, 7d2) and 7e fit into the through holes 5d to 5f of clamping plate 4a. At least a portion of connectors 70d to 70f pass through the through holes 5d to 5f and fit into the through holes 3d to 3f of cover chip 2b.

[0034] The tubes 7a to 7e are made of, but are not limited to, plastic such as polytetrafluoroethylene (PTFE). The tubes 7d1 and 7d2 are made of a single bifurcated tube 7d (FIG. 3), but may be made of separate tubes. The connectors 70a to 70f are made of, but are not limited to, plastic or the like.

[0035] Three screw holes 6 are formed at regular intervals along the X axis at one end of clamping plate 4a in the Y axis direction. Three screw holes 6 are formed at regular intervals along the X axis at the other end of clamping plate 4a in the Y axis direction. Furthermore, three screw holes 6 are formed at regular intervals along the X axis at one end of clamping plate 4b in the Y axis direction. Three screw holes 6 are formed at regular intervals along the X axis at the other end of clamping plate 4b in the Y axis direction.

[0036] With the base chip 2a and cover chip 2b sandwiched between the clamping plates 4a and 4b from above and below, the clamping plates 4a and 4b are fixed together with six screws 8a and six nuts 8b. The screws 8a pass through the screw holes 6 formed in the clamping plates 4a and also pass through the screw holes 6 formed in the clamping plates 4b. The nuts 8b are threaded onto the screws 8a on the underside of the clamping plates 4b. The means for fixing the clamping plates 4a and 4b are not limited to the screws 8a and nuts 8b, and other fasteners such as rivets or tacks may also be used.

[0037] The clamping plates 4a and 4b clamp the base chip 2a and cover chip 2b from above and below, and the clamping plates 4a and 4b are further fixed with screws 8a and nuts 8b, thereby improving the airtightness of the closed space (groove portion 9) formed between the base chip 2a and the cover chip 2b.

[0038] As shown in FIG. 2 , the groove portion 9 has at least a first fluid inlet flow path 10a, a second fluid inlet flow path 10b, a third fluid inlet flow path 10c, and a mixing flow path 20. In addition to these flow paths, the groove portion 9 further has a fluid outlet flow path 30 and terminal ends 40a to 40f, although the configuration of the groove portion 9 is not limited thereto. The depth of the groove portion 9 is not particularly limited, but is 0.05 to 0.5 mm, or 0.1 to 0.3 mm. In this embodiment, the mixing flow path 20 is a flow path with a rectangular cross section. The Reynolds number, which will be described later, is calculated assuming a flow in a flow path with a rectangular cross section.

[0039] The terminal portions 40a to 40c are formed at one end of the base chip 2a in the X-axis direction and are arranged at regular intervals along the Y-axis. The terminal portions 40d to 40f are formed at the other end of the base chip 2a in the X-axis direction and are arranged at regular intervals along the Y-axis. The terminal portions 40a to 40f have a circular shape in a planar view. However, the planar shape of the terminal portions 40a to 40f may be elliptical, rectangular, other polygonal, or other shapes.

[0040] As shown in Fig. 1, the terminal ends 40a to 40f are located directly below the through-holes 3a to 3f. The through-holes 5a to 5f formed in the clamping plate 4a, the through-holes 3a to 3f formed in the cover chip 2b, and the terminal ends 40a to 40f formed in the base chip 2a are connected to each other. Therefore, the fluid flowing through the tubes 7a to 7c flows into the first fluid inlet flow channel 10a to the third fluid inlet flow channel 10c (Fig. 2) through the terminal ends 40a to 40c.

[0041] Fluid flowing through fluid outlet flow path 30 (FIG. 2) flows out through terminal portions 40d-40f into tubes 7d (7d1, 7d2) and 7e. The lengths of tubes 7d-7e are not particularly limited, but are generally 0.2-2.0 m. The inner diameters of tubes 7d-7e are not particularly limited, but are generally 0.1-1.5 mm.

[0042] 2, the first fluid inlet channel 10a has at least a main fluid inlet channel 11a. In addition to the main fluid inlet channel 11a, the first fluid inlet channel 10a also has a downstream extension channel 12a and an upstream extension channel 13a, but the configuration of the first fluid inlet channel 10a is not limited to this.

[0043] The second fluid inlet channel 10b has at least a main fluid inlet channel 11b. In addition to the main fluid inlet channel 11b, the second fluid inlet channel 10b has a downstream extension channel 12b and an upstream extension channel 13b, but the configuration of the second fluid inlet channel 10b is not limited to this.

[0044] The third fluid inlet channel 10c has at least a main fluid inlet channel 11c. The third fluid inlet channel 10c has a downstream extension channel 12c and an upstream extension channel 13c in addition to the main fluid inlet channel 11c, but the configuration of the third fluid inlet channel 10c is not limited to this.

[0045] The first fluid inlet channel 10a is formed in the center of the base chip 2a in the Y-axis direction and is located between the second fluid inlet channel 10b and the third fluid inlet channel 10c. The second fluid inlet channel 10b and the third fluid inlet channel 10c are located on both sides (outside) of the first fluid inlet channel 10a in the Y-axis direction. The second fluid inlet channel 10b and the third fluid inlet channel 10c have shapes that are symmetrical (mirror symmetrical) with respect to the first fluid inlet channel 10a.

[0046] The fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c each extend linearly along the X-axis. The fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c have the same width (channel width), but may have different widths. The widths of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c are not particularly limited, but are typically 0.02 to 0.3 mm.

[0047] The lengths (channel lengths) of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c are the same, but may be different. The lengths of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c are not particularly limited, but are in the range of 2.0 to 8.0 mm.

[0048] The distance D1 (center-to-center distance) between the fluid inlet main channel 11a and the fluid inlet main channel 11b is greater than the maximum width W1 among the widths of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c. The ratio D1 / W1 of D1 to W1 is not particularly limited, but may be, for example, 1 < D1 / W1 ≦ 7. The distance D1 represents the center-to-center distance between the channels. A distance D2, which will be described later, also represents the center-to-center distance between the channels.

[0049] Similarly, the distance D2 (center-to-center distance) between the fluid inlet main channel 11a and the fluid inlet main channel 11c is greater than the maximum width W1 among the widths of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c. The ratio D2 / W1 of D2 to W1 is not particularly limited, but may be, for example, 1<D2 / W1≦7.

[0050] The downstream extension channel 12a is connected to the upstream end of the fluid inlet main channel 11a. The downstream extension channel 12a extends linearly along the X-axis. The length of the downstream extension channel 12a is longer than the length of the fluid inlet main channel 11a, and the width of the downstream extension channel 12a is greater than the width of the fluid inlet main channel 11a.

[0051] The downstream extension channel 12b is connected to the upstream end of the fluid inlet main channel 11b. Most of the downstream extension channel 12b extends linearly along the X-axis. However, the downstream end of the downstream extension channel 12b is bent at a right angle. The length of the downstream extension channel 12b (the length from one end of the downstream extension channel 12b to the other end) is longer than the length of the fluid inlet main channel 11b, and the width of the downstream extension channel 12b is greater than the width of the fluid inlet main channel 11b.

[0052] The downstream extension channel 12c is connected to the upstream end of the fluid inlet main channel 11c. Most of the downstream extension channel 12c extends linearly along the X-axis. However, the downstream end of the downstream extension channel 12c is bent at a right angle. The length of the downstream extension channel 12c (the length from one end of the downstream extension channel 12c to the other end) is longer than the length of the fluid inlet main channel 11c, and the width of the downstream extension channel 12c is greater than the width of the fluid inlet main channel 11c.

[0053] The upstream expansion channel 13a is connected to the upstream end of the downstream expansion channel 12a. The upstream expansion channel 13a extends linearly along the X-axis. The length of the upstream expansion channel 13a is shorter than the length of the downstream expansion channel 12a, and the width of the upstream expansion channel 13a is greater than the width of the downstream expansion channel 12a. The upstream end of the upstream expansion channel 13a is connected to the terminal portion 40b.

[0054] The upstream expansion channel 13b is connected to the upstream end of the downstream expansion channel 12b. Most of the upstream expansion channel 13b extends linearly along the Y axis. However, the upstream end of the upstream expansion channel 13b is bent at a right angle. The length of the upstream expansion channel 13b (the length from one end to the other of the upstream expansion channel 13b) is shorter than the length of the downstream expansion channel 12b (the length from one end to the other of the downstream expansion channel 12b). The lateral width of the upstream expansion channel 13b is greater than the lateral width of the downstream expansion channel 12b. The upstream end of the upstream expansion channel 13b is connected to the terminal portion 40a.

[0055] The upstream expansion channel 13c is connected to the upstream end of the downstream expansion channel 12c. Most of the upstream expansion channel 13c extends linearly along the Y axis. However, the upstream end of the upstream expansion channel 13c is bent at a right angle. The length of the upstream expansion channel 13c (the length from one end to the other of the upstream expansion channel 13c) is shorter than the length of the downstream expansion channel 12c (the length from one end to the other of the downstream expansion channel 12c). The width of the upstream expansion channel 13c is greater than the width of the downstream expansion channel 12c. The upstream end of the upstream expansion channel 13c is connected to the terminal portion 40c.

[0056] By adding the downstream extension channel 12a and the upstream extension channel 13a to the main fluid inlet channel 11a, the width of the first fluid inlet channel 10a gradually narrows from the upstream side to the downstream side, which prevents clogging of the fluid flowing through the first fluid inlet channel 10a and allows the fluid to be introduced into the mixing channel 20 at a desired flow rate through the first fluid inlet channel 10a.

[0057] Similarly, by adding the downstream extension channel 12b and the upstream extension channel 13b to the main fluid inlet channel 11b, the width of the second fluid inlet channel 10b narrows in stages from the upstream side to the downstream side, which prevents clogging of the fluid flowing through the second fluid inlet channel 10b and allows the fluid to be introduced into the mixing channel 20 at a desired flow rate through the second fluid inlet channel 10b.

[0058] Similarly, by adding the downstream extension channel 12c and the upstream extension channel 13c to the main fluid inlet channel 11c, the width of the third fluid inlet channel 10c gradually narrows from the upstream side to the downstream side, which prevents clogging of the fluid flowing through the third fluid inlet channel 10c and allows the fluid to be introduced into the mixing channel 20 at a desired flow rate through the third fluid inlet channel 10c.

[0059] The mixing channel 20 is connected to the downstream ends of each of the first to third fluid inlet channels 10a to 10c. A fluid outlet channel 30 is connected to the downstream end of the mixing channel 20. The width of the mixing channel 20 is greater than the width of each of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c. The width of the mixing channel 20 is not particularly limited, but is 0.2 to 3 mm, or 0.5 to 1.5 mm.

[0060] The length of the mixing channel 20 is not particularly limited, but is 5 to 20 mm, or 5 to 15 mm. The depth of the mixing channel 20 is not particularly limited, but is 0.05 to 0.5 mm, or 0.1 to 0.3 mm. The fluids flowing out from the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c join and are mixed in the mixing channel 20.

[0061] The ratio w / h of the width (width along the Y-axis) w of the mixing channel 20 to the depth h along the Z-axis of the mixing channel 20 is not particularly limited, but is preferably 1 or greater, or 2 or greater, or 3 to 7. However, this numerical range applies when the fluid inlet main channels 11a to 11c are arranged along the Y-axis, and when the fluid inlet main channels 11a to 11c are arranged along the Z-axis upstream of the mixing channel 20, the h / w ratio preferably falls within this range. Furthermore, in this embodiment, it is preferable that the Z-axis approximately coincides with the direction of gravity, but the X-axis, Y-axis, or other intermediate axis may also approximately coincide with the direction of gravity.

[0062] The above dimensional relationships are those in one embodiment, and may differ from the above numerical ranges in the case of industrial production, etc. However, it is important that the configuration is such that the flow of the first liquid entering the mixing channel 20 from the fluid inlet main channel 11 a is made asymmetric inside the mixing channel 20.

[0063] The fluid that has flowed into the mixing channel 20 is discharged from the mixing channel 20 through the fluid outlet channel 30. The fluid outlet channel 30 has multiple fluid outlet main channels (in this embodiment, fluid outlet main channels 31a to 31e). However, the number of fluid outlet main channels is not limited to five, and may be one, two to four, or six or more. In addition to these channels, the fluid outlet channel 30 further has multiple outlet extension channels (in this embodiment, outlet extension channels 32a to 32c), but the configuration of the fluid outlet channel 30 is not limited to this. The number of outlet extension channels 32a to 32c is not limited to three, and may be one, two, four or more.

[0064] The fluid outlet main channels 31a to 31e all extend linearly along the X axis and are arranged at regular intervals along the Y axis. The fluid outlet main channels 31a to 31e are connected to the downstream end of the mixing channel 20.

[0065] The width of each of the fluid outlet main channels 31a to 31e is smaller than the width of the mixing channel 20. The width of each of the fluid outlet main channels 31a to 31e is equal to the width of each of the fluid inlet main channels 11a to 11c, but may be smaller or larger than this.

[0066] The width of each of the fluid outlet main channels 31a to 31e is not particularly limited, but is 0.05 to 0.3 mm. The length of each of the fluid outlet main channels 31a to 31e is shorter than the length of the mixing channel 20, but is not particularly limited. The ratio W2 / W3 of the width W2 of each of the fluid outlet main channels 31a to 31e to the width W3 of the mixing channel 20 is not particularly limited, but is 1 / 10≦W2 / W3≦1 / 5.

[0067] The outlet extension channel 32a is connected to the main fluid outlet channels 31a and 31b on its upstream side and to the terminal end 40d on its downstream side. The fluids flowing out of the main fluid outlet channels 31a and 31b join together in the outlet extension channel 32a and flow to the terminal end 40d. The width of the outlet extension channel 32a is larger than the width of each of the main fluid outlet channels 31a and 31b.

[0068] The outlet extension channel 32b is connected to the main fluid outlet channel 31c on its upstream side and to the terminal end 40e on its downstream side. The fluid flowing out of the main fluid outlet channel 31c flows through the outlet extension channel 32b to the terminal end 40e. The width of the outlet extension channel 32b is larger than the width of the main fluid outlet channel 31c.

[0069] The outlet extension channel 32c is connected to the main fluid outlet channels 31d and 31e on its upstream side and to the terminal end 40f on its downstream side. The fluids flowing out of the main fluid outlet channels 31d and 31e join together in the outlet extension channel 32c and flow to the terminal end 40f. The width of the outlet extension channel 32c is greater than the width of each of the main fluid outlet channels 31d and 31e.

[0070] As shown in FIG. 3 , the microfluidic device 1, with the tubes 7a-7e attached, is immersed in a constant-temperature water bath. This heats the microfluidic device 1, the tubes 7a-7e, and the fluids flowing therethrough to a predetermined temperature. The temperature of the liquids stored in the constant-temperature water bath (the temperature of the microfluidic device 1 (more specifically, the first fluid inlet channel 10a-third fluid inlet channel 10c, the mixing channel 20, and the fluid outlet channel 30), the tubes 7a-7e, and the fluids flowing therethrough) is room temperature or higher (e.g., 10°C or higher, preferably 15°C or higher, or 20°C or higher), preferably 45°C or higher, and more preferably 55°C or higher.

[0071] As shown in FIG. 2, the method for producing nanoparticles of this embodiment includes the steps of introducing a first liquid containing at least one material into a mixing channel 20 through a first fluid inlet channel 10a of a microfluidic device 1, and introducing a second liquid into the mixing channel 20 through a second fluid inlet channel 10b and a third fluid inlet channel 10c on either side of the first fluid inlet channel 10a.

[0072] The first liquid is a solution containing at least one material. Examples of the material contained in the first liquid include lipids, lipids modified with polyethylene glycol, and biodegradable synthetic polymers. Examples of lipids include cholesterol, fatty acids, phospholipids, glycerolipids, sphingolipids, sterol lipids, prenol lipids, and saccharolipids, with phospholipids being preferred.

[0073] Examples of phospholipids include phosphatidylglycerol, phosphatidylserine, phosphatidylcholine, and phosphatidylethanolamine, with phosphatidylcholine being preferred. Examples of phosphatidylcholine include dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), diarachidoylphosphatidylcholine (DAPC), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), and dioleoylphosphatidylcholine (DOPC). These lipids are dissolved in an appropriate solvent, such as methanol, ethanol, or chloroform. Examples of biodegradable synthetic polymers include lactic acid-glycolic acid copolymer (PLGA), polylactic acid, polyglycolic acid, and polydioxanone.

[0074] The second liquid is an aqueous solvent. The second liquid may contain at least one water-soluble material. Examples of the water-soluble material include proteins, peptides, and lipids modified with polyethylene glycol. Preferably, the second liquid contains at least one of proteins and peptides.

[0075] Examples of proteins include apolipoproteins and genetically modified apolipoproteins thereof. Examples of apolipoproteins include apolipoproteins belonging to the group of apolipoproteins A to E, such as at least one selected from the group consisting of apolipoprotein AI (apoA-I), apolipoprotein A-II (apoA-II), apolipoprotein C (apoC), apolipoprotein E (apoE), and genetically modified apolipoproteins thereof. Among these, apolipoprotein AI (apoA-I) and genetically modified apolipoproteins thereof are preferred.

[0076] Genetically modified apolipoproteins refer to mutants or analogs (i.e., functional equivalents) that have the same function as apolipoproteins (e.g., lipid-binding function). Examples of genetically modified apolipoproteins include partial fragments of apolipoproteins and apolipoproteins formed by combining these fragments. An example of a genetically modified apolipoprotein of apolipoprotein AI is apoA-I, which lacks 43 amino acids at the N-terminus.

[0077] Examples of peptides include apoA-I partial peptides and apoA-I mimetic peptides.

[0078] 2 and 3, a first liquid is introduced into the microfluidic device 1 through tube 7b, for example, by a syringe pump. A second liquid is introduced into the microfluidic device 1 through tubes 7a and 7c, for example, by a syringe pump. The first liquid flows into the first fluid inlet channel 10a through terminal end 40b. The second liquid flows into the second fluid inlet channel 10b and the third fluid inlet channel 10c through terminal ends 40a and 40c.

[0079] The first liquid is introduced into the mixing channel 20 through the first fluid inlet channel 10a (upstream expansion channel 13a, downstream expansion channel 12a, and main fluid inlet channel 11a). The second liquid is introduced into the mixing channel 20 through the second fluid inlet channel 10b (upstream expansion channel 13b, downstream expansion channel 12b, and main fluid inlet channel 11b) and the third fluid inlet channel 10c (upstream expansion channel 13c, downstream expansion channel 12c, and main fluid inlet channel 11c).

[0080] Furthermore, the first liquid and the second liquid are mixed in the mixing channel 20 and are discharged from the mixing channel 20 through the fluid outlet channel 30. More specifically, a portion of the fluid flowing through the mixing channel 20 is discharged from the mixing channel 20 through the fluid outlet main channels 31a and 31b, and further flows into the tube 7d1 through the outlet extension channel 32a and the terminal end 40d.

[0081] A portion of the fluid flowing through mixing channel 20 is discharged from mixing channel 20 through fluid outlet main channel 31c, and flows into tube 7e through outlet extension channel 32b and terminal end 40e. A portion of the fluid flowing through mixing channel 20 is discharged from mixing channel 20 through fluid outlet main channels 31d and 31e, and flows into tube 7d2 through outlet extension channel 32c and terminal end 40f.

[0082] Next, as shown in FIG. 3, the fluids (first liquid and second liquid) that have flowed into the tubes 7d and 7e are stored in a container such as a test tube.

[0083] In this embodiment, the Reynolds number in the mixing channel 20 shown in Fig. 2 is set to a predetermined value or greater. When the Reynolds number in the mixing channel 20 is set to a first predetermined value, it is believed that a fluid flow as described below occurs in the mixing channel 20. As shown in Fig. 4A , immediately after entering the mixing channel 20 through the first fluid inlet channel 10a, the first liquid flows on average in the direction indicated by arrow A1, i.e., toward one of the side walls of the mixing channel 20.

[0084] Furthermore, immediately after entering the mixing channel 20 through the second fluid inlet channel 10b, the second liquid flows in the direction indicated by arrow B1, i.e., along the extension direction of the mixing channel 20. Also, immediately after entering the mixing channel 20 through the third fluid inlet channel 10c, the second liquid flows in the direction indicated by arrow C1, i.e., along the extension direction of the mixing channel 20.

[0085] As a result, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes asymmetric with respect to the first fluid inlet channel 10a, so as to be biased toward one of the side walls of the mixing channel 20. In the example shown in Fig. 4A, the flow of the first liquid is biased toward the side wall of the mixing channel 20 on the positive side of the Y axis, but it may also be biased toward the side wall of the mixing channel 20 on the negative side of the Y axis.

[0086] In the microvortex region 50, a microvortex (a minute whirlpool) asymmetric with respect to the first fluid inlet channel 10a is generated, and the first liquid and the second liquid are mixed. Here, a first boundary 60 asymmetric with respect to the first fluid inlet channel 10a is formed between the flow mainly containing the first liquid (around arrow A1) and the microvortex region 50. In addition, a second boundary 70 is formed between the microvortex region 50 and the flows mainly containing the second liquid on both sides of the microvortex region 50 in the Y-axis direction.

[0087] The flow of the first liquid becomes asymmetric immediately after it enters the mixing channel 20 through the first fluid inlet channel 10a, and the average flow directions of the first liquid and the second liquid are no longer parallel, which is thought to make it easier for the first liquid and the second liquid to mix even in the early stages when the first liquid and the second liquid flow into the mixing channel 20.

[0088] That is, the first liquid and the second liquid are mixed not only in the microvortex region 50 but also upstream of the mixing channel 20 (near the first fluid inlet channel 10a, the second fluid inlet channel 10b, and the third fluid inlet channel 10c). This mixing can sometimes be observed as extremely small vortices (vortices smaller than the above-mentioned microvortexes).

[0089] While the above-mentioned first predetermined value is 151 as an example, it is not particularly limited as long as it is possible to make the flow of the first liquid in the mixing channel 20 asymmetric in the manner shown in Fig. 4A . Furthermore, air bubbles may be mixed into the mixing channel 20. Even if air bubbles are mixed into the mixing channel 20, since the fluid flow is generally as shown in Fig. 4A , the same effect as when no air bubbles are present in the mixing channel 20 can be obtained (the first liquid and the second liquid are more easily mixed in the early stage when they flow into the mixing channel 20).

[0090] In other words, even if air bubbles are mixed into the mixing channel 20, since a fluid flow as shown in FIG. 4A is generally observed, the flow of the first liquid is considered to be asymmetric with respect to the first fluid inlet channel 10a so as to be biased toward one of the side walls of the mixing channel 20. The Reynolds number Re in the mixing channel 20 is calculated based on the following equation (1): where ρ is the fluid density, U is the average fluid velocity, μ is the fluid viscosity, w is the channel width of the mixing channel 20, h is the channel depth of the mixing channel 20, and Q is the flow rate.

[0091]

[0092] Furthermore, when the Reynolds number in the mixing channel 20 is set to a second predetermined value (a value higher than the first predetermined value), it is believed that a fluid flow as described below occurs in the mixing channel 20. As shown in Figure 4B, the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a flows on average in the direction indicated by arrow A2, i.e., toward one of the side walls of the mixing channel 20.

[0093] Furthermore, immediately after entering the mixing channel 20 through the second fluid inlet channel 10b, the second liquid flows in the direction indicated by arrow B2, i.e., along the extension direction of the mixing channel 20. Also, immediately after entering the mixing channel 20 through the third fluid inlet channel 10c, the second liquid flows in the direction indicated by arrow C2, i.e., along the extension direction of the mixing channel 20.

[0094] As a result, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes asymmetric with respect to the first fluid inlet channel 10a, biased toward one of the side walls of the mixing channel 20. In the microvortex region 50, the first and second liquids are mixed, but a microvortex may not be observable.

[0095] Here, a first boundary 60 that is asymmetric with respect to the first fluid inlet channel 10a is formed between the flow mainly containing the first liquid (around arrow A2) and the microvortex region 50. In addition, a second boundary 70 is formed between the microvortex region 50 and the flows mainly containing the second liquid on both sides of the microvortex region 50 in the Y-axis direction.

[0096] It is believed that the flow of the first liquid becomes asymmetric immediately after entering the mixing channel 20 through the first fluid inlet channel 10a, and the average flow directions of the first liquid and the second liquid are no longer parallel, which makes it easier for the first liquid and the second liquid to mix even in the early stages of their flow into the mixing channel 20. In other words, the first liquid and the second liquid are mixed not only in the microvortex region 50, but also on the upstream side of the mixing channel 20 (near the first fluid inlet channel 10a, the second fluid inlet channel 10b, and the third fluid inlet channel 10c). This mixing can sometimes be observed as extremely small vortices.

[0097] In addition, the second boundary 70 disappears (becomes unclear or disturbed) midway through the mixing channel 20. This indicates that uniform mixing is promoted between the inside and outside in the Y-axis direction beyond the second boundary 70, further improving the mixing efficiency of the first and second liquids. At the same temperature, the disappearance of the second boundary 70 occurs further upstream in the mixing channel 20 as the Reynolds number increases above a second predetermined value. Furthermore, as the Reynolds number increases, the microvortex becomes unclear, and extremely small vortices different from conventional microvortexes are observed. This indicates that the mixing of the first and second liquids proceeds more quickly.

[0098] The second predetermined value is 281 as an example, but is not particularly limited to this value as long as it is possible to make the flow of the first liquid in the mixing channel 20 asymmetric in the manner shown in Fig. 4B. Also, air bubbles may be mixed into the mixing channel 20.

[0099] Even if air bubbles are mixed into the mixing channel 20, since a fluid flow generally as shown in Fig. 4B is confirmed, the same effects as when no air bubbles are present in the mixing channel 20 are obtained ((i) the first liquid and the second liquid are more likely to mix in the early stage when they flow into the mixing channel 20, and (ii) the mixing efficiency of the first liquid and the second liquid is improved as the second boundary 70 disappears midway through the mixing channel 20). In other words, since a fluid flow generally as shown in Fig. 4B is confirmed even when air bubbles are mixed into the mixing channel 20, the flow of the first liquid is considered to be asymmetric with respect to the first fluid inlet channel 10a so as to be biased toward one of the side walls of the mixing channel 20.

[0100] In particular, the nanoparticle manufacturing method of this embodiment is suitable for manufacturing minute nanoparticles, for example, with a particle size of approximately 8 to 15 nm, and can reconstitute rHDL using DSPC as the phospholipid, which has been difficult to manufacture using conventional nanoparticle manufacturing methods using microfluidic devices (details will be described in the Examples).

[0101] Furthermore, the nanoparticle manufacturing method of this embodiment is suitable for reconstituting rHDL using a phospholipid other than DSPC that has a higher phase transition temperature than DMPC, such as HSPC (hydrogenated soy phosphatidylcholine).

[0102] Therefore, using microfluidic devices, it is possible to produce various nanoparticles (e.g., rHDL, liposomes, lipid nanoparticles, polymer nanoparticles, polymer-lipid hybrids) that can be used as carriers in drug delivery systems, pharmaceuticals, cosmetics, or quasi-drugs.

[0103] If the Reynolds number in the mixing channel 20 is set to be less than a first predetermined value, it is believed that a fluid flow as described below will occur in the mixing channel 20. As shown in Figure 4C, the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a flows, on average, in the direction indicated by arrow A3, i.e., along the extension direction of the mixing channel 20.

[0104] Furthermore, immediately after entering the mixing channel 20 through the second fluid inlet channel 10b, the second liquid flows in the direction indicated by arrow B3, i.e., along the extension direction of the mixing channel 20. Also, immediately after entering the mixing channel 20 through the third fluid inlet channel 10c, the second liquid flows in the direction indicated by arrow C3, i.e., along the extension direction of the mixing channel 20.

[0105] As a result, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes symmetrical with respect to the first fluid inlet channel 10a. Furthermore, in the microvortex region 50, a microvortex is generated that is symmetrical with respect to the first fluid inlet channel 10a, and the first liquid and the second liquid are mixed.

[0106] Here, a first boundary 60 is formed symmetrically with respect to the first fluid inlet channel 10a between the flow mainly containing the first liquid (around arrow A3) and the microvortex region 50. Also, a second boundary 70 is formed between the microvortex region 50 and the flows mainly containing the second liquid on both sides of the microvortex region 50 in the Y-axis direction.

[0107] In this case, in the initial stage of the flow of the first and second liquids into the mixing channel 20, the average flow directions of the first and second liquids are parallel, making it difficult for the first and second liquids to mix. That is, the first and second liquids are mixed mainly by microvortexes in the microvortex region 50, and are not easily mixed on the upstream side of the mixing channel 20 (near the first fluid inlet channel 10a, the second fluid inlet channel 10b, and the third fluid inlet channel 10c). Therefore, the mixing efficiency of the first and second liquids may not be sufficient, making it difficult to achieve rapid and uniform mixing of the first and second liquids.

[0108] 2, the distance between the fluid inlet main channel 11a and the fluid inlet main channel 11b is greater than the width of each of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c. The distance between the fluid inlet main channel 11a and the fluid inlet main channel 11c is also greater than the width of each of the fluid inlet main channel 11a, the fluid inlet main channel 11b, and the fluid inlet main channel 11c.

[0109] Therefore, the boundary (second boundary 70) between the microvortex region 50 and the flows mainly containing the second liquid on both sides of the microvortex region 50 in the Y-axis direction tends to disappear as far upstream as possible, which promotes rapid and uniform mixing of the first and second liquids and allows for the easy production of minute nanoparticles.

[0110] The present invention is not limited to the above-described embodiment, and various modifications can be made within the scope of the present invention. For example, the following modifications are possible.

[0111] 2, the fluid outlet channel 30 has five main fluid outlet channels 31a to 31e, but it may have only a single main fluid outlet channel having a width narrower than the width of the mixing channel 20. For example, the fluid outlet channel 30 may have only the main fluid outlet channel 31c. In this case, the fluid is discharged from the mixing channel 20 through the single main fluid outlet channel.

[0112] Furthermore, the fluid outlet main channels 31a, 31b, 31d, and 31e may be blocked by a jig or the like to prevent fluid from flowing therethrough. Alternatively, the outlet extension channels 32a and 32c may be blocked by a jig or the like to prevent fluid from flowing through the fluid outlet main channels 31a, 31b, 31d, and 31e. Alternatively, the tube 7d (tubes 7d1 and 7d2) shown in FIG. 1 may be blocked by a jig or the like to prevent fluid from flowing through the fluid outlet main channels 31a, 31b, 31d, and 31e.

[0113] In this case, the fluid outlet flow path 30 essentially has only a single fluid outlet main flow path (fluid outlet main flow path 31c). Note that the damming positions of the fluid outlet main flow paths 31a, 31b, 31d, and 31e may be the upstream end or the downstream end of the fluid outlet main flow paths 31a, 31b, 31d, and 31e.

[0114] Alternatively, the damming positions of the fluid outlet main channels 31 a, 31 b, 31 d, and 31 e may be any positions between the upstream end and the downstream end of the fluid outlet main channels 31 a, 31 b, 31 d, and 31 e. The ratio W2 / W3 of the width W2 of the single fluid outlet main channel 31 c to the width W3 of the mixing channel 20 is not particularly limited, but is 1 / 20≦W2 / W3<1, or 1 / 10≦W2 / W3≦1 / 2.

[0115] In this way, when the fluid outlet channel 30 has a single main fluid outlet channel 31c, the first liquid and the second liquid are concentrated in the single main fluid outlet channel 31c, which increases the Reynolds number in the main fluid outlet channel 31c and promotes mixing of the first liquid and the second liquid in the main fluid outlet channel 31c. As a result, the first liquid and the second liquid are mixed not only in the mixing channel 20 but also in the main fluid outlet channel 31c, further increasing the mixing efficiency of the first liquid and the second liquid.

[0116] The number of main fluid outlet channels is not limited to five, and may be four or less, or six or more. When the number of main fluid outlet channels is four or less, for example, the fluid outlet channel 30 may have any two, three, or four of the main fluid outlet channels 31a to 31e. In this case, the main fluid outlet channel 31c may be required among the main fluid outlet channels 31a to 31e.

[0117] 2, the first fluid inlet channel 10a includes the downstream extension channel 12a and the upstream extension channel 13a, but these channels may be omitted. In this case, the first fluid inlet channel 10a may be composed of only the main fluid inlet channel 11a. The length of the main fluid inlet channel 11a may be extended as necessary. The same applies to the second fluid inlet channel 10b and the third fluid inlet channel 10c.

[0118] 2, the fluid outlet channel 30 includes the outlet extension channels 32a to 32c, but these channels may be omitted. The groove 9 only needs to include the first to third fluid inlet channels 10a to 10c (main fluid inlet channels 11a to 11c) and the mixing channel 20, and does not necessarily need to include the fluid outlet channel 30 and the terminal ends 40a to 40f.

[0119] When the fluid outlet flow channel 30 and / or the terminal portions 40a to 40f are omitted from the groove portion 9, the first liquid and the second liquid may be discharged directly or indirectly (through a tube or the like) from the downstream side of the mixing flow channel 20. In this case as well, the effects described in the above embodiment can be obtained.

[0120] 12 is an apparatus used in a method for producing nanoparticles according to another embodiment of the present invention. A description of parts common to the above-described embodiment will be omitted, and the following description will mainly focus on parts that differ from the above-described embodiment. In the drawings of the above-described embodiment and the following embodiment, common components are designated by the same reference numerals.

[0121] Furthermore, at least a portion of the configuration of the embodiments (second embodiment and onward) described below can be replaced with or combined with at least a portion of the configuration of the embodiments described above, and at least a portion of the configuration of the embodiments described above can be replaced with or combined with at least a portion of the configuration of the embodiments described below.

[0122] As shown in Figures 12 and 13, the microfluidic device 1a of this embodiment differs from the microfluidic devices of the previously described embodiments mainly in that the configuration of the groove portion 9a is different from the configuration of the groove portion 9 shown in Figures 1 and 2.

[0123] 13 , the groove 9a has at least a first fluid inlet channel 10a, a second fluid inlet channel 10b, a third fluid inlet channel 10c, and a mixing channel 20. The first fluid inlet channel 10a has a main fluid inlet channel 11a and an extension channel 14a with a larger channel cross section than the main channel 11a. Similarly, the second fluid inlet channel 10b has a main fluid inlet channel 11b and an extension channel 14b with a larger channel cross section than the main channel 11b. Similarly, the third fluid inlet channel 10c has a main fluid inlet channel 11c and an extension channel 14c with a larger channel cross section than the main channel 11c.

[0124] As in the first embodiment, the flow path is not formed only by the groove 9 a, but the opening of the groove 9 a is closed by the underside of the cover chip along the Z axis, thereby forming a flow path that is sealed except for the inlet and outlet. In this embodiment, as shown in Figure 13, one downstream end of each of the fluid inlet main channels 11 a, 11 b, and 11 c communicates with the mixing channel 20, and the other upstream end of each of the fluid inlet main channels 11 a, 11 b, and 11 c communicates with the extension channels 14 a, 14 b, and 14 c, respectively.

[0125] The cross-sectional areas of the extension channels 14a, 14b, and 14c may be different from one another, but are preferably approximately the same, and may be, for example, approximately the same as the cross-sectional area of ​​the mixing channel 20. The cross-sectional area of ​​the mixing channel 20 is, for example, 0.1 to 1 mm 2 The cross-sectional areas of the fluid inlet main channels 11a, 11b, and 11c may be different from one another, but are preferably approximately equal, and each is preferably within the range of 1 / 20 to 1 / 5 of the cross-sectional area of ​​the mixing channel 20.

[0126] Terminal portions 40a to 40c for introducing fluids are formed at the upstream ends of the extension channels 14a to 14c. Furthermore, terminal portion 40g for extracting fluids is formed at the downstream end of the mixing channel 20. These terminal portions 40a to 40c and 40g are formed in part of the groove 9a and communicate with through-holes 3a to 3c and 3g, respectively, formed in the cover chip 2b shown in FIG.

[0127] These through holes 3a to 3c and 3g communicate with through holes 5a to 5c and 5g, respectively, formed in the clamping plate 4a. Tubes 7a to 7c and 7g are attached to the through holes 5a to 5c and 5g via adapters 70a to 70c and 70g, respectively, and communicate with the through holes 5a to 5c and 5g.

[0128] 13, in this embodiment, the terminal portions 40a and 40c are formed at one end of the base chip 2a in the X-axis direction and are spaced apart at regular intervals along the Y-axis, while the terminal portions 40b and 40g are formed at the other end of the base chip 2a in the X-axis direction and are spaced apart at regular intervals along the Y-axis.

[0129] 12, the terminal portions 40a to 40c and 40g are located directly below the through-holes 3a to 3c and 3g. The through-holes 5a to 5c and 5g formed in the clamping plate 4a, the through-holes 3a to 3c and 3g formed in the cover chip 2b, and the terminal portions 40a to 40c and 40g formed in the base chip 2a are all in communication with each other.

[0130] Therefore, fluids flowing from tubes 7a to 7c toward device 1a flow into first fluid inlet flow channel 10a to third fluid inlet flow channel 10c (FIG. 13) through terminal ends 40a to 40c. Fluids flowing through mixing flow channel 20 (FIG. 13) flow out into tube 7g through terminal end 40g.

[0131] The main channel 11a of the first fluid inlet channel 10a is formed in the center of the base chip 2a in the Y-axis direction and is located between the main channel 11b of the second fluid inlet channel 10b and the main channel 11c of the third fluid inlet channel 10c. The main channel 11b of the second fluid inlet channel 10b and the main channel 11c of the third fluid inlet channel 10c are located on both sides (outside) of the main channel 11a of the first fluid inlet channel 10a in the Y-axis direction. The main channel 11b of the second fluid inlet channel 10b and the main channel 11c of the third fluid inlet channel 10c have shapes that are symmetrical (mirror symmetrical) with respect to the main channel 11a of the first fluid inlet channel 10a.

[0132] The main fluid inlet channels 11a, 11b, and 11c each extend linearly along the X-axis, but the main channel 11a is formed longer along the X-axis than the main channels 11b and 11c. In this embodiment, the extension channel 14a of the first fluid inlet channel 10a is formed to be longer than the extension channel 14b of the second fluid inlet channel 10b and the extension channel 14c of the third fluid inlet channel 10c.

[0133] The flow resistance from the terminal end 40b through the first fluid inlet channel 10a to the inlet of the mixing channel 20 may be made larger than the flow resistance from each of the terminal ends 40a and 40c through the fluid inlet channels 10b and 10c to the inlet of the mixing channel 20. Alternatively, the flow resistances may be the same.

[0134] It is also possible to change the flow rate of the fluids blown into the mixing channel 20 from each of the flow channels 11a to 11c. The flow rate of the fluids blown into the mixing channel 20 can also be changed by changing the fluid pressure applied to each of the upstream terminal portions 40a to 40c. The flow rates of the fluids blown into the mixing channel 20 from each of the flow channels 11a to 11c may be the same or different. For example, when the first liquid is a liquid containing phospholipids and the second liquid is a liquid containing a water-soluble material, it is preferable that the flow rate of the second liquid flowing into the mixing channel 20 from flow channels 11b and 11c is greater than the flow rate of the first liquid flowing into the mixing channel 20 from flow channel 11a, for example, preferably two or more times, three or more times, five or more times, or eight or more times.

[0135] In this embodiment, the fluids that have flowed into the mixing channel 20 and been mixed can be taken out from the terminal portion 40g shown in Fig. 13 through the tube 7g shown in Fig. 12 without passing through the fluid outlet channel 30 (see Fig. 2) of the first embodiment. The microfluidic device 1a of this embodiment can be attached to, for example, a constant temperature water bath shown in Fig. 3, in the same manner as in the first embodiment.

[0136] This allows the microfluidic device 1a and the fluids flowing therein to be maintained at a predetermined temperature. The temperature of the liquids stored in the constant temperature water bath (the temperature of the microfluidic device 1a (more specifically, the first fluid inlet channel 10a to the third fluid inlet channel 10c, the mixing channel 20), the tubes 7a to 7c, 7g, and the fluids flowing therein) is maintained at room temperature or higher (for example, 10°C or higher, preferably 15°C or higher, or 20°C or higher). The upper limit of the temperature of the fluid in the mixing channel 20 is not particularly limited, but is preferably below the temperature at which the first liquid changes in quality, for example, 65°C or lower when the first liquid contains lipids as a material.

[0137] As shown in Figure 13, in the method for producing nanoparticles of this embodiment, a first liquid containing at least one material is introduced into a mixing channel 20 through a first fluid inlet channel 10a of a microfluidic device 1a, and a second liquid is introduced into the mixing channel 20 through a second fluid inlet channel 10b and a third fluid inlet channel 10c on both sides of the first fluid inlet channel 10a.

[0138] The first liquid is a solution containing at least one material. The material contained in the first liquid is the same as in the first embodiment. The second liquid is, for example, an aqueous solvent, specifically the same as in the first embodiment. The first liquid is introduced into the microfluidic device 1a through the tube 7b, for example, by a syringe pump. The second liquid is introduced into the microfluidic device 1a through the tubes 7a and 7c, for example, by a syringe pump.

[0139] The first liquid flows into the first fluid inlet channel 10a through the terminal end 40b. The second liquid flows into the second fluid inlet channel 10b and the third fluid inlet channel 10c through the terminal ends 40a and 40c. The first liquid is introduced into the mixing channel 20 through the first fluid inlet channel 10a. The second liquid is introduced into the mixing channel 20 through the second fluid inlet channel 10b and the third fluid inlet channel 10c.

[0140] Furthermore, the first liquid and the second liquid are mixed in the mixing channel 20 and are discharged from the mixing channel 20. More specifically, all of the fluid flowing through the mixing channel 20 flows into the tube 7g through the terminal portion 40g. The fluid that has flowed into the tube 7g is stored in a container such as a test tube.

[0141] In this embodiment, the Reynolds number in the mixing channel 20 is set to a predetermined value or greater. When the Reynolds number in the mixing channel 20 is set to the first or second predetermined value, a fluid flow such as that shown in Figure 4A or 4B occurs in the mixing channel 20, as in the first embodiment. In this embodiment, the same effects as those of the first embodiment described above are achieved, and the following effects can also be expected.

[0142] In this embodiment, it has been confirmed that the Reynolds number can be increased even further than in the first embodiment, and may be, for example, 600 or more, 800 or more, 1000 or more, or 2000 or more. Rapid and uniform mixing of the first liquid and the second liquid is promoted even at room temperature, and minute nanoparticles can be easily produced. While there is no particular upper limit to the Reynolds number, from the viewpoint of the pressure resistance of the microfluidic device 1a, connectors 70a-70c, and 70g, etc., a value of 6000 or less, preferably 4000 or less, or 2400 or less is preferred.

[0143] 15 or 17, in the base chip 2a1 or 2a2, the fluid inlet main channel 11a, which is located between the two fluid inlet main channels 11b and 11c located at both ends along the Y axis, is offset from the center line of the mixing channel so as to approach one of the fluid inlet main channels 11b or 11c. The difference between the embodiment shown in Fig. 15 and the embodiment shown in Fig. 17 is that the amount of offset from the center line of the mixing channel is greater in the embodiment shown in Fig. 17 than in the embodiment shown in Fig. 15.

[0144] In this embodiment, the other configurations are the same as those of the second embodiment described above, and similar functions and effects are achieved. A description of the parts common to the previous embodiment will be omitted, and the following description will mainly focus on the parts that are different from the previous embodiment. In the previous embodiment, the main fluid inlet channel 11a of the first fluid inlet channel 10a is configured to be located on an extension of the center line that passes through the center of the mixing channel 20 along the Y axis and extends along the X axis. In this embodiment, the main fluid inlet channel 11a of the first fluid inlet channel 10a is shifted from the center line of the mixing channel 20 to the vicinity of either the main fluid inlet channel 11b or 11c.

[0145] In this embodiment as well, the flow of the first liquid immediately after entering the mixing channel 20 through the fluid inlet main channel 11a of the first fluid inlet channel 10a is asymmetric with respect to the center line of the fluid inlet main channel 11a, so as to be biased toward one of the side walls of the mixing channel 20. In the above-mentioned embodiment, the direction of bias of the flow of the first liquid immediately after entering the mixing channel 20 could be toward either side wall of the mixing channel 20. However, in this embodiment, the direction of bias of the flow of the first liquid immediately after entering the mixing channel 20 is often a direction in which the flow is biased toward the side wall of the mixing channel 20 that is positioned near the fluid inlet main channel 11a, offset from the center line. The other configurations and effects are similar to those of the above-mentioned embodiment.

[0146] In the first to third embodiments described above, the mixing channel 20 is a channel with a rectangular cross section, but this is not a limitation in the embodiments of the present invention. For example, there is no particular limitation as long as the configuration is devised to make the flow of the first liquid in the mixing channel asymmetric so that the flow is biased toward one of the side walls of the mixing channel, in a sealed space. For example, the cross section of the mixing channel 20 is not limited to a rectangle, and may be a quadrangle other than a rectangle, a polygon with more sides than a triangle, a circle, an ellipse, or any other irregular cross-sectional shape.

[0147] The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

[0148] Example 1 To fabricate the microfluidic device 1 shown in FIGS. 1 and 2, a silicon wafer was processed using a deep reactive ion etching apparatus (Sumitomo Precision Products Co., Ltd.: MUC-21 ASE-SRE) to create a mold for the microchannel pattern (groove portion 9) of the base chip 2a. The depth of the groove portion 9 was 0.2 mm. Each of the main fluid inlet channels 11a-11c had a width of 0.1 mm and a length of 5 mm. The spacing (center-to-center distance) between each of the main fluid inlet channels 11a-11c was 0.45 mm. The width of the mixing channel 20 was 1 mm and a length of 10 mm. Each of the main fluid outlet channels 31a-31e had a width of 0.1 mm and a length of 6 mm. The spacing (center-to-center distance) between each of the main fluid outlet channels 31a-31e was 0.2 mm.

[0149] A base chip 2a with a transferred microchannel pattern and a cover chip 2b were fabricated by injection molding at Richell Corporation. The base chip 2a and the cover chip 2b were then stacked and pressed together at 80°C for 1 hour to bond them together. In this manner, the microfluidic device 1 shown in Figures 1 and 2 was fabricated.

[0150] As shown in Figure 1, connectors 70a to 70f were attached to the through-holes 3a to 3f of the cover chip 2b, and the microfluidic device 1 was fixed by sandwiching it from above and below between clamping plates 4a and 4b. Tubes 7a to 7e were connected to the connectors 70a to 70f. Tubes 7a to 7c were PTFE tubes with an inner diameter of 0.5 mm and a length of 1.5 m.

[0151] Tubes 7d and 7e were made of PTFE tubes with an inner diameter of 1 mm and a length of 25 cm. Tube 7d had two branches, 7d1 and 7d2. Tubes 7a to 7c were connected to a syringe pump (Chemyx: Fusion 200).

[0152] 1 and 2, using the fabricated microfluidic device 1, a first liquid was introduced into the mixing channel 20 via the first fluid inlet channel 10a through tube 7b, a second liquid was introduced into the mixing channel 20 via the second fluid inlet channel 10b through tube 7a, and the second liquid was introduced into the mixing channel 20 via the third fluid inlet channel 10c through tube 7c, thereby mixing the first and second liquids. Ethanol was used as the first liquid, and pure water was used as the second liquid. The fluids discharged from the mixing channel 20 via tubes 7d and 7e were collected in a container.

[0153] When introducing the first liquid and the second liquid into the microfluidic device 1, the flow rate ratio of the second liquid (the sum of the second liquid flowing through each of the second fluid inlet flow path 10b and the third fluid inlet flow path 10c) to the first liquid was 10:1, the flow rate ratio of the second liquid flowing through the second fluid inlet flow path 10b to the second liquid flowing through the third fluid inlet flow path 10c was 10:10, the temperature of the mixing flow path 20 was approximately 20°C (room temperature), the total flow rate of the first liquid and the second liquid (the flow rate of the fluid flowing through the mixing flow path 20) was 7.7 mL / min, and the Reynolds number in the mixing flow path 20 was 151.

[0154] The Reynolds number Re in the mixing channel 20 was calculated based on the above-mentioned formula (1). In formula (1), w was set to 1 mm and h was set to 0.2 mm. For ρ and μ, values ​​(literature values ​​or calculated values ​​based on literature values) when ethanol (first liquid) and pure water (second liquid) were completely mixed were used.

[0155] Under the above conditions, the flow state of the fluid flowing through the mixing channel 20 was observed. Observation and photography of the mixing channel 20 were performed using an inverted phase-contrast microscope (Olympus Corporation: CKX-53) equipped with a digital video camera (Sony Corporation: HDR-PJ630). The results are shown in FIG. 5A. As shown in FIG. 5A, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was asymmetric, biased toward one of the side walls of the mixing channel 20.

[0156] This result shows that the first liquid and the second liquid are more easily mixed and the mixing efficiency of the first liquid and the second liquid is improved in the early stage of their flow into the mixing channel 20. Note that in Fig. 5A and subsequent figures, in order to prevent the drawings from becoming too complicated, the reference numerals indicating the first fluid inlet channel 10a, the second fluid inlet channel 10b, the third fluid inlet channel 10c, and the mixing channel 20, and the X, Y, and Z coordinate axes are omitted.

[0157] Example 2 The total flow rate of the first liquid and the second liquid was 8.8 mL / min, and the Reynolds number in the mixing channel 20 was 173. The other experimental conditions were the same as those in Example 1. The results are shown in Figure 5B. As shown in Figure 5B, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20.

[0158] This result indicates that the same effect as in Example 1 can be obtained. The direction of the bias of the first liquid was different (opposite) from the direction of the bias of the first liquid shown in Fig. 5A. However, there were also cases where the direction of the bias of the first liquid was the same as the direction of the bias of the first liquid shown in Fig. 5A.

[0159] Example 3 The total flow rate of the first liquid and the second liquid was 14.3 mL / min, and the Reynolds number in the mixing channel 20 was 281. Other experimental conditions were the same as those in Example 1. The results are shown in FIG. 5C. As shown in FIG. 5C, the flow of the first liquid immediately after it entered the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so that it was biased toward one of the side walls of the mixing channel 20. Furthermore, extremely small vortices were observed immediately after the first liquid entered the mixing channel 20 through the first fluid inlet channel 10a.

[0160] These results indicate that the first and second liquids are more easily mixed in the early stages of flowing into the mixing channel 20, improving the mixing efficiency of the first and second liquids. In addition, the boundaries between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear). This result indicates that mixing of the first and second liquids is promoted even between these regions, further improving the mixing efficiency of the first and second liquids.

[0161] 5C, the microvortex is also unclear. A region where the microvortex is unclear and the boundaries between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) are also unclear is hereinafter referred to as a uniformly mixed region.

[0162] Example 4 The total flow rate of the first liquid and the second liquid was 16.5 mL / min, the Reynolds number in the mixing channel 20 was 306, and the flow rate ratio of the second liquid to the first liquid was 8:1. The other experimental conditions were the same as in Example 1. The results are shown in Figure 5D.

[0163] 5D , the flow of the first liquid immediately after it entered the mixing channel 20 through the first fluid inlet channel 10a was asymmetric so as to be biased more sharply toward one of the side walls of the mixing channel 20. Furthermore, immediately after the first liquid entered the mixing channel 20 through the first fluid inlet channel 10a, two microscopic vortices were observed aligned approximately in the X-axis direction.

[0164] Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappears (becomes unclear) shifted upstream of the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results indicate that the mixing of the first and second liquids progresses at an earlier stage (further upstream of the mixing channel 20).

[0165] Example 5 The total flow rate of the first liquid and the second liquid was 18.5 mL / min, and the Reynolds number in the mixing channel 20 was 345. The other experimental conditions were the same as those in Example 4. The results are shown in Figure 5E. As shown in Figure 5E, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so that it was more sharply biased toward one of the side walls of the mixing channel 20.

[0166] Furthermore, microscopic vortices were observed immediately after the first liquid entered the mixing channel 20 through the first fluid inlet channel 10a. Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear) moved further upstream in the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results indicate that mixing of the first and second liquids proceeds at an earlier stage (further upstream in the mixing channel 20).

[0167] Example 6 The total flow rate of the first liquid and the second liquid was 8.8 mL / min, and the Reynolds number in the mixing channel 20 was 383. As shown in FIG. 3 , the microfluidic device 1, together with the tubes 7a to 7e, was immersed in a constant temperature water bath heated to 55°C, and the temperature of the mixing channel 20 was heated to 55°C. The microfluidic device 1 was removed from the constant temperature water bath to observe and photograph the mixing channel 20. The other experimental conditions were the same as those in Example 1.

[0168] The results are shown in Figure 5F. As shown in Figure 5F, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was asymmetric, biased toward one of the side walls of the mixing channel 20. In addition, the boundaries between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear). These results demonstrate that mixing of the first and second liquids is promoted even between these regions, further improving the mixing efficiency of the first and second liquids.

[0169] Example 7 The total flow rate of the first liquid and the second liquid was 11 mL / min, and the Reynolds number in the mixing channel 20 was 479. The other experimental conditions were the same as those in Example 6. The results are shown in Figure 5G. As shown in Figure 5G, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so that it was biased more sharply toward one of the side walls of the mixing channel 20.

[0170] Furthermore, immediately after the first liquid entered the mixing channel 20 through the first fluid inlet channel 10a, two micro-vortices were formed side by side in approximately the X-axis direction. Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear) shifted toward the upstream side of the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results demonstrate that mixing of the first and second liquids proceeds at an earlier stage (more upstream of the mixing channel 20).

[0171] Example 8 The total flow rate of the first liquid and the second liquid was 13.2 mL / min, and the Reynolds number in the mixing channel 20 was 575. The other experimental conditions were the same as those in Example 6. The results are shown in Figure 5H. As shown in Figure 5H, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so that it was more sharply biased toward one of the side walls of the mixing channel 20.

[0172] Furthermore, microscopic vortices were observed immediately after the first liquid entered the mixing channel 20 through the first fluid inlet channel 10a. Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear) moved further upstream in the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results indicate that mixing of the first and second liquids proceeds at an earlier stage (further upstream in the mixing channel 20).

[0173] Comparative Example 1 The total flow rate of the first liquid and the second liquid was set to 5.5 mL / min, and the Reynolds number in the mixing channel 20 was set to 108. The other experimental conditions were the same as those in Example 1. The results are shown in Figure 5I. As shown in Figure 5I, the flow of the first liquid immediately after it entered the mixing channel 20 through the first fluid inlet channel 10a was symmetrical with respect to the first fluid inlet channel 10a.

[0174] Furthermore, a microvortex symmetrical with respect to the first fluid inlet channel 10a was formed in the mixing channel 20. The above results indicate that the mixing efficiency of the first liquid and the second liquid cannot be sufficient, and that it may be difficult to achieve rapid and uniform mixing of the first liquid and the second liquid.

[0175] Comparative Example 2 The total flow rate of the first liquid and the second liquid was set to 6.6 mL / min, and the Reynolds number in the mixing channel 20 was set to 130. The other experimental conditions were the same as in Example 1. The results are shown in Figure 5J. As shown in Figure 5J, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was symmetrical with respect to the first fluid inlet channel 10a.

[0176] Furthermore, a microvortex symmetrical with respect to the first fluid inlet channel 10a was formed in the mixing channel 20. The above results indicate that the mixing efficiency of the first liquid and the second liquid cannot be sufficient, and that it may be difficult to achieve rapid and uniform mixing of the first liquid and the second liquid.

[0177] Example 9 The flow rate ratio between the second liquid flowing through the second fluid inlet channel 10b and the second liquid flowing through the third fluid inlet channel 10c was 10:7 (more precisely, 10:6.77), the flow rate ratio between the second liquid (the total of the second liquid flowing through each of the second fluid inlet channel 10b and the third fluid inlet channel 10c) and the first liquid was 8.5:1, the total flow rate of the first liquid and the second liquid was 9.4 mL / min, and the Reynolds number in the mixing channel 20 was 175. The other experimental conditions were the same as those in Example 1.

[0178] The results are shown in Fig. 6A. As shown in Fig. 6A, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20. This result showed that in the early stage when the first liquid and the second liquid flow into the mixing channel 20, the first liquid and the second liquid are more easily mixed, improving the mixing efficiency of the first liquid and the second liquid.

[0179] Example 10 The total flow rate of the first liquid and the second liquid was 4.7 mL / min, and the Reynolds number in the mixing channel 20 was 197. The temperature of the mixing channel 20 was 55°C. Other experimental conditions were the same as those in Example 9. The results are shown in FIG. 6B. As shown in FIG. 6B, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was asymmetric, biased toward one of the side walls of the mixing channel 20. This result indicated that the same effect as in Example 9 could be obtained.

[0180] Example 11 The total flow rate of the first liquid and the second liquid was set to 7.5 mL / min, and the Reynolds number in the mixing channel 20 was set to 316. The other experimental conditions were the same as those in Example 10. The results are shown in Figure 6C. As shown in Figure 6C, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20.

[0181] Furthermore, the boundaries between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappeared (became unclear). These results demonstrate that mixing of the first and second liquids is promoted even between these regions, further improving the mixing efficiency of the first and second liquids.

[0182] Example 12 The total flow rate of the first liquid and the second liquid was 9.4 mL / min, and the Reynolds number in the mixing channel 20 was 395. The other experimental conditions were the same as those in Example 10. The results are shown in Figure 6D. As shown in Figure 6D, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20.

[0183] Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappears (becomes unclear) moved further upstream in the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results indicate that the mixing of the first and second liquids proceeds at an earlier stage (further upstream in the mixing channel 20).

[0184] Example 13 The total flow rate of the first liquid and the second liquid was 11.3 mL / min, and the Reynolds number in the mixing channel 20 was 474. The other experimental conditions were the same as those in Example 10. The results are shown in Figure 6E. As shown in Figure 6E, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20.

[0185] Furthermore, the position where the boundary between the microvortex region 50 and the regions on either side of it (regions mainly containing the second liquid) disappears (becomes unclear) moved further upstream in the mixing channel 20. It was also confirmed that a uniformly mixed region was obtained from the upstream side. These results indicate that mixing of the first and second liquids progresses at an earlier stage (upstream of the mixing channel 20).

[0186] Example 14 The total flow rate of the first liquid and the second liquid was 5.5 mL / min, and the Reynolds number in the mixing channel 20 was 240. The temperature of the mixing channel 20 was 55°C. Other experimental conditions were the same as those in Example 1. The results are shown in FIG. 7. As shown in FIG. 7, when the temperature of the mixing channel 20 was set to 55°C, unlike Comparative Example 1 in which the temperature of the mixing channel 20 was set to 20°C, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric, biased toward one of the side walls of the mixing channel 20.

[0187] These results show that when the total flow rates of the first liquid and the second liquid are approximately the same, by heating the temperature of the mixing flow path 20 to 55°C, the first liquid and the second liquid become more easily mixed in the initial stage when they flow into the mixing flow path 20, and the mixing efficiency of the first liquid and the second liquid is improved.

[0188] Example 15 As described below, a first liquid containing distearoyl phosphatidylcholine (DSPC) as a phospholipid and a second liquid containing apolipoprotein AI (apoA-I) as a protein were mixed using microfluidic device 1 to prepare reconstituted high-density lipoprotein (rHDL).

[0189] The first liquid was a 5 mg / mL lipid solution containing DSPC dissolved in ethanol. The second liquid was a 200 μg / mL protein solution containing apolipoprotein AI dissolved in phosphate-buffered saline (PBS) containing 2 M urea. A mutant apolipoprotein AI lacking 43 amino acids at its N-terminus was used. The density of the second liquid (2 M urea solution) at 25°C was approximately 1.03, and the viscosity was approximately 1.09. As shown in Figure 3, the microfluidic device 1, together with tubes 7a-7e, was immersed in a thermostatic water bath heated to 55°C, and the temperature of the mixing channel 20 was raised to 55°C, the phase transition temperature of DSPC.

[0190] As shown in FIGS. 1 to 3 , using the fabricated microfluidic device 1 (the same microfluidic device 1 as in Example 1), a first liquid was introduced into mixing channel 20 via tube 7b and first fluid inlet channel 10a. Furthermore, a second liquid was introduced into mixing channel 20 via tube 7a and second fluid inlet channel 10b. Furthermore, the second liquid was introduced into mixing channel 20 via tube 7c and third fluid inlet channel 10c. In this way, the first liquid and the second liquid were mixed in mixing channel 20. Then, the fluids discharged from mixing channel 20 via tubes 7d and 7e were collected in a container.

[0191] When the first liquid and the second liquid were introduced into the microfluidic device 1, the flow rate ratio of the second liquid to the first liquid was set to 10:1, the flow rate ratio of the second liquid flowing through the second fluid inlet channel 10b to the second liquid flowing through the third fluid inlet channel 10c was set to 10:10, and the temperature of the mixing channel 20 was set to approximately 55° C. The total flow rates of the first liquid and the second liquid were set to 5.5 mL / min, 7.7 mL / min, 8.8 mL / min, 11 mL / min, and 13.2 mL / min, respectively.

[0192] When the total flow rate of the first liquid and the second liquid is 5.5 mL / min, the Reynolds number in the mixing channel 20 is 228. When the total flow rate of the first liquid and the second liquid is 7.7 mL / min, the Reynolds number in the mixing channel 20 is 319. When the total flow rate of the first liquid and the second liquid is 8.8 mL / min, the Reynolds number in the mixing channel 20 is 364. When the total flow rate of the first liquid and the second liquid is 11 mL / min, the Reynolds number in the mixing channel 20 is 456. When the total flow rate of the first liquid and the second liquid is 13.2 mL / min, the Reynolds number in the mixing channel 20 is 547.

[0193] The recovered solution was dialyzed against 2 L of PBS at room temperature for 2 hours using a dialysis membrane with a molecular weight cutoff of 3 kDa (Spectra / Por 6 Pre-Wetted RC dialysis membrane, Spectrum Chemical). The resulting sample was centrifuged at 20,000 x g and 24°C for 15 minutes to obtain the supernatant. This supernatant was purified and analyzed by size exclusion chromatography (SEC). Purification and analysis were performed by passing the supernatant through a column (GE Healthcare: HiLoad 16 / 60 Superdex 200 prepgrade) attached to a high-performance liquid chromatograph (Shimadzu: Prominence HPLC system) at a flow rate of 1 mL / min with PBS at room temperature, and measuring the absorbance at 280 nm of the eluate from the column.

[0194] The mean hydrodynamic diameter of the rHDL samples was measured using a particle size analyzer (Microtrac Bell: Nanotrac UPA-UT151), and the protein content of the rHDL was analyzed using a Bio-Rad DC Protein Assay Kit and a BioTek Instrument: Synergy HTX Multi-Mode Microplate Reader, using bovine serum albumin (Thermo Fisher Scientific) as a standard.

[0195] The lipid content of rHDL was analyzed using a phospholipid assay kit (Fujifilm Wako Pure Chemical Corporation: Phospholipid C assay kit) and a multimode plate reader (BioTek Instrument: Synergy HTX Multi-Mode Microplate Reader). The particle surface structure was also observed using an atomic force microscope (AFM). AFM observations were performed using a Shimadzu SPM-9700HT with a cantilever (Olympus: BL-AC40TS). A drop of rHDL sample was placed on the mica surface and allowed to stand for 4 minutes. The suspended sample was then rinsed with pure water, and the rHDL sample adsorbed on the mica surface in pure water was observed using AFM.

[0196] The results of size exclusion chromatography at the total flow rates (TFR) of the first and second liquids were set to 5.5 mL / min, 7.7 mL / min, 8.8 mL / min, 11 mL / min, and 13.2 mL / min, respectively, are shown in Figure 8. Table 1 also shows the mean hydrodynamic diameter, yield, and percentage of free protein (protein remaining unbound to lipids) for each flow rate.

[0197] Table 2 also shows the mean hydrodynamic diameter, yield, and lipid to protein molar ratio (lipid / protein) for each peak (peak at retention time around 50 minutes and peak at retention time around 60 minutes) when the total flow rate of the first and second liquids was set to 13.2 mL / min. Note that the results when the total flow rate of the first and second liquids was set to 13.2 mL / min are the average values ​​of three experiments (n = 3).

[0198]

[0199]

[0200] As shown in Figure 8, as the total flow rate of the first and second liquids increased, the number of large particles, represented by a peak around 45 minutes of retention time, decreased, while the number of small particles, represented by a peak around 60 minutes of retention time, increased. Furthermore, as shown in Table 1, as the total flow rate of the first and second liquids increased, the average hydrodynamic diameter decreased. Furthermore, as shown in Table 1, when the total flow rate of the first and second liquids was 13.2 mL / min, the average hydrodynamic diameter was 14.4 ± 0.2 nm.

[0201] Furthermore, as shown in Table 2, when the total flow rate of the first and second liquids was 13.2 mL / min, the average hydrodynamic diameter was 15.4±1.2 nm at a retention time of around 50 minutes (retention time 43 to 55 minutes). On the other hand, the average hydrodynamic diameter was 9.8±0.8 nm at a retention time of around 60 minutes (retention time 55 to 70 minutes). Furthermore, AFM confirmed that the small particles represented by the peaks at retention times around 50 and 60 minutes had a disk-like structure.

[0202] These results confirmed that the small particles represented by the peaks at retention times around 50 and 60 minutes were rHDL containing DSPC as the lipid (DSPC-rHDL). Thus, it was confirmed that the nanoparticle production method of the present invention can reconstitute DSPC-rHDL, which was difficult to produce using conventional nanoparticle production methods using microfluidic devices.

[0203] It was also confirmed that the nanoparticle production method of the present invention is suitable for producing minute nanoparticles with a particle size of about 10 nm. Furthermore, it is believed that the nanoparticle production method of the present invention produces minute disk-shaped fragments (small pieces of lipid bilayer membrane) with a size of about 10 nm, and that apolipoprotein AI rapidly binds to these disk-shaped fragments to produce DSPC-rHDL.

[0204] Furthermore, as shown in Figure 8, it was confirmed that as the total flow rate of the first and second liquids increased, the number of small particles, represented by the peak at retention time around 77 minutes, decreased. It was also confirmed that the small particles, represented by the peak at retention time around 77 minutes, were free protein. Furthermore, as shown in Table 1, it was confirmed that as the total flow rate of the first and second liquids increased, the yield increased while the proportion of free protein decreased. This confirmed that the method for producing nanoparticles according to the present invention improved the mixing efficiency of the first and second liquids and promoted rapid and uniform mixing of the first and second liquids.

[0205] Example 16 The efficiency of DSPC-rHDL production was evaluated when the temperature of the mixing channel 20 was set to approximately 45°C and approximately 55°C as follows. As the first liquid, a 2.5 mg / mL lipid solution was prepared by dissolving DSPC in ethanol. As the second liquid, a 100 μg / mL protein solution was prepared by dissolving apolipoprotein AI in PBS containing 2 M urea.

[0206] However, a mutant apolipoprotein AI with 43 amino acids deleted at the N-terminus was used. The density of the second liquid (2 M urea solution) at 25°C was approximately 1.03, and the viscosity was approximately 1.09. As shown in Figure 3, the microfluidic device 1, together with tubes 7a to 7e, was immersed in a thermostatic water bath heated to approximately 45°C or approximately 55°C, and the temperature of the mixing channel 20 was heated to approximately 45°C or approximately 55°C.

[0207] 1 to 3, using the fabricated microfluidic device 1 (similar to the microfluidic device 1 in Example 1), a first liquid was introduced at a flow rate of 1 mL / min through the tube 7b and via the first fluid inlet channel 10a into the mixing channel 20. Furthermore, a second liquid was introduced at a flow rate of 5 mL / min through the tube 7a and via the second fluid inlet channel 10b into the mixing channel 20.

[0208] Furthermore, the second liquid was introduced into the mixing channel 20 at a flow rate of 5 mL / min through the tube 7c and via the third fluid inlet channel 10c. This caused the first liquid and the second liquid to mix in the mixing channel 20. Then, the fluid discharged from the mixing channel 20 through the tubes 7d and 7e was collected in a container. The Reynolds number in the mixing channel 20 when the temperature of the mixing channel 20 was approximately 45°C was 377. The Reynolds number in the mixing channel 20 when the temperature of the mixing channel 20 was approximately 55°C was 456.

[0209] The recovered solution was dialyzed against PBS for 2 hours using the same method as in Example 15. The resulting sample was centrifuged at 20,000 x g and 24°C for 15 minutes to obtain the supernatant. This supernatant was then analyzed by size exclusion chromatography (SLC). The analysis was performed by passing the supernatant through a column (Cytiva: Superdex 200 Increase 5 / 150 GL) attached to a high-performance liquid chromatograph (Shimadzu: Prominence HPLC System) at a flow rate of 0.15 mL / min at room temperature using PBS, and measuring the absorbance at 280 nm of the eluate. The results of SLC analysis when the temperature of the mixing channel 20 was set to approximately 45°C and approximately 55°C are shown in Figure 9.

[0210] 9, when the temperature of the mixing flow channel 20 was about 45° C. and about 55° C., a large peak was observed near the retention time of 10 minutes, confirming that small particles were generated, as represented by the peak near the retention time of 10 minutes. Furthermore, when the temperature of the mixing flow channel 20 was about 55° C., the peak near the retention time of 10 minutes was larger than when the temperature of the mixing flow channel 20 was about 45° C.

[0211] In the above column, the peak at a retention time of approximately 10 minutes is attributed to DSPC-rHDL of approximately 10 nm. Therefore, it was confirmed that DSPC-rHDL of approximately 10 nm was produced when the temperature of the mixing channel 20 was approximately 45°C or approximately 55°C. It was also confirmed that the efficiency of DSPC-rHDL production was improved by setting the temperature of the mixing channel 20 to approximately 55°C.

[0212] In the nanoparticle manufacturing method of the present invention, by setting the temperature of the mixing channel 20 to approximately 45°C or higher, it becomes less likely that clumps of DSPC contained in the first liquid will form in the mixing channel, and it becomes easier to form tiny nanoparticles called DSPC-rHDL.

[0213] Example 17 A microfluidic device 1 similar to that of Example 1 was fabricated, except for the following points. In the microfluidic device 1 of this example, PTFE tubes with an inner diameter of 0.25 mm and a length of 25 cm were used as the tubes 7d and 7e shown in Figures 1 and 3. Furthermore, as shown in Figures 1 to 3, tube clamps were fixed to the tubes 7d1 and 7d2 to block the main fluid outlet channels 31a, 31b, 31d, and 31e so that the fluid discharged from the mixing channel 20 would not flow through the main fluid outlet channels 31a, 31b, 31d, and 31e.

[0214] The first liquid was a 1 mg / mL lipid solution prepared by dissolving DSPC in ethanol. The second liquid was a 40 μg / mL protein solution prepared by dissolving apolipoprotein AI in PBS containing 2 M urea. A mutant apolipoprotein AI lacking 43 amino acids at its N-terminus was used. The density of the second liquid (2 M urea solution) at 25°C was approximately 1.03, and the viscosity was approximately 1.09. As shown in Figure 3, the microfluidic device 1, together with tubes 7a-7e, was immersed in a thermostatic water bath heated to approximately 55°C, and the temperature of the mixing channel 20 was heated to approximately 55°C.

[0215] Using the fabricated microfluidic device 1, a first liquid was introduced at a flow rate of 0.5 mL / min through the tube 7b and via the first fluid inlet channel 10a into the mixing channel 20. Furthermore, a second liquid was introduced at a flow rate of 2.5 mL / min through the tube 7a and via the second fluid inlet channel 10b into the mixing channel 20.

[0216] Furthermore, the second liquid was introduced into the mixing channel 20 through the tube 7c and the third fluid inlet channel 10c at a flow rate of 2.5 mL / min. This caused the first liquid and the second liquid to be mixed in the mixing channel 20. Then, the fluid discharged from the mixing channel 20 through the tube 7e and the main fluid outlet channel 31c was collected in a container. The Reynolds number in the mixing channel 20 was set to 228.

[0217] Under these conditions, the flow state of the fluid flowing through the mixing channel 20 was observed. The results are shown in Figure 10. Furthermore, in the same manner as in Example 15, the recovered solution was dialyzed against PBS for 2 hours, and the resulting sample was centrifuged at 20,000 xg and 24°C for 15 minutes to obtain a supernatant. This supernatant was then analyzed by size exclusion chromatography using a column (GE Healthcare: HiLoad 16 / 60 Superdex 200 prepgrade). The results are shown in Figure 11.

[0218] 10 , it was confirmed that the fluids (first liquid and second liquid) flowing through the mixing channel 20 are discharged from the mixing channel 20 through the fluid outlet main channel 31c (see FIG. 2 ). When the fluid outlet main channels 31a, 31b, 31d, and 31e are blocked, the flow velocity of the fluid in the fluid outlet main channel 31c increases by approximately 10 times, and the Reynolds number in the fluid outlet main channel 31c becomes approximately 911.

[0219] Although detailed illustration is omitted, even when the main fluid outlet channels 31a, 31b, 31d, and 31e were blocked, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a was made asymmetric so as to be biased toward one of the side walls of the mixing channel 20. On the other hand, the boundaries between the microvortex region 50 and the regions on both sides thereof (regions mainly containing the second liquid) did not disappear.

[0220] 11, a large peak was observed near the retention time of 50 minutes, confirming that particles with small particle diameters, represented by the peak near the retention time of 50 minutes, were generated. Also, a large peak was observed near the retention time of 60 minutes, confirming that particles with small particle diameters, represented by the peak near the retention time of 60 minutes, were generated.

[0221] In the above column, the peak at a retention time of approximately 50 minutes is attributed to DSPC-rHDL of approximately 15 nm, and the peak at a retention time of approximately 60 minutes is attributed to DSPC-rHDL of approximately 10 nm. Thus, it was confirmed that narrowing the fluid outlet channel 30 (i.e., blocking the main fluid outlet channels 31a, 31b, 31d, and 31e so that the fluid flows only through the single main fluid outlet channel 31c) produces DSPC-rHDL of approximately 10 nm. This is thought to be due to an increase in the Reynolds number in the fluid outlet channel 30 (main fluid outlet channel 31c).

[0222] Example 18: Instead of the microfluidic device 1 shown in FIGS. 1 and 2, a microfluidic device 1a shown in FIGS. 12 and 13 was fabricated, and the flow state of the fluid flowing through the mixing channel 20 was observed in the same manner as in Example 1, except that the following conditions were adopted.

[0223] In this example, the depth of the groove 9a was 0.2 mm, as in Example 1, the width of each of the fluid inlet main channels 11a to 11c was 0.1 mm, and the length of each of the fluid inlet main channels 11b and 11c was approximately 5 mm, but the fluid inlet main channel 11a was longer than each of the fluid inlet main channels 11b and 11c. The width of each of the extension channels 14a to 14c was 1 mm. The spacing (center-to-center distance) between each of the fluid inlet main channels 11a to 11c was 0.45 mm. The width of the mixing channel 20 was 1 mm, and the length was 24 mm. No fluid outlet channel 30 was provided.

[0224] When the first and second liquids were introduced into the microfluidic device 1a, the flow rate ratio of the second liquid (the sum of the second liquids flowing through the second fluid inlet channel 10b and the third fluid inlet channel 10c) to the first liquid was 10:1, the flow rate ratio of the second liquid flowing through the second fluid inlet channel 10b to the second liquid flowing through the third fluid inlet channel 10c was 10:10, the temperature of the mixing channel 20 was approximately 55°C, the total flow rate of the first and second liquids (the flow rate of the fluids flowing through the mixing channel 20) was 33 mL / min, and the Reynolds number in the mixing channel 20 was 1437. The first liquid, consisting of ethanol, was delivered using a syringe pump (Chemyx: Fusion 200), and the second liquid, consisting of pure water, was delivered using a syringe pump (Chemyx: Fusion 6000). The results are shown in FIG. 14A.

[0225] 14A is similar to the flow shown in Fig. 5H, and it was confirmed that the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes asymmetric, biasing more sharply toward one of the side walls of the mixing channel 20. This result shows that, similar to Example 8 and the like, the mixing of the first and second liquids proceeds at an earlier stage (further upstream of the mixing channel 20).

[0226] 4B disappears and a uniformly mixed region is easily formed early on the upstream side in the initial stage when the first liquid and the second liquid flow into the mixing channel 20. This indicates that the first liquid and the second liquid are easily mixed, improving the mixing efficiency of the first liquid and the second liquid.

[0227] Example 19 The total flow rate of the first liquid and the second liquid was set to 55 mL / min, and the Reynolds number in the mixing channel 20 was set to 2395. The other experimental conditions were the same as those in Example 18. The results are shown in FIG. 14B. As shown in FIG. 14B, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a is made asymmetric so as to be biased more sharply toward one of the side walls of the mixing channel 20 compared to FIG. 14A.

[0228] 4B disappears, and a uniformly mixed region is easily formed early on the upstream side, in an even earlier stage when the first and second liquids flow into the mixing channel 20. This indicates that the first and second liquids are easily mixed, improving the mixing efficiency of the first and second liquids.

[0229] Example 20 The total flow rate of the first liquid and the second liquid was 55 mL / min, the temperature of the mixing channel 20 was approximately 20°C, and the Reynolds number in the mixing channel 20 was 1081. The other experimental conditions were the same as those in Example 18. The results are shown in Figure 14C. As shown in Figure 14C, the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a is asymmetrical, being sharply biased toward one of the side walls of the mixing channel 20 in a manner similar to that shown in Figure 14A.

[0230] Furthermore, even at around room temperature of 20° C., it was confirmed that by setting the Reynolds number to, for example, 1000 or more, the boundary 70 as shown in Fig. 4B disappears and a uniformly mixed region is easily formed early on the upstream side in the early stage when the first liquid and the second liquid flow into the mixing channel 20. Therefore, it was shown that the first liquid and the second liquid are easily mixed, and the mixing efficiency of the first liquid and the second liquid is improved.

[0231] Example 21 A microfluidic device was fabricated using the base chip 2a1 shown in Figure 15 instead of the base chip 2a shown in Figure 13, and the flow state of the fluid flowing through the mixing channel 20 was observed in the same manner as in Example 18, except that the conditions shown below were adopted.

[0232] In this example, the center line of the fluid inlet main channel 11a (which is long along the X-axis) was shifted by approximately 0.1 mm in the Y-axis direction relative to the center line of the mixing channel 20 so as to be closer to the fluid inlet main channel 11c. The temperature of the mixing channel 20 was set to approximately 20°C, the total flow rate of the first and second liquids (the flow rate of the fluids flowing through the mixing channel 20) was set to 55 mL / min, and the Reynolds number in the mixing channel 20 was set to 1081. The results are shown in FIG.

[0233] 16 is similar to the flow shown in Fig. 14C , and it was confirmed that the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes asymmetric, biasing more sharply toward one of the side walls of the mixing channel 20. This result shows that, similar to Fig. 14C etc., mixing of the first and second liquids proceeds at an earlier stage (further upstream of the mixing channel 20).

[0234] 4B disappears and a uniformly mixed region is easily formed early on the upstream side in the initial stage when the first liquid and the second liquid flow into the mixing channel 20. This indicates that the first liquid and the second liquid are easily mixed, improving the mixing efficiency of the first liquid and the second liquid.

[0235] Example 22 A microfluidic device was fabricated using the base chip 2a2 shown in Figure 17 instead of the base chip 2a1 shown in Figure 15, and the flow state of the fluid flowing through the mixing channel 20 was observed in the same manner as in Example 21, except that the conditions shown below were adopted.

[0236] In this example, the center line of the main fluid inlet channel 11a (long along the X-axis) was shifted by approximately 0.25 mm in the Y-axis direction with respect to the center line of the mixing channel 20 so as to approach the main fluid inlet channel 11c. The results are shown in Figure 18.

[0237] The flow shown in Figure 18 is similar to the flow shown in Figure 16, and it was confirmed that the flow of the first liquid immediately after entering the mixing channel 20 through the first fluid inlet channel 10a becomes asymmetric, biasing more sharply toward one of the side walls of the mixing channel 20. This result shows that, similar to Figure 16 etc., mixing of the first liquid and the second liquid proceeds at an earlier stage (further upstream of the mixing channel 20).

[0238] 4B disappears and a uniformly mixed region is easily formed early on the upstream side in the initial stage when the first liquid and the second liquid flow into the mixing channel 20. This indicates that the first liquid and the second liquid are easily mixed, improving the mixing efficiency of the first liquid and the second liquid.

[0239] Example 23: A microfluidic device 1a shown in FIGS. 12 and 13 was prepared instead of the microfluidic device 1 shown in FIGS. 1 and 2, and reconstituted high-density lipoprotein (rHDL) was prepared in the same manner as in Example 15, except that the following conditions were employed:

[0240] The first liquid was a 5 mg / mL lipid solution prepared by dissolving DSPC in ethanol. The second liquid was a 200 μg / mL protein solution prepared by dissolving apolipoprotein AI in phosphate-buffered saline (PBS) containing 2 M urea. The density of the second liquid (2 M urea solution) at 25°C was approximately 1.03, and the viscosity was approximately 1.09. The temperature of the mixing channel 20 was approximately 20°C. The flow rates of the first liquid and the second liquid were 5 mL / min and 50 mL / min, respectively, resulting in a total flow rate of 55 mL / min. The Reynolds number in the mixing channel 20 was approximately 1028.

[0241] The recovered solution was placed in a centrifugal filter unit (Merck: Amicon Ultra-15, 50 kDa) and centrifuged at 4°C, 5000 g for 20 minutes to concentrate it approximately 22-fold, and then diluted approximately 27-fold with PBS to reduce the ethanol concentration to approximately 0.3% before analysis. The results of size exclusion chromatography are shown in Figure 19.

[0242] The column used was an empty column (Cytiva: XK 16 / 70 column) packed with a carrier (Cytiva: Superdex 200 prepgrade). The retention times were divided by 1.06 to correct for the difference in retention time compared to the HiLoad 16 / 60 Superdex 200 prepgrade. The peak at a retention time of approximately 60 minutes was dominant, confirming the efficient production of small particles. The mean hydrodynamic diameter was measured using a particle size analyzer and found to be 9.6 nm. The yield based on protein was 96%.

[0243] The results of Example 15 confirm that the small particles represented by the peak at a retention time of approximately 60 minutes are rHDL using DSPC as the lipid (DSPC-rHDL). Therefore, the small particles represented by the peak at a retention time of approximately 60 minutes in Figure 19 obtained in this embodiment are thought to be rHDL using DSPC as the lipid (DSPC-rHDL).

[0244] Thus, it was confirmed that the nanoparticle manufacturing method of this example makes it possible to reconstitute DSPC-rHDL even at near room temperature, which was difficult to produce using conventional nanoparticle manufacturing methods using microfluidic devices.

[0245] Example 24 Lipid nanoparticles other than DSPC-rHDL, lipid nanoparticles composed of DSPC and cholesterol, were prepared in the same manner as in Example 23, except for the following conditions. Ethanol containing 10 mg / mL DSPC and 4.89 mg / mL cholesterol was flowed at 5 mL / min as the first liquid, and PBS was flowed at 50 mL / min as the second liquid. The total flow rate of the first and second liquids was 55 mL / min, and the temperature of the mixing channel was approximately 20°C. After dialysis, the average hydrodynamic diameter was measured to be 30.1 nm. This demonstrated that lipid nanoparticles composed of DSPC and cholesterol can be prepared at around room temperature.

[0246] DESCRIPTION OF SYMBOLS 1, 1a...Microfluidic device 2a, 2a1, 2a2...Base chip 2b...Cover chip 3a to 3g...Through-hole 4a, 4b...Clip plates 5a to 5g...Through-hole 6...Screw hole 7a to 7d, 7d1, 7d2, 7e, 7g...Tube 70a to 70g...Connector 8a...Screw 8b...Nut 9, 9a...Groove portion 10a to 10c...First fluid inlet channel to third fluid inlet channel 11a to 11c...Fluid inlet main channel 12a to 12c...Downstream expansion channel 13a to 13c...Upstream expansion channel 14a to 14c...Expansion channel 20...Mixing channel 30...Fluid outlet channel 31a to 31e...Fluid outlet main channel 32a to 32c...Outlet expansion channel 40a to 40g...End portion 50...Microvortex region 60...First boundary 70...Second boundary

Claims

1. A method for producing nanoparticles, comprising the steps of: introducing a first liquid containing at least one material into a mixing channel through a first fluid inlet channel of a microfluidic device; and introducing a second liquid into the mixing channel through a second fluid inlet channel and a third fluid inlet channel on either side of the first fluid inlet channel; setting the Reynolds number in the mixing channel to a predetermined value or greater; and making the flow of the first liquid in the mixing channel asymmetric so that the flow of the first liquid immediately after entering the mixing channel through the first fluid inlet channel is biased toward one of the side walls of the mixing channel.

2. The method for producing nanoparticles according to claim 1, wherein the Reynolds number is 151 or more.

3. The method for producing nanoparticles according to claim 2, wherein the Reynolds number is 281 or more.

4. A method for producing nanoparticles described in any of claims 1 to 3, wherein the distance between the first fluid inlet flow path and the second fluid inlet flow path is greater than the width of each of the first fluid inlet flow path, the second fluid inlet flow path and the third fluid inlet flow path, and the distance between the first fluid inlet flow path and the third fluid inlet flow path is greater than the width of each of the first fluid inlet flow path, the second fluid inlet flow path and the third fluid inlet flow path.

5. A method for producing nanoparticles described in any one of claims 1 to 3, further comprising a step of discharging the first liquid and the second liquid from the mixing channel through a fluid outlet channel located downstream of the mixing channel.

6. The method for producing nanoparticles according to claim 5, wherein the fluid outlet channel comprises a single fluid outlet channel having a width narrower than a width of the mixing channel.

7. A method for producing nanoparticles according to any one of claims 1 to 3, wherein the temperature of the mixing channel is 45°C or higher.

8. A method for producing nanoparticles according to any one of claims 1 to 3, wherein the temperature of the mixing channel is room temperature or higher.

9. A method for producing nanoparticles according to any one of claims 1 to 3, wherein at least one of said materials comprises a lipid.

10. A method for producing nanoparticles according to any one of claims 1 to 3, wherein the second liquid is an aqueous solvent and contains at least one water-soluble material.

11. The method of claim 10, wherein the at least one water-soluble material comprises at least one of a protein and a peptide.

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