WO2023153404A1 - Structure de trajet d'écoulement et procédé de fabrication de particules de matériau à auto-organisation l'utilisant - Google Patents

Structure de trajet d'écoulement et procédé de fabrication de particules de matériau à auto-organisation l'utilisant Download PDF

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WO2023153404A1
WO2023153404A1 PCT/JP2023/004007 JP2023004007W WO2023153404A1 WO 2023153404 A1 WO2023153404 A1 WO 2023153404A1 JP 2023004007 W JP2023004007 W JP 2023004007W WO 2023153404 A1 WO2023153404 A1 WO 2023153404A1
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channel
self
flow path
flow
channel structure
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PCT/JP2023/004007
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English (en)
Japanese (ja)
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正寿 真栄城
学 渡慶次
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国立大学法人北海道大学
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Publication of WO2023153404A1 publication Critical patent/WO2023153404A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J13/00—Colloid chemistry, e.g. the production of colloidal materials or their solutions, not otherwise provided for; Making microcapsules or microballoons
    • B01J13/02—Making microcapsules or microballoons
    • B01J13/04—Making microcapsules or microballoons by physical processes, e.g. drying, spraying
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J19/00—Chemical, physical or physico-chemical processes in general; Their relevant apparatus

Definitions

  • the present invention relates to a channel structure and a method for producing self-organizing material particles using the same.
  • CROSS REFERENCE TO RELATED APPLICATIONS This application claims priority from Japanese Patent Application No. 2022-17619 filed on February 8, 2022, the entire disclosure of which is specifically incorporated herein by reference.
  • Amphiphilic molecules such as lipids self-assemble multiple lipid molecules in a polar solvent such as water, exposing the polar groups of each lipid molecule to the solvent side (nonpolar groups are isolated from the solvent). ) particles (lipid nanoparticles).
  • lipid nanoparticles In recent years, such self-assembling material particles have been put to practical use as nucleic acid delivery carrier particles.
  • SARS-CoV-2 novel coronavirus
  • mRNA vaccine that was granted emergency use in 2020 also uses lipid nanoparticles as mRNA delivery carriers. Recently, it has been reported that nucleic acid delivery efficiency and activity differ depending on the particle size of lipid nanoparticles.
  • microfluidic devices have attracted attention as a fabrication method for precisely controlling the particle size of lipid nanoparticles (Patent Documents 1 and 2).
  • Patent Documents 1 and 2 By using a microfluidic device, it is possible to control the particle size of lipid nanoparticles in a flow-dependent manner, and it is also used in the development of Onpattro (registered trademark), the world's first siRNA preparation.
  • Patent Document 1 WO2018/190423
  • Patent Document 2 WO2021/064998
  • Patent Document 3 Special table 2019-503271 (WO2017/117647)
  • the microfluidic devices reported so far have the problem that the particle size becomes small when the particles are produced under high flow conditions for mass production.
  • the average particle size is large, it is also possible to selectively deliver drugs to different tissues than small particles. be. Therefore, there is a great demand for lipid nanoparticles with a relatively large average particle size and high size uniformity.
  • a similar demand is expected in the future for self-assembled material particles made from materials other than lipids.
  • lipid nanoparticles of around 100 nm can be prepared at a total flow rate of 100 ⁇ L/min under the conditions used in the study by the present inventors.
  • the average particle size of the lipid nanoparticles obtained at a total flow rate of 500 ⁇ L/min was around 30 nm.
  • the particle size of the lipid nanoparticles obtained at a total flow rate of 2 to 10 mL/min was all about 80 nm or less.
  • the microfluidic devices described in Patent Documents 1 and 2 have a structure in which the channel width (cross-sectional area) intermittently changes by half or less midway. Therefore, under high flow conditions, the pressure loss increases and the device may be damaged, making it unsuitable for mass production of lipid nanoparticles, which requires a high flow rate.
  • the problem to be solved by the present invention and the object of the present invention are self-organization such as relatively large lipid nanoparticles with an average particle size of 100 nm or more even when the flow rate is relatively high, for example, the total flow rate is 2 mL / min.
  • An object of the present invention is to provide a fluid structure capable of producing organic material particles, and a method for producing self-organizing material particles such as lipid nanoparticles using this fluid structure.
  • the average particle diameter in the specification of the present application means the Z average particle diameter defined in ISO 13321.
  • the present invention is based on the principle of forming self-assembled material particles, and does not intend to shorten the time required for complete mixing of two liquids as much as possible as in Patent Document 3, for example. This is the result of an investigation into a channel shape that can achieve a relatively high flow rate while considering the dilution time of the substance-containing solution. Specifically, it is as follows.
  • the cross-sectional area of the straight portion of the flow path B (20) is constant or varies periodically from the inlet (11) to the confluence (30) or the outlet (22), [1]- The channel structure according to any one of [7].
  • the crossing angle of the flow paths A and B (10, 20) is such that the angle ⁇ formed by the center line of the flow path A and the center line of the flow path B is in the range of 30° to 150°.
  • the widths of channels A and B (10, 20) each independently range from 10 to 2000 ⁇ m, and the heights of channels A and B (10, 20) each independently range from 10 to 1000 ⁇ m.
  • the channel structure according to any one of [1] to [9].
  • the planar shape of the outer edge and inner edge of the bent portion of the flow path A (10) that is two-dimensionally or three-dimensionally bent is polygonal or substantially circular, According to any one of [1] to [10], wherein the planar shape of the outer edge and inner edge of the bent portion of the flow path B (20) that is two-dimensionally or three-dimensionally bent is polygonal or substantially circular. channel structure.
  • a substrate A having a continuous groove A for the flow channel A and at least one main surface of the substrate having a flat main surface, for the flow channel B It has a structure in which a substrate B having a continuous groove B is laminated so that the continuous groove A and the continuous groove B face each other,
  • the flow path A is two-dimensionally regularly or irregularly curved or straight
  • the flow path B is two-dimensionally regularly or irregularly curved or straight.
  • a method for producing self-assembling material particles comprising the step of diluting a self-assembling material-containing solution with a diluent medium to obtain a liquid containing self-assembling material particles, The manufacturing method, wherein the steps are performed using the channel structure according to any one of [1] to [13].
  • the solution containing the self-assembling substance is introduced from the inlet of the channel A and the dilution medium is introduced from the inlet of the channel B, or the solution containing the self-assembling substance is introduced from the inlet of the channel B and the dilution medium is introduced from the inlet of the flow path A.
  • At least a portion of the self-assembling substance-containing solution flow diverges and merges with the dilution medium flow at the common hole of the intersection region of the channel structure, and at least a portion of the dilution medium flow diverges into the self-assembling substance-containing solution flow.
  • a diluted layer of the self-assembling substance-containing solution is formed at the interface between the self-assembling substance-containing solution flow and the dilution medium flow, and self- 16.
  • the total flow rate of the solution containing the self-assembling substance and the dilution medium to channel A and channel B is in the range of 1 ⁇ l/min to 1000 ml/min.
  • Production method. Any one of [14] to [17], wherein the ratio of the flow rate V2 of the dilution medium to the flow rate V1 of the self-assembling substance-containing solution (V2/V1) is in the range of 1:1 to 1:20. manufacturing method.
  • the diluent medium is selected from aqueous solutions, buffer solutions, nucleic acid-containing aqueous solutions, protein-containing aqueous solutions, peptide-containing aqueous solutions, adjuvant-containing aqueous solutions, and mixtures thereof.
  • Production method [21] The production method according to any one of [14] to [20], wherein the self-assembling material particles contained in the liquid containing self-assembling material particles are nano-sized.
  • the nano-sized self-assembled material particles have a Z-average particle diameter in the range of 10 to 1000 nm.
  • the nano-sized self-assembled material particles have a Z-average particle size in the range of 20 to 200 nm.
  • the present invention it is possible to provide a fluid structure capable of producing self-organizing substance particles such as lipid nanoparticles having a relatively large average particle size of 100 nm or more even under relatively high flow conditions. can. Furthermore, according to the present invention, by using the fluid structure of the present invention, self-organizing substance particles such as lipid nanoparticles having a relatively large average particle size of 100 nm or more can be obtained even under conditions of a relatively large flow rate. can be manufactured.
  • FIG. 1-1 is a perspective view showing only the flow channel of the flow channel structure, showing an example of the flow channel structure of the present invention.
  • 1-2 are a plan view, a front view, and a side view showing only the channels of the channel structure of FIG. 1-1.
  • FIG. 1-3 is a plan view showing only the channels of the channel structure of FIG. 1-1, and indicates dimensions W1 to W8.
  • 2A and 2B are a perspective view, a plan view, a front view and a side view showing only the flow path of one example of the flow path structure of the present invention.
  • 3A and 3B are a perspective view, a plan view, a front view and a side view showing only the flow path of one example of the flow path structure of the present invention.
  • FIG. 4A and 4B are a perspective view, a plan view, a front view, and a side view showing only the flow path of one example of the flow path structure of the present invention.
  • FIG. 5-1 is a schematic explanatory diagram showing an example of the flow channel structure of the present invention.
  • FIG. 5-2 is a schematic explanatory diagram showing an example of the flow channel structure of the present invention.
  • FIG. 5-3 is a schematic explanatory diagram showing an example of the flow channel structure of the present invention.
  • FIG. 5-4 is a schematic explanatory diagram showing an example of the flow channel structure of the present invention.
  • FIG. 5-5 is a schematic explanatory diagram showing an example of the flow channel structure of the present invention.
  • 5-6 are schematic explanatory diagrams showing an example of the flow channel structure of the present invention.
  • FIG. 5-7 are schematic explanatory diagrams showing an example of the flow path structure of the present invention.
  • FIG. 5-8 are schematic explanatory diagrams showing an example of the flow channel structure of the present invention.
  • FIG. 6 is a plan view of an example of the flow channel structure of the present invention.
  • FIG. 7 is an explanatory diagram of a flow path structure used in Examples. 8 shows the experimental results of Example 1.
  • FIG. 9 shows the experimental results of Example 1.
  • FIG. 10 shows the experimental results of Example 1.
  • FIG. 11 shows the experimental results of Example 2.
  • FIG. 12 shows the experimental results of Example 3.
  • FIG. FIG. 13 shows the simulation results of Reference Example 2-1.
  • FIG. 14 shows the simulation results of Reference Example 2-2.
  • FIG. 15 shows the simulation results of Reference Example 2-3.
  • FIG. 16 shows simulation results of Reference Example 2-4.
  • FIG. 17 shows the simulation results of Reference Example 2-5.
  • 18 shows the particle size measurement results of Example 5.
  • FIG. 19 shows the results of relative luminescence intensity measurement of Example
  • the flow channel structure of the present invention is Having a channel A (10) having an inlet (11) and a channel B (20) having an inlet (21), Channels A and B (10, 20) three-dimensionally intersect at one or more locations downstream of inlets (11, 21), and communicate with channel walls of channels A and B (10, 20), respectively.
  • Flow paths A and B (10, 20) join at a confluence (30) located in the most downstream intersection region, and have at least one outlet (31) downstream of the confluence (30) (however, A part or the whole from the most upstream intersection area to the outlet port (31) is a site for diluting the fluid flowing through the channel) (hereinafter sometimes referred to as a dilution site 1), or the flow Paths A and B (10, 20) have outlets (12) and (22) independently without merging (however, from the intersection area to outlets (12) and (22) A part or the whole of each is a part for diluting the fluid flowing through the channel) (hereinafter sometimes referred to as a dilution part 2).
  • the flow path structure of the present invention is preferably for producing self-assembled material particles, and includes self The assembling material-containing solution is diluted by the diluent medium introduced from the other inlet to form self-assembling material particles.
  • Channel A (10) is regularly or irregularly curved or straight in two or three dimensions and Channel B (20) is regularly or irregularly in two or three dimensions. It can be curved or straight.
  • the flow path structure of the present invention has one or more intersection areas, and may have two or more intersection areas. If there is a single intersection area, one or both of Channel A (10) and Channel B (20) can be linear, and if not linear, regularly in two or three dimensions or It may be irregularly curved. An embodiment in which both Channel A (10) and Channel B (20) are straight is shown in FIGS. 5-7 and 5-8.
  • FIG. 1-1 is a perspective view showing only the channels of the channel structure, and the channels are incorporated in the main body of the structure (not shown).
  • 1-2 are a plan view, a front view, and a side view showing only the channels of the channel structure of FIG. 1-1.
  • FIG. 1-3 is a plan view showing only the channels of the channel structure of FIG. 1-1, and indicates dimensions W1 to W8.
  • a channel structure having a dilution site 1 has a channel A indicated by 10 having an inlet 11 and a channel B indicated by 20 having an inlet 21 .
  • the flow path A and the flow path B merge at a junction 30 located downstream of the inlets 11 and 21 and can have at least one outlet 31 downstream of the junction 30 .
  • the confluence portion 30 is located at the most downstream intersection region of the flow path A and the flow path B. As shown in FIG. A portion or the entire portion between the most upstream intersection region and the outlet port 31 is a portion for diluting the fluid flowing through the channel.
  • dilution may be completed in a portion between the intersection region at the most upstream and the outlet port 31, or may be completed at the maximum. Dilution may continue all the way from the upstream intersection region to outlet 31 .
  • a portion between the most upstream intersection region and the outlet port 31 is a portion for diluting the fluid flowing through the channel. be.
  • the entire area from the most upstream intersection area to the outlet port 31 is a site for diluting the fluid flowing through the channel.
  • the channel A and the channel B each have an outlet without merging.
  • a part or the whole of each section from the intersection region to the outlet 12 and the outlet 22 is a section for diluting the fluid flowing through the channel.
  • dilution site 1 depending on the fluid flow rate, the ratio of the fluid to be diluted and the diluent medium, and the length of the dilution site, the distance from the intersection region to outlet 12 and outlet 22 respectively. Dilution is completed in part, dilution continues in the entire area from the intersection area to outlet 12 and outlet 22, and dilution is completed in part from the intersection area to outlet 12.
  • dilution is completed in the entire area from the intersection area to the outlet 22, dilution is completed in the entire area from the intersection area to the outlet 12, and in a part from the intersection area to the outlet 22 It may be the case that the dilution is complete.
  • the distance between the intersection region and the outlet port 12 and between the intersection region and the outlet port 22 is A portion is a portion for diluting the fluid flowing through each channel.
  • flow path A and flow path B have independent outlets 12 and 22 without merging, and outlets 12 and outlets 12 and 22 are drawn from the intersection area. Each of them has a dilution site up to the outlet 22 .
  • the number of crossings of the channels A and B is not particularly limited, and can be appropriately determined in consideration of the purpose of use of the channel structure and the dimensions of the channels A and B.
  • the minimum number of times can be, for example, 1, 2, 3, 4, 5 times, and the maximum number of times, for example, 30, 25 , 20 times, 15 times, 10 times.
  • the flow path A and the flow path B three-dimensionally intersect at two or more points between the introduction ports 11 and 21 and the confluence portion 30, or three-dimensionally intersect at one point between the introduction ports 11 and 21 and the outlet ports 12 and 22. and has a common hole 32 that communicates the flow path A and the flow path B in the flow path side wall at each intersection region.
  • the opening surface of the common hole of the flow channel A and the opening surface of the common hole of the flow channel B can constitute substantially the same plane.
  • the common hole can be a passage (connecting passage) that connects the opening of the common hole of the flow path A and the opening of the common hole of the flow path B.
  • part of the fluid flowing through the flow path A and part of the fluid flowing through the flow path B flow into the communication flow path to form a contact surface between the two fluids.
  • channels A and B can intersect to have a common channel with each other at a common hole. In this case, since they have a common flow path, the fluids flowing through the two flow paths mix at that portion.
  • the cross-sectional area of the flow path A can be constant from the inlet 11 to the confluence portion 30 or the outlet 12, or can vary periodically at least in part. From the viewpoint of maintaining laminar flow, it is preferable to be constant, and from the viewpoint of promoting diffusion in the fluid, it is preferable to vary periodically.
  • the cross-sectional area of the flow path B can be constant from the introduction port 21 to the confluence portion 20 or the outlet port 22, or can vary periodically at least in part. From the viewpoint of maintaining a laminar flow, it is preferable to be constant, and from the viewpoint of promoting diffusion without a fluid, it is preferable to vary periodically.
  • the dimensions of the channels A and B can be determined as appropriate in consideration of the purpose of use of the channel structure, and are not intended to be limited.
  • the width and height of channel A can range, for example, from 1 ⁇ m to 10 mm and 1 ⁇ m to 10 mm, respectively, and the width and height of channel B can range, for example, from 1 ⁇ m to 10 mm and 1 ⁇ m to 10 mm, respectively.
  • the width and height of channel A are preferably in the range of 10 ⁇ m to 2 mm (2000 ⁇ m) and 10 ⁇ m to 1 mm (1000 ⁇ m) respectively, the width and height of channel B are preferably in the range of 10 ⁇ m to 2 mm (2000 ⁇ m) and 10 ⁇ m to 1 mm respectively.
  • the widths of the channels A and B are both 200 ⁇ m, and the heights of the channels A and B are both 100 ⁇ m.
  • the larger the size the more suitable for mass production at a large flow rate, but the size can be appropriately selected as long as particles having the desired particle size (average particle size) can be obtained.
  • FIGS. 3 and 4 show examples of channel structures having channel dimensions different from the channel structure in FIG.
  • the flow channel structure shown in FIGS. 3 and 4 is an example of a flow channel structure in which the cross-sectional area of the straight portion of the flow channel A varies periodically.
  • the flow path B is straight, the flow path A is regularly bent in two dimensions, and the bent portion has a curved planar shape.
  • the flow path B is straight, the flow path A is regularly bent in two dimensions, the plane shape of the bent portion is a bent shape, and the crossing angle of the flow paths A and B is This is an example that is not 90°.
  • the flow path B is straight, the flow path A is regularly bent in two dimensions, and the bent portion has a curved planar shape as in FIG. The path A is discontinued, and the path A joins with the path B in the intersection region.
  • the flow path B is straight, the flow path B is regularly bent in two dimensions, the plane shape of the bent portion is curved, and the crossing angle of the flow paths A and B is This is an example that is not 90°.
  • the flow path B is straight, the flow path A is regularly curved in three dimensions, and the curved portion has a curved planar shape as in FIG. 5-6
  • channel B is straight
  • channel A consists of two channels, both of which are regularly bent in two dimensions, and the bent portions have a planar shape similar to that in FIG. It is a refraction shape.
  • the two channels A have a sequential intersecting area in the longitudinal direction of the channel B. As shown in FIG.
  • the two channels A each have an inlet and an outlet.
  • channel B is straight and channel A consists of three channels, all of which are straight.
  • Each of the three channels A has one intersecting area in succession in the longitudinal direction of the channel B.
  • Each of the three channels A has an inlet and an outlet.
  • channel B is straight and channel A is also straight.
  • Channel A and channel B have one intersection area.
  • Channel A and channel B each have an inlet and an outlet.
  • the angle ⁇ formed by the center line of flow path A and the center line of flow path B is, for example, 30°. It can range from ⁇ 150°. Typically 90°, but preferably in the range 45° to 120°. Typically, it is 90° as shown in FIG. 1, but if the angle ⁇ is other than 90°, such as alternating between about 60° and about 120°, as shown in FIGS. There is also a mode that appears in
  • the flow path A is two-dimensionally or three-dimensionally or irregularly curved
  • the flow path B is two-dimensionally or three-dimensionally regularly or irregularly curved or linear.
  • the flow path A is two-dimensionally curved
  • the flow path B is straight
  • the flow paths A and B intersect periodically.
  • Channels A and B can intersect irregularly.
  • FIG. 5-5 shows an example in which the channel A is straight and the channel B is regularly curved in three dimensions.
  • the planar shape of the outer edge and inner edge of the curved portion of the flow path A that is bent two-dimensionally or three-dimensionally can be polygonal or substantially circular, and the curved portion of the flow path B that is bent two-dimensionally or three-dimensionally
  • the planar shape of the outer and inner edges of can be polygonal or substantially circular.
  • FIG. 5-1 shows a case of a substantially circular shape
  • FIG. 5-2 shows a case of a polygonal shape.
  • the channel structure of the present invention comprises, for example, a substrate having flat surfaces and a continuous groove A for the flow channel A on at least one main surface of the substrate and at least one of the substrate having flat main surfaces. It has a structure in which a substrate B having a continuous groove B for the flow channel B is attached to the main surface so that the continuous groove A and the continuous groove B face each other, and the flow channel A is two-dimensionally regular or irregular. It can be a channel structure in which the channel B is regularly or irregularly curved or linear in two dimensions.
  • FIG. 6 is a plan view showing the main bodies and channels of four types of channel structures (1) to (4) with different Ws.
  • the flow channel A is two-dimensionally curved, and the flow channel B is linear.
  • inlet ports (two places) for each of the flow paths A and B, and one outlet port for the flow path where the flow paths A and B join are shown.
  • (1), (2), (3) and (4) are examples in which the width W of the flow path A that is regularly curved two-dimensionally is 200 ⁇ m, 400 ⁇ m, 600 ⁇ m and 800 ⁇ m, respectively.
  • the flow channel structure of the present invention can further have a second flow channel structure downstream of the outlet port of the first flow channel structure.
  • the channel structures may be the same or different.
  • the outlet port of the first channel structure is connected to the inlet port of the channel A of the second channel structure or connected to the inlet port of the channel B of the second channel structure. More specifically, the outlet of the channel structure having the dilution part 1 and the inlet of the channel A or the channel B of the channel structure having the dilution part 2 are connected, and the dilution part 2 is provided.
  • 5-6 shows a configuration in which the outlet of the channel B of the channel structure having the dilution portion 2 and the inlet of the channel B of the channel structure having the dilution portion 1 are connected.
  • 5-7 shows an example in which three channel structures having dilution sites 1 and 2 are connected, the most upstream channel structure has dilution site 1, and the outlet of channel B is Connected to the inlet of the intermediate channel B, the intermediate channel structure has the dilution part 1, and the outlet of the channel B is the most downstream channel structure having the dilution part 2 It is the form connected with the inlet of the flow path B.
  • the present invention provides a method for producing self-assembling material particles, comprising a step of diluting a self-assembling material-containing solution with a diluent medium to obtain a liquid containing self-assembling material particles, wherein It includes a method implemented with a tract structure.
  • the solution containing the self-assembling substance is introduced from the inlet of the channel A and the dilution medium is introduced from the inlet of the channel B, or the solution containing the self-assembling substance is introduced from the inlet of the channel B and the dilution medium is introduced from the inlet of the channel A.
  • the self-assembling substance-containing solution fluid and the dilution medium fluid introduced into the channel from each inlet come into contact with the common hole of the channel structure, and the self-assembling substance-containing solution is diluted with the dilution medium to self-assemble. form particles of matter. More specifically, at least a portion of the self-assembling substance-containing solution flow branches into the dilution medium flow and merges with the dilution medium flow at the common hole of the intersection region of the channel structure, and at least the dilution medium flow A portion of the flow branches into the self-assembling substance-containing solution flow and merges with the self-assembling substance-containing solution flow.
  • a diluted layer of the self-assembling material-containing solution is formed at the interface between the converged self-assembling material-containing solution flow and the diluent medium flow, and self-assembling material particles are formed in the diluted layer.
  • a continuous dilution layer in the flow direction suitable for forming relatively large-diameter particles can be formed.
  • the flow channel structure of the present invention is a structure capable of realizing the above fluid behavior and thus particle formation.
  • the mechanism by which a diluted layer of a self-assembled substance-containing solution is formed and self-assembled substance particles are formed in the diluted layer is described in PLoS ONE 12(11): e0187962. (2017) (hereafter abbreviated as PLoS literature), the mechanism described in FIG.
  • the EtOH stream contacts the water stream at the first common hole, partially diverges and joins the intersecting water stream, and flows into the water stream flow path, while the water stream also contacts the EtOH stream at the first common hole. , diverges and joins the intersecting EtOH streams and flows into the EtOH stream flow path.
  • the EtOH stream entrained in the water streams that intersect at the common hole flows into the water stream flow path.
  • the cross-sectional view it can be seen that the water flow drawn into the EtOH flow crossing at the common hole flows into the flow path of the EtOH flow.
  • the self-assembled material particles are formed in the concentration range in which the solvent that dissolves the self-assembled material is in the dilution layer (the literature In the case of POPC lipid nanoparticles, they are formed when the EtOH concentration is generally in the range of 60 to 80%), and the longer the residence time in the diluted layer in this solvent concentration range, the larger the particle size, and the shorter the residence time, the larger the particle size.
  • the diameter tends to be smaller. From the results of FIG.
  • Reference Example 2-2 is a simulation result using a channel structure similar to that of Reference Example 1 with a total flow rate of 1000 ⁇ L/min. Results are shown in FIG. It is presumed that the dilution of the EtOH flow progresses rapidly, the time during which the solvent concentration capable of forming particles can be maintained becomes shorter than in Reference Example 1, and the particle size tends to become smaller.
  • Reference Example 2-3 is a simulation result using a channel structure similar to Reference Examples 1 and 2, with a total flow rate of 2000 ⁇ L/min. Results are shown in FIG. It is presumed that the EtOH stream is diluted more rapidly, the time during which the solvent concentration capable of forming particles can be maintained becomes shorter than in Reference Examples 1 and 2, and the particle size tends to become smaller.
  • Reference Example 2-5 is a simulation result using a channel structure similar to that of Reference Example 2-4 with a total flow rate of 2000 ⁇ L/min. Results are shown in FIG. Dilution of the EtOH flow progresses rapidly, and the time during which the solvent concentration capable of forming particles can be maintained is shorter than in Reference Example 2-4, and the particle size tends to be smaller.
  • the channel structure of the present invention by controlling the width and depth of the two channels, the flow rate ratio of the self-assembling substance-containing solution flow and the dilution medium flow, and the total flow rate, particles It can be seen that the diameter can be controlled and the uniformity of the particle size can also be controlled. Furthermore, in the examples, when the actual self-assembling substance-containing solution flow and the diluent medium flow are used, by controlling the above conditions, self-assembly having a highly uniform particle size and a desired particle size It shows that substance particles can be manufactured.
  • the formed self-assembled material particles are particles containing the self-assembled material as a particle constituent.
  • Particles containing a self-assembled substance as a particle constituent are particles obtained by association of the self-assembled substances to form particles. can be taken inside.
  • the constituent components of the particles formed under the condition where the substance to be encapsulated coexist are at least the self-assembling substance and the substance to be encapsulated.
  • the solution containing the self-assembling substance and the diluent medium to channel A and channel B can be flowed into the channel structure so that the total flow rate is, for example, 1 ⁇ l/min to 1000 ml/min.
  • the total flow rate is not intended to be limited to this range. It can be determined as appropriate in consideration of the encapsulation efficiency of the substance and the like.
  • the total flow rate of the self-assembling material-containing solution and the dilution medium is, for example, in the range of 100 ⁇ l/min to 1000 ml/min. From the viewpoint of obtaining substance particles, it can be in the range of 100 ⁇ l/min to 100 ml/min.
  • the ratio (V1:V2) between the flow rate V1 of the self-assembling substance-containing solution and the flow rate V2 of the dilution medium can be, for example, in the range of 1:1 to 1:20. However, it is not intended to be limited to this range, and can be appropriately selected within a range in which the desired particles can be obtained.
  • a self-assembling substance can be, for example, a lipid or an amphiphilic substance.
  • the self-assembling substance-containing solution can be, for example, any solution selected from the group consisting of a neutral lipid-containing solution, an anionic lipid-containing solution, a cationic lipid-containing solution, and a polymer-containing solution. It is not intended to be limiting.
  • the self-assembling substance in the present invention may be any substance as long as it has a self-assembling function and can associate with each other and form particles as described above.
  • Examples of lipids that are self-organizing substances include, but are not limited to, soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, phosphatidylcholines (eg, egg-derived eggPC), phosphatidylserines, and phosphatidyl.
  • Ethanolamines phosphatidylinositols, phosphasphingomyelins, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, sphingolipids, sterols, naturally-derived lipids such as lysophospholipids, and N-(2 ,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA); N,N-distearyl-N,N-dimethylammonium bromide (DDAB); oiloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP); 3-(N-(N',N'-dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol) and N-(1, 2-Dimyristyloxyprop-3-yl)-N,N-d
  • Amphiphilic substances which are another example of self-assembling substances, include, but are not particularly limited to, amphiphilic polymer compounds such as polystyrene-polyethylene oxide block copolymers, polyethylene oxide-polypropylene oxide block copolymers, Examples include polymers, amphiphilic block copolymers such as polylactic acid polyethylene glycol copolymers, polycaprolactone-polyethylene glycol copolymers, and the like.
  • the substance to be encapsulated is not particularly limited, but includes substances such as biopolymers such as nucleic acids, peptides, proteins, and sugar chains, metal ions, low-molecular-weight or middle-molecular-weight organic compounds, organometallic complexes, and metal particles.
  • drugs such as anticancer agents, antioxidants, antibacterial agents, anti-inflammatory agents, vitamins, artificial blood (hemoglobin), vaccines, hair growth agents, moisturizers, pigments, whitening agents, pigments, etc.
  • Physiologically active substances, cosmetics and the like can be exemplified.
  • These encapsulating substances can be included in the aqueous phase of the formed particles if they are water soluble.
  • the particles are included in the hydrophobic part of the self-assembled film formed by the self-assembled material, or as aggregates that are combined with the hydrophobic part of the self-assembled material and aggregated.
  • the substance to be encapsulated can be made water-soluble or sparingly soluble in advance in a pretreatment step, or can be made into an aggregate, and the pretreatment step can also be performed in an appropriate channel for the treatment.
  • the discharge port of the channel in which the pretreatment process is carried out can be connected to the first or second introduction channel of the channel structure of the present invention.
  • the water-miscible organic solvent used for dissolving the self-assembling material to prepare the particle solution is not particularly limited, but examples thereof include alcohols, ethers, esters, ketones, and acetals. and water-miscible organic solvents such as Alcohols such as methanol, ethanol, t-butanol, butanediols, 1-propanol, 2-propanol, and 2-butoxyethanol, particularly alkanols having 1 to 6 carbon atoms, are particularly preferred. Ethers such as tetrahydrofuran, acetonitrile, and acetone are also included.
  • diluent medium water or an aqueous solution containing essentially water as a main component, for example, physiological saline, phosphate buffer, acetate buffer, citrate buffer, malate buffer, etc., is to be formed. It is used as appropriate depending on the intended use of the particles.
  • Dilution media can also be aqueous solutions, buffers, as well as solutions that further contain water-soluble substances in these solutions.
  • the water-soluble substance can be low molecular weight, middle molecular weight, high molecular weight, nucleic acid, protein, peptide, and physiologically active substances containing them, pharmaceuticals, cosmetic materials, vaccines, adjuvants, and the like.
  • the diluent medium further containing a water-soluble substance is, for example, a low-molecular-weight aqueous solution, an intermediate-molecular-weight aqueous solution, a high-molecular-weight aqueous solution, a nucleic acid-containing aqueous solution, a protein-containing aqueous solution, a peptide-containing aqueous solution, a physiologically active substance-containing aqueous solution, a drug-containing aqueous solution, It may be an aqueous solution containing cosmetic material, an aqueous solution containing vaccine, an aqueous solution containing adjuvant, or a mixture thereof.
  • the self-assembled material particles encapsulating the material to be encapsulated obtained by the method of the present invention can be nano-sized, and can have a Z-average particle size in the range of, for example, 10 to 1000 nm, such as 20 to 200 nm. can range from However, it is not intended to be limited to this range.
  • the Z-average particle size is also called cumulative average (harmonic intensity obtained by averaging particle diameters), and is defined in ISO 13321.
  • Example 1 Lipid particles were prepared using six channel structures shown in Tables 1-1 and 1-2 below.
  • W5, W7, W2, and W4 in Table 1-2 are the dimensions of the portion shown in FIG. and B from the confluence to the outlet (mm).
  • ⁇ Upper channel buffer flow [25 mM acetate buffer (pH 4.0)] - Lower flow path: lipid EtOH liquid flow - Lipid solution: 10 mM DOTAP/DSPC/cholesterol/DMG-PEG 2K (40/11.5/47.5/1%) ⁇ Dialysis: D-PBS (-) ⁇ Particle size measurement: DLS
  • the results are shown in Figures 8-10.
  • the particle size on the vertical axis in FIGS. 8 to 10 means the Z-average particle size, and the particle size in the following description also means the average particle size.
  • the upper channel width increases from 200 nm to 800 nm in FRR3.
  • the flow rate increases, particles with a diameter of 100 nm or more are formed even at a high flow rate of 1000 ⁇ L/min or more.
  • FRR5 and FRR7 show the same tendency.
  • channel No. 6 With FRR3, particles with a particle diameter of 100 nm or more can be obtained even at 2000 ⁇ L/min. Smaller FRR tends to make it easier to obtain particles with a large particle size at a high flow rate.
  • the width of the upper flow path is increased, it tends to become easier to form particles having a particle diameter of 100 nm or more.
  • the chamfering of the channel is a structure in which the corners of the inner surface of the side wall of the channel are curved, and is intended to smooth the flow of the fluid.
  • the results are shown in FIG.
  • the particle size on the vertical axis of FIG. 11 means the Z-average particle size.
  • the particle size of the obtained lipid particles did not change significantly depending on whether the upper channel of the channel structure was chamfered or not, but the particle size was larger in the case of having the chamfered structure.
  • Example 4 Lipid particles encapsulating nucleic acids were prepared using three types of channel structures shown in Table 2 below.
  • Z-average indicates Z-average particle size
  • Number indicates number-average particle size
  • PDI indicates polydispersity.
  • the Z-average particle size is the average particle size determined based on the measured scattering intensity (light intensity), and the number-average particle size is the average particle size weighted by the number of particles.
  • ⁇ Nucleic acid solution 60 (FRR7) or 70 (FRR3) ⁇ g/mL siRNA in 25 mM acetate buffer (pH 4.0) - Lipid solution: 10 mM DOTAP/DSPC/cholesterol/DMG-PEG 2K (40/11.5/47.5/1%) ⁇ Dialysis: D-PBS (-) - Particle size measurement: A dynamic light scattering method (DLS) was used. - Measurement of nucleic acid encapsulation rate: Ribogreen (registered trademark) assay was used.
  • the channel structure used in the examples of the present invention can produce lipid particles encapsulating nucleic acid with a Z-average particle size in the range of 100 to 200 nm.
  • Nucleic acid encapsulation was as high as 96% or more.
  • channel no. 4 flow rate 300 ⁇ L/min, 2000 ⁇ L/min
  • channel No. 6 flow rate of 2000 ⁇ L/min
  • particles with a PDI of 0.1 or less could be produced.
  • Example 5 Using the channel (W800) in FIG. 6(4), mRNA-LNP was produced.
  • the dialyzed three types of mRNA-LNP were diluted with medium to a concentration of 200 ng/mL mRNA and dosed to HeLa cells. After incubation for 24 hours, the luminescence intensity and total protein concentration of Luciferase were measured using ONE-Glo and BCA assay kit. From this, the relative luminescence intensity (RLU/mg protein) was obtained. The results are shown in FIG. In all three cases, the Z-average particle size was 100 nm or more (Fig. 18), and the relative emission intensity did not change significantly (Fig. 19).
  • Example 6 mRNA-LNP was prepared using the channel (W800) in FIG. .
  • Reference example 2-1 The upper channel with a width (W) of 200 ⁇ m and a depth (D) of 100 ⁇ m shown in FIG. This is an image of the formation of a diluted layer when the total flow rate is 500 ⁇ L/min and the water/EtOH flow rate ratio is 5 in a flow channel structure having six crossing regions and a structure that merges to reach an inlet. be. Results are shown in FIG.
  • Reference example 2-2 This is the result of a simulation using a channel structure similar to that of Reference Example 2-1 and with a total flow rate of 1000 ⁇ L/min. Results are shown in FIG.
  • Reference example 2-3 This is the result of a simulation using a channel structure similar to that of Reference Example 2-1 and with a total flow rate of 2000 ⁇ L/min. Results are shown in FIG.
  • Reference example 2-4 The upper channel with a width (W) of 400 ⁇ m and a depth (D) of 100 ⁇ m shown in FIG. This is an image of the formation of a diluted layer when the total flow rate is 500 ⁇ L/min and the water/EtOH flow rate ratio is 5 in a flow channel structure having seven intersection regions and a structure that merges to reach an inlet. be. Results are shown in FIG.
  • Reference example 2-5 This is the result of a simulation using a channel structure similar to that of Reference Example 2-4 and with a total flow rate of 2000 ⁇ L/min. Results are shown in FIG.
  • the present invention is useful in fields related to production of channel structures and self-assembled material nanoparticles using channel structures.

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Abstract

La présente invention concerne une structure de trajet d'écoulement qui inclut un trajet d'écoulement A10 ayant un orifice d'entrée 11 et un trajet d'écoulement B20 ayant un orifice d'entrée 21. Les trajets d'écoulement A10, B20 se croisent en trois dimensions au niveau d'un ou plusieurs emplacements en aval des orifices d'entrée 11, 21 et incluent, dans les régions d'intersection, un trou mutuel 32 dans des parois de trajet d'écoulement respectives à travers lesquelles les trajets d'écoulement A10, B20 communiquent. Les trajets d'écoulement (A10, B20) fusionnent au niveau d'une section de fusion (30) positionnée en aval de la région d'intersection la plus en aval et ont au moins un orifice de sortie (31) en aval de la section de fusion (30). En variante, les trajets d'écoulement A10, B20 ne fusionnent pas et ont indépendamment un orifice de sortie 12 et un orifice de sortie 22. La présente invention concerne un procédé de fabrication de particules de matériau à auto-organisation, le procédé utilisant ladite structure de trajet d'écoulement et incluant une étape d'obtention d'un liquide qui contient des particules de matériau à auto-organisation par dilution, dans un milieu de dilution, d'une solution contenant un matériau à auto-organisation. La présente invention concerne : une structure de fluide avec laquelle il est possible de produire des particules de matériau à auto-organisation ayant un diamètre de particule moyen de 100 nm ou plus même dans des conditions dans lesquelles le débit est relativement élevé ; et un procédé de fabrication de particules de matériau à auto-organisation, le procédé utilisant ladite structure de fluide.
PCT/JP2023/004007 2022-02-08 2023-02-07 Structure de trajet d'écoulement et procédé de fabrication de particules de matériau à auto-organisation l'utilisant WO2023153404A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005054023A (ja) * 2003-08-01 2005-03-03 Nippon Paint Co Ltd ポリマー粒子の製造方法
JP2014231038A (ja) * 2013-05-29 2014-12-11 株式会社フジクラ マイクロミキサ
JP2016028031A (ja) * 2009-11-04 2016-02-25 ザ ユニバーシティ オブ ブリティッシュ コロンビア 核酸含有脂質粒子および関連方法
JP2017081954A (ja) * 2011-10-25 2017-05-18 ザ ユニバーシティ オブ ブリティッシュ コロンビア 限界サイズ脂質ナノ粒子および関連方法
WO2020095927A1 (fr) * 2018-11-09 2020-05-14 国立大学法人北海道大学 Procédé de production d'une solution aqueuse contenant des particules
WO2021064998A1 (fr) * 2019-10-04 2021-04-08 国立大学法人北海道大学 Corps de structure de chemin d'écoulement tridimensionnel et procédé pour produire des nanoparticules l'utilisant

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2005054023A (ja) * 2003-08-01 2005-03-03 Nippon Paint Co Ltd ポリマー粒子の製造方法
JP2016028031A (ja) * 2009-11-04 2016-02-25 ザ ユニバーシティ オブ ブリティッシュ コロンビア 核酸含有脂質粒子および関連方法
JP2017081954A (ja) * 2011-10-25 2017-05-18 ザ ユニバーシティ オブ ブリティッシュ コロンビア 限界サイズ脂質ナノ粒子および関連方法
JP2014231038A (ja) * 2013-05-29 2014-12-11 株式会社フジクラ マイクロミキサ
WO2020095927A1 (fr) * 2018-11-09 2020-05-14 国立大学法人北海道大学 Procédé de production d'une solution aqueuse contenant des particules
WO2021064998A1 (fr) * 2019-10-04 2021-04-08 国立大学法人北海道大学 Corps de structure de chemin d'écoulement tridimensionnel et procédé pour produire des nanoparticules l'utilisant

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