WO2020095927A1

WO2020095927A1 - Method for producing particle-containing aqueous solution

Info

Publication number: WO2020095927A1
Application number: PCT/JP2019/043420
Authority: WO
Inventors: 正寿真栄城; 学渡慶次; 笑木村; 悠介佐藤; 原島　秀吉
Original assignee: 国立大学法人北海道大学
Priority date: 2018-11-09
Filing date: 2019-11-06
Publication date: 2020-05-14
Also published as: JPWO2020095927A1

Abstract

The present invention relates to a method comprising diluting aqueous solution A, which contains particles Z comprising self-organizing molecules as a particle-constituting ingredient and which contains a water-miscible organic solvent in a concentration of 20 vol% or higher, with aqueous solution B₁ to thereby obtain aqueous solution C, which contains the particles Z having a number-average particle diameter of 200 nm or smaller. For the dilution is used a channel structure for dilution which comprises a D1 introduction channel and a D2 introduction channel that have been disposed on the upstream side, a dilution channel disposed downstream from a confluence of the D1 introduction channel and the D2 introduction channel, and one or more Dn introduction channels provided somewhere to the dilution channel. The dilution comprises supplying aqueous solution A and aqueous solution B₁ respectively to the D1 introduction channel and the D2 introduction channel, adding one or more aqueous solutions B_n through the Dn introduction channels to the aqueous solutions that are running through the dilution channel, and obtaining aqueous solution C, which contains the water-miscible organic solvent having been diluted to or below a concentration at which the particles can be stably present, through the outlet of the dilution channel. The present invention provides: a method for producing a nanoparticle-containing aqueous solution in which the particle diameters of particles comprising self-organizing molecules as a particle-constituting ingredient can be maintained as much as possible; and a device for use in the production method.

Description

Method for producing particle-containing aqueous solution

The present invention relates to a method for producing a particle-containing aqueous solution.
Cross Reference of Related Applications This application claims the priority of Japanese Patent Application No. 2018-211841 filed on Nov. 9, 2018, the entire description of which is specifically incorporated herein by reference.

Nanoparticles containing self-assembling molecules as particle constituents such as lipid nanoparticles and polymer micelles are most practically used as nanocarriers for drug delivery systems (DDS), and have already been clinically applied. Recently, it has been revealed that the delivery efficiency of a drug to a cancer tissue varies depending on the particle size of the nanocarrier. Therefore, in order to precisely control the particle size of nanoparticles, a particle size control method using a microfluidic device has been developed [Non-patent documents 2-4]. Furthermore, the present inventors have developed a microfluidic device that is easy to manufacture or process and has high particle size controllability, and a method for producing nanoparticles using the same [Non-Patent Document 1 and Patent Document 1].

Patent Document 1: WO2018 / 190423
Patent Document 2: Japanese Patent Laid-Open No. 2009-505957 Patent Document 3: Japanese Patent Laid-Open No. 2013-51096 (WO2011 / 001766)

Non-Patent Document 1: "Development of the iLiNP Device: Fine Tuning the Lipid Nanoparticle Size within 10 nm for Drug Delivery", N. Kimura, M. Maeki, Y. Sato, T. Note, A. Ishida, H. Tani, H. Harashima, and M. Tokeshi, ACS Omega, 3, 5044, (2018).
Non-Patent Document 2: “Understanding the Formation Mechanism of Lipid Nanoparticles in Microfluidic Devices with Chaotic Micromixers”, M. Maeki, Y. Fujishima, Y. Sato, T. Yasui, N. Kaji, A. Ishida, H. Tani, Y. Baba, H. Harashima, and M. Tokeshi, PLOS ONE, 12, e0187962, (2017).
Non-Patent Document 3: "Bottom-Up Design and Synthesis of Limit Size Lipid Nanoparticle Systems with Aqueous and Triglyceride Cores Using Millisecond Microfluidic Mixing", IV Zhigaltsev, N. Belliveau, I. Hafez, AKK Leung, C. Hansen, and PR Cullis. , Langmuir, 38, 3633, (2012).
Non-Patent Document 4: “Rapid Discovery of Protein siRNA-Containing Lipid Nanoparticles Enabled by Controlled Microfluidic Formation”, D. Chen, KT Love, Y. Chen, AA Eltoukhy, C. Kastrup, G. Sahay, A. Jeon, Y. Dong, KA Whitehead, and DG Anderson, Journal of the American Chemical Society, 134, 6948, (2012).
The entire descriptions of Patent Documents 1 to 3 and Non-Patent Documents 1 to 4 are specifically incorporated herein by reference.

In the method for producing nanoparticles described in Non-Patent Document 1 and Patent Document 1, a lipid / alcohol solution, which is a raw material for producing lipid nanoparticles in a microchannel, is rapidly diluted with a buffer solution, etc. This enables fine particle size control. However, since high-concentration ethanol remains in the lipid nanoparticle solution immediately after preparation, the residual alcohol is usually diluted by a post-treatment such as dialysis (overnight).

However, in dialysis and the like, fusion of particles occurs during post-treatment, and lipid nanoparticle suspension having a desired particle size may not be obtained, although prepared with precise particle size control. .. This is because in the above method, lipid / alcohol solution is diluted with a buffer etc. to form lipid nanoparticles, but the alcohol concentration in the diluted aqueous solution is still high, promoting fusion of particles formed by dilution. It was supposed to be to do so.

Therefore, the problem to be solved by the present invention is to provide means / method for avoiding fusion of formed nanoparticles, and an object of the present invention is to maintain the particle diameter of prepared nanoparticles as much as possible. It is intended to provide a method for producing the obtained aqueous solution containing nanoparticles and a device used therefor.

The present invention is as follows.
[1]
An aqueous solution A containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent at a concentration of 20 vol% or more is diluted with an aqueous solution B ₁ to have a number average particle diameter of 200 nm or less. A method comprising obtaining an aqueous solution C containing particles Z, comprising:
The dilution has a D1 introduction path and a D2 introduction path on the upstream side, a dilution flow path on the downstream side from the confluence of the D1 introduction path and the D2 introduction path, and one or more in the middle of the dilution flow path. Using a diluting channel structure having a Dn introducing channel (for example, n = 3 to 7),
The aqueous solution A is supplied to the D1 introduction path and the aqueous solution B ₁ is supplied to the D2 introduction path, and the aqueous solution B _n (for example, n = 2 to 6) is added to the aqueous solution in the dilution flow path from the Dn introduction path to obtain a diluted flow. A method for producing an aqueous solution containing particles, which comprises obtaining an aqueous solution C diluted with a water-miscible organic solvent concentration to a concentration at which particles can stably exist from an outlet of a passage.
[2]
An aqueous solution A containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent at a concentration of 20 vol% or more is diluted with an aqueous solution B ₁ to have a number average particle diameter of 200 nm or less. A method comprising obtaining an aqueous solution C containing particles Z, comprising:
The dilution has a D1 introduction path and a Dn (for example, n = 2 to 7) introduction path on the upstream side, and the Dn introduction paths are arranged such that all the introduction paths sandwich the D1 introduction path at a point p. Using a diluting flow channel structure having a diluting flow channel on the downstream side from p,
An aqueous solution A is supplied to the D1 introduction path and an aqueous solution B _n (for example, n = 1 to 6) is supplied to the Dn introduction path, and the water-miscible organic solvent is supplied from the outlet of the dilution flow path to a concentration at which the particles can stably exist. A method for producing an aqueous solution containing particles, which comprises obtaining an aqueous solution C having a diluted concentration.
[3]
The production method according to [1] or [2], wherein the self-assembling molecule comprises either A) a lipid, B) an amphipathic substance, or both A) and B).
[4]
The production method according to any one of [1] to [3], wherein the water-miscible organic solvent is an alkanol.
[5]
The production method according to any one of [1] to [4], wherein the particles Z are liposomes, lipid micelles, or polymer micelles.
[6]
The D2 introduction path is composed of a plurality of D2 introduction paths m, and the plurality of D2 introduction paths m are arranged so as to sandwich the dilution flow path at the same point p of the dilution flow path. [1] to [5] The production method described in Crab.
[7]
Any one of [1] to [6], wherein the Dn introducing passage is composed of a plurality of Dn introducing passages m, and the plurality of Dn introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The production method described in Crab.
[8]
The manufacturing method according to any one of [1] to [7], wherein the D1 introducing passage is branched into two or more, and the D2 introducing passage is arranged for each branched D1 passage.
[9]
The production method according to any one of [1] to [8], wherein the diluting flow path is branched into two or more on the way, and a Dn introducing path is arranged for each branched diluting flow path.
[10]
The production method according to any one of [1] to [9], wherein the residence time of the aqueous solution in the dilution flow channel is 1000 milliseconds or less.
[11]
The production method according to any one of [1] to [10], wherein the concentration of the water-miscible organic solvent contained in the aqueous solution C is 3 vol% or less.
[12]
The aqueous solution A is prepared by using a particle-preparing flow channel structure for preparing a particle-containing aqueous solution by mixing a water-miscible organic solvent containing a self-assembling molecule and a particle-preparing aqueous solution, and immediately after preparation, The manufacturing method according to any one of [1] to [11], which is supplied to the D1 introduction path of the diluting flow path structure.
[13]
The particle-preparing flow path structure has an Sq introduction path consisting of at least two or more on the upstream side, has a particle preparation flow path on the downstream side from the joining portion of each introduction path, and has an aqueous solution from the outlet of the particle preparation flow path. The production method according to [12], wherein A is discharged, and the discharged aqueous solution A is supplied to the D1 introducing passage.
[14]
The production method according to [13], wherein the particle preparation channel and the dilution channel each include a micromixer in one or one of the channels.
[15]
The production method according to any one of [1] to [14], wherein the number average particle size of the lipid particles in the aqueous solution C is 100 nm or less or 60 nm or less.
[16]
The production method according to any one of [1] to [15], wherein the concentration of the lipid particles in the aqueous solution A is 20 mg / mL or less and the concentration of the lipid particles in the aqueous solution C is 5 mg / mL or less.
[17]
The production method according to any one of [1] to [16], wherein the aqueous solution C discharged from the outlet of the dilution flow channel is further subjected to dialysis to obtain an aqueous solution from which the water-miscible organic solvent is removed.
[18]
The production method according to any one of [1] to [17], wherein the particles Z include an encapsulated material.
[19]
A flow channel structure for diluting an aqueous solution containing a particle Z containing a self-assembling molecule as a particle constituent and containing a water-miscible organic solvent, the upstream side being a D1 introduction path and a D2 introduction A diluting flow path downstream from the confluence of the D1 introducing path and the D2 introducing path, and one or more Dn introducing paths (for example, n = 3 to 7) in the middle of the diluting path. A diluting flow channel structure having.
[20]
A flow channel structure for diluting an aqueous solution containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent with a D1 introducing path and a Dn ( For example, n = 2 to 7) has an introduction path, all of the Dn introduction paths are arranged so as to sandwich the D1 introduction path at the point p, and a dilution flow path is provided downstream from the point p for dilution. A diluting flow channel structure having a flow channel structure.
[21]
A particle production flow channel structure having a particle preparation flow channel structure and a dilution flow channel structure,
The particle-preparing flow channel structure has an Sq introducing passage consisting of at least two or more on the upstream side, and has a particle preparing passage on the downstream side from the joining portion of each introducing passage,
The diluting flow path structure has a D1 introducing path and a D2 introducing path on the upstream side, a diluting flow path on the downstream side from the confluence of the D1 introducing path and the D2 introducing path, and in the middle of the diluting path. Having one or more Dn introduction paths (eg, n = 3 to 7),
A particle production flow channel structure in which the outlet of the preparation flow channel is directly connected to the D1 introduction channel.
[22]
The D2 introducing passage is composed of a plurality of D2 introducing passages, and the plurality of D2 introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. [19] to [21] The dilution flow channel structure or the flow channel structure for producing particles according to item 1.
[23]
Any of [19] to [22], wherein the Dn introducing passage is composed of a plurality of Dn introducing passages m, and the plurality of Dn introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The dilution flow channel structure or the flow channel structure for producing particles as described in 1.
[24]
The diluting flow channel structure according to any one of [19] to [23], wherein the D1 introducing channel is branched into two or more, and the D2 introducing channel is arranged for each branched D1 channel. A flow channel structure for producing particles.
[25]
The dilution flow channel structure according to any one of [19] to [24], wherein the dilution flow channel has two or more branches on the way, and a Dn introduction channel is arranged for each branched dilution flow channel. A flow channel structure for producing a body or particles.

According to the present invention, it is possible to provide a method for producing a particle-containing aqueous solution that can maintain the particle size of particles containing a self-assembling molecule as a particle constituent component, and a device used therefor.

FIG. 1 is a schematic view of one embodiment of a diluting flow channel structure (with a mixer) used in the present invention. FIG. 2 is a schematic view of one embodiment of the dilution flow channel structure (with a mixer) used in the present invention. FIG. 3 is a schematic diagram of (A) one embodiment of the particle preparation flow channel structure used in the present invention, and (B) one mode of the dilution flow channel structure (with a mixer). FIG. 4 is a schematic view of one embodiment of the diluting flow channel structure (without mixer) used in the present invention. FIG. 5 is a schematic view of a specific example of the mixer provided in the flow channel structure. FIG. 6 is a schematic view of a specific example of the confluence portion of the flow channel structure. FIG. 7 is a schematic view of one embodiment of a device in which the particle preparation channel structure and the dilution channel structure used in the present invention are directly connected. 8A to 8D are schematic views of one embodiment of the dilution flow channel structure used in the present invention. FIG. 9 shows the results of Example 1 and Comparative Examples 1 and 2. FIG. 10 shows the results of Example 2 and Comparative Examples 3 and 4. FIG. 11 shows the results of Example 3 and Comparative Examples 5 and 6. FIG. 12 shows the results of Example 4. FIG. 13 shows the results of Example 4. FIG. 14 shows the results of Example 5. FIG. 15 shows the results of Example 6.

<Method for producing aqueous solution containing lipid particles>
The first aspect of the present invention is a method for producing a particle-containing aqueous solution.
This production method comprises diluting an aqueous solution A containing a particle Z containing a self-assembling molecule as a particle constituent component and a water-miscible organic solvent at a concentration of 20 vol% or more with an aqueous solution B ₁ to obtain number-average particles. A method comprising obtaining an aqueous solution C containing particles Z having a diameter of 200 nm or less,
The dilution has a D1 introduction path and a D2 introduction path on the upstream side, a dilution flow path on the downstream side from the confluence of the D1 introduction path and the D2 introduction path, and one or more in the middle of the dilution flow path. Using a diluting channel structure having a Dn introducing channel (for example, n = 3 to 7),
The aqueous solution A is supplied to the D1 introduction path and the aqueous solution B ₁ is supplied to the D2 introduction path, and the aqueous solution B _n (for example, n = 2 to 6) is added to the aqueous solution in the dilution flow path from the Dn introduction path to obtain a diluted flow. From the outlet of the passage, obtaining an aqueous solution C diluted with a water-miscible organic solvent concentration below a concentration at which the particles Z can stably exist.

<Aqueous solution A>
The aqueous solution A containing the particles Z containing the self-assembling molecule as a particle constituent and containing the water-miscible organic solvent at a concentration of 20 vol% or more is a raw material in the production method of the present invention. The aqueous solution A is a product prepared by any method using a self-assembling molecule, a water-miscible organic solvent and an arbitrary aqueous solution, and for example, the self-assembling molecule prepared by the method described in Patent Document 1. A particle Z-containing aqueous solution containing as a particle constituent component can be used. For example, it can be prepared by using the flow path structure described in Patent Document 1 and introducing a self-assembled molecule-containing water-miscible organic solvent solution and a diluting solvent which is an arbitrary aqueous solution. Alternatively, it can be prepared by adding (for example, dropping) the self-assembled molecule-containing water-miscible organic solvent while optionally stirring a diluting solvent that is an arbitrary aqueous solution without using the flow channel structure. The self-assembling molecule-containing water-miscible organic solvent is introduced or dropped into a diluting solvent that is an aqueous solution, the self-assembling molecule forms particles, and the water-miscible organic solvent is a diluting solvent that is an arbitrary aqueous solution. Upon mixing, a water-miscible organic solvent aqueous solution is formed, and the self-assembled molecular particles are dispersed in the water-miscible organic solvent aqueous solution. In the present invention, the concentration of the water-miscible organic solvent in the aqueous solution A is 20 vol% or more, preferably in the range of 20 to 60 vol%. The higher the concentration of the water-miscible organic solvent in the aqueous solution A, the lower the stability of the self-assembled molecular particles, and the larger the particle stabilizing effect by dilution in the method of the present invention, the more preferable. The particle diameter of the particles Z contained in the aqueous solution A is preferably 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, from the viewpoint that the number average particle diameter of the particles Z in the aqueous solution C is 200 nm or less. It is less than 100 nm.

The compositions of the self-assembled molecule solution and the dilution medium, which are the raw materials of the aqueous solution A used in the production method of the present invention, and the dilution ratios thereof are not particularly limited. In principle, the method for forming the particle Z in which the self-assembling molecule is composed of either A) lipid, B) one of the amphiphiles, or both A) and B) is based on the principle that the self-assembling molecule is water-miscible. It is carried out by adding and diluting a solution dissolved in an organic solvent to an aqueous solution (diluting medium) under heating conditions, if necessary, and a conventionally known composition or the like can be used in such a method. .. Examples of the particle Z containing a self-assembling molecule as a particle constituent component include monolayer or multilayer liposomes, lipid micelles, polymer micelles, and composites of monolayer or multilayer liposomes and polymer micelles (for example, polymer micelles having liposome particles as a core are exemplified. But not limited thereto), and a combination of lipid micelles and polymer micelles (for example, but not limited to, polymer micelles having a lipid micelle particle as a core).

The lipid, which is an example of a self-assembling molecule, is not particularly limited, for example, soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, Phosphasphingomyelins, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, sterols, and other naturally occurring lipids, as well as cations considered to be suitable as components of liposomes for nucleic acid delivery Non-naturally occurring lipids N, N-dioleoyl-N, N-dimethyl ammonium chloride (DODAC); N- (2,3-dioleyloxy) propyl) -N, N, N-trimethyl ammonium chloride (DOTMA) ); N, N-this Allyl-N, N-dimethylammonium bromide (DDAB); N- (2,3-dioleoyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTAP); 3- (N- (N ', N′-dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol) and N- (1,2-dimyristyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE), Lipofectin®, Lipofectamine®, Transfectam®, DODAP, DODMA, DMDMA, 1,2-dilinoleyloxy-N, N-dimethylaminopropane (DLinDMA), 1,2- Dilinolenyloxy-N, N-dimethylaminopropane (DLenDM ), 1,2-Dilinoleooxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1,2-Dilinoreoxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane ( DLinDAP), 1,2-dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-dili Noreyloxy-3-trimethylaminopropane chloride salt (DLin-TMA-Cl), 1,2-dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP-Cl), 1,2-dilinoleyloxy -3- (N-methylpiperazino) propane (DLin-MPZ) , 3- (N, N-dilinoleylamino) -1,2-propanediol (DLinAP), 3- (N, N-dioleylamino) -1,2-propanedio (dio) (DOAP), 1 , 2-Dilinoleyloxo-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA) and 2,2-dilinoleyl-4-dimethylaminomethyl- [1,3] -dioxolane ( DLin-K-DMA) and the like can be used.

The amphipathic substance which is another example of the self-assembling molecule is not particularly limited, but an amphipathic polymer compound such as polystyrene-polyethylene oxide block copolymer or polyethylene oxide-polypropylene oxide block copolymer is available. Examples thereof include amphiphilic block copolymers such as polymers, polylactic acid polyethylene glycol copolymers, polycaprolactone-polyethylene glycol copolymers, and the like.

The water-miscible organic solvent used to prepare the nanoparticle solution by dissolving the self-assembling molecule as described above is not particularly limited, and examples thereof include alcohols, ethers, esters, Water-miscible organic solvents such as ketones and acetals, especially alcohols such as ethanol, t-butanol, 1-propanol, 2-propanol, and 2-butoxyethanol, especially alkanols having 1 to 6 carbon atoms are used. Preferably. The same water-miscible organic solvent may be used to prepare the amphipathic substance solution, but preferred examples include ethers such as tetrahydrofuran and chloroform.

As the dilution medium, water or basically water as a main component, for example, physiological saline, phosphate buffer solution, acetate buffer solution, aqueous solution such as citrate buffer solution, etc. It is used as appropriate.

The particles Z contained in the aqueous solution A can further contain an encapsulated material. It is possible to mix a physiologically active substance or the like into the particles, as is known, depending on the intended use of the obtained particles. The enclosed material is not particularly limited, and examples thereof include metal ions, low-molecular or medium-molecular organic compounds, organometallic complexes, nucleic acids, peptides, proteins, biopolymers such as sugar chains, and substances such as metal particles. In addition, from the viewpoint of use, agents such as anticancer agents, antioxidants, antibacterial agents, anti-inflammatory agents, vitamin agents, artificial blood (hemoglobin), vaccines, hair growth agents, moisturizers, pigments, whitening agents, pigments, etc. , Physiologically active substances, cosmetics and the like. These encapsulated substances can be included in the aqueous phase of the particles to be formed, as long as they are water-soluble. Also, if it is oil-soluble, it can be incorporated into the lipophilic phase such as in the lipid membrane of the particles. Further, if it has some interaction such as electrostatic interaction with the particle surface, it can be incorporated into the particle surface. Furthermore, the encapsulated material is, for example, an insoluble particle (core particle) obtained by dispersing a drug (for example, a drug, a test agent), a physiologically active substance, a cosmetic, etc. in an aqueous phase in the particle formed by the present invention. It can be incorporated in the form.

<Aqueous solutions B ₁ and B _n >
The aqueous solution B ₁ and the aqueous solution B _n are diluting solvents used to reduce the concentration of the water-miscible organic solvent of the aqueous solution A, the first diluting solvent used is the aqueous solution B ₁ , and the nth diluting solvent is Aqueous solution B _n . The aqueous solution B ₁ and the aqueous solution B _n are preferably any aqueous solution containing no water-miscible organic solvent. The aqueous solution may be any buffer solution. The buffer solution can be appropriately selected depending on the type of the particles, the encapsulated material encapsulated in the particles, or the core particles.

<Dilution flow path structure>
Dilution of the aqueous solution A in the production method of the present invention has a D1 introduction path and a D2 introduction path on the upstream side, a dilution flow path on the downstream side from the confluence of the D1 introduction path and the D2 introduction path, and a dilution flow It is carried out using a diluting channel structure having one or more Dn introducing channels (for example, n = 3 to 7) in the middle of the channel.

An example of the diluting flow channel structure is shown in FIG. FIG. 1 shows an example in which only one D3 introduction path is provided as the Dn introduction path. From the left side (upstream) side, the aqueous solution A and the aqueous solution B ₁ are supplied to the D1 introduction path and the D2 introduction path, respectively, and the aqueous solution A and the aqueous solution B ₁ are merged at the merging portion 1 and are supplied to the upstream dilution flow path DF1. The mixing of the aqueous solution A and the aqueous solution B ₁ proceeds, and the aqueous solution A is diluted. Next, the aqueous solution B ₂ is added to the aqueous solution in the dilution flow path from the Dn introduction path, which is the Dn introduction path, and the aqueous solution B ₂ merges with the aqueous solution from the dilution flow path DF1 at the merging portion 2 to generate the diluted flow path DF2. Further mixing progresses, and dilution progresses. From the outlet of the dilution flow path DF2, an aqueous solution C in which the concentration of the water-miscible organic solvent is diluted to a concentration at which the particles can stably exist is obtained.

The diluting flow path structure used in the present invention has a D1 introduction path and a Dn (for example, n = 2 to 7) introduction path on the upstream side, and all of the Dn introduction paths are D1 introduction paths at point p. It can be a diluting channel structure having a diluting channel on the downstream side from the point p. In this diluting flow channel structure, for example, as shown in FIG. 4, the D2 introduction path is composed of a plurality of D2 introduction paths m (m is, for example, 1 to 5, and in FIG. 4, m is 1 and 2, and D2 is introduced. The plurality of D2 introduction paths m can be arranged so as to sandwich the dilution flow path at the same point p of the dilution flow path. With such a structure, it is possible to increase the number of interfaces between the aqueous solution flowing from the D1 introducing passage and the diluting liquid flowing from the diluting passage, and to increase the dilution rate. Further, in the example shown in FIG. 4, the flow passage width of the D1 introduction passage is 200 μm, whereas the flow passage width of the D2 introduction passage 1 and the D2 introduction passage 2 is 500 μm, and the passage width of the diluted passage after the merging. Is 1 mm. With such a structure, a large amount of diluting liquid can be flowed and the dilution ratio can be increased. FIG. 8A shows an example in which the D3 introduction passage merges after the D1 introduction passage and the D2 introduction passage merge at the point p in the dilution flow channel structure shown in FIG.

In the diluting flow channel structure used in the present invention, the Dn introducing passage is composed of a plurality of Dn introducing passages m, and the plurality of Dn introducing passages m (m is, for example, 1 to 5) are at the same point p of the diluting passage. It may be arranged so as to sandwich the dilution flow channel. In the example shown in FIG. 3B, the D5 introduction path 1 and the D5 introduction path 2 are arranged so as to sandwich the dilution flow path at the same point p of the dilution flow path. With such a structure, it is possible to increase the number of interfaces between the diluting liquid flowing from the Dn (D5 in FIG. 3) introducing passage and the aqueous solution flowing from the diluting passage, and to increase the dilution speed. Further, the example shown in FIG. 8B is the dilution flow channel structure of the aspect of FIG. 4, but is arranged so that the D3 introduction channel 1 and the D3 introduction channel 2 sandwich the dilution channel at the same point p of the dilution channel. Has been done.

In the diluting flow channel structure used in the present invention, the diluting flow channel may be branched into two or more in the middle, and in this case, the Dn introduction channel is arranged for each branched diluting flow channel. You can also stay. In the example shown in FIG. 2, the dilution flow passage is branched into two downstream of the D3 introduction passage, and the branched D4 introduction passage is arranged in each of the branched dilution passages. With such a structure, a large amount of diluting liquid can be flowed and the dilution ratio can be increased. In the example shown in FIG. 8D, the dilution flow path is branched into two downstream, and the D3 introduction path is arranged in each of the branched dilution flow paths.

The diluting flow channel structure used in the present invention may have two or more D1 introducing passages, and in this case, a D2 introducing passage should be arranged for each branched D1 passage. You can also Further, as in the example shown in FIG. 8C, the D1 introducing passage may be branched into two or more, and the D2 introducing passage may be arranged for each branched D1 passage.

<Aqueous solution C>
The aqueous solution C is an aqueous solution obtained by diluting the aqueous solution A, and the dilution in the diluting flow channel structure is performed by adjusting the water-miscible organic solvent to a concentration at which the particles Z in the aqueous solution can stably exist. Done. The concentration of the water-miscible organic solvent in which the particles in the aqueous solution can exist stably varies depending on the components constituting the particles, the particle size of the particles, the concentration of the particles in the aqueous solution C, the temperature, etc. The solvent concentration is 3 vol% or less, preferably 2 vol% or less, more preferably 1.5 vol% or less, and further preferably 1 vol% or less.

Immediately after the particles are formed, the particles contained in the aqueous solution A are in a dispersed state with the individual particles isolated in the aqueous solution. However, since the aqueous solution A contains a water-miscible organic solvent that is easily mixed with the self-assembling molecule which is a constituent component of the particles, and the concentration thereof is as high as 20 vol% or more, if the particles are left as it is, the particles will be aged. Destabilization of the particles occurs, which results in the fusion of particles and the division of particles. As a result, when the particles fuse, the particle diameter increases and the number of particles per unit volume decreases. Heretofore, the aqueous solution A containing particles has been diluted to further reduce the concentration of the water-miscible organic solvent, or subjected to dialysis to remove the water-miscible organic solvent. For example, in the method described in Patent Document 2, the particle-containing water-miscible organic solvent solution corresponding to the aqueous solution A prepared in the distribution system is diluted after preparation as shown in FIG. 3A of Patent Document 2. To the batch 360 of the above, or as described in FIG. 3B, the same amount (flow rate) of the diluent containing the particle-containing water-miscible organic solvent solution corresponding to the prepared aqueous solution A in the flow system. Were combined at 340 and then diluted by addition to batch 350 of dilute solution. However, in the method described in Patent Document 2, the water-miscible organic solvent concentration of the particle-containing water-miscible organic solvent solution after dilution is 10 to 40 vol%, which is too high to suppress the fusion of particles. In addition, it takes some time for the water-miscible organic solvent concentration in the diluted solution to reach this concentration evenly, and if there is a locally high water-miscible organic solvent concentration, the Fusion inhibition is limited.

On the other hand, in the production method of the present invention, the concentration of the water-miscible organic solvent in the aqueous solution C after dilution in the dilution channel is set to a concentration equal to or lower than a concentration at which particles in the aqueous solution can stably exist, for example, 3 vol% or less, and The dilution in the dilution flow path is not performed in one step, but is performed sequentially in two or more steps. According to the common sense of those skilled in the art, in order to lower the concentration of the water-miscible organic solvent and stabilize the particles as soon as possible, for example, as described in Patent Document 2, an excessive amount of a diluting solution is used to perform dilution in one step. It was thought that it should be done naturally, but in practice, as in the present invention, in two or more stages, dilution is performed such that the concentration is gradually decreased in each stage, and the same rate is obtained in one stage. It is possible to effectively prevent an increase in particle diameter due to fusion of particles during dilution, as compared with the case of performing dilution.

Multi-stage dilution is performed by combining aqueous solutions B _n (B ₂ to B ₆ ) from one or more Dn introduction channels (for example, n = 3 to 7) provided between the inlet and the outlet of the dilution channel. Can be implemented by The aqueous solution B _n may be an aqueous solution having the same composition as the aqueous solution B ₁ or an aqueous solution having a different composition. Even if the number of stages is too large, there is no great difference in preventing the particle size from increasing due to the fusion of particles during dilution. Therefore, n = 3 to 6, preferably n = 3 to 5, more preferably It can be in the range of n = 3-4. For example, in the case of n = 6, the introduction passages provided in the middle of the dilution passages are D3 to D6 introduction passages, and the total of D2 introduction passages has five stages. In the case of four stages, the introduction passages provided in the middle of the dilution passage are the introduction passages D3 to D5. FIG. 2 shows an example in the case of three stages. D2-D4 introduction paths are provided.

The water-miscible organic solvent concentration in the aqueous solution after merging with the first-stage D2 introducing passage is particularly limited, and the water-miscible organic solvent concentration in the aqueous solution after merging the aqueous solution from the second-stage and subsequent D3 introducing passages is particularly limited. However, the final concentration may be appropriately determined in consideration of the number of stages of dilution so that the final concentration is equal to or lower than the concentration at which the particles in the aqueous solution can exist stably. For example, the water-miscible organic solvent concentration of the aqueous solution A is 25 vol%, the water-miscible organic solvent concentration of the aqueous solution C is 1 vol%, and when diluting in three steps, for example, 25 vol% to 10 vol% (dilution ratio 2.5 It can be from 4 times (vol.) To 4 vol% (2.5 times dilution) to 1 vol% (4 times dilution).

The shorter the residence time of the aqueous solution in the dilution flow channel, that is, the faster the flow rate, the faster the dilution is performed, and the more rapidly the aqueous solution C having a concentration below the concentration at which particles in the aqueous solution can stably exist can be obtained. The particle size of the particles in the obtained aqueous solution tends to be small. In the present invention, the retention time of the aqueous solution in the dilution flow channel is preferably in the range of, for example, 1000 milliseconds or less from the above viewpoint. The residence time of the aqueous solution in the dilution channel varies depending on the volume of each introduction channel and the volume of the dilution channel, and further, the amount of the aqueous solution introduced from each introduction channel per unit, for example, 1000 milliseconds or less. , Preferably 800 milliseconds or less, more preferably 700 milliseconds or less.

In the diluting channel structure used in the present invention, the D1 introducing channel, the D2 introducing channel, the diluting channel and the Dn introducing channel have a size in the depth direction of the channel (paper thickness direction in FIG. 1). Although not particularly limited, for example, it is set to about 10 to 1000 μm, more preferably about 50 to 200 μm, and the channel width is set to about 50 to 400 μm, more preferably about 50 to 200 μm. However, these are merely examples and are not intended to be limiting.

The flow rates of the respective solutions in the D1 introducing passage, the D2 introducing passage, the diluting passage and the Dn introducing passage are the particle content of the aqueous solution A and the water-miscible organic solvent concentration, the desired water-miscible organic solvent concentration of the aqueous solution C, and It can be appropriately determined in consideration of the residence time of the aqueous solution in the dilution flow channel and the like.

The concentration of the particles in the aqueous solution A is not particularly limited, but can be, for example, 20 mg / mL or less, and preferably 1 to 15 mg / mL.

The concentration of particles in the aqueous solution C is not particularly limited, but can be, for example, 5 mg / mL or less, and preferably 0.1 to 4 mg / mL.

The number average particle size of the particles in the aqueous solution C is 200 nm or less, preferably 180 nm or less, 160 nm or less, 140 nm or less, 120 nm or less, 100 nm or less. The lower limit of the number average particle size of the particles in the aqueous solution C is not particularly limited, but can be, for example, 10 nm or 20 nm.

In the diluting flow channel structure used in the present invention, the structure of the confluent portion of the D1 introducing passage and the D2 introducing passage and the structure of the diluting passage after the confluence, and each of the diluting passages after the Dn introducing passage are an aqueous solution of the aqueous solution A. It is preferable that the dilution with B ₁ and B _n has a structure that can be performed more uniformly and rapidly. Therefore, the dilution flow channel can be a micromixer having a flow channel portion that is two-dimensionally bent in at least a part thereof described in Patent Document 1. By having the two-dimensionally bent channel portion, the inflowing aqueous solution A and aqueous solution B1 are mixed with the flow in the dilution channel. An example is shown in FIG. In FIG. 1, each of the dilution channels DF1 and DF2 has 20 baffles, and the baffles form a channel portion that is two-dimensionally bent. However, the number of baffles provided in each dilution flow channel can be appropriately determined, and FIG. 1 is merely an example. Further, FIG. 5 shows an example of a flow path portion in which three-dimensionally bent or uneven portions are arranged. The arrows in FIG. 5 indicate the respective introduction paths.

The diluting flow channel may have a straight tubular shape or a smooth curved tubular shape without having a flow channel portion in which two-dimensionally bent or three-dimensionally bent or irregularly arranged. Even if the shape of the dilution flow path is a straight tube shape or a smooth curved tube shape, in the confluence part 1 of the D1 introduction path and the D2 introduction path, due to the difference in the flow velocity of the aqueous solution A and the aqueous solution B1, etc. , A mixed state occurs. Mixing also occurs due to molecular diffusion and the like at the liquid-liquid interface generated after the aqueous solution A and the aqueous solution B1 merge. Similarly, due to differences in the flow rate of the aqueous solution and the aqueous solution B ₂ from dilute channel DF1, mixed state occurs at the merging portion 2 of the dilution channel DF1 and D3 introduction path.

The structure of the confluence of the D1 introduction path and the D2 introduction path in the dilution flow channel structure used in the present invention, and the structure of the confluence with Dn in the dilution flow channel promotes rapid and uniform mixing of the respective aqueous solutions that merge. It is preferably a structure. An example of such a structure is shown in FIG. The structure before and after the merging portion shown in FIG. 6 can be appropriately applied to each merging portion.

The aqueous solution C discharged from the outlet of the dilution flow passage can be further subjected to dialysis to remove the water-miscible organic solvent.

The aqueous solution A may be prepared by using a particle-preparing flow channel structure for preparing a particle-containing water-miscible organic solvent aqueous solution by mixing the self-assembled molecule-containing water-miscible organic solvent and the particle-preparing aqueous solution. it can. Further, it is preferable that the particles are supplied to the D1 introduction passage of the diluting flow channel structure of the present invention immediately after the preparation using the particle preparing flow channel structure. For example, as shown in FIG. 2, a particle-containing water-miscible organic solvent aqueous solution A is prepared using a particle-preparing flow channel structure, and after the preparation, a D1 introducing path of the diluting flow channel structure of the present invention is continuously provided. It is preferable that the particles are supplied because the particles can be immediately diluted by the method of the present invention without giving time for fusing particles to each other and a particle-containing solution having a desired particle size can be obtained.

<Particle manufacturing flow path structure>
Another aspect of the present invention is a particle manufacturing channel structure having a diluent channel structure, a particle preparation channel structure, and a diluent channel structure. The flow path structure for producing particles is basically the same as that described in the production method of the present invention, and the above description can also be referred to.

The diluting flow channel structure of the present invention is a flow channel structure for diluting an aqueous solution containing particles Z containing a self-assembling molecule as a particle constituent component and a water-miscible organic solvent with another aqueous solution. Has a D1 introduction path and a D2 introduction path on the upstream side, has a dilution flow path on the downstream side from the confluence of the D1 introduction path and the D2 introduction path, and introduces one or more Dn in the middle of the dilution flow path. A diluting channel structure having channels (for example, n = 3 to 7).

The flow path structure for producing particles of the present invention has at least two Sq introduction paths (for example, q = 1 to 5) on the upstream side, and the particle preparation flow path on the downstream side from the confluence of each introduction path. And the diluting flow path structure has a D1 introducing path and a D2 introducing path on the upstream side, a diluting flow path on the downstream side from the confluence of the D1 introducing path and the D2 introducing path, and There is one or more Dn introduction passages (for example, n = 3 to 7) in the middle of the passage, and the outlet of the preparation passage and the D1 introduction passage are directly connected.

An example of a particle preparation flow channel structure having two Sq introduction paths has an S1 introduction path and an S2 introduction path on the upstream side, and a particle formation flow path on the downstream side from the confluence of the S1 introduction path and the S2 introduction path. Can have An example of this is shown in FIG. The diluting flow passage structure has the D1 introducing passage and the D2 introducing passage on the upstream side as described above, the diluting passage on the downstream side from the confluence of the D1 introducing passage and the D2 introducing passage, and the diluting flow passage. One or more Dn introduction paths (for example, n = 3 to 7) are provided in the middle of the path. An example of the diluting flow channel structure having the D2 introducing path to the D6 introducing path is shown in B of FIG. Then, the outlet of the particle forming flow path of the particle preparing flow path structure is directly connected to the D1 introduction path. By directly connecting the outlet of the particle formation flow path and the D1 introduction path, the aqueous solution A prepared in the particle preparation flow path structure is rapidly introduced into the dilution flow path structure after synthesis, and the water-miscible organic solvent It is possible to obtain an aqueous solution C in which the concentration is diluted stepwise, and the concentration of the water-miscible organic solvent is diluted to a concentration equal to or less than the concentration at which the particles can be stably present very quickly. FIG. 7 shows one mode of the particle production flow channel structure in which the outlet of the particle formation flow channel of the particle preparation flow channel structure is directly connected to the D1 introduction channel.

As shown in FIG. 4, the diluting flow channel structure constituting the diluting flow channel structure and the particle producing flow channel structure of the present invention has a plurality of D2 introducing passages m (m is, for example, 1 to 5, m is 1 and 2 in FIG. 4, and is composed of D2 introducing passage 1 and D2 introducing passage 2), and the plurality of D2 introducing passages m sandwich the dilution passage at the same point p of the dilution passage. Can be located in. With such a structure, it is possible to increase the number of interfaces between the aqueous solution flowing from the D1 introducing passage and the diluting liquid flowing from the diluting passage, and to increase the dilution rate. In the example shown in FIG. 4, the flow passage width of the D1 introduction passage is 200 μm, whereas the flow passage width of the D2 introduction passage 1 and the D2 introduction passage 2 is 500 μm, and the passage width of the dilution passage after joining is 1 mm. Is. With such a structure, a large amount of diluting liquid can be flowed and the dilution ratio can be increased.

In the diluting flow channel structure constituting the diluting flow channel structure and the particle producing flow channel structure of the present invention, the Dn introducing path is composed of a plurality of Dn introducing paths m, and the plurality of Dn introducing paths m (m For example, 1 to 5) can be arranged so as to sandwich the dilution flow path at the same point p of the dilution flow path. In the example shown in FIG. 3B, the D5 introduction path 1 and the D5 introduction path 2 are arranged so as to sandwich the dilution flow path at the same point p of the dilution flow path. With such a structure, the interface between the diluting liquid flowing from the Dn introducing passage and the diluting liquid flowing from the diluting passage can be increased, and the dilution speed can be increased.

In the diluting flow path structure used in the present invention, which constitutes the diluting flow path structure and the particle producing flow path structure, the diluting flow path is divided into two or more in the middle, and each branched diluting flow path. It is also possible to arrange a Dn introduction path with respect to. In the example shown in FIG. 2, the dilution flow path is branched into two downstream of the D3 introduction path, and the branched D4 introduction path is arranged in each of the branched dilution flow paths.

In the diluting flow channel structure used in the present invention and the diluting flow channel structure constituting the particle manufacturing flow channel structure, the D1 introduction channel may be branched into two or more, and in this case, The D2 introducing passage may be arranged with respect to the branched D1 passage.

Hereinafter, the present invention will be described in more detail based on examples. However, the examples are exemplifications of the present invention, and the present invention is not intended to be limited to the examples.

Example 1
The lipid particle-containing aqueous solution A prepared under the following particle preparation conditions is used to directly connect the particle preparation channel structure and the dilution channel structure (20 baffles) shown in FIG. Aqueous solution C was prepared by diluting in the order of 25 → 5 → 2.5 → 1% by three-stage multi-stage dilution. After the preparation, the particle size distribution of the particles in the aqueous solution C was measured using Zetasizer Nano ZS (Malvern). The results are shown in Fig. 9.
Particle preparation conditions: 10 mg / mL POPC + saline iLiNP device, 50 μL / min FRR3

Comparative Example 1
The lipid particle-containing aqueous solution A prepared under the same particle preparation conditions as in Example 1 was directly connected to the particle preparation channel structure at one stage of the dilution channel structure (20 baffles) to produce a particle production channel structure. (See FIG. 7) was used to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 9.

Comparative example 2
The lipid particle-containing aqueous solution A prepared by using the same particle preparation channel structure (no dilution channel structure) as in Example 1 under the same particle preparation conditions as in Example 1 was dialyzed after particle preparation. , And diluted by pipetting (1 step or 3 steps) to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 9.

The particle ratio within the number average ± 10% was as low as 20% with the one-step dilution of Comparative Example 1, whereas the three-step dilution of Example 1 was 30%. The average particle size of the lipid particles obtained by the dialysis of Comparative Example 2 and the dilution by pipetting (1 step or 3 steps) was significantly larger than that of Example 1.

Example 2
The lipid particle-containing aqueous solution A prepared under the following particle preparation conditions is used to directly connect the particle preparation channel structure and the dilution channel structure (20 baffles) shown in FIG. An aqueous solution C was prepared by diluting in the order of 25 → 10 → 4 → 1% by three-stage multi-stage dilution. After the preparation, the particle size distribution of the particles in the aqueous solution C was measured in the same manner as in Example 1. The results are shown in Fig. 10.
Particle preparation conditions: Lipid: YSK05 / ethanol Water system: MES buffer

Comparative Example 3
The lipid particle-containing aqueous solution A prepared under the same particle preparation conditions as in Example 2 was directly connected to the particle preparation channel structure at one stage of the dilution channel structure (20 baffles) to produce a particle production channel structure. (See FIG. 7) was used to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 10.

Comparative Example 4
The lipid particle-containing aqueous solution A prepared using the same particle preparation flow channel structure (no dilution flow channel structure) as in Example 2 under the same particle preparation conditions as in Example 2 was prepared by the following procedure. Dilution by petting (one step) was performed to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 10.

In Example 1, the particle ratio within the number average ± 10% was higher than that of Comparative Example 1, but the average particle size of Comparative Example 1 was smaller. On the other hand, in Example 2 (three-step dilution), the particle ratio within the number average ± 10% was higher and the average particle diameter was smaller than in Comparative Example 2 (one-step dilution). This result shows that the optimal dilution rate (ethanol% / s) was obtained by setting the dilution step to 25 → 10 → 4 → 1%, which enabled more precise control of particle size. ..

Example 3
The lipid particle-containing aqueous solution A prepared under the following particle production conditions is used as a particle production flow channel structure in which the particle preparation flow channel structure and the dilution flow channel structure (without baffles) shown in FIG. 4 are directly connected. Then, an aqueous solution C was prepared by diluting in the order of 25 → 10 → 4 → 1% by three-step multi-step dilution by the following operation. After the preparation, the particles were subjected to the following post-treatment method, and then the particle size distribution of the particles in the aqueous solution C was measured in the same manner as in Example 1. The results are shown in Fig. 11.

(1) The nucleic acid / buffer solution was introduced from the buffer solution or the nucleic acid / buffer solution supply port of the integrated device without a mixer in FIG. 4, and YSK05 / ethanol was introduced from the lipid solution supply port. In addition, the MES buffer solution was introduced from the D2 introduction paths 1 and 2.
Particle preparation: 50 μL / min (FRR3) D1 In the introduction path, particles are generated upstream from point p, and the ethanol concentration in the particle suspension is 25%. Ethanol in the particle suspension is diluted to 10% by the MES buffer solution that flows in from the D2 introduction paths 1 and 2 at the following flow rates. Diluted solution 1: 75 μL / min (introduced from D2 introduction channels 1 and 2) → recovery (10% EtOH)
(2) The particle suspension diluted previously was introduced from both the nucleic acid / lipid solution inlet of the integrated device without a mixer. The MES buffer solution (diluted solution 2) was introduced from D2 introduction paths 1 and 2 under the following conditions.
Particle suspension: 125 μL / min, diluted solution 2: 187.5 μL / min → recovery (4% EtOH)
(3) The previously diluted particle suspension was introduced from both the nucleic acid / lipid solution inlet of the integrated device without a mixer. The MES buffer solution (diluted solution 2) was introduced from D2 introduction paths 1 and 2 under the following conditions.
Particle suspension: 312.5 μL / min, diluted solution 2: 937.5 μL / min → recovery (1% EtOH)

Post-treatment method (immediately after dilution with MES buffer)
2. Dialysis with PBS (pH 7.4) (overnight, 4 ℃) ・・・ Replacement with PBS

Comparative Example 5
A particle production channel structure in which a lipid particle-containing aqueous solution A prepared under the following particle preparation conditions is directly connected to a particle preparation channel structure with one-step dilution channel structure (without baffle) (see FIG. 4). Was used to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the same post-treatment method as in Example 3 was applied, and then the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 11.
Particle preparation: 50 μL / min (FRR3), Diluted solution: 1200 μL / min

Comparative Example 6
Under the same particle preparation conditions as in Example 3, the lipid particle-containing aqueous solution A prepared using the same particle preparation channel structure as in Example 3 (without a diluent channel structure) was prepared by the following procedure. Dilution by petting (one step) was performed to prepare a diluted solution of the lipid particle-containing aqueous solution. After the preparation, the same post-treatment method as in Example 3 was applied, and then the particle size distribution of the particles in the aqueous solution was measured in the same manner as in Example 1. The results are shown in Fig. 11.

From the results shown in FIG. 11, the lipid particles in the aqueous solution prepared by multi-step dilution of Example 3 showed the smallest change in particle size after MES substitution and PBS substitution.

Example 4
The lipid nanoparticle composed of pH-responsive cationic lipid (YSK05), cholesterol, PEG lipid, and siRNA was directly connected to the particle preparation channel structure and the dilution channel structure (20 baffles) shown in FIG. Aqueous solution C was prepared by diluting in the order of 25 → 10 → 4 → 1% by three-step multi-step dilution using the flow channel structure for particle production. The performance of the particles was evaluated using mice using the aqueous solution, and the results are shown in FIGS. 12 and 13. We succeeded in knocking down the target gene and confirmed that the delivery efficiency depends on the particle size.

Example 5
A particle production flow channel structure (iLiNP device) in which a lipid particle-containing aqueous solution A prepared under the following particle production conditions is directly connected to a particle preparation flow channel structure and a dilution flow channel structure (20 baffles) shown in FIG. Was used to prepare an aqueous solution C by diluting in the order of 25 → 1% by two-step multi-step dilution. After the preparation, the particle size distribution of the particles in the aqueous solution C was measured using Zetasizer Nano ZS (Malvern). The results are shown in FIG. 14 (the device in the figure is the result of this example). For comparison, the results of dialysis instead of using the diluting flow channel structure and diluting with a pipette are also shown.
Particle preparation conditions:
Lipid solution 65 mM POPC / ethanol aqueous solution physiological saline (154 mM NaCl aqueous solution)
Primary dilution Total flow rate 100 μL / min (FRR3)
Secondary dilution Total flow rate 2500 μL / min (FRR24)

Since the lipid solution was a high-concentration lipid solution, conventional post-treatments (dialysis and pipette) resulted in non-uniform particle size (the difference between the number average and the Z average was large). On the other hand, in the present example (device), the continuous treatment of particle production and dilution improved the particle uniformity and also improved the dispersion (standard deviation).

Example 6
A particle production flow channel structure (iLiNP device) in which a lipid particle-containing aqueous solution A prepared under the following particle production conditions is directly connected to a particle preparation flow channel structure and a dilution flow channel structure (20 baffles) shown in FIG. Was used to prepare an aqueous solution C by diluting in the order of 25 → 1% by two-step multi-step dilution. After the preparation, the particle size distribution of the particles in the aqueous solution C was measured using Zetasizer Nano ZS (Malvern). The results are shown in Fig. 15. (The device in the figure is the result of this example.). For comparison, the results of dialysis instead of using the diluting flow channel structure and diluting with a pipette are also shown.
Particle preparation conditions:
Lipid solution 15 mM DPPC / cholesterol / DSPE-PEG2K / ethanol (67/33/5 mol%)
Aqueous solution PBS
Primary dilution Total flow rate 100 μL / min (FRR3)
Secondary dilution Total flow rate 2500 μL / min (FRR24)

In this example (device), the variation (standard deviation) was improved. Furthermore, in this example (device), particles having both a number average particle diameter and a Z average particle diameter of 100 nm or less could be produced.

The present invention is useful in the field of technology for preparing particles containing a self-assembling molecule as a particle constituent while controlling the particle size.

Claims

An aqueous solution A containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent at a concentration of 20 vol% or more is diluted with an aqueous solution B 1 to have a number average particle diameter of 200 nm or less. A method comprising obtaining an aqueous solution C containing particles Z, comprising:
The dilution has a D1 introduction path and a D2 introduction path on the upstream side, a dilution flow path on the downstream side from the confluence of the D1 introduction path and the D2 introduction path, and one or more in the middle of the dilution flow path. Using a diluting channel structure having a Dn introducing channel (for example, n = 3 to 7),
The aqueous solution A is supplied to the D1 introduction path and the aqueous solution B 1 is supplied to the D2 introduction path, and the aqueous solution B n (for example, n = 2 to 6) is added to the aqueous solution in the dilution flow path from the Dn introduction path to obtain a diluted flow. A method for producing an aqueous solution containing particles, which comprises obtaining an aqueous solution C diluted with a water-miscible organic solvent concentration to a concentration at which particles can stably exist from an outlet of a passage.
An aqueous solution A containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent at a concentration of 20 vol% or more is diluted with an aqueous solution B 1 to have a number average particle diameter of 200 nm or less. A method comprising obtaining an aqueous solution C containing particles Z, comprising:
The dilution has a D1 introduction path and a Dn (for example, n = 2 to 7) introduction path on the upstream side, and the Dn introduction paths are arranged such that all the introduction paths sandwich the D1 introduction path at a point p. Using a diluting flow channel structure having a diluting flow channel on the downstream side from p,
An aqueous solution A is supplied to the D1 introduction path and an aqueous solution B n (for example, n = 1 to 6) is supplied to the Dn introduction path, and the water-miscible organic solvent is supplied from the outlet of the dilution flow path to a concentration at which the particles can stably exist. A method for producing an aqueous solution containing particles, which comprises obtaining an aqueous solution C having a diluted concentration.
The method according to claim 1 or 2, wherein the self-assembling molecule is composed of either A) a lipid, B) an amphipathic substance, or both A) and B).
The production method according to any one of claims 1 to 3, wherein the water-miscible organic solvent is an alkanol.
The production method according to any one of claims 1 to 4, wherein the particles Z are liposomes, lipid micelles, or polymer micelles.
The D2 introducing passage is composed of a plurality of D2 introducing passages m, and the plurality of D2 introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The manufacturing method described.
7. The Dn introducing passage is composed of a plurality of Dn introducing passages m, and the plurality of Dn introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The manufacturing method of statement x.
8. The manufacturing method according to claim 1, wherein the D1 introducing passage is branched into two or more, and the D2 introducing passage is arranged for each branched D1 flow passage.
The manufacturing method according to any one of claims 1 to 8, wherein the dilution flow channel is branched into two or more on the way, and a Dn introduction channel is arranged for each branched dilution flow channel.
The production method according to claim 1, wherein the residence time of the aqueous solution in the dilution flow channel is 1000 milliseconds or less.
The production method according to any one of claims 1 to 10, wherein the concentration of the water-miscible organic solvent contained in the aqueous solution C is 3 vol% or less.
The aqueous solution A is prepared by using a particle-preparing flow channel structure for preparing a particle-containing aqueous solution by mixing a water-miscible organic solvent containing a self-assembling molecule and a particle-preparing aqueous solution, and immediately after preparation, The manufacturing method according to any one of claims 1 to 11, which is supplied to a D1 introduction path of the diluting flow path structure.
The particle-preparing flow path structure has an Sq introduction path consisting of at least two or more on the upstream side, has a particle preparation flow path on the downstream side from the joining portion of each introduction path, and has an aqueous solution from the outlet of the particle preparation flow path. The manufacturing method according to claim 12, wherein A is discharged, and the discharged aqueous solution A is supplied to the D1 introducing passage.
The manufacturing method according to claim 13, wherein the particle preparation channel and the dilution channel each include a micromixer in one or one of the channels.
15. The production method according to claim 1, wherein the number average particle diameter of the lipid particles in the aqueous solution C is 100 nm or less or 60 nm or less.
The method according to any one of claims 1 to 15, wherein the concentration of the lipid particles in the aqueous solution A is 20 mg / mL or less and the concentration of the lipid particles in the aqueous solution C is 5 mg / mL or less.
The production method according to any one of claims 1 to 16, wherein the aqueous solution C discharged from the outlet of the dilution channel is further subjected to dialysis to obtain an aqueous solution from which the water-miscible organic solvent is removed.
The production method according to any one of claims 1 to 17, wherein the particles Z include an encapsulated material.
A flow channel structure for diluting an aqueous solution containing a particle Z containing a self-assembling molecule as a particle constituent and containing a water-miscible organic solvent, the upstream side being a D1 introduction path and a D2 introduction A diluting flow path downstream from the confluence of the D1 introducing path and the D2 introducing path, and one or more Dn introducing paths (for example, n = 3 to 7) in the middle of the diluting path. A diluting flow channel structure having.
A flow channel structure for diluting an aqueous solution containing particles Z containing self-assembled molecules as a particle constituent and containing a water-miscible organic solvent with a D1 introducing path and a Dn ( For example, n = 2 to 7) has an introduction path, all of the Dn introduction paths are arranged so as to sandwich the D1 introduction path at the point p, and a dilution flow path is provided downstream from the point p for dilution. A diluting flow channel structure having a flow channel structure.
A particle production flow channel structure having a particle preparation flow channel structure and a dilution flow channel structure,
The particle-preparing flow channel structure has an Sq introducing passage consisting of at least two or more on the upstream side, and has a particle preparing passage on the downstream side from the joining portion of each introducing passage,
The diluting flow path structure has a D1 introducing path and a D2 introducing path on the upstream side, a diluting flow path on the downstream side from the confluence of the D1 introducing path and the D2 introducing path, and in the middle of the diluting path. Having one or more Dn introduction paths (eg, n = 3 to 7),
A flow channel structure for producing particles, wherein the outlet of the preparation flow channel is directly connected to the D1 introduction channel.
The D2 introducing passage is composed of a plurality of D2 introducing passages, and the plurality of D2 introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The diluting flow channel structure or the flow channel structure for producing particles.
The Dn introducing passage is composed of a plurality of Dn introducing passages m, and the plurality of Dn introducing passages m are arranged so as to sandwich the dilution passage at the same point p of the dilution passage. The diluted flow channel structure or the flow channel structure for producing particles as described above.
The dilution flow channel structure or particle production according to any one of claims 19 to 23, wherein the D1 introduction passage is branched into two or more, and the D2 introduction passage is arranged for each branched D1 passage. Flow channel structure.
The dilution flow channel structure according to any one of claims 19 to 24, wherein the dilution flow channel has two or more branches on the way, and a Dn introduction channel is arranged for each branched dilution flow channel. A flow channel structure for producing particles.