Disk-based microfluidic platform for manufacturing lipid nanoparticles using centrifugal force and method using same

Abstract

The present invention relates to a microfluidic platform for manufacturing lipid nanoparticles, provided as a disk member and including: a microfluidic channel that uses centrifugal force and manufactures lipid nanoparticles (LNPs) therein; at least one inlet through which a fluid is supplied to the microfluidic channel; and at least one outlet through which the fluid is discharged from the microfluidic channel.

Description

Microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force and method using the same

[0001] The present invention relates to a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force and a method using the same, and more particularly, to a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force capable of manufacturing lipid nanoparticles with high efficiency and a method using the same.

[0002] Lipid nanoparticles (LNPs) can be used as delivery vehicles for biologically active substances, such as small molecule drugs, proteins, and nucleic acids, into cells and / or intracellular compartments. Lipid nanoparticles are manufactured by physically mixing key components. Depending on the mixing method, the size, physical properties, and encapsulation efficiency of the lipid nanoparticles vary.

[0003] Methods used to manufacture lipid nanoparticles include pipette mixing and microfluidic mixing. Pipette mixing, which simply mixes two solutions using pipetting, offers the advantages of easy accessibility and low cost, and is primarily used in small-volume research. However, pipette mixing has the disadvantages of making it difficult to control the size of lipid nanoparticles and low encapsulation efficiency of the manufactured lipid nanoparticles. Microfluidic mixing is performed by connecting a mixing chip, where two solutions are mixed, to a syringe pump, or using a mixing system such as a nano-assembler. Microfluidic mixing has the advantages of reproducibly and uniformly manufacturing lipid nanoparticles, allowing for controllable small size, and exhibiting relatively high encapsulation efficiency. On the other hand, microfluidic mixing has the disadvantage of generating a large amount of dead volume within devices such as mixing chips and making it difficult to control manufacturing capacity, making it unsuitable for mass production or even ultra-small quantities. Furthermore, the requirement for a mixing system results in relatively high costs.

[0004] Accordingly, various studies are being conducted to ensure that lipid nanoparticles, which are being used in various ways recently, have properties and sizes that do not differ from batch to batch, while facilitating mass production.

[0005] The purpose of the present invention is to provide a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force, which manufactures lipid nanoparticles having a controlled size with high production efficiency, and a method using the same.

[0006] In addition, another object of the present invention is to provide a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force, which can mass-produce lipid nanoparticles of various uses with uniform sizes, and a method using the same.

[0007] According to one aspect of the present invention, embodiments of the present invention include a microfluidic platform for manufacturing lipid nanoparticles and a method using the microfluidic platform for manufacturing lipid nanoparticles.

[0008] In one embodiment, the microfluidic platform for manufacturing lipid nanoparticles may include a microfluidic channel that uses centrifugal force to manufacture lipid nanoparticles (LNPs) therein; one or more inlets that supply fluid to the microfluidic channel; one or more outlets through which fluid is discharged from the microfluidic channel; and a disk member that rotates the fluid.

[0009] In one embodiment, the microfluidic channel extends in one direction, the inlet portion and the outlet portion are connected to one end and the other end of the microfluidic channel, respectively, and extend upward with respect to the microfluidic channel, the inlet portion may be provided at a first angle with respect to a first plane parallel to the ground, and the outlet portion may be provided at a second angle with respect to the first plane.

[0010] In one embodiment, the first angle is 20° to 85°, the second angle is 85° or less, and as the first angle and the second angle decrease, the size of the lipid nanoparticle may decrease.

[0011] In one embodiment, the first angle and the second angle may be provided at the same angle within a range of 45° to 70°.

[0012] In one embodiment, the inner diameter of the microfluidic channel may be 100 μm to 300 μm, and the Reynolds number of the fluid in the microfluidic channel may be 5 to 1520.

[0013] In one embodiment, the disk member rotates to generate the centrifugal force inside the microfluidic channel, the rotation speed of the disk member is 500 rpm to 4000 rpm, the average diameter of the lipid nanoparticles is 10 nm to 300 nm, and the polydispersity index (PDI) may be 0.20 or less.

[0014] In one embodiment, the inlet portion is provided in two or more numbers so that a lipid mixture and an active material mixture are introduced into the microfluidic channel, and the average velocity of the fluid in the inlet portion may be 1 μL / s to 200 μL / s.

[0015] In one embodiment, the lipid mixture comprises an ionizable lipid, a helper lipid, a stabilizing lipid, and a PEG-lipid, the active substance mixture comprises at least one selected from the group consisting of a chemotherapeutic agent, a small molecule drug, a protein, and a nucleic acid, and the N / P ratio of the lipid mixture and the active substance mixture may be 1 to 30.

[0016] In one embodiment, the microfluidic chip comprises at least one microfluidic chip formed by including the microfluidic channel, the at least one inlet portion, and the at least one outlet portion, and mounted on one surface of the disk member, wherein the microfluidic chip may be provided to be reversibly detachable from the disk member.

[0017] In one embodiment, the microfluidic chip includes a body portion in which the microfluidic channel, the inlet portion, and the outlet portion are formed; and at least one cover portion covering at least one surface and the other surface of the body portion; wherein the microfluidic channel is formed to be concave inward on one surface of the body portion, and the inlet portion and the outlet portion can be formed to penetrate the body portion.

[0018] In one embodiment, the cover portion includes a first cover and a second cover, the first cover is provided to cover one surface of the body portion, and includes one or more holes provided at positions corresponding to the inlet portion and the outlet portion, and the second cover can cover the other surface of the body portion.

[0019] In one embodiment, one surface of the disk member further includes one or more mounting portions on which the microfluidic chip is mounted, and the mounting portions are provided in a size corresponding to the microfluidic chip and are provided inwardly concave on one surface of the disk member so that the lower surface of the microfluidic chip can be inserted.

[0020] In one embodiment, the microfluidic chip may be provided in a box shape and arranged radially based on the center of the disk member, the inlet portion may be provided adjacent to the center of the disk member, and the outlet portion may be provided adjacent to the edge of the disk member.

[0021] In one embodiment, the microfluidic channel may include at least one of a herringbone mixer pattern, a serpentine mixer pattern, and a toroidal mixer pattern.

[0022] In one embodiment, a method of using the microfluidic platform for manufacturing lipid nanoparticles as described above may include the steps of preparing a lipid mixture and an active substance mixture; injecting the lipid mixture and the active substance mixture into an inlet; and rotating a disk member.

[0023] In one embodiment, the inlet portion is provided in two or more numbers so that a lipid mixture and an active material mixture are introduced into the microfluidic channel, the average velocity of the fluid in the inlet portion may be 1 μl / s to 200 μl / s, and the rotational speed of the disk member may be 500 rpm to 4000 rpm.

[0024] In one embodiment, the inlet portion is connected to one end of a microfluidic channel, and the Reynolds number of the fluid inside the microfluidic channel may be 5 to 1520.

[0025] In one embodiment, the lipid mixture and the active material mixture are injected into the inlet portion so that the N / P ratio is 1 to 30, the lipid mixture and the active material mixture are mixed by centrifugal force generated inside the microfluidic channel to produce lipid nanoparticles, and the lipid nanoparticles are discharged through an outlet connected to the other end of the microfluidic channel, and the average diameter of the lipid nanoparticles may be 10 nm to 300 nm.

[0026] According to the present invention as described above, a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force and a method using the same can be used to manufacture lipid nanoparticles with high encapsulation efficiency and a uniform size.

[0027] In addition, according to the present invention, a microfluidic platform for manufacturing disk-based lipid nanoparticles using centrifugal force and a method using the same enable easy mass production of lipid nanoparticles, and provide lipid nanoparticles that can be used for various purposes.

[0028] FIG. 1 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to one embodiment of the present invention.

[0029] Figure 2 is a drawing showing the angles of the inlet and outlet of the microfluidic chip of Figure 1.

[0030] Figure 3 is a drawing showing the upper surface of the disk member of Figure 1.

[0031] Figure 4 is a drawing showing a microfluidic channel according to an embodiment of the present invention.

[0032] FIG. 5 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to another embodiment of the present invention.

[0033] FIG. 6 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to another embodiment of the present invention.

[0034] Figure 7 is a drawing manufactured using 3D printing of Figure 6.

[0035] Figure 8 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles used in the examples.

[0036] Figure 9 shows the size of lipid nanoparticles according to the angle of the inlet portion of a microfluidic chip according to an embodiment of the present invention.

[0037] Figure 10 shows the results of comparing lipid nanoparticles manufactured according to each of the pipetting mixing methods according to the microfluidic platform of the present invention and the comparative example.

[0038] Figure 11 shows the results of comparing lipid nanoparticles manufactured according to each of the microfluidic platform according to an embodiment of the present invention and syringe mixing according to a comparative example.

[0039] Figure 12 shows the results of confirming the intracellular delivery efficiency according to the manufacturing method of lipid nanoparticles including eGFP-mRNA.

[0040] Figure 13 shows the results of confirming the intracellular delivery efficiency according to the manufacturing method of lipid nanoparticles including F-Luc-mRNA.

[0041] Figure 14 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to HEK293 cells.

[0042] Figure 15 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to CHO-K1 cells.

[0043] Figure 16 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to Jurkat cells.

[0044] Figure 17 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles used in another embodiment of the present invention.

[0045] Figure 18 shows the size of lipid nanoparticles manufactured with a microfluidic chip having inverted microfluidic channels formed in a serpentine mixer pattern.

[0046] Figure 19 shows the size of lipid nanoparticles manufactured with a microfluidic chip having inverted microfluidic channels formed in a toroidal mixer pattern.

[0047] Figure 20 is a schematic drawing of a microfluidic platform for manufacturing lipid nanoparticles used in another embodiment of the present invention.

[0048] Figure 21 is a photograph of the microfluidic platform for manufacturing lipid nanoparticles of Figure 20.

[0049] Figure 22 shows the size and encapsulation efficiency of lipid nanoparticles fabricated in a microfluidic chip including microfluidic channels formed in a serpentine mixer pattern using a 3D printer.

[0050] Specific details of other embodiments are included in the detailed description and drawings.

[0051] The advantages and features of the present invention, and the methods for achieving them, will become clear with reference to the embodiments described below in detail together with the accompanying drawings. However, the present invention is not limited to the embodiments disclosed below, but can be implemented in various different forms, and unless otherwise specified in the following description, all numbers, values, and / or expressions expressing components, reaction conditions, and contents of components in the present invention are to be understood as being modified in all cases by the term "about" because such numbers are approximations that reflect various uncertainties of measurement that occur in obtaining such values, among other things. In addition, when a numerical range is disclosed herein, such range is continuous and includes every value from the minimum value to the maximum value inclusive, unless otherwise indicated. Furthermore, when such a range refers to an integer, every integer from the minimum value to the maximum value inclusive, unless otherwise indicated, is included.

[0052] Additionally, when a range is described for a variable in the present invention, it will be understood that the variable includes all values ​​within the described range including the described endpoints of the range. For example, the range "5 to 10" will be understood to include the values ​​5, 6, 7, 8, 9, and 10, as well as any subranges such as 6 to 10, 7 to 10, 6 to 9, 7 to 9, etc., and also any value between integers that fall within the described range, such as 5.5, 6.5, 7.5, 5.5 to 8.5, and 6.5 to 9. For example, a range of "10% to 30%" would be understood to include values ​​such as 10%, 11%, 12%, 13%, etc., and all integers up to and including 30%, as well as any subranges such as 10% to 15%, 12% to 18%, 20% to 30%, and any value between reasonable integers within the stated range, such as 10.5%, 15.5%, 25.5%, etc.

[0053]

[0054] FIG. 1 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to one embodiment of the present invention. FIG. 2 is a diagram illustrating the angles of the inlet and outlet of the microfluidic chip of FIG. 1. FIG. 3 is a diagram illustrating the upper surface of the disk member of FIG. 1. FIG. 4 is a diagram illustrating a microfluidic channel according to an embodiment of the present invention.

[0055] A microfluidic platform (100) for producing lipid nanoparticles according to one embodiment of the present invention may include a microfluidic channel (110) that uses centrifugal force to produce lipid nanoparticles (LNPs) therein; one or more inlet portions (120) that supply fluid to the microfluidic channel (110); one or more outlet portions (130) through which fluid is discharged from the microfluidic channel (110); and a disk member (150) that rotates the fluid.

[0056] Conventional methods for manufacturing lipid nanoparticles, such as pipetting mixing or syringe pumps, have the disadvantage that the physical properties of lipid nanoparticles vary depending on the operating environment, equipment performance, and operator. Furthermore, while demand for lipid nanoparticles has recently increased alongside the development of RNA vaccines, mass production of these lipid nanoparticles with uniform physical properties remains challenging.

[0057] A microfluidic platform (hereinafter, “microfluidic platform”) (100) for manufacturing lipid nanoparticles according to an embodiment of the present invention can uniformly manufacture lipid nanoparticles in small sizes. Furthermore, the physical properties of lipid nanoparticles can be controlled to be uniform regardless of the operator or work environment, and lipid nanoparticles can be efficiently mass-produced. Lipid nanoparticles manufactured using the microfluidic platform according to an embodiment of the present invention can be utilized for various purposes, such as intracellular drug delivery, gene therapy, and RNA delivery.

[0058] A microfluidic platform (100) according to an embodiment of the present invention may include a microfluidic channel (110) through which a fluid passes and receives a centrifugal force, and lipid nanoparticles are manufactured by the centrifugal force. The fluid is injected into the microfluidic channel (110) through the inlet portion (120), and the lipid nanoparticles manufactured in the microfluidic channel (110) may be discharged together with the fluid to the outlet portion (130). The fluid may include a lipid mixture and an active substance mixture for manufacturing the lipid nanoparticles. The fluid including the lipid mixture and the active substance mixture may be manufactured into lipid nanoparticles while passing through the microfluidic channel (110).

[0059] The microfluidic channel (110) extends in one direction, and the inlet portion (120) and the outlet portion (130) are respectively connected to one end and the other end of the microfluidic channel (110), and may extend upward with respect to the microfluidic channel (110). The inlet portion (120) may be provided at a first angle (θ1) with respect to a first plane parallel to the ground, and the outlet portion (130) may be provided at a second angle (θ2) with respect to the first plane. For example, when the microfluidic channel (110) is provided in a first plane formed by the xy axes, the inlet portion (120) and the outlet portion (130) may be provided to extend in the z-axis direction and have a first angle (θ1) and a second angle (θ2).

[0060] The above microfluidic channel (110) is provided so that a fluid can flow in the first plane, and can form a flow path of various shapes such as a straight or curved shape. The inlet portion (120) and the outlet portion (130) are formed in an upper direction with respect to the microfluidic channel (110), and are provided at a first angle (θ1) and a second angle (θ2), respectively, so that the fluid can be introduced into or discharged from the microfluidic channel (110).

[0061] The first angle (θ1) may be 20° to 85°, and the second angle (θ2) may be 85° or less. When the first angle (θ1) and the second angle (θ2) are within the specified ranges, a stable lipid nanoparticle size is formed, and at this time, no change in the size of the particles may occur. Each of the first angle (θ1) and the second angle (θ2) may be an acute angle with respect to an angle measured clockwise or counterclockwise with respect to the first plane.

[0062] If the first angle (θ1) is less than 20°, excessive shear stress occurs between the inner surface of the inlet portion (120) and the solvent and solute constituting the fluid, which may be problematic, and as the space occupied by the inlet portion increases, the length of the chip may increase, making it unsuitable for manufacturing a small-sized circular chip as shown in Fig. 6. If it is greater than 85°, the size of the manufactured lipid nanoparticles may be large and the particle size may not be uniform, which may be problematic.

[0063] Specifically, the first angle (θ1) may be 20° to 80°, or 20° to 75°, or 20° to 70°, or 30° to 85°, or 40° to 85°, or 45° to 85°, or 45° to 80°, or 45° to 70°.

[0064] If the second angle (θ2) exceeds 85°, it is difficult to effectively recover lipid nanoparticles, which is problematic. Specifically, the second angle (θ2) may be 80° or less, or 75° or less, or 70° or less, or 45° to 70°.

[0065] More specifically, the first angle and the second angle may be provided at the same angle within a range of 45° to 70°. In addition, as the first angle and the second angle decrease, the size of the lipid nanoparticle may decrease, and by being provided within the above-described range, lipid nanoparticles having a uniform and small size may be produced.

[0066] The above inlet portion (120) is provided in two or more numbers so that a lipid mixture and an active material mixture are introduced into the microfluidic channel (110), and the average velocity of the fluid in the inlet portion (120) can be 1 μl / s to 200 μl / s. By providing two or more inlet portions (120), the mixing volume ratio of the lipid mixture and the active material mixture constituting the fluid can be controlled more accurately.

[0067] When the average velocity of the fluid in the inlet portion (120) is less than 1 μL / s, the mixing speed of the lipid mixture and the active material decreases, preventing uniform mixing, which is undesirable. When it exceeds 200 μL / s, further size reduction is not achieved due to the limit size of the lipid nanoparticles, which is an unnecessary condition. Specifically, the average velocity of the fluid in the inlet portion (120) may be 1.5 μL / s to 200 μL / s, or 1.5 μL / s to 150 μL / s, or 1.5 μL / s to 132.5 μL / s.

[0068] The inner diameter of the microfluidic channel may be 100 μm to 300 μm. If the inner diameter of the microfluidic channel is less than 100 μm, a very high rotational speed is required to achieve a desired flow rate within the microfluidic channel, which may make it difficult to produce stable lipid nanoparticles. If the inner diameter of the channel exceeds 300 μm, the mixing efficiency is reduced due to the enlarged fluid volume, which causes a problem in that the efficiency of producing lipid nanoparticles is reduced. Here, since the microfluidic channel has a rectangular cross-section, the inner diameter of the microfluidic channel is a hydraulic diameter.

[0069] The Reynolds number of the fluid in the microfluidic channel may be 5 to 1520. By providing the Reynolds number of the fluid within the aforementioned range, lipid nanoparticles having uniform physical properties can be manufactured with high encapsulation efficiency.

[0070] The above disk member (150) can rotate to generate the centrifugal force inside the microfluidic channel (110). The rotation speed of the disk member (150) may be 500 rpm to 4000 rpm. If the rotation speed of the disk member (150) is less than 500 rpm, the size of the lipid nanoparticles becomes large and uneven, which is problematic. In addition, since the size of the lipid nanoparticles does not decrease any further after 4000 rpm, if the process is performed at more than 4000 rpm, unnecessary energy may be wasted, which may lower the process efficiency. Specifically, the rotation speed of the disk member (150) may be 700 rpm to 3500 rpm, 800 rpm to 3000 rpm, or 900 rpm to 3000 rpm, or 1000 rpm to 3000 rpm.

[0071] The average diameter of the lipid nanoparticles may be 10 nm to 300 nm. It is difficult to manufacture the lipid nanoparticles when the size is less than about 10 nm due to the influence of the internal delivery substance, and when the size exceeds 300 nm, a problem of reduced intracellular delivery efficiency may occur. In addition, the polydispersity index (PDI) of the lipid nanoparticles may be 0.20 or less. Specifically, the average diameter of the lipid nanoparticles may be 10 nm to 250 nm, or 10 nm to 200 nm, or 20 nm to 300 nm, or 40 nm to 300 nm, or 60 nm to 300 nm, or 80 nm to 300 nm, or 80 nm to 200 nm.

[0072] The fluid may comprise a lipid mixture and an active substance mixture. The lipid mixture may comprise an ionizable lipid, a helper lipid, a stabilizing lipid, and a PEG-lipid, and the active substance mixture may comprise at least one selected from the group consisting of chemotherapeutic agents, small molecule drugs, proteins, and nucleic acids. The nucleic acid may be at least one selected from the group consisting of genes, recombinant genes, plasmid genes, DNA molecules, RNA molecules, antisense oligonucleotides, siRNA, rRNA, cDNA, mRNA, shRNA, lncRNA, miRNA, and tRNA.

[0073] The above-mentioned ionizable lipids (ionizable lipids) are the main constituents that control the properties of lipid nanoparticles. (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (D-Lin MC3-DMA or MC3), 9-heptadecanyl 8-{(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate (SM-102), [(4-hydroxybutyl)azanediyl]di(hexane-6,1-diyl)bis(2-hexyldecanoate)] (ALC-0315), 1,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1,2-dioleyloxy-N,Ndimethyl-3-aminopropane (DODMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (D-Lin-DMA), 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (D-Lin-KC2-DMA), N,N-dimethyl-2,3-bis(((9Z,12Z,15Z)-octadeca-9,12,15-trien-1-yl)oxy)propan-1-amine(D-Len-DMA), (Z)-N,N-dimethyl-N-((Z)-octadec-9-en-1-yl)octadec-9-en-1-aminium chloride (DODAC), N,N-dimethyl-N-octadecyloctadecan-1-aminium bromide (DDAB), N-(2-hydroxyethyl)-N,N-dimethyl-2,3-bis(tetradecyloxy)propan-1-aminium bromide (DMRIE), N,Ndimethyl-3,4-dioleyloxybenzylamine (DMOBA), 1,2-N,It may be at least one selected from the group consisting of N'-dioleylcarbamyl-3-dimethylaminopropane (DOcarbDAP), 2,3-dilinoleoyloxy-N,N-dimethylpropylamine (D-Lin-DAP), 1,2-N,N'-dilinoleylcarbamyl-3-dimethylaminopropane (DLincarbDAP), 1,2-dilinoleoylcarbamyl-3-dimethylaminopropane (DLinCDAP) and 2,2-dilinoleyl-4-dimethylaminoethyl[1,3]-dioxolane (DLin-K-XTC2-DMA).

[0074] The above helper lipid may be provided to improve the stability of lipid nanoparticles while enhancing the delivery efficiency of the active substance (e.g., nucleic acid, etc.). The above helper lipid may be at least one selected from the group consisting of distearoylphosphatidylcholine (DSPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-Dipalmitoyl-snglycero-3-Phospho-rac-(1-glycerol) (DPPG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), and dioleoyl-sn-glycero-3-phosphoethanolamine-N-(maleimidomethyl) (DOPEmal).

[0075] The above stabilizing lipid can improve the structural stability of lipid nanoparticles. The stabilizing lipid can include cholesterol (CHOL).

[0076] The PEG-lipid may be a PEGylated lipid and may include a PEG derivative bound to a lipid moiety. The PEG-lipid may improve the circulation time of lipid nanoparticles as nucleic acid delivery vehicles and reduce nonspecific absorption. The PEG-lipid may be at least one selected from the group consisting of pegylated diacylglycerol lipid (PEG-DAG), pegylated ceramide lipid (PEG-Cer), pegylated phosphatidylethanolamine lipid (PEG-PE), pegylated succinate diacylglycerol lipid (PEG-S-DAG), pegylated dialkoxypropylcarbamate lipid, 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (PEG-DMG), and 1,2-dicapryl-rac-glycero-3-methylpolyoxyethylene glycol (C10 diacylglycerol PEG).

[0077] The N / P ratio of the lipid mixture and the active substance mixture may be 1 to 30. By providing the N / P ratio of the lipid mixture and the active substance mixture within the above-described range, the encapsulation efficiency and intracellular delivery efficiency of lipid nanoparticles can be improved. Specifically, the N / P ratio of the active substance mixture may be 1 to 25, or 1 to 20, or 3 to 25, or 3 to 20.

[0078] In the above microfluidic platform (100), the microfluidic channel (110), one or more inlet portions (120) and one or more outlet portions (130) may be formed integrally on the disk member (150) or may be formed in the form of separate detachable microfluidic chips (101, 102). The microfluidic chips (101, 102) may be provided individually, one or more of which may be reversibly detachable from the disk member (150).

[0079] The above microfluidic chip (101, 120) may include a body portion (101) in which the microfluidic channel (110), the inlet portion (120) and the outlet portion (130) are formed; and one or more cover portions (102) covering at least one of one surface and the other surface of the body portion (101).

[0080] In the above body part (101), the microfluidic channel (110) may be formed concavely inward on one surface of the body part (101), and the inlet part (120) and the outlet part (130) may be formed to penetrate the body part (101).

[0081] Referring to FIG. 3, one surface of the disk member (150) may further include one or more mounting portions (151) on which the microfluidic chip (101, 102) is mounted. The mounting portions (151) are provided in a size corresponding to the microfluidic chip (101, 102) and are provided concavely inwardly on one surface of the disk member (150) so that the lower surface of the microfluidic chip (101, 102) can be inserted. By the mounting portions (151) provided on the disk member (150), the microfluidic chip (101, 102) can be stably fixed on the disk member (150) while the disk member (150) rotates, and also the position of the microfluidic chip (101, 102) can be guided.

[0082] The above microfluidic chips (101, 102) are provided in a box shape and can be arranged radially based on the center (160) of the disk member (150). The inlet portion (120) can be provided adjacent to the center (160) of the disk member (150), and the outlet portion (130) can be provided adjacent to the edge of the disk member (150).

[0083] The above disk member (150) can be rotated by means of a rotating shaft or the like provided in the central portion (160). By radially arranging the microfluidic chips (101, 102) on the disk member (150), a centrifugal force can be uniformly provided to each of the arranged microfluidic chips (101, 102) without any difference. In addition, by arranging the inlet portion (120) of the microfluidic chip (101, 102) adjacent to the central portion (160) of the disk member (150), a fluid introduced through the inlet portion (120) can pass through the microfluidic channel (110) to produce lipid nanoparticles of uniform size.

[0084] Referring to FIG. 4, the microfluidic channel (110) may include at least one of a herringbone mixer pattern, a serpentine mixer pattern, and a toroidal mixer pattern. In addition, the microfluidic channel (110) may include two or three inlet portions (120). The microfluidic channel (110) may be provided in various shapes, so that the size and properties of the lipid nanoparticles can be controlled to suit various purposes.

[0085] Hereinafter, another embodiment of the present invention will be described with reference to FIGS. 5 to 21. Except for the contents described below, the contents are similar to those described in the embodiments described in FIGS. 1 to 4, and thus detailed descriptions thereof will be omitted.

[0086] FIG. 5 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to another embodiment of the present invention.

[0087] The above microfluidic chip (201, 202, 203) may include a body portion (201) in which the microfluidic channel (210), the inlet portion (220), and the outlet portion (230) are formed; and one or more cover portions (202, 203) covering at least one of one surface and the other surface of the body portion (201). The microfluidic channel (210) may be provided on one surface of the body portion (201) and may be formed in an inwardly concave shape, and the inlet portion (220) and the outlet portion (230) may be formed to be connected to the microfluidic channel (210) and penetrate the other surface of the body portion (201).

[0088] The above cover portion (202, 203) may include a first cover (202) and a second cover (203). The first cover (202) may be provided to cover one surface of the body portion (201), and may include one or more holes (202a) provided at positions corresponding to the inlet portion (220) and the outlet portion (230), and the second cover (203) may be provided to cover the other surface of the body portion (201).

[0089] The microfluidic chip (201, 202, 203) according to an embodiment of the present invention may be provided in an inverted form in which the microfluidic channel (210) is positioned on the upper side and the inlet portion (220) and outlet portion (230) are provided in a direction facing the lower side.

[0090] The fluid can be introduced into the inlet portion (210) through the hole (202a) provided in the first cover (202). In addition, the hole (202a) provided in the first cover (202) can function as a vent hole to allow the fluid to flow by preventing the inlet portion (220) and the outlet portion (230) from being sealed. When the coefficients of friction of the lipid mixture and the active material mixture constituting the fluid are different, the speed of the material introduced through the inlet portion (220) can be different. However, the microfluidic chips (201, 202, 203) according to the embodiment of the present invention are provided in an inverted form, thereby reducing the influence on the inflow speed of the material constituting the fluid generated by the materials constituting the microfluidic chips (202, 202, 203).

[0091] Figure 6 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles according to another embodiment of the present invention. Figure 7 is a diagram of Figure 6 manufactured using 3D printing.

[0092] Referring to FIGS. 6 and 7, a microfluidic platform (300) according to an embodiment of the present invention may be provided with a microfluidic chip (301, 302) that is not provided in multiple pieces but may be provided in an integrated form with a circular cross-section. The microfluidic chip (301, 302) may include a body part (301) having a plurality of microfluidic channels (310), an inlet part (320), and an outlet part (320), and a cover part (302) that covers one surface of the body part (301).

[0093] The microfluidic channel (310), inlet portion (320), and outlet portion (330) provided in the body portion (301) may be provided as a set and may be arranged radially based on the center of the body portion (301). At this time, the inlet portion (320) may be provided adjacent to the center of the body portion (301), and the outlet portion (330) may be provided adjacent to a corner of the body portion (301).

[0094] The above microfluidic chip (301, 302) can be mounted and rotated on the disk member (350). The microfluidic chip (301, 302) can be mounted on the disk member (350) by being fixed by an upper frame (351a) and a lower frame (351b) that fix the microfluidic chip (301, 302). The upper frame (351a) and the lower frame (351b) can be combined by screws, and the microfluidic chip (301, 302) can be accommodated therein.

[0095] The microfluidic platform (300) according to the present embodiment can be manufactured by 3D printing.

[0096] According to another aspect of the present invention, an embodiment of the present invention includes a method for manufacturing lipid nanoparticles using the microfluidic platform for manufacturing lipid nanoparticles described above.

[0097] A method of using the above microfluidic platform for manufacturing lipid nanoparticles may include the steps of preparing a lipid mixture and an active substance mixture; injecting the lipid mixture and the active substance mixture into an inlet; and rotating a disk member.

[0098] The above inlet portion is provided in two or more numbers so that a lipid mixture and an active material mixture can be introduced into the microfluidic channel.

[0099] The average velocity of the fluid in the inlet portion may be 1 μl / s to 200 μl / s, and the rotational speed of the disk member may be 500 rpm to 4000 rpm. In addition, the inlet portion may be connected to one end of a microfluidic channel, and the Reynolds number of the fluid inside the microfluidic channel may be 5 to 1520. Specifically, the average velocity of the fluid in the inlet portion (120) may be 1.5 μl / s to 132.5 μl / s.

[0100] The lipid mixture and the active material mixture are injected into the inlet portion so that the N / P ratio is 1 to 30, and the lipid mixture and the active material mixture are mixed by centrifugal force generated inside the microfluidic channel to produce lipid nanoparticles. The produced lipid nanoparticles can be discharged through an outlet connected to the other end of the microfluidic channel.

[0101] The fluid may include a lipid mixture and an active material mixture, and the fluid may be delivered to the microfluidic channel through the inlet portion, and lipid nanoparticles may be produced by centrifugal force within the microfluidic channel.

[0102] In addition, the lipid nanoparticles can exhibit high encapsulation efficiency and have a uniform size due to the centrifugal force generated by the rotational speed in the aforementioned range and the Reynolds number within the microfluidic channel. The average diameter of the lipid nanoparticles can be 10 nm to 300 nm.

[0103] Hereinafter, examples and comparative examples of the present invention are described. However, the following examples are only preferred embodiments of the present invention and the scope of the present invention is not limited by the following examples.

[0104] 1. Preparation of lipid mixture and nucleic acid solution

[0105] To prepare lipid nanoparticles, a lipid mixture and a nucleic acid solution were prepared as follows.

[0106] The lipid mixture used D-Lin-MC3-DMA as an ionizable amino acid, distearoylphosphatidylcholine (DSPC) as a helper lipid, cholesterol as a stabilizing lipid, and DMG-PEG 2000 as a PEG-lipid. These were mixed in ethanol at a molar ratio of Dlin-MC3-DMA: DSPC: Cholesterol: DMG-PEG 2000 = 50:10:38.5:1.5 to prepare a lipid mixture.

[0107] The nucleic acid solution was prepared by mixing ssDNA in a 10 mM citrate buffer solution (pH 3), and F-Luc-mRNA and eGFP-mRNA in a 25 mM sodium acetate buffer solution (pH 5).

[0108] The lipid mixture was prepared with the same composition as the aforementioned materials, and the nucleic acid solution was prepared as described in Table 1 below. The lipid mixture and nucleic acid solution were separately injected into the inlet port so that the volume ratio was 1:3, and mixing was performed using the rotational force of the disk. In addition, the N / P ratio of the lipid mixture and nucleic acid solution were adjusted and mixed as specified in Table 1 below.

[0109] Lipid concentration (mM) of the nucleic acid solution lipid mixture and the mixed nucleic acid solution lipid mixture N / P ratio ssDNA ssDNA Citrate buffer solution 6.25 6 F-Luc F-Luc-mRNA Sodium acetate buffer solution 12.5 5.57 eGFP eGFP-mRNA Sodium acetate buffer solution 12.5 5.57

[0110] 2. Preparation of lipid nanoparticles

[0111] (1) Manufacturing of lipid nanoparticles using a microfluidic platform

[0112] Figure 8 is a schematic diagram of a microfluidic chip used in a microfluidic platform for manufacturing lipid nanoparticles used in the examples. As illustrated in Figure 8, the microfluidic chip was manufactured by covering a glass plate with a box-shaped PDMS (polydimethylsiloxane) having a microfluidic channel, an inlet, and an outlet formed therein. Here, the microfluidic channel was designed using AutoCAD, and a herringbone mixer pattern was manufactured by photolithography using SU-8 photoresist from Microchem. The microfluidic channel was formed on the PDMS using a soft lithography technique. Subsequently, the inlet and outlet were formed using a hole punch. At this time, the inlet and outlet were manufactured to have the same angle so as to be parallel to each other, and were manufactured and evaluated at a 70° and 90° incline, respectively. The fabricated microfluidic chip was used to fabricate lipid nanoparticles together with a disk member as illustrated in Fig. 1. The disk member was fabricated using a 3D printer.

[0113] The previously prepared lipid mixture and nucleic acid solution were injected into the microfluidic chip's inlet, respectively, to prepare the microfluidic chip. The microfluidic chip, into which the lipid mixture and nucleic acid solution were injected, was then placed on a disk member, which was then rotated. After rotation, the prepared lipid nanoparticles were recovered through the outlet.

[0114] (2) Preparation of lipid nanoparticles by pipetting mixing

[0115] Lipid nanoparticles were prepared using the same lipid mixture and nucleic acid solution as in the examples, at the same ratios, using a commonly used pipetting mixing method. Pipetting mixing was performed by pipetting 60 times to ensure uniform mixing of the lipid mixture and nucleic acid solution. After mixing was completed, the mixture was left at room temperature for 10 minutes.

[0116] (3) Manufacturing of lipid nanoparticles by syringe pump mixing

[0117] Syringe pump mixing was performed using the following device. An injection syringe pump was connected to the syringe, and the syringe was pumped at a controlled flow rate. The lipid solution and nucleic acid solution were injected into the PDMS chip containing the herringbone microfluidic mixer at a flow rate of 1:3 using the injection syringe pump, and mixed. The syringe and inlet were connected by a peek tube, and the outlet was connected by a peek tube to obtain the discharged mixture. Syringe pump mixing is a disk-like mixing method, but the fluid flow is controlled by the syringe pump. Here, the herringbone microfluidic mixer was manufactured with reference to a prior art (Abraham D. Stroock (2002), Chaotic Mixer for Microchannels, February 2002 Science 295(5555):647-51).

[0118] The same lipid mixture and nucleic acid solution as in the examples were each injected into 1 ml syringes to prepare. The syringes containing the prepared lipid mixture and the syringes containing the nucleic acid solution were injected using a syringe pump commonly used in microfluidic chips. At this time, mixing was performed by connecting to the microfluidic chip using a peek tube. After mixing was completed, the mixture was left at room temperature for 10 minutes.

[0119] 3. Measurement of lipid nanoparticles

[0120] The size of lipid nanoparticles (LNPs) was measured using a digital laser sizing analyzer (DLS; Zetasizer Nano S90, Malvern). Samples were prepared by diluting 10 μl of lipid nanoparticles in 990 μl of PBS, and the results were confirmed using this.

[0121] The encapsulation efficiency of lipid nanoparticles refers to the gene capture efficiency of lipid nanoparticles and was quantified using the Quant-iT RiboGreen assay (Thermo Fisher).

[0122] 4. Evaluation according to the angle of the inlet and outlet

[0123] Figure 9 shows the size of lipid nanoparticles according to the angle of the inlet portion of a microfluidic chip according to an embodiment of the present invention.

[0124] In Fig. 9, the angles of the inlet portion in the microfluidic chip were manufactured to be 70° and 90° inclinations (θ, see Fig. 3), respectively, and the outlet portion was also made to have the same angle as the inlet portion. Table 2 shows the size and polydispersity index (PDI) of lipid nanoparticles manufactured according to the angle of the inlet portion.

[0125] Manufacturing methodInlet angle (°)Disk member rotation speed (rpm)Nucleic acid solutionAverage size of lipid nanoparticles (nm)PDIMicrofluidic platform701600ssDNA118.20.195901600ssDNA242.20.452

[0126] Referring to Fig. 9 and Table 2, the size and PDI of the lipid nanoparticles manufactured differed depending on the inlet slope. It was confirmed that when the inlet slope decreased from 90° to 70°, the size of the lipid nanoparticles became uniform and the PDI decreased.

[0127] 4. Lipid nanoparticles manufactured by microfluidic platform, pipetting, and syringe

[0128] Figure 10 shows the results of comparing lipid nanoparticles manufactured using a microfluidic platform according to an embodiment of the present invention and pipetting mixing according to a comparative example. Figure 11 shows the results of comparing lipid nanoparticles manufactured using a microfluidic platform according to an embodiment of the present invention and syringe mixing according to a comparative example.

[0129] Table 3 shows the size, PDI, and encapsulation efficiency of lipid nanoparticles according to Fig. 10, and Table 4 shows the size, PDI, and encapsulation efficiency of lipid nanoparticles according to Fig. 11. In Figs. 10 and 11, the angle of the inlet portion in the microfluidic platform was 70°, and the rotation speed of the disk member was 1600 rpm.

[0130] Manufacturing method Nucleic acid solution Z-average (nm) PDI Encapsulation efficiency (%) Microfluidic platform (Disc mixing) ssDNA 125.10.13177.7 Pipetting (Pipette mixing) ssDNA 156.40.13056.1

[0131] Manufacturing method Nucleic acid solution Z-average (nm) PDI Encapsulation efficiency (%) Microfluidic platform (Disc mixing) ssDNA 125.10.13 177.7 Syringe (Syring mixing) ssDNA 115.30.12 173.7

[0132] Referring to FIG. 10 and Table 2, the lipid nanoparticles manufactured according to the examples of the present invention had an average size of about 125.1 nm and a PDI of about 0.131, whereas the lipid nanoparticles manufactured by the commonly used pipetting method had an average size of about 156.4 nm, and despite the large size, the PDI was similar to that of the lipid nanoparticles manufactured by the present invention. Here, the PDI is a value related to size, and even if the distribution is the same, the PDI becomes smaller as the size increases. Therefore, it can be confirmed that the lipid nanoparticles manufactured by the present invention with a smaller size when the PDI is the same are manufactured more uniformly. That is, the lipid nanoparticles manufactured according to the present invention were also superior to the lipid nanoparticles manufactured by pipetting in terms of encapsulation efficiency.

[0133] Referring to FIG. 11 and Table 3, the lipid nanoparticles manufactured according to the embodiment of the present invention and the lipid nanoparticles manufactured using syringe mixing showed similar average sizes and PDIs, but the lipid nanoparticles manufactured according to the present invention showed better encapsulation efficiency.

[0134] That is, it was confirmed that the lipid nanoparticles manufactured according to the present invention had a smaller average size and were manufactured uniformly, and also exhibited superior encapsulation efficiency, compared to the case where pipetting or syringes were commonly used in manufacturing lipid nanoparticles.

[0135] 5. Confirmation of intracellular delivery efficiency according to the manufacturing method of lipid nanoparticles

[0136] The intracellular delivery efficiency of lipid nanoparticles manufactured by the microfluidic platform of the present invention and pipetting was confirmed using HEK293 T cells.

[0137] Figure 12 shows the results of confirming the intracellular delivery efficiency according to the manufacturing method of lipid nanoparticles containing eGFP-mRNA. Figure 13 shows the results of confirming the intracellular delivery efficiency according to the manufacturing method of lipid nanoparticles containing F-Luc-mRNA. In the microfluidic platform of Figures 12 and 13, the angle of the inlet part was 70°, and the rotation speed of the disk member was 1600 rpm.

[0138] Table 5 shows the results comparing the intracellular delivery efficiencies of Figures 12 and 13.

[0139] Manufacturing method Cell Nucleic acid solution Z-average (nm) PDI Encapsulation efficiency (%) Transfection efficiency (%) MFI Relative light unit Microfluidic platform (Disc mixing) HEK293 eGFP 143.40.10 460.898.45 28.2 - Pipetting (Pipette mixing) HEK293 eGFP 187.40.08 255.198.64 25.2 - Microfluidic platform (Disc mixing) HEK293 F-Luc 138.10.17 973.7 - - 67719 Pipetting (Pipette mixing) HEK293 F-Luc 196.30.06 457.0 - - 26563

[0140] Referring to Figure 12 and Table 5, when the sizes and PDIs of lipid nanoparticles containing eGFP-mRNA prepared using the microfluidic platform and pipetting were similar, it was confirmed that the lipid nanoparticles prepared using the microfluidic platform of the present invention had superior encapsulation efficiency. In addition, the intracellular delivery (transfection) efficiencies of the lipid nanoparticles prepared using the microfluidic platform and pipetting were similar.

[0141] Referring to Figure 13 and Table 5, lipid nanoparticles containing F-Luc-mRNA prepared using the microfluidic platform and pipetting were smaller and more uniform in size and PDI when using the microfluidic platform. Furthermore, lipid nanoparticles prepared using the microfluidic platform exhibited superior encapsulation efficiency compared to pipetting, but showed slightly lower intracellular delivery compared to pipetting.

[0142] 6. Confirmation of intracellular delivery efficiency of lipid nanoparticles according to cell type

[0143] Figure 14 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to HEK293 cells. Figure 15 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to CHO-K1 cells. Figure 16 shows the results of confirming the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to Jurkat cells. In Figures 14 and 15, the angle of the inlet part was 70°, and the rotation speed of the disk member was 2800 rpm.

[0144] Table 6 shows the results showing the delivery efficiency of lipid nanoparticles manufactured using the microfluidic platform of the present invention to various cells.

[0145] Manufacturing method Cell Nucleic acid solution Z-average (nm) PDI Encapsulation efficiency (%) Transfection efficiency (%) MFI Microfluidic platform (Disc) HEK293 eGFP 10 4.9 0.179 84.2% 95.0 285.1 Microfluidic platform (Disc) CHO-K1 eGFP 10 4.9 0.179 84.2% 99.96 79.1 Microfluidic platform (Disc) Jurkate GFP 10 4.9 0.179 84.2% 98.96 26.7

[0146] In FIGS. 14 to 15 and Table 6, lipid nanoparticles were manufactured using the microfluidic platform of the present invention, and it was confirmed that the manufactured lipid nanoparticles exhibited high delivery efficiency to various cells.

[0147] In Fig. 14, HEK293 cells were seeded in each well of a 12-well plate at a density of 4e5 cells / well, and after 24 h, lipid nanoparticles (LNPs) loaded with eGFP mRNA were diluted in serum-free media to an eGFP mRNA concentration of 500 ng / mL, and then the existing culture medium was replaced with the lipid nanoparticle mixed medium to treat the cells with lipid nanoparticles. After treating with lipid nanoparticles, the delivery of eGFP mRNA was confirmed using a fluorescence microscope and flow cytometry 24 h later. Referring to Fig. 14 and Table 6, it was confirmed that the lipid nanoparticles manufactured according to the present invention exhibited a high delivery efficiency of 95.02% in HEK293 cells.

[0148] In Fig. 15, CHO-K1 cells were seeded in each well of a 12-well plate at a density of 2e5 cells / well and maintained for 24 h. Thereafter, lipid nanoparticles (LNPs) loaded with eGFP mRNA were diluted in serum-free medium to an eGFP mRNA concentration of 500 ng / mL, and the existing culture medium was replaced with the lipid nanoparticle mixed medium to treat the cells with the lipid nanoparticles. After treating with the lipid nanoparticles, the delivery of eGFP mRNA was analyzed using fluorescence microscopy and flow cytometry after another 24 h. Referring to Fig. 15 and Table 6, it was confirmed that the lipid nanoparticles manufactured according to the present invention exhibited a high delivery efficiency of 99.96% in CHO-K1 cells.

[0149] In Fig. 16, Jurkat cells were seeded in each well of a 24-well plate at a density of 1e5 cells / well, and after 24 h, lipid nanoparticles (LNPs) loaded with eGFP mRNA were diluted in serum-free medium to an eGFP mRNA concentration of 500 ng / mL, and then the existing culture medium was replaced with the lipid nanoparticle mixed medium to treat the cells with lipid nanoparticles. After 24 h of lipid nanoparticle (LNP) treatment, the delivery of eGFP mRNA was analyzed using fluorescence microscopy and flow cytometry. Referring to Fig. 16 and Table 6, it was confirmed that the lipid nanoparticles manufactured according to the present invention exhibited a high delivery efficiency of 98.96% in Jurkat cells.

[0150] That is, it was confirmed that lipid nanoparticles using the microfluidic platform of the present invention exhibit high delivery efficiency regardless of cell type.

[0151] 7. Confirmation of lipid nanoparticle size according to microfluidic chip type

[0152] (1) Preparation of lipid mixture and nucleic acid solution

[0153] To prepare lipid nanoparticles, a lipid mixture and a nucleic acid solution were prepared as follows.

[0154] The lipid mixture used D-Lin-MC3-DMA as an ionizable amino acid, distearoylphosphatidylcholine (DSPC) as a helper lipid, cholesterol as a stabilizing lipid, and DMG-PEG 2000 as a PEG-lipid. These were mixed in ethanol at a molar ratio of Dlin-MC3-DMA: DSPC: Cholesterol: DMG-PEG 2000 = 50:10:38.5:1.5 to prepare a lipid mixture.

[0155] The nucleic acid solution was prepared by mixing ssDNA in a 10 mM citrate buffer solution (pH 3). The nucleic acid solution was prepared with a 6.25 mM lipid mixture and an N / P ratio of 6, and the lipid mixture and nucleic acid solution were mixed in a volume ratio of 1:3.

[0156] (2) Manufacturing of lipid nanoparticles using a microfluidic platform

[0157] Figure 17 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles used in another embodiment of the present invention.

[0158] Figure 17 is manufactured similarly to the microfluidic platform for manufacturing lipid nanoparticles described in Figure 8, but the microfluidic channels are manufactured so that they do not come into direct contact with the glass. At this time, the microfluidic channels of the microfluidic chip are manufactured so that the microfluidic channels are positioned on the upper side, so that the shape is inverted with respect to the shape of the microfluidic channels of Figure 8.

[0159] As illustrated in Fig. 17, a microfluidic chip was manufactured by manufacturing a box-shaped primary PDMS (polydimethylsiloxane) having a microfluidic channel, an inlet, and an outlet, and a box-shaped secondary PDMS with holes. Here, the microfluidic channel was designed using AutoCAD, and a serpentine mixer pattern or a toroidal mixer pattern was manufactured by performing photolithography using SU-8 photoresist from Microchem. The microfluidic channel was formed on the primary PDMS using a soft lithography technique. Subsequently, the inlet and outlet were formed using a hole punch. At this time, the inlet and outlet were manufactured to have the same angle so as to be parallel to each other, and were manufactured at a 70° incline and evaluated. In addition, three holes were formed in the secondary PDMS using a hole punch. At this time, the three holes formed in the secondary PDMS were formed in positions corresponding to those in the primary PDMS. After the microfluidic channel of the primary PDMS was positioned at the top, the secondary PDMS was covered on top of the primary PDMS, and a glass plate was provided on the bottom of the primary PDMS to manufacture a microfluidic chip. The manufactured microfluidic chip was used together with a disk member as illustrated in Fig. 1 to manufacture lipid nanoparticles. The disk member was manufactured using a 3D printer.

[0160] The previously prepared lipid mixture and nucleic acid solution were then injected through the inlet of a microfluidic chip, respectively, to prepare a microfluidic chip. The microfluidic chip, into which the lipid mixture and nucleic acid solution were injected, was placed on a disk member, which was then rotated at 2,200 rpm. After completing the rotation, the prepared lipid nanoparticles were recovered through the outlet.

[0161] (3) Evaluation according to microfluidic channel pattern and number of inlets

[0162] Figure 18 shows the sizes of lipid nanoparticles fabricated using a microfluidic chip having inverted microfluidic channels formed in a serpentine mixer pattern. Figure 19 shows the sizes of lipid nanoparticles fabricated using a microfluidic chip having inverted microfluidic channels formed in a toroidal mixer pattern.

[0163] Table 7 shows the shape of the pattern of the microfluidic channel and the size of the lipid nanoparticles according to the number of inlets.

[0164] Manufacturing methodDisk member rotation speed (rpm)Microfluidic channel patternInletNucleic acid solutionAverage size of lipid nanoparticles (nm)PDIMicrofluidic platform (inverted microfluidic channel)22002 serpentine mixersInlet ssDNA155.60.23322003 serpentine mixersInlet ssDNA119.50.24622002 toroidal mixersInlet ssDNA97.050.10022003 toroidal mixersInlet ssDNA97.050.078

[0165] In this example, we utilized a microfluidic chip with inverted microfluidic channels to determine differences from non-inverted microfluidic channels. In the non-inverted microfluidic channel experiment previously conducted, the lipid mixture was preferentially introduced into the microfluidic channel before the disk member was rotated due to the ethanol used as a solvent for the lipid mixture. To mitigate this effect, the microfluidic channel was fabricated in an inverted configuration to produce lipid nanoparticles.

[0166] Referring to Figure 18, the average sizes of lipid nanoparticles in cases with two and three inlets were found to be almost similar. Furthermore, in Figure 19, where the microfluidic pattern is different from Figure 18, the average sizes of lipid nanoparticles according to the number of inlets were found to be almost identical.

[0167] Referring to Figures 18 and 19, it was confirmed that microfluidic channels formed with a toroidal mixer pattern formed smaller lipid nanoparticles compared to microfluidic channels formed with a serpentine mixer pattern. This is believed to be because the fluid flow rate within the microfluidic channel varies depending on the shape of the pattern.

[0168] 8. Confirmation of lipid nanoparticle size according to microfluidic chip manufacturing method

[0169] (1) Preparation of lipid mixture and nucleic acid solution

[0170] To prepare lipid nanoparticles, a lipid mixture and a nucleic acid solution were prepared as follows.

[0171] The lipid mixture used D-Lin-MC3-DMA as an ionizable amino acid, distearoylphosphatidylcholine (DSPC) as a helper lipid, cholesterol as a stabilizing lipid, and DMG-PEG 2000 as a PEG-lipid. These were mixed in ethanol at a molar ratio of Dlin-MC3-DMA: DSPC: Cholesterol: DMG-PEG 2000 = 50:10:38.5:1.5 to prepare a lipid mixture.

[0172] The nucleic acid solution was prepared by mixing ssDNA in a 10 mM citrate buffer solution (pH 3). The nucleic acid solution was prepared with a 6.25 mM lipid mixture and an N / P ratio of 6, and the lipid mixture and nucleic acid solution were mixed in a volume ratio of 1:5.

[0173] (2) Manufacturing of lipid nanoparticles using a microfluidic platform

[0174] Figure 20 is a schematic diagram of a microfluidic platform for manufacturing lipid nanoparticles used in another embodiment of the present invention. Figure 21 is a photograph of the microfluidic platform for manufacturing lipid nanoparticles of Figure 20. Figure 21 shows photographs of the top layer and the bottom layer, respectively, in order, with the top layer positioned above the bottom layer, and the photograph showing the bottom surface of the bottom layer. The microfluidic chip of Figure 21 was manufactured in an inverted configuration.

[0175] In Figs. 20 and 21, unlike the previous microfluidic platform for manufacturing lipid nanoparticles, a microfluidic chip was manufactured using a 3D printer rather than photolithography. The microfluidic channel was designed to have a serpentine mixer pattern and was manufactured using a PC-like resin (V-FT resin) for 3D printing using the SLA (Stereo Lithography Apparatus) method. After loading the lipid mixture and nucleic acid solution in an appropriate ratio into the bottom layer, the reservoir, and then covering and attaching the upper layer with a hole for a vent, the manufactured microfluidic chip was placed on a disk member. Subsequently, the disk member was rotated at various speeds, and after the rotation was completed, the manufactured lipid nanoparticles were recovered through the outlet.

[0176] (3) Evaluation according to microfluidic chip manufacturing method and disk member rotation speed

[0177] Figure 22 shows the size and encapsulation efficiency of lipid nanoparticles fabricated in a microfluidic chip including microfluidic channels formed in a serpentine mixer pattern using a 3D printer.

[0178] Table 8 shows the size and encapsulation efficiency of lipid nanoparticles evaluated by varying the rotational speed of the disk member in a microfluidic chip manufactured using a 3D printer.

[0179] Manufacturing methodDisk member rotation speed (rpm)Microfluidic channel patternNucleic acid solutionAverage size of lipid nanoparticles (nm)PDIEncapsulation efficiency (%)Microfluidic platform (3D printing)700serpentine mixerssDNA122.50.129561000serpentine mixerssDNA103.60.11460.21600serpentine mixerssDNA99.100.02483.32200serpentine mixerssDNA94.840.07290.82800serpentine mixerssDNA93.240.06687

[0180] Referring to Figure 21 and Table 8, it was confirmed that lipid nanoparticles were uniformly produced in small sizes even in microfluidic chips manufactured using a 3D printer. Furthermore, the manufactured lipid nanoparticles exhibited high encapsulation efficiency.

[0181] Examining the effect of the rotational speed of the disk element, we found that as the rotational speed increased, the size of the lipid nanoparticles decreased and the encapsulation efficiency increased. After approximately 1600 rpm, the disk element's rotational speed exhibited similar lipid nanoparticle size and encapsulation efficiency. In other words, it was confirmed that the microfluidic platform according to an embodiment of the present invention can control the size of lipid nanoparticles to be uniformly small.

[0182] Those skilled in the art will appreciate that the present invention can be implemented in other specific forms without altering its technical spirit or essential characteristics. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention is defined by the claims below rather than the detailed description above, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included within the scope of the present invention.

Claims

1. Using centrifugal force, Microfluidic channels for manufacturing lipid nanoparticles (LNPs) inside; One or more inlets for supplying fluid into the microfluidic channel; One or more outlet portions through which fluid is discharged from the microfluidic channel; and A microfluidic platform for manufacturing lipid nanoparticles, comprising a disk member for rotating the fluid.

2. In paragraph 1, The above microfluidic channel extends in one direction, The above inlet portion and the above outlet portion are respectively connected to one end and the other end of the microfluidic channel, and extend upward with respect to the microfluidic channel. A microfluidic platform for manufacturing lipid nanoparticles, wherein the inlet portion is provided at a first angle with respect to a first plane parallel to the ground, and the outlet portion is provided at a second angle with respect to the first plane.

3. In paragraph 2, The first angle is between 20° and 85°, The second angle is less than or equal to 85°, A microfluidic platform for manufacturing lipid nanoparticles, wherein the size of the lipid nanoparticles decreases as the first angle and the second angle decrease.

4. In paragraph 3, A microfluidic platform for manufacturing lipid nanoparticles, wherein the first angle and the second angle are provided at the same angle within a range of 45° to 70°.

5. In paragraph 1, The inner diameter of the above microfluidic channel is 100 ㎛ to 300 ㎛, A microfluidic platform for manufacturing lipid nanoparticles, wherein the Reynolds number of the fluid in the microfluidic channel is 5 to 1520.

6. In paragraph 1, The above disk member rotates to generate centrifugal force inside the microfluidic channel, The rotation speed of the above disk member is 500 rpm to 4000 rpm, A microfluidic platform for manufacturing lipid nanoparticles, wherein the average diameter of the lipid nanoparticles is 10 nm to 300 nm and the polydispersity index (PDI) is 0.20 or less.

7. In paragraph 6, The above inlet portion is provided in two or more numbers so that a lipid mixture and an active material mixture are introduced into the microfluidic channel. A microfluidic platform for manufacturing lipid nanoparticles, wherein the average velocity of the fluid in the inlet portion is 1 μl / s to 200 μl / s.

8. In paragraph 7, The lipid mixture comprises ionized lipids, helper lipids, stabilizing lipids and PEG-lipids, The above active substance mixture comprises at least one selected from the group consisting of chemotherapeutic agents, small molecule drugs, proteins and nucleic acids, A microfluidic platform for manufacturing lipid nanoparticles, wherein the N / P ratio of the lipid mixture and the active material mixture is 1 to 30.

9. In paragraph 1, comprising at least one microfluidic chip formed including the microfluidic channel, at least one inlet portion, and at least one outlet portion, and mounted on one surface of the disk member; A microfluidic platform for manufacturing lipid nanoparticles, wherein the microfluidic chip is provided to be reversibly detachable from the disk member.

10. In paragraph 9, The above microfluidic chip, A body part in which the microfluidic channel, inlet part, and outlet part are formed; and Including at least one cover part covering at least one of one side and the other side of the above body part; A microfluidic platform for manufacturing lipid nanoparticles, wherein the microfluidic channel is formed concavely inwardly on one surface of the body portion, and the inlet portion and the outlet portion are formed to penetrate the body portion.

11. In paragraph 10, The above cover part includes a first cover and a second cover, The first cover is provided to cover one side of the body portion, and includes one or more holes provided at positions corresponding to the inlet portion and the outlet portion. The second cover is a microfluidic platform for manufacturing lipid nanoparticles that covers the other surface of the body part.

12. In paragraph 9, One side of the above disk member further includes one or more mounting portions on which the microfluidic chip is mounted, A microfluidic platform for manufacturing lipid nanoparticles, wherein the above-mentioned mounting portion is provided in a size corresponding to the above-mentioned microfluidic chip and is provided concavely inwardly on one surface of the above-mentioned disk member, into which the lower surface of the above-mentioned microfluidic chip is inserted.

13. In paragraph 9, The above microfluidic chip is provided in a box shape and is arranged radially based on the center of the disk member. A microfluidic platform for manufacturing lipid nanoparticles, wherein the inlet portion is provided adjacent to the center of the disk member, and the outlet portion is provided adjacent to the edge of the disk member.

14. In paragraph 1, A microfluidic platform for producing lipid nanoparticles, wherein the microfluidic channel comprises at least one of a herringbone mixer pattern, a serpentine mixer pattern, and a toroidal mixer pattern.

15. Step of preparing a lipid mixture and an active substance mixture; A step of injecting the lipid mixture and the active material mixture into the inlet; and comprising a step of rotating a disc member; A method using a microfluidic platform for manufacturing lipid nanoparticles according to any one of claims 1 to 14.

16. In paragraph 15, The above inlet portion is provided in two or more numbers so that a lipid mixture and an active material mixture are introduced into the microfluidic channel. The average velocity of the fluid in the above inlet is 1 ㎕ / s to 200 ㎕ / s, A method for manufacturing lipid nanoparticles using a microfluidic platform, wherein the rotation speed of the above disk member is 500 rpm to 4000 rpm.

17. In paragraph 15, The above inlet portion is connected to one end of the microfluidic channel, A method for manufacturing lipid nanoparticles using a microfluidic platform, wherein the Reynolds number of the fluid inside the microfluidic channel is 5 to 1520.

18. In paragraph 15, The N / P ratio of the above lipid mixture and the above active material mixture is injected into the inlet portion so as to be 1 to 30, The above lipid mixture and the above active material mixture are mixed by centrifugal force generated inside the microfluidic channel to produce lipid nanoparticles. A method using a microfluidic platform for manufacturing lipid nanoparticles, wherein the lipid nanoparticles are discharged through an outlet connected to the other end of the microfluidic channel, and the lipid nanoparticles have an average diameter of 10 nm to 300 nm.

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