Chip for producing lipid nanoparticles containing a spiral structure and method for producing lipid nanoparticles using the same

Abstract

The present invention relates to a chip for producing lipid nanoparticles having a spiral structure and a method for producing lipid nanoparticles using the chip, which can increase the mixing efficiency of an aqueous phase solution containing nucleic acids and an oil phase solution containing lipids, thereby forming uniform lipid nanoparticles through a self-assembly process at the interface between the aqueous phase solution and the oil phase solution.The present invention also relates to a production method that enables the efficient production of lipid nanoparticles having a uniform shape and diameter using the chip for producing lipid nanoparticles having the spiral structure.

Description

[Technical Field]

[0001] The present invention relates to a chip for producing lipid nanoparticles having a spiral structure and a method for producing lipid nanoparticles using the chip. [Background technology]

[0002] Lipid nanoparticles (LNPs) are effective drug delivery systems for biologically active compounds such as therapeutic nucleic acids, proteins, and peptides that are cell-impermeable.

[0003] Vaccines are usually subdivided into "first-generation," "second-generation," and "third-generation" vaccines, and genetic vaccines, i.e., vaccines for genetic vaccination, are usually considered "third-generation" vaccines. Genetic vaccines typically consist of genetically engineered nucleic acid molecules that enable the expression of peptides or protein (antigen) fragments characteristic of pathogens or tumor antigens in vivo. Upon administration to a patient, genetic vaccines are expressed after uptake by target cells. Expression of the administered nucleic acid leads to the production of the encoded protein. If such a protein is recognized as foreign by the patient's immune system, an immune response is triggered.

[0004] From the viewpoint of gene vaccination, not only DNA but also RNA can be used as the nucleic acid molecule for administration, as DNA is known to be relatively stable and easy to handle.

[0005] However, the use of DNA carries the risk of unwanted insertion of the administered DNA fragment into the patient's genome, potentially resulting in mutagenic events such as loss of function of the damaged gene.

[0006] By using RNA instead of DNA for genetic vaccination, the risk of unwanted genome integration and the generation of anti-DNA antibodies can be minimized or prevented. However, RNA can be easily degraded by ubiquitous RNases and has the problems of extreme instability, impermeability, fragility, and immunogenicity.

[0007] Although many advances have been achieved in the past few years, lipid nanoparticle formulations have been used as an efficient method for mRNA vaccination capable of eliciting adaptive immune responses.

[0008] The lipid nanoparticle formulations can improve the delivery of nucleic acids in vivo.

[0009] The lipid nanoparticle-based drug delivery system is a multi-component formulation containing ionizable lipids, non-ionizable lipids, neutral lipids, and fusogenic lipids. The cationic ionizable lipids bind to anionic nucleic acids, while the other components support stable self-assembly of lipid nanoparticles.

[0010] The lipid nanoparticles can be manufactured with an optimal drug:lipid ratio to protect nucleic acids from degradation and removal in serum, be suitable for systemic or local delivery, and provide intracellular delivery of nucleic acids.

[0011] When the lipid nanoparticles are produced by conventional methods, the size of the produced particles is not uniform, which requires a separate classification process, resulting in a decrease in production efficiency.

[0012] It is necessary to develop a chip and a manufacturing method for producing lipid nanoparticles that can solve these problems, produce lipid nanoparticles with uniform diameters, and increase production efficiency. [Prior art documents] [Patent documents]

[0013] [Patent Document 1] US10,835,878 Summary of the Invention [Problem to be solved by the invention]

[0014] An object of the present invention is to provide a chip for producing lipid nanoparticles having a spiral structure and a method for producing lipid nanoparticles using the chip.

[0015] Another object of the present invention is to provide a chip for producing lipid nanoparticles that includes a spiral structure that can increase the mixing efficiency of an aqueous phase solution containing nucleic acids and an oil phase solution containing lipids, and form uniform lipid nanoparticles through a self-assembly process at the interface between the aqueous phase solution and the oil phase solution.

[0016] Another object of the present invention is to provide a method for efficiently producing lipid nanoparticles having a uniform shape and diameter using the lipid nanoparticle production chip having the spiral structure. [Means for solving the problem]

[0017] In order to achieve the above object, the present invention relates to a chip for producing lipid nanoparticles, which includes an oil phase solution supply unit, an aqueous phase solution supply unit, and a mixer unit having a spiral structure, and the mixer unit is connected to the oil phase solution supply unit and the aqueous phase solution supply unit, and the oil phase solution supplied via the oil phase solution supply unit and the aqueous phase solution supplied via the aqueous phase solution supply unit are mixed to form lipid nanoparticles.

[0018] The mixer portion may include peaks and valleys formed alternately along a spiral locus on the circumferential surface.

[0019] The cross section of the mixer portion may have an outer diameter of 1 mm to 2 mm due to peaks and an inner diameter of 0.5 mm to 0.6 mm due to valleys.

[0020] The mixer section may have a pitch of 1 mm to 3 mm between the spiral locus on the circumferential surface.

[0021] Furthermore, the mixer section may have peaks and valleys with a depth of 0.1 mm to 0.5 mm.

[0022] Furthermore, the mixer portion may have a valley formed by the spiral locus on the circumferential surface, the width of which may be 0.1 mm to 0.5 mm.

[0023] The mixer section may include a mixing section in which the oil phase solution and the aqueous phase solution are mixed, and a stabilization section in which the mixed solution in which the oil phase solution and the aqueous phase solution are completely mixed is stabilized, and the length ratio of the mixing section to the stabilization section may be 1:1 to 1:5.

[0024] The mixer unit may be connected to a discharge unit, and the discharge unit may be connected to the other end of the mixer unit connected to the oil phase solution supply unit and the aqueous phase solution supply unit.

[0025] A method for producing lipid nanoparticles according to another embodiment of the present invention includes the steps of producing an aqueous phase solution containing nucleic acids, dissolving lipids in an organic solution to produce an oil phase solution, supplying the aqueous phase solution and the oil phase solution to an oil phase solution supply unit and an aqueous phase solution supply unit on the lipid nanoparticle production chip described in claim 1, and mixing the oil phase solution and the aqueous phase solution supplied to the oil phase solution supply unit and the aqueous phase solution supply unit in a mixer unit to form lipid nanoparticles.

[0026] Furthermore, the oil phase solution and the aqueous phase solution flow and mix along a spiral trajectory on the circumferential surface of the mixer section to form a mixed solution, and the mixed solution flows along the spiral trajectory and can be stabilized.

[0027] Furthermore, the oil phase solution and the aqueous phase solution form an interface along a spiral trajectory on the peripheral surface of the mixer section, and the lipids in the oil phase solution and the nucleic acids in the aqueous phase solution can form lipid nanoparticles at the interface through a self-assembly process.

[0028] In addition, the oil phase solution and the aqueous phase solution may flow through a mixer section to form a mixed solution, and move through a stabilization section in which the mixed solution is stabilized, and the length ratio of the mixing section to the stabilization section may be 1:1 to 1:5.

[0029] The oil phase solution and the aqueous phase solution may be supplied to the oil phase solution supply section and the aqueous phase solution supply section at a flow rate ratio of 1:1 to 1:5.

[0030] The total flow rate of the oil phase solution and the aqueous phase solution may be 5 mL / min to 50 mL / min.

[0031] Furthermore, after the step of forming the lipid nanoparticles, a post-treatment step of dilution and filtration may be additionally included. [Effects of the Invention]

[0032] The present invention can increase the mixing efficiency of an aqueous phase solution containing nucleic acids and an oil phase solution containing lipids, thereby forming uniform lipid nanoparticles through a self-assembly process at the interface between the aqueous phase solution and the oil phase solution.

[0033] Furthermore, lipid nanoparticles having a uniform shape and diameter can be efficiently produced using the lipid nanoparticle production chip having the spiral structure. [Brief explanation of the drawings]

[0034] [Figure 1] FIG. 1 is a diagram showing a conventional chip for producing lipid nanoparticles according to an embodiment of the present invention. [Figure 2] 1 is a diagram showing a chip for producing lipid nanoparticles according to an embodiment of the present invention. [Figure 3] FIG. 1 is an enlarged view of the portion where the chip for producing lipid nanoparticles and the mixer unit are joined together according to one embodiment of the present invention. [Figure 4] FIG. 1 is a diagram showing a mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 5] FIG. 1 is a cross-sectional view of a mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 6] FIG. 10 is a diagram showing the mixing section and stabilization section in the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 7]FIG. 10 is a diagram showing the flow of the oil phase solution and the aqueous phase solution injected into the mixer section of the chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 8] FIG. 1 is a diagram showing the flow of an oil phase solution and an aqueous phase solution in a cross section of a mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 9] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 10] 10 shows experimental results of fluid flow depending on the change in the total flow rate of the oil phase solution and the aqueous phase solution in the mixer section of the chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 11] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 12] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 13] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 14] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. [Figure 15] 10 shows experimental results regarding the fluid flow within the mixer section of a chip for producing lipid nanoparticles according to one embodiment of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0035] The present invention relates to a chip for producing lipid nanoparticles, which includes an oil phase solution supply unit, an aqueous phase solution supply unit, and a mixer unit including a spiral structure, and the mixer unit is connected to the oil phase solution supply unit and the aqueous phase solution supply unit, and the oil phase solution supplied via the oil phase solution supply unit and the aqueous phase solution supplied via the aqueous phase solution supply unit are mixed to form lipid nanoparticles. [Example]

[0036] Although the present invention may be embodied in various different forms, it is not intended to be limited to the embodiments set forth herein, and the present invention is not limited to the embodiments set forth herein.

[0037] mRNA is an abbreviation for messenger ribonucleic acid, and is an intermediate that links DNA and proteins in the process of protein synthesis, when DNA containing genetic information becomes mRNA.

[0038] The COVID-19 pandemic has sparked interest and development in mRNA vaccines. mRNA vaccines have several advantages over other types of vaccines. The greatest advantage of mRNA vaccines is that lipid nanoparticles (LNPs) containing mRNA serve as a platform technology, enabling rapid technological development to combat viruses that mutate frequently, such as COVID-19.

[0039] Specifically, a protective protein antigen can be identified, and the gene corresponding to the antigen can be sequenced to produce mRNA. When new mRNA is produced using this method and the formulation and manufacturing processes of conventional mRNA vaccines are used, rapid production of mRNA vaccines becomes possible. This means that because mRNAs encoding different antigens are very similar chemically and physically, the formulation and manufacturing processes of new mRNA vaccines can be carried out using the same steps as the formulation and manufacturing processes of conventional mRNA vaccines.

[0040] Due to the negative charge of the phosphate group, mRNA is a polyanionic macromolecule in the pH range typically used for parenteral administration. As mentioned above, the electrical properties of negatively charged mRNA can be utilized to prepare lipid nanoparticles using positively charged ionizable lipids (ionisable lipids or cationic lipids). Specifically, ionizable lipids are positively charged lipids that strongly bind to negatively charged mRNA through electrical attraction. In addition to the ionizable lipids, lipid nanoparticles can be formed using non-ionizable lipids, neutral lipids, and fusogenic lipids.

[0041] The nucleic acid-lipid particles of US9364435B2 comprise (a) a nucleic acid, (b) a cationic lipid, (c) a non-cationic lipid, and (d) a fusogenic lipid, and disclose that the particles contain 50 mol% to 85 mol% of the cationic lipid, 13 mol% to 49.5 mol% of the non-cationic lipid, and 0.5 mol% to 2 mol% of the fusogenic lipid, based on the total content of lipids in the particles.

[0042] Furthermore, EP2279254B1 discloses that the nucleic acid-lipid particles contain 50 mol% to 65 mol% of the cationic lipid, 49.5 mol% or less of the non-cationic lipid, 30 mol% to 40 mol% of cholesterol or a derivative thereof, and 0.5 mol% to 2 mol% of a fusogenic lipid, based on the total lipid content within the particle.

[0043] As described above, lipid nanoparticles containing nucleic acids are produced using an aqueous phase solution containing nucleic acids and an oil phase solution containing lipids, and a lipid nanoparticle production chip capable of mixing the aqueous phase solution and the oil phase solution is used.

[0044] The chip for producing lipid nanoparticles is used to mix the aqueous and oily phase solutions using a microfluidic method, and lipid nanoparticles can be formed at the interface where the aqueous and oily phase solutions contact through a self-assembly process. Specifically, as described below, by mixing the flow of the aqueous solution with the flow of the oily phase solution, a mixed solution containing lipid nanoparticles can be produced by self-assembly of lipids and mRNA at the interface between the two fluids.

[0045] The conventional chip for producing the lipid nanoparticles may have a channel structure with two-dimensionally curved channels and a structure in which baffles are arranged alternately, as in WO2018 / 190423.

[0046] The flow channel structure with the baffles arranged alternately is characterized in that it is possible to control the dilution rate of the solution and adjust the diameter of the nanoparticles by adjusting the width, length, and arrangement of the baffles.

[0047] Alternatively, a method using a fluid mixer with bifurcated fluid flow using a torus mixing element has also been proposed, such as in KR10-2361123B1. This fluid mixer has a chip structure as shown in Figure 1, and compared to the chip for producing lipid nanoparticles of the present invention described below, the oil phase solution and the aqueous phase solution do not mix completely, resulting in relatively low lipid nanoparticle production efficiency and differences in particle uniformity.

[0048] That is, although various chips for producing lipid nanoparticles have been developed, the conventional chips for producing lipid nanoparticles have a problem in that the oil phase solution and the aqueous phase solution must be mixed by forming an interface, and it is relatively difficult to mix the oil phase solution and the aqueous phase solution.

[0049] Therefore, the present invention provides a chip for producing lipid nanoparticles containing a novel spiral structure. When the chip for lipid nanoparticles containing the spiral structure is used, the mixing efficiency of an aqueous phase solution containing nucleic acids and an oil phase solution containing lipids can be increased, and uniform lipid nanoparticles can be formed at the interface between the aqueous phase solution and the oil phase solution through a self-assembly process.

[0050] Specifically, a chip for producing lipid nanoparticles according to one embodiment of the present invention includes an oil phase solution supply unit, an aqueous phase solution supply unit, and a mixer unit having a spiral structure, and the mixer unit is connected to the oil phase solution supply unit and the aqueous phase solution supply unit, and the oil phase solution supplied through the oil phase solution supply unit and the aqueous phase solution supplied through the aqueous phase solution supply unit are mixed to form lipid nanoparticles.

[0051] The mixer unit may be connected to a discharge unit, and the discharge unit may be connected to the other end of the mixer unit connected to the oil phase solution supply unit and the aqueous phase solution supply unit.

[0052] More specifically, the chip for producing lipid nanoparticles of the present invention is as shown in Figure 2. As shown in Figure 2, it can include an oil phase solution supply unit 100, an aqueous phase solution supply unit 200, a mixer unit 300 including a spiral structure, and a discharge unit 400.

[0053] The oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 can each be connected to one end of the mixer unit 300. The oil phase solution and the aqueous phase solution can flow into the mixer unit 300 through the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200, and the oil phase solution and the aqueous phase solution can be mixed in the mixer unit 300 to form lipid nanoparticles.

[0054] More specifically, the chip for producing lipid nanoparticles of the present invention shown in Figure 2 is configured such that the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 are positioned based on the z-axis, extend vertically in the y-axis direction, and are then connected to the mixer unit 300 in the x-axis direction.

[0055] The oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 are positioned on the same z-axis to allow the oil phase solution and the aqueous phase solution to be injected, respectively. The oil phase solution and the aqueous phase solution flow through the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200, respectively, and are connected vertically to the mixer unit 300 at the point where the oil phase solution flow and the aqueous phase solution flow meet, and the oil phase solution flow and the aqueous phase solution flow all move into the mixer unit 300.

[0056] FIG. 3 shows an enlarged view of the portion where the oil phase solution flow and the aqueous phase solution flow meet, and the portion vertically connected to the mixer unit 300.

[0057] The flow of each of the oil phase solution and the aqueous phase solution, and the flow of the mixed solution in the mixer section 300 will be described later.

[0058] Referring to FIG. 3, the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 are arranged facing each other and are connected to the mixer unit 300 vertically at the point where the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 meet.

[0059] The mixer section 300 of the present invention is structurally characterized by including a spiral structure as described above. Specifically, the spiral structure is characterized by including peaks 310 and valleys 320 alternately formed along a spiral trajectory on the circumferential surface.

[0060] The spiral structure is characterized in that the peripheral surface has repeated peaks 310 and valleys 320 formed along a spiral locus, resulting in a difference between the outer diameter due to the peaks 310 and the inner diameter due to the valleys 320. That is, as shown in Fig. 5, the diameter (330) of the cross-sectional circle formed by the peaks 310 can be determined, and the valleys 320 are recessed portions formed along the spiral locus, so the diameter 340 of the cross-sectional circle formed by the valleys 320 can also be determined.

[0061] The cross section of the mixer section 300 may have an outer diameter 330 of 1 mm to 2 mm due to peaks 310 and an inner diameter 340 of 0.5 mm to 0.6 mm due to valleys 320, or an outer diameter 330 of 1 mm to 1.8 mm due to peaks 310 and an inner diameter 340 of 0.51 mm to 0.59 mm due to valleys 320, or an outer diameter 330 of 1 mm to 1.5 mm due to peaks 310 and an inner diameter 340 of 0.52 mm to 0.58 mm due to valleys 320. As will be described later, when flows of the oil phase solution and the aqueous phase solution occur within the mixer section 300, a flow may occur along the outer diameter 330 of the mixer section 300 and a flow may occur along the inner diameter 340. That is, when the oil phase solution and the aqueous phase solution mix in the mixer unit 300, some of them flow along the outer diameter 330 of the mixer unit 300, and some of them flow along the inner diameter 340. The flows along the outer diameter 330 and the inner diameter 340 mix due to the movement of the fluid, forming a completely mixed solution. This means that the flows of the oil phase solution and the aqueous phase solution injected into the mixer unit 300 of the present invention do not mix only due to the shape of the spiral structure, but also mix between the flow along the periphery of the mixer unit 300 and the flow that flows in a straight line at the center.

[0062] As shown in FIG. 4, the mixer unit 300 of the present invention can specify the pitch (350) of the spiral locus on the circumferential surface, the depth (370) of the peaks 310 and valleys 320, and the width (360) of the valleys formed by the spiral locus on the circumferential surface. Specifically, the spacing (Pitch, 350) of the spiral locus on the circumferential surface may be 1 mm to 3 mm, the depth (Depth, 370) of the peaks 310 and valleys 320 may be 0.1 mm to 0.5 mm, and the width (Width, 360) of the valleys formed by the spiral locus on the circumferential surface may be 0.1 mm to 0.5 mm, or the spacing (Pitch, 350) of the spiral locus on the circumferential surface may be 1.5 mm to 2.5 mm, the depth (Depth, 370) of the peaks 310 and valleys 320 may be 0.2 mm to 0.4 mm, and the width (Width, 360) of the valleys formed by the spiral locus on the circumferential surface may be 0.2 mm to 0.4 mm.

[0063] The pitch (350) means the length to the same point as the starting point when rotating along the circumferential surface from a specific point on the circumferential surface according to the spiral structure. The depth (370) means the distance between the peaks 310 and the valleys 320 on the cross section of the mixer unit 300. The valley width (360) means the interval at which the spiral structure is repeated, and the valley width 360 determines the number of times the spiral locus is repeated within a specific section.

[0064] The characteristics of the spiral structure change depending on the differences in the spacing 350, depth 370, and width 360 of the mixer section 300, which has the above-mentioned spiral structure. When the characteristics of the spiral structure change, the flow of the fluid flowing within the mixer section 300 changes, making it possible to form uniform lipid nanoparticles by mixing the oil phase solution and the aqueous phase solution within the range of the present invention for the spacing 350, depth 370, and width 360 of the mixer section 300 and the length ratio of the mixing section and stabilization section of the mixer section 300 described below.

[0065] The mixer unit 300 includes a mixing section 380 in which the oil phase solution and the aqueous phase solution are mixed, and a stabilization section 390 in which the mixed solution obtained by completely mixing the oil phase solution and the aqueous phase solution is stabilized, and the length ratio of the mixing section 380 to the stabilization section 390 may be 1:1 to 1:5.

[0066] The specific flow of the fluid is as shown in FIGS.

[0067] Specifically, as shown in FIG. 7, when the oil phase solution and the aqueous phase solution are injected via the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 as described above, the oil phase solution and the aqueous phase solution flow in opposite directions, and are connected to the mixer unit 300 at the point where the oil phase solution flow and the aqueous phase solution flow meet, and the oil phase solution flow and the aqueous phase solution flow form an interface and move to the mixer unit 300.

[0068] The oil phase solution and the aqueous phase solution that have moved to the mixer unit 300 form an interface, and lipid nanoparticles are formed on the interface. At the same time, the oil phase solution and the aqueous phase solution are mixed to form a mixed solution. At this time, the oil phase solution and the aqueous phase solution can be mixed more effectively in the mixer unit 300 due to the fluid flow as shown in FIG. 8.

[0069] That is, as described above, the mixer unit 300 of the present invention is characterized by a spiral structure, and a fluid flow is generated along the periphery of the mixer unit 300 having a spiral structure, and the fluid flow can increase the mixing efficiency of the oil phase solution and the aqueous phase solution and promote the formation of uniform lipid nanoparticles on the interface.

[0070] The oil phase solution and the aqueous phase solution mixed along the mixer unit 300 can form uniform lipid nanoparticles by passing through a mixing section 380 in which the oil phase solution and the aqueous phase solution are mixed within the mixer unit 300, and a stabilization section 390 in which the mixed solution in which the oil phase solution and the aqueous phase solution are completely mixed is stabilized.

[0071] As described above, the oil phase solution and the aqueous phase solution are thoroughly mixed in the mixer unit 300 to form a mixed solution. When the mixed solution is formed, the lipids in the oil phase solution and the nucleic acids in the aqueous phase solution self-assemble to form lipid nanoparticles. To stabilize the lipid nanoparticles, a stabilization step is performed within the flow of the thoroughly mixed mixed solution in the mixer unit 300.

[0072] The length ratio of the mixing section 380 to the stabilization section 390 may be 1:1 to 1:5, 1:1 to 1:4, or 1:2 to 1:4. The oil phase solution and the aqueous phase solution are completely mixed within this length range, and then the completely mixed solution moves within the stabilization section. Within this fluid flow, lipid nanoparticles are simultaneously formed and stabilized.

[0073] The mixing of the fluids will be explained in more detail in Figure 9. Figure 9 shows the mixing of the oil phase solution and the aqueous phase solution in each section of the mixer unit 300 in the chip for producing lipid nanoparticles of the present invention, confirmed by using dye solutions. A yellow solution was used instead of the oil phase solution, and a blue solution was used instead of the aqueous phase solution. When the solutions were completely mixed, the solution turned green, and the presence or absence of mixing was confirmed by the color.

[0074] Specifically, these are the experimental results of the flow at the three locations shown in Figure 9. The working fluids used in the experiment were deionized water as the aqueous phase solution, a solution of yellow dye diluted to a concentration of 50 μM, and a solution of blue dye diluted to a concentration of 50 μM in ethanol as the oil phase solution. Images of the contact surface between the two solutions inside the mixer were taken in a direction perpendicular to the top surface of the mixer. The intensity based on the pixels of the captured images was analyzed by dividing the horizontal direction (x-axis) of the image into n numbers and the vertical direction (y-axis) into m numbers, using n-by-m pixel analysis for each image. When the fluid flow at each flow rate at the measurement location stabilized in a steady state where it did not change over time, an image was taken. Additionally, for more accurate relative analysis, images were taken when the oil phase solution was flowing and when only deionized water was flowing. When only the oil phase solution was flowing, this was reflected as 100% mixing, i.e., a state where mixing was complete, and when only deionized water was flowing, this was reflected as 0% mixing, i.e., a state where mixing had not yet occurred.

[0075] Section A in Figure 9 shows the mixing of solutions at the start of the mixer section 300. Section A is the section where the yellow solution and the blue solution are mixed, and the yellow and blue solutions are also observed, as well as a section where the two solutions are mixed together to produce a green color.

[0076] Section B in FIG. 9 is used to check the degree of mixing of the two solutions through section A. Compared to section A, section B shows a more greenish area, indicating that a relatively more complete mixed solution is included.

[0077] Section C in FIG. 9 indicates the stabilization section, and it can be seen from the color that the solution is in a completely mixed state.

[0078] In contrast, Figure 1 shows the mixing of fluids in the chip for producing lipid nanoparticles in KR10-2361123B1. It can be seen that, unlike the present invention, complete mixing does not occur within the entire section of the chip when the oil phase solution and the aqueous phase solution are injected and mixed, and there is a difference in the degree of complete mixing of the oil phase solution and the aqueous phase solution compared to the chip for producing lipid nanoparticles of the present invention.

[0079] When using the chip for producing lipid nanoparticles shown in Figure 1, as described above, complete mixing of the oil phase solution and the aqueous phase solution does not occur, or a torus-shaped mixing element must be added to the chip to achieve complete mixing. That is, if the oil phase solution and the aqueous phase solution are not completely mixed, lipid nanoparticles are formed during the mixing process of the oil phase solution and the aqueous phase solution. However, unlike the chip for producing lipid nanoparticles of the present invention, the process does not go through a stabilization section, which can lead to collisions between the lipid nanoparticles or the formation of clumps, making it difficult to produce uniform lipid nanoparticles. Furthermore, if a torus-shaped mixing element is added to achieve complete mixing, the chip size increases, which is undesirable from a cost perspective.

[0080] A method for producing lipid nanoparticles according to another embodiment of the present invention includes step S100 of producing an aqueous phase solution containing nucleic acids, step S200 of dissolving lipids in an organic solution to produce an oil phase solution, step S300 of supplying the aqueous phase solution and the oil phase solution to the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 on the above-mentioned lipid nanoparticle production chip, and step S400 of mixing the oil phase solution and the aqueous phase solution supplied to the oil phase solution supply unit 100 and the aqueous phase solution supply unit 200 in the mixer unit 300 to form lipid nanoparticles.

[0081] Specifically, in step S100, nucleic acid is mixed with a solvent to prepare an aqueous solution. The solvent is a citric acid solution with a pH of 3.0, but the present invention is not limited to this example, and any solvent that can be mixed with nucleic acid to prepare lipid nanoparticles can be used without limitation.

[0082] The nucleic acid can be selected from the group consisting of RNA, DNA, siRNA (short interfering RNA), mRNA (messenger RNA), aptamer, antisense ODN (antisense oligodeoxynucleotide), antisense RNA, ribozyme, DNAzyme, and mixtures thereof, and is preferably, but not limited to, mRNA.

[0083] The nucleic acid is used for preventing or treating a disease, for example, synthesizing a spike protein to combat the coronavirus 19 virus, such as in a coronavirus 19 vaccine. The nucleic acid is not limited to the above examples, and any nucleic acid for preventing or treating a disease can be used.

[0084] The step S200 is to prepare an oil phase solution by dissolving an ionizable lipid, a non-ionizable lipid, a neutral lipid, and a fusogenic lipid in an organic solution.

[0085] The ionizable lipid may be ALC-0315 (Genevant), ALC-0159 (Genevant), DLinDAP, Dlin-MC3-DMA, or SM102 (Arbutus). The ionizable lipid is not limited to the examples, and any ionizable lipid used in the production of lipid nanoparticles may be used without limitation.

[0086] The non-ionizable lipid may be included together with the fusogenic lipid to enhance the stability of the lipid nanoparticles. Lipid nanoparticles are designed to deliver nucleic acids to target tissues or organs, but they can be destroyed before reaching the target tissues or organs after infusion. To prevent this problem, the non-ionizable lipid and the fusogenic lipid may be included. Specifically, the non-ionizable lipid is selected from the group consisting of distearoylphosphatidylcholine (DSPC), dioleolphosphatidyl ethanolamine (DOPE), bis(diphenylphosphino)ethane (DPPE), diacylphosphatidylcholine, diacylphosphatidylethanolamine, diacylphosphatidylserine, and mixtures thereof, preferably DSPC, but not limited to these examples.

[0087] The fusogenic lipid may be selected from the group consisting of cholesterol, tocopherol, and mixtures thereof, preferably cholesterol, but is not limited to the above examples.

[0088] The neutral lipid is included to adjust the particle size and act as a steric barrier to prevent aggregation during storage, and may be specifically selected from the group consisting of polyethylene glycol 2000 distearoylphosphatidylethanolamine (PEG(2000)DSPE), DMG-PEG, PEG-DMPE, DPPE-PEG, DPG-PEG, PEG-DOPE, and mixtures thereof, preferably DMG-PEG, but not limited to the above examples.

[0089] The organic solution used to prepare the oil phase solution is an alcohol, specifically, methanol, ethanol, isopropanol, n-propanol, etc., but is preferably ethanol. However, the organic solvent is not limited to the above examples, and any organic solvent that can uniformly dissolve the ionized lipid can be used without any restrictions.

[0090] Step S300 involves injecting the aqueous phase solution and oil phase solution prepared in steps S100 and S200 into oil phase solution supply unit 100 and aqueous phase solution supply unit 200 to allow them to flow. As described above, the oil phase solution and aqueous phase solution injected into oil phase solution supply unit 100 and aqueous phase solution supply unit 200, respectively, flow in opposite directions and are connected to mixer unit 300 at the intersection of the two flows, forming a laminar flow within mixer unit 300, allowing them to be mixed within mixer unit 300.

[0091] In step S400, the aqueous phase solution and the oil phase solution injected into the mixer unit 300 form laminar flows at the intersection and are mixed within the mixer unit 300, and the nucleic acids in the aqueous phase solution and the ionized lipids, non-ionized lipids, neutral lipids, and fusogenic lipids in the oil phase solution are bound together through a self-assembly process caused by electrostatic attraction to form lipid nanoparticles. That is, lipid nanoparticles are prepared by mixing the aqueous phase solution and the oil phase solution, and the oil phase solution can be used in a state where all four types of lipids are mixed.

[0092] The nucleic acid is specifically mRNA, which, as mentioned above, is anionic, while the ionized lipid is cationic, and a binding force is generated between them due to electrostatic attraction, thereby forming lipid nanoparticles.

[0093] In step S400, the oil phase solution and the aqueous phase solution flow along a spiral trajectory on the circumferential surface of the mixer unit 300 and mix to form a mixed solution. At this time, the oil phase solution and the aqueous phase solution pass through a mixing section 380 where they mix, and a stabilization section 390 where the mixed solution, which has passed through the mixing section 380 to form lipid nanoparticles, flows along a spiral trajectory and is stabilized. That is, within the mixing section 380, the oil phase solution and the aqueous phase solution form an interface along the spiral trajectory on the circumferential surface of the mixer unit 300, and lipids in the oil phase solution and nucleic acids in the aqueous phase solution can form lipid nanoparticles at this interface through a self-assembly process. The formation of an interface between the oil phase solution and the aqueous phase solution along a spiral trajectory on the circumferential surface of the mixer unit 300 can be achieved by the flow shown in FIG. 8, as described above.

[0094] The flow rate ratio of the oil phase solution and the aqueous phase solution when supplied to the oil phase solution supply unit and the aqueous phase solution supply unit, respectively, may be 1:1 to 1:5, 1:1 to 1:4, or 1:2 to 1:4. When mixed within this range, lipid nanoparticles with uniform particle size can be produced. More specifically, if the ethanol content is less than this range, the lipid nanoparticles may have difficulty maintaining their shape during subsequent processes due to the high ethanol content. If the ethanol content exceeds this range, the excessive aqueous phase solution may restrict the movement of lipid particles in the aqueous phase solution, resulting in the formation of particles with excessively small diameters.

[0095] The total flow rate of the oil phase solution and the aqueous phase solution may be 5 mL / min to 50 mL / min, 10 mL / min to 45 mL / min, or 15 mL / min to 40 mL / min. Within this total flow rate range, the oil phase solution and the aqueous phase solution can be quickly and completely mixed in the mixing section 380 of the mixer unit 300, and uniform lipid nanoparticles can be produced.

[0096] FIG. 10 shows the length of the mixing section 380 in the mixer section 300 depending on the total flow rate of the oil phase solution and the aqueous phase solution of the present invention. It can be seen that within the above range, a complete mixed solution is formed when the length ratio of the mixing section 380 to the stabilization section 390 of the mixer section 300 is within the range of 1:1 to 1:5.

[0097] 11 to 15 show the results of an experiment in which the total flow rate was changed from 4 mL / min to 68 mL / min and the flow was confirmed in the same sections A to C as in Fig. 9. When checked together with the experimental results in Fig. 14, when the total flow rate of the oil phase solution and the aqueous phase solution is 4 mL / min, the mixing index becomes significantly low in the section from 0 mm to 2 mm, which is the inlet section of the mixer unit 300, until a completely mixed solution is formed, which makes it difficult to form lipid nanoparticles in the mixed solution.

[0098] Furthermore, even when the total flow rate of the oil phase solution and the aqueous phase solution is 52 mL / min or 68 mL / min, the speed at which the oil phase solution and the aqueous phase solution are mixed in part A of the mixer unit 300 is relatively slow, which causes the problem that lipid nanoparticles are not easily formed in the mixed solution.

[0099] In contrast, when the total flow rate of the oil phase solution and the aqueous phase solution is 20 mL / min or 36 mL / min, it can be seen that explosive mixing of the two solutions occurs in part A of the mixer section 300, and they are mixed quickly.The section in which a completely mixed solution is formed is short, and lipid nanoparticles with a uniform diameter can be formed within the mixing section.

[0100] The lipid nanoparticles formed in step S400 may further include post-treatment steps of dilution and filtration.

[0101] The lipid nanoparticle production chip of the present invention may be formed from a material selected from the group consisting of a glass substrate, a silicon wafer, or a polymer film, but examples of the material are not limited to the above examples, and any material capable of forming a microchannel can be used.

[0102] The polymer film may be selected from the group consisting of polyimide, polyethylene, fluorinated ethylene propylene, polypropylene, polyethylene terephthalate, polyethylene naphthalate, polysulfone, and mixtures thereof, but is not limited to these examples.

[0103] For example, aluminum is deposited on a silicon wafer using an e-beam evaporator, and photoresist is patterned on the aluminum using photolithography.Then, the aluminum is etched using the photoresist as a mask, and after removing the photoresist, the silicon is etched using DRIE (deep ion reactive etching) using the aluminum as a mask.After removing the aluminum, glass is anodically bonded onto the wafer and sealed.

[0104] Although the preferred embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and improvements made by those skilled in the art using the basic concept of the present invention defined in the following claims also fall within the scope of the present invention. [Industrial Applicability]

[0105] The present invention relates to a chip for producing lipid nanoparticles having a spiral structure and a method for producing lipid nanoparticles using the chip.

Claims

1. an oil phase solution supply unit; an aqueous phase solution supply section; a mixer portion including a spiral structure, The mixer unit is connected to the oil phase solution supply unit and the aqueous phase solution supply unit, and the oil phase solution supplied through the oil phase solution supply unit and the aqueous phase solution supplied through the aqueous phase solution supply unit are mixed to form lipid nanoparticles. A chip for producing lipid nanoparticles.

2. The mixer portion includes peaks and valleys alternately formed along a spiral trajectory on a circumferential surface thereof. The chip for producing lipid nanoparticles according to claim 1.

3. The cross section of the mixer portion has an outer diameter of 1 mm to 2 mm due to the peaks and an inner diameter of 0.5 mm to 0.6 mm due to the valleys. The chip for producing lipid nanoparticles according to claim 2.

4. The mixer section has a pitch of 1 mm to 3 mm between the spiral locus on the circumferential surface. The chip for producing lipid nanoparticles according to claim 1.

5. The mixer section has a depth of the peaks and valleys of 0.1 mm to 0.5 mm. The chip for producing lipid nanoparticles according to claim 2.

6. The mixer portion has a valley formed by a spiral locus on the peripheral surface, the width of which is 0.1 mm to 0.5 mm. The chip for producing lipid nanoparticles according to claim 2.

7. The mixer unit includes a mixing section in which the oil phase solution and the aqueous phase solution are mixed, and a stabilization section in which a mixed solution obtained by completely mixing the oil phase solution and the aqueous phase solution is stabilized, The length ratio of the mixing section and the stabilization section is 1:1 to 1:

5. The chip for producing lipid nanoparticles according to claim 1.

8. The mixer section is connected to a discharge section, The discharge unit is connected to the other end of the mixer unit connected to the oil phase solution supply unit and the aqueous phase solution supply unit. The chip for producing lipid nanoparticles according to claim 1.

9. preparing an aqueous phase solution containing nucleic acids; dissolving lipids in an organic solution to produce an oil phase solution; Supplying the aqueous phase solution and the oil phase solution to an oil phase solution supply unit and an aqueous phase solution supply unit on the lipid nanoparticle production chip according to claim 1; The oil phase solution and the aqueous phase solution supplied to the oil phase solution supply unit and the aqueous phase solution supply unit are mixed in a mixer unit to form lipid nanoparticles. Method for producing lipid nanoparticles.

10. the oil phase solution and the aqueous phase solution flow and mix along a spiral trajectory on the circumferential surface of the mixer unit to form a mixed solution; The mixed solution flows along a spiral trajectory and is stabilized. The method for producing lipid nanoparticles according to claim 9.

11. The oil phase solution and the aqueous phase solution form an interface along a spiral locus on the peripheral surface of the mixer unit, and the lipids in the oil phase solution and the nucleic acids in the aqueous phase solution form lipid nanoparticles at the interface through a self-assembly process. The method for producing lipid nanoparticles according to claim 10.

12. The oil phase solution and the aqueous phase solution move through a mixing section in which they flow through a mixer section to form a mixed solution, and a stabilization section in which the mixed solution is stabilized; The length ratio of the mixing section and the stabilization section is 1:1 to 1:

5. The method for producing lipid nanoparticles according to claim 10.

13. the oil phase solution and the aqueous phase solution are supplied to the oil phase solution supply unit and the aqueous phase solution supply unit at a flow rate ratio of 1:1 to 1:5; The method for producing lipid nanoparticles according to claim 9.

14. The total flow rate of the oil phase solution and the aqueous phase solution is 5 mL / min to 50 mL / min. The method for producing lipid nanoparticles according to claim 9.

15. After the step of forming the lipid nanoparticles, additionally comprising post-processing steps of dilution and filtration; The method for producing lipid nanoparticles according to claim 9.

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