Waveform microstructure mixing unit and its use

Abstract

The present invention provides a corrugated microstructure mixing unit and uses thereof. The present invention relates to a corrugated microstructure mixing unit, a microfluidics device comprising said unit, and the use of said unit or device in the preparation of nanoparticles, such as lipid nanoparticles or self-assembling particles.

Description

[Technical Field]

[0001] (Cross-reference of related applications) This application claims priority to Chinese Patent Application No. 202210119774.0, filed on 8 February 2022, entitled “Waveform Microstructure Mixing Unit and its Use.”

[0002] The present invention relates to the field of microfluidics, and more particularly to a multilayer corrugated microstructure mixing unit and a mixing apparatus having the mixing unit. The present invention also relates to the use of the mixing unit and mixing apparatus in the production of nanoparticles such as nucleic acid molecule-borne lipid nanoparticles. The mixing unit and mixing apparatus of the present invention are particularly suitable for the production of self-assembling lipid nanoparticles as pharmaceuticals. [Background technology]

[0003] Microfluidic technology is used in the production of nano-sized delivery carriers, particularly drug delivery carriers such as LNPs, polymer nanoparticles, and liposomes.

[0004] Lipid nanoparticles (LNPs) are nanoparticles formed using lipids. Currently, the technology of encapsulating nucleic acids such as mRNA using lipid nanoparticles is being used in the manufacture of nucleic acid-based drugs and gene therapy. LNP technology solves the problems of nucleic acid delivery as an innovative delivery technology and makes the realization of mRNA vaccines possible. Compared to viral delivery systems, LNP delivery systems, as typical organic nanoparticles, have advantages such as strong permeability, high nucleic acid load capacity, low toxicity, good long-term stability, and good biocompatibility.

[0005] In the manufacturing process of nanoparticles such as LNPs, mixing the ethanol phase and the aqueous phase leads to a rapid mixing process that causes supersaturation of lipid or polymer molecules, promoting the self-assembly of nanoparticles and forming nano-sized particles.

[0006] Rapid mixing of the two solutions during this process is key to limiting the resulting particle size to less than 100 nm, eliminating the need for top-down size reduction methods employed in other conventional manufacturing processes, such as extrusion and ultrasonic methods.

[0007] Since nanoparticles are formed by self-assembly, their manufacturing methods are considered bottom-up methods. Compared to conventional top-down methods, the main advantages of rapid mixing processes are enhanced control over physicochemical properties, improved encapsulation efficiency, and increased feasibility of process scaling.

[0008] Although the 3D structures of different microstructure mixing devices differ, they can induce rapid mixing of organic and aqueous phases in a controlled environment. The principle of these devices is to rapidly mix two mutually dissolving phases and rapidly increase the polarity of the solvent environment for materials such as lipids or polymers, thereby forming lipid or polymer nanoparticles.

[0009] Taking the preparation of lipid nanoparticles (LNPs) used for mRNA delivery as an example, the mixing process typically involves rapidly mixing an ethanol phase containing hydrophobic lipids with a buffered aqueous phase containing mRNA using a microfluidic chip or T-mixer. In this process, LNP formation is achieved through hydrophobic interactions, and mRNA encapsulation is completed simultaneously through electrostatic action.

[0010] In a typical process for producing mRNA-carrying lipid nanoparticles, four lipid materials (ionizable lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated lipids) are dissolved in an ethanol solution. The ionizable lipids remain in a non-ionized state and are electrically neutral. Typically, the ethanol solution containing the lipids is mixed with a weakly acidic buffer (pH=3-5.5) containing mRNA. While the lipids become insoluble when exposed to the aqueous buffer, the ionizable lipids ionize and become positively charged. The positive charge of the ionizable lipids then drives the negative charge of the ionizable lipids and the mRNA phosphate backbone, causing an electrostatic interaction. This allows the lipid materials to complete self-assembly in a supersaturated state, forming mRNA-carrying lipid nanoparticles.

[0011] The key to mixing techniques in LNP fabrication is creating turbulent flow, or chaotic flow, when mixing the ethanol phase and the aqueous phase. At low flow velocities or low Reynolds numbers, the solution in the fluid channel generally forms laminar flow, and the mixing in this case is diffusive mixing, a relatively slow process. In diffusive mixing, the degree of mixing depends on the length of the channel and the contact surface area of ​​the two phases. When the ethanol phase and aqueous phase come into contact and mix in laminar flow, a phospholipid bilayer is formed at the interface, eventually forming structures similar to liposomes or multilayer vesicles, affecting particle yield and particle uniformity. Under turbulence, lipid materials quickly become supersaturated and can self-assemble to form a uniform solid structure. At high Reynolds numbers (Re) or flow velocities, turbulence improves mixing efficiency and shortens mixing time, and shorter mixing times reduce mass transfer effects. Mass transfer effects increase lipid aggregation and particle morphological heterogeneity. In LNP preparation, the mixing channel has a low mixing rate and Reynolds number, and under these conditions, turbulence is unlikely to form. The self-assembly process of LNPs is based on the fact that when the ethanol phase is diluted with the aqueous phase, the polarity of the solvent increases, leading to lipid precipitation.

[0012] Patent Document 1 discloses a channel structure similar to a fishbone shape, called a staggered herringbone mixer (SHM). This structure has uncertainties when scaling up from the laboratory level to the industrial level, which may make it difficult to use at the industrial level.

[0013] Hirota et al. first reported the use of T-tube mixing in the fabrication of lipid material drug delivery systems in 1999. As a method for generating DNA-lipid complexes, T-tube mixing provides an alternative to macroscopic mixing methods. As the name suggests, a T-tube is a three-sided T-shaped tube, with fluids flowing in from both ends in the transverse direction of the "T" and out from the end in the longitudinal direction of the "T". Compared to macroscopic mixing methods (e.g., vortexing or dropwise mixing), the T-junction-shaped mixer provides a relatively controlled mixing environment, enabling the reproducible generation of nanoparticles. When the two input fluids of the T-junction collide, rapid mixing occurs, creating a turbulent or chaotic flow in the outflow fluid.

[0014] T-tube mixing is one of the recommended methods for large-scale LNP production by mRNA vaccine companies. However, in the early stages of drug development, the use of T-tube mixing for small-scale laboratory production is very limited. The main reason for this limited use is that the relatively simple T-shaped structure requires high flow rates to ensure effective mixing. Generally, high flow rates accommodate larger material usage and allow materials to flow faster through the apparatus. However, in the case of nucleic acid drugs, raw material costs are high and only small amounts are required for the product, making it unsuitable for situations where high flow rates are met for large doses. For example, if the flow rate is 60 mL / min and the minimum production time is only 20 seconds, at least 20 mL of feed solution is required. Taking mRNA as an example, such a quantity would contain mg-level RNA molecules, but in studies such as cytology, only μg-level RNA may be required, resulting in high costs and waste.

[0015] In 2002, Stroock et al. found that adding a herringbone structure to the flow channel improved mixing efficiency at low Reynolds numbers, enabling high-speed mixing in milliseconds at lower flow velocities. Pieter Cullis's team used a staggered herringbone mixer (SHM) to produce LNPs via chaotic convection mixing. This technique was subsequently commercialized by Precision Nanosystems. The method was developed to enhance control over the mixing process and reduce mixing time. While SHMs are widely used in laboratories, such mixers are difficult to fabricate and have certain performance limitations. The difficulty in scaling and process scaling of SHM designs limits their translation into clinical applications and large-scale applications.

[0016] Patent documents disclose a branch mixer that utilizes Dean vortexing, including multiple rings arranged in series.

[0017] There is still a need to design improved microfluidic mixing systems that can be widely applied to pharmaceutical manufacturing in the pharmaceutical field, particularly the production of self-assembling nanoparticles, by improving mixing efficiency and reducing manufacturing costs. [Prior art documents] [Patent Documents]

[0018] [Patent Document 1] US Patent No. US 10,843,194 B2 [Patent Document 2] Chinese Patent No. CN108778477A Publication [Patent Document 3] U.S. Patent Application Publication No. 2004 / 0091546 [Overview of the Initiative] [Problems that the invention aims to solve]

[0019] In the design of a simple curved or helical shape, a very high flow rate is required for high-speed mixing of fluids. When the flow rate is very high, the formation of secondary Dean vortices can be induced, so this situation that changes the fluid into a chaotic convection state usually corresponds to a very high Reynolds number and is not suitable for many practical scenarios.

[0020] The multi-layered waveform micro-structure mixing unit of the present invention designs the geometric shape of each layer of the flow path to bend alternately in different directions, and by combining such flow paths in multiple layers, more flows are generated, which helps to induce chaotic convection. Therefore, the waveform micro-structure mixing unit of the present invention can induce stronger chaotic convection in the generation of self-assembled lipids or polymer nanoparticles, enabling two fluids to surround each other and effectively wrap each other, so the interface between the fluids is exponentially increased, ensuring high-speed mixing of the fluids.

[0021] Specifically, the waveform micro-structure of the present invention is a multi-layered waveform micro-structure, each layer has the same or different widths, and the projected shapes between two adjacent layers on the entire flow path do not completely overlap, but always overlap. Also, by providing one or more flow restriction ribs along the flow path with an original rectangular cross-section, it is conceivable that at least a part of the cross-section of the flow path forms one or more recesses with respect to the original rectangle. In particular, the cross-section of the curved part of the waveform structure is no longer rectangular. This design can further promote the generation of vortices and improve the mixing effect. Based on these designs, the present invention has been completed.

Means for Solving the Problems

[0022] In a first aspect, the present invention provides a waveform multi-layer (sinuous multilayer; SML) micro-structure mixing unit including two or more waveform flow paths, and the projected shapes between two adjacent layers on the entire flow path always overlap, but do not completely overlap. Preferably, the projected shapes between two adjacent layers are such that the semi-circular ring part or semi-elliptical ring part of the waveform always partially overlaps.

[0023] In a second aspect, the present invention further provides a microfluidic device comprising the waveform microstructure mixing unit or waveform multilayer microstructure mixing unit of the first aspect described above. In the device, the waveform microstructure mixing unit or waveform multilayer microstructure mixing unit is combined in parallel and / or in series.

[0024] In a third aspect, the present invention relates to a method for producing self-assembling nanoparticles using the microstructure mixing unit of the first aspect or the apparatus of the second aspect. In a specific embodiment, the self-assembling nanoparticles are nucleic acid-carrying lipid nanoparticles. [Effects of the Invention]

[0025] The present invention has at least the following advantages.

[0026] The microstructured mixing unit of the present invention can increase the intensity of mixing by creating turbulence in the liquid during mixing at lower mixing velocities (such as total flow velocity) and Reynolds numbers (Re). Due to the multilayer corrugated channels of different widths or staggered configurations, the relative direction of fluid flow between different layers repeatedly changes, causing the main center of rotation of the Dean vortex to shift between mixing units.

[0027] The combination of corrugated microstructure mixing units in the apparatus of the present invention can provide precise liquid partial pressure. By using a curved or helical design, the microstructure mixing unit of the present invention provides a method for promoting convective transport within microchannels through a simple geometric design.

[0028] The waveform microstructure mixing unit of the present invention can maintain excellent mixing effects even when proportionally scaled up (for example, by proportionally increasing the cross-sectional area and length of the flow path) or when its scale is increased (for example, by connecting multiple mixing units in parallel), thus expanding the range of applicable scenarios for the mixing unit.

[0029] The waveform microstructure mixing unit of the present invention and the microfluidic chip containing the unit are particularly suitable for the production of nanoparticles, especially for the self-assembly of drug molecule-laden nanoparticles, such as the self-assembly of nucleic acid-laden lipid nanoparticles.

[0030] The waveform multilayer microstructure mixing unit of the present invention can achieve millisecond-level mixing and form nanoparticles, with effects similar to those of a herringbone SHM mixer. This effect is achieved without requiring complex 3D surface structures such as the ridge structure of a staggered herringbone mixer. Given that complex 3D surface structures can induce high local shear forces, the channels formed by the unit of the present invention are more suitable for production processes involving biomacromolecules (particularly nucleic acid molecules). At the same time, because there are no complex 3D surface structures, such mixing devices (such as chips) are easy to clean and reusable. [Brief explanation of the drawing]

[0031] [Figure 1] This is a schematic diagram of the D-B1 waveform microstructure mixing unit. The two small diagrams on the left are a top view of the repeating unit of the waveform channel in the mixing section (the small diagram above; including two semicircular and two short straight channels), and a cross-sectional view of this channel (the small diagram below). [Figure 2] This is a 3D schematic diagram of the D-B1 waveform microstructure mixing unit. [Figure 3A-3E] This diagram shows a mixing apparatus with four parallel-connected waveform multilayer microstructure mixing units. (A) Schematic diagram of the inlet layer as the first layer, (B) Schematic diagram of the flow divider layer as the second layer, (C) Schematic diagram of the mixing layer as the third layer, (D) Perspective view of all layers combined, (E) Photograph of the actual apparatus. [Figure 4A-4D] This diagram shows a mixing apparatus with 16 parallel stages of waveform multilayer microstructure mixing units. (A) Schematic diagram of the inlet layer as the first layer, (B) Schematic diagram of the flow divider layer as the second layer, (C) Schematic diagram of the mixing layer as the third layer, (D) Perspective view of all layers merged. [Figure 4E-4F] This diagram shows a mixing apparatus with 16 parallel-connected waveform multilayer microstructure mixing units. (E~F) Actual photographs. [Figure 5A-5B] These are top and cross cross views of the repeating units in the mixing section of the four waveform multilayer microstructure mixing units D-A1, D-B1, D-C1, and T-B1 of the present invention. (A) Two-layer structure D-A1 in which the inner edge of the narrow channel layer on the semi-ring portion always overlaps with the inner edge of the wide channel layer, (B) Two-layer structure D-B1 in which the outer edge of the narrow channel layer on the semi-ring portion always overlaps with the outer edge of the wide channel layer. The figures show the dimensions of each layer, where W: width, H: height, Lc: length of the straight channel, Do: length of the outer diameter of the semi-ring in the projected shape, and Di: length of the inner diameter of the semi-ring in the projected shape. [Figure 5C-5D] These are top and cross-sectional views of the repeating units in the mixing section of the four waveform multilayer microstructure mixing units D-A1, D-B1, D-C1, and T-B1 of the present invention. (C) Two-layer structure D-C1 with the same width, where the widths of the two-layer channels on the semi-ring portion are offset, forming a cross section similar to the Z shape of Tetris. (D) Three-layer structure T-B1. The figures show the dimensions of each layer, where W: width, H: height, Lc: length of the straight channel, Do: length of the outer diameter of the semi-ring in the projected shape, and Di: length of the inner diameter of the semi-ring in the projected shape. [Figure 6A-6B] These are schematic diagrams of the waveform single-layer microstructure mixing units S1(A) and S2(B). The small diagram in the upper left shows a top view (left; including two semicircular and two short straight channels) and a cross-sectional view (right) of the repeating waveform channel of the mixing section. [Figure 6C-6D] These are schematic diagrams of the waveform single-layer microstructure mixing units S3(C) and S4(D). The small diagrams in the upper left show a top view (left; including two semicircular and two short straight channels) of the repeating waveform channel in the mixing section and a cross-sectional view (right) of this channel. [Figure 7] This is a schematic diagram of the T-shaped mixing unit T1. The small diagram on the left is a cross-sectional view of the flow path in the mixing section. [Figures 8A-8B] This is a schematic diagram of the Y-shaped mixing units Y1(A) and Y2(B). The small diagram on the left is a cross-sectional view of the flow path in the mixing section. [Figure 9] This is a histogram of particle size (volume-based particle size) and PDI of particles obtained by mixing with the wave-like microstructure mixing units S1, D-A1, D-B1, D-C1, and T-B1. [Figure 10] This is a histogram of particle size (volume-based particle size) and PDI obtained by the wave-type microstructure mixing units Y1, T1, S1, S2, and D-B1. [Figure 11] This is a histogram of particle size (volume-based particle size) and PDI obtained by the wave-type microstructure mixing units Y2, S3, and D-B4. [Figure 12] This is a histogram of particle size (volume-based particle size) and PDI obtained by wave-type microstructure mixing units D-B4 and D-B5. [Figure 13] This is a histogram of particle size (volume-based particle size) and PDI obtained by the wave-like microstructure mixing units D-B1, D-B2, S1, and S2. [Figure 14] Figure A shows the particle size (volume-based particle diameter) and PDI histogram of particles obtained by the wave-microstructure mixing units S2 and D-B2. Figure B shows the particle diameter (volume-based particle diameter) and PDI histogram of particles obtained by the wave-microstructure mixing units S3 and D-B2. Figure C shows the particle diameter (volume-based particle diameter) and PDI histogram of particles obtained by the wave-microstructure mixing units S4 and D-B7. [Figure 15] Figure A shows the particle size (volume-based particle diameter) and PDI histogram of particles obtained by the corrugated microstructure mixing units D-B2, D-B3, D-B4, and D-B6, and Figure B shows the particle diameter (volume-based particle diameter) and PDI histogram of particles obtained by the corrugated microstructure mixing units D-B1, D-B5, and D-B7. [Figure 16] This is a schematic diagram of a three-layer corrugated microstructure mixing unit. A to F are schematic diagrams of different embodiments. [Figure 17] This is a schematic diagram of a three-layer corrugated microstructure mixing unit. A and B are schematic diagrams of different embodiments. [Figure 18] This is a schematic diagram of a two-layer mixing unit with a T-shaped mixing section. [Figure 19] This is a schematic diagram of a two-layer mixing unit equipped with an in-line dilution channel at the outlet. [Figure 20]This figure shows the results of Example 5, comparing the PDI and particle size (volume-based particle size) histograms of nanoparticles prepared using a two-layer mixed unit with a different number of semicircular structures. [Figure 21] This is a schematic diagram of the mixing unit (A) of the present invention and the annular mixing unit (B) of the prior art in Example 10. [Figure 22] This figure shows the size and DPI measurements of RNA-carrying self-assembling LNPs produced using the mixed unit of the present invention in Example 10 and the cyclic mixed unit of the prior art. [Figures 23A-23B] This is a photograph showing the mixing effect when an ethanol solution containing phenol red and a phosphate buffer containing trypan blue are mixed using the Y-1, S-1, and D-B1 mixing units (A) and a 16-stage parallel mixing apparatus (B), illustrating the difference in color between the two fluids before mixing and the fluids after mixing. [Figure 24] This is a schematic diagram of the turbulence direction in a two-layer mixing unit such as D-B1. [Modes for carrying out the invention]

[0032] <Detailed description of the invention> The terms used in this application are defined below. Any undefined term has the same meaning as it would be understood by a person of ordinary skill in the art.

[0033] (definition) In the context of this invention, a "microstructure mixing unit" refers to a fluid mixing device having a one-dimensional, sub-millimeter-sized structure. Specifically, the microstructure mixing unit is a part of a microfluidic chip, or the microfluidic chip itself.

[0034] In the context of this invention, "flow channel" or "flow passage" have the same meaning and refer to a cavity through which fluid can pass within a microfluidic chip, which is usually an elongated passage. Flow channels in conventional microfluidic chips generally have a rectangular cross-section. Preferably, the flow channels of this invention do not have a rectangular cross-section along their entire length. Such flow channels of this invention can be considered as consisting of multiple layers of flow channels with rectangular cross-sections arranged vertically. Therefore, the reference to "multilayer flow channels" is merely to more clearly describe the structure of the mixing unit of this invention and does not mean that the fluids between these flow channels are physically isolated from each other to form multiple independent flow channels. In fact, the "multilayer flow channels" ultimately form a single fluid flow channel.

[0035] A "waveform" channel is a channel composed of multiple circular arcs connected from end to end, curving alternately in opposite directions, so that the entire channel is wave-shaped. In a specific embodiment of the present invention, the circular arcs are semicircular or semielliptical. Since the bending directions of two adjacent semicircular or semielliptical rings are opposite, if each semicircular or semielliptical ring is the same size, two consecutive semicircular or semielliptical rings can be considered a "repeating structure" or "repeating unit". The direction of the wave motion of the "waveform" is similar to the direction of wave vibration in physics. The direction in which the "waveform" extends is similar to the direction of wave propagation in physics. Therefore, the direction of the wave motion and the direction in which the wave extends are two orthogonal directions.

[0036] A "cross-section" refers to a cross-section perpendicular to the direction of liquid flow in a fluid channel.

[0037] "Projected shape" refers to the shape of a microfluidic chip or corrugated microstructure mixing unit when viewed from directly above while placed horizontally. "Overlap of projected shapes" means that the size and shape completely overlap, and does not include cases where one projected shape is smaller and fits inside the other.

[0038] "Fluid communication" or "fluid-connected" refers to a connection between structures, parts, or elements that allows fluid to flow from one to the other.

[0039] "Parallelization" means that two elements or components are connected end to end, or end to end. In the context of the present invention, "parallelization" generally means the parallelization of two or more mixing units, that is, these mixing units are connected via a confluence at their inlet ends or share one confluence, and their outlet ends are connected via the same outlet or share one outlet.

[0040] In the context of this invention, "flow velocity" refers to the total flow rate (TFR) unless otherwise specified. TFR is the sum of the velocities at which the fluids to be mixed are pumped through the inlets, for example, the sum of the velocities at which a fluid containing lipids and a fluid containing nucleic acid molecules pass through two inlets. TFR is usually expressed in units of mL / min. For the sake of explanation, in the context of this invention, ultra-low flow velocity is considered to be less than 1 ml / min, low flow velocity is considered to be 1 ml / min or more and less than 4 ml / min, medium flow velocity is considered to be 4 ml / min or more and less than 12 ml / min, and high flow velocity is considered to be 12 ml / min or more.

[0041] A "Dean vortex" is a phenomenon that occurs when a fluid flows through a curved channel. The fluid flowing towards the center of the channel is pushed outwards by centripetal forces and the higher velocity of the fluid at that point (due to the no-slip boundary condition). As a result of these forces, the fluid rotates perpendicular to the channel.

[0042] The Reynolds number represents the state of fluid flow and is a dimensionless number that takes the ratio of inertial forces to viscous forces within the fluid. Laminar flow occurs when the Reynolds number is low, while turbulent flow occurs when the Reynolds number is high.

[0043] "Nanoparticles" refer to particulate matter with a diameter of submicron size, typically less than 100 nm in diameter. In the context of this invention, nanoparticles generally refer to nanocarriers on which molecules having pharmaceutically active components are mounted.

[0044] "LNP" is an abbreviation for lipid nanoparticle, which refers to lipid nanoparticles.

[0045] In the context of this application, "carrier material" refers to the main material that forms nanoparticles, which is generally a biocompatible component that can form nanoparticles on its own. In the present invention, the "carrier material" generally does not possess the desired pharmaceutical efficacy, such as therapeutic efficacy.

[0046] In the context of this application, "carrying material" means a substance having pharmaceutical efficacy, such as therapeutic efficacy, that can be delivered by a carrier material.

[0047] "Nucleic acid" refers to nucleotides in any polymeric form, including deoxyribonucleotides, ribonucleotides, or their analogues. "Nucleic acid" can be used interchangeably with "polynucleotide" and "oligonucleotide." Nucleic acid can be single-stranded, double-stranded, or multi-stranded. Polynucleotides can be DNA. Polynucleotides can also be RNA.

[0048] In the context of this specification, "particle diameter" refers to the diameter of spherical particulate matter. The particle diameter of submicron-sized particles is typically measured by dynamic light scattering (DLS). DLS estimates the particle size distribution by irradiating submicron-sized particles with laser light to induce scattering and analyzing the fluctuations in the scattered light. The measurement results include volume-based particle diameter, intensity-based particle diameter, and number-based particle diameter, as well as the average particle diameter calculated based on these three parameters. Unless otherwise specified, when "particle diameter" is referred to in the context of this invention, it refers to the volume-based particle diameter measured by dynamic light scattering.

[0049] "PDI" stands for Polydispersity Index, which is a parameter representing the width of the particle size distribution of nanoparticles. A larger PDI indicates a wider distribution and higher polydispersity, while a smaller PDI indicates a narrower distribution and better uniformity.

[0050] (Waveform multilayer (SML) microstructure mixing unit) Self-assembly of nanoparticles typically requires turbulence. The typical flow characteristics at low Reynolds numbers are laminar, not turbulent. Therefore, for microstructure mixing units (such as microfluidic chips) used for self-assembly of nanoparticles, either the flow velocity is accelerated to increase the Reynolds number and turbulence formation, or turbulence is achieved at low velocity / Reynolds number through special velocity design.

[0051] The present invention utilizes corrugated channels consisting of alternating semicircular or semielliptical rings that curve in different directions, with multiple layers of such corrugated channels in the mixing section. By inducing alternating flow and chaotic convection within the mixer, the mixing of components in the fluid is promoted (Figure 24). By using a curved or helical design, a simple geometric design promotes convective transport within the microchannels and generates Dean vortices during the mixing process, improving the mixing effect.

[0052] The present invention provides a corrugated multilayer microstructure mixing unit, the mixing unit comprising a fluid-communicated inlet, confluence, multilayer mixing section, and outlet, wherein each layer of the multilayer mixing section is a corrugated channel, the corrugated channel comprising n semicircular or semielliptical rings, any two adjacent semicircular or semielliptical rings having opposite curvature directions and connected to each other by a linear channel. The projected shapes of two adjacent layers of the multilayer mixing section always overlap at least partially but not completely on the flow path of the entire mixing section. The inlet section comprises at least two inlets, the inlets being in fluid communication with the confluence section, so that different fluids flowing in from the inlets merge at the confluence section.

[0053] The aforementioned inlet section is used to allow different fluids to be mixed to flow in, and therefore includes at least two inlets, for example, two inlets. For example, when producing nucleic acid-carrying liposome nanoparticles, at least one inlet is for the flow of a lipid-containing fluid, and at least one other inlet is for the flow of a nucleic acid-containing fluid.

[0054] After different fluids flow in through the inlets, they preferably flow through the flow path sections before converging. Therefore, in a preferred embodiment, the inlets include flow path sections between each inlet and the converging section. In a preferred embodiment, the length of the flow path between the inlets and the converging section is at least 2000 μm. In a specific embodiment, the flow path between the inlets and the converging section is a straight flow path.

[0055] Different liquids flowing in from different inlets pass through the inlet section and then merge at the confluence section. The length of the confluence section can be arbitrary. The length and shape of the confluence section can be adjusted according to the needs of chip manufacturing and flow path design.

[0056] In some embodiments, the mixing unit of the present invention does not include a confluence. When there is no confluence, fluids flowing in from different inlets merge directly in the mixing unit.

[0057] Fluids flowing in from different inlets can merge at the same location in a confluence. In this case, taking a scheme with two inlets as an example, the inlets and confluence can form a T-shaped or Y-shaped structure. In this case, the confluence is the vertical portion of the T or Y, and the flow path of the inlet is the two upper branches. In a preferred embodiment, the angle α between the straight flow path of the inlet and the straight flow path of the confluence is between 90° and less than 180°.

[0058] After passing through the confluence, the different fluids are roughly mixed. Subsequently, the fluid mixture enters the mixing section, where turbulence is formed due to the special structure of the mixing section, resulting in thorough mixing and promoting the formation of desired products such as self-assembling nanoparticles.

[0059] The straight channel in the confluence is perpendicular to or coincides with the wave direction of the waveform channel in the mixing section. In a specific embodiment, the straight channel in the confluence is perpendicular to or coincides with the extending direction of the waveform channel in the mixing section.

[0060] While there are many implementation forms of multilayer mixing units, the main purpose is to generate more turbulence under the same conditions by forming a more complex flow channel structure than conventional simple rectangular channels. Therefore, corrugated multilayer microstructure mixing units can produce better mixing effects. Specifically, fluids can become turbulent even at low flow velocities and Reynolds numbers.

[0061] For example, a multilayer mixed unit may contain 2 to 20 layers. Considering the manufacturing process and cost, it is preferable not to exceed 5 layers, for example, 2, 3, 4, or 5 layers. However, considering future developments in manufacturing processes, it may be possible to include even more layers.

[0062] The widths of the channels in each layer of a multilayer mixing unit may be the same or different. For example, each layer may have one or more widths. The only requirement is that the projected shapes of two adjacent layers do not completely overlap. Imperfect overlap of projected shapes can be achieved by using different widths or by offsetting the channels of two layers of the same width.

[0063] For example, the narrowest width of each layer can be 20% to 100% of the widest layer's width, such as 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 2 / 3, 3 / 4, 2 / 5, 3 / 5, 4 / 5, etc.

[0064] The heights of the flow channels in each layer of the multilayer mixing unit may be the same or different. In a specific embodiment, the heights of each layer are the same.

[0065] In a preferred embodiment, in the corrugated multilayer microstructure mixing unit, the flow channels in the semicircular or semielliptical ring portions of each layer are always parallel. In other words, each semicircular flow channel in each layer may form concentric circles, or each semielliptical flow channel in each layer may form concentric ellipses of the same orientation.

[0066] Based on this, when two adjacent layers have different widths, it is preferable that the outer edges of the flow channels in the semicircular or semielliptical ring portions of each layer always overlap. Similarly, when two adjacent layers have different widths, the orientation of the flow channels can be adjusted by connecting the straight sections of the two semicircular or semielliptical rings, thereby achieving the above arrangement for each semicircular or semielliptical ring. Specifically, when each layer has a different width, the straight sections of layers with different widths will form different angles with respect to the direction of extension of the corrugated structure. In such an arrangement, the cross-section of the multilayer mixed portion in the semicircular or semielliptical ring portion may be, for example, L-shaped (two layers), or it may be, for example, three layers B, three layers C, and three layers F as shown in Figure 16.

[0067] In another embodiment, if two adjacent layers have different widths, the inner edges of the flow channels in the semicircular or semielliptical ring portions of each layer always overlap. In such an arrangement, the cross-section of the multilayer mixing portion in the semicircular or semielliptical ring portion may be, for example, L-shaped (two layers), or it may be three layers A as shown in Figure 16.

[0068] In another embodiment, when two adjacent layers have different widths, for at least three-layer mixing units, there are two adjacent layers whose outer edges always overlap the flow channels of the semicircular or semielliptical portions, and there are also two adjacent layers whose inner edges always overlap the flow channels of the semicircular or semielliptical portions. In such an arrangement, the cross-section of the multilayer mixing portion in the semicircular or semielliptical portion may be, for example, the cross-sections of three layers C and three layers E shown in Figure 16 and the cross-section of four layers B in Figure 17.

[0069] If each layer has the same width, or if two adjacent layers have the same width, then to ensure that the projected shapes of two adjacent layers are different and the flow paths within the semicircular or semielliptical ring are always parallel, the widths of two adjacent layers of the same width can be offset in the semicircular or semielliptical ring. Thus, the direction of the flow paths is adjusted by connecting the straight sections of the two semicircular or semielliptical rings. In one embodiment, when two adjacent layers have the same width and are offset in width in the semicircular or semielliptical ring, the outer layer of each semicircular or semielliptical ring (i.e., the one with the larger inner and outer diameters of the ring) is always on the outside, and the inner layer is always on the inside. In such an arrangement, the cross-section of the multilayer mixed section in the semicircular or semielliptical ring may be, for example, two layers in Figure 5C, three layers D in Figure 16, and four layers A in Figure 17.

[0070] In various configurations, a preferred technical means is that the flow paths of the semi-ringular or semi-elliptical ring portions are always parallel, and the outer edges of the flow paths of the semi-ringular or semi-elliptical ring portions of each layer (such as each of two or three layers) always overlap. Specific examples include the structures of the mixing units D-B1, T-B1, etc., in the present invention.

[0071] In the mixing section, the width of the waveform channel in the mixing section of the multilayer mixing unit is denoted as W, and its height as H. Preferably, the height of the channel in the mixing section is always constant and unchanging. In the case of a multilayer mixing unit, the width and height of each layer can be expressed separately; for example, the width and height of a narrow layer can be expressed as W, respectively. narrow and H narrow This is expressed as W, and the width of the wide layer is W wide and H wide This is expressed as follows: If only the width W is given without specifying which layer it is, W is measured based on the projected shape of the flow channel in the semicircular or semielliptical ring portion. If only the height H is given without specifying which layer it is, it is the total width of the superimposed multi-layered flow channels.

[0072] In a preferred embodiment, the width (W) of the waveform channel portion is any value between 200 μm and 3000 μm, preferably 250 μm to 2000 μm, more preferably 300 μm to 1500 μm, and even more preferably 400 μm to 1000 μm, for example, to the extent of 400 μm, 500 μm, 600 μm, 700 μm, 800 μm, 900 μm, or 1000 μm.

[0073] In a preferred embodiment, the height (H) of the waveform channel portion is any value between 100 μm and 1500 μm, preferably 125 μm to 1000 μm, more preferably 150 μm to 750 μm, and even more preferably 200 μm to 500 μm, for example, to the extent of 200 μm, 250 μm, 300 μm, 350 μm, 400 μm, 450 μm, or 500 μm.

[0074] In a preferred embodiment, the cross-sectional area of ​​the waveform channel portion is 0.01 to 1 mm². 2 It is between 0.04 and 0.8 mm. 2 , more preferably 0.06~0.5mm 2 More preferably 0.08 to 0.4 mm 2 That is the case.

[0075] In a preferred embodiment, the width of each layer in the multilayer mixing section is less than or equal to the width of the flow path at the connection between the confluence and outlet sections of the multilayer microstructure mixing unit and the mixing unit. In a preferred embodiment, the width of the widest layer in the multilayer mixing section is equal to the flow path at the connection between the confluence and outlet sections of the multilayer microstructure mixing section and the mixing unit.

[0076] In the mixing section, the outer diameter of each semicircular ring or the outer axis length of each elliptical ring in the direction of the extension of the wave is denoted as Do, and the inner diameter of each semicircular ring or the inner axis length of each elliptical ring in the direction of the extension of the wave is denoted as Di. Since Do and Di are the inner and outer diameters, respectively, Do is equal to Di plus W x 2. For example, the length of Do may be 600 μm to 8000 μm, preferably 800 μm to 6000 μm, more preferably 1000 μm to 4000 μm, and even more preferably 1500 μm to 3000 μm. For example, the length of Di may be 200 μm to 5000 μm, preferably 400 μm to 4000 μm, more preferably 600 μm to 3000 μm, and even more preferably 800 μm to 2000 μm. For example, the length of Do may be about 3 to 5 times that of W, for example, about 4 times, and the length of Di may be about 1 to 3 times that of W, for example, about 2 times. In the case of a multilayer mixed unit, the possible values ​​of Do and Di can be described for each layer. If only Do and Di are described without specifying which layer it is, the values ​​of Do and Di are measured based on the projected shape of the flow path in the semi-ringular or semi-elliptical ring.

[0077] In the mixing section, it is preferable that the semicircular or semielliptical rings with opposite curvature directions are connected by a straight channel. The length of the straight channel is represented by the parameter Lc. Note that Lc means the straight-line distance between the two semicircular or semielliptical rings in the direction of the wave of the waveform channel, as shown in the drawings of the present invention, and does not take into account the angle between this straight channel and the direction of the wave of the waveform channel. For example, the length of the straight channel (Lc) is 0.5W to 4W, preferably 0.5W to 3W, more preferably 0.5W to 2W, and even more preferably 0.5W to 1W. For example, the length of the straight channel can be any value between 200 μm and 6000 μm, preferably 300 μm to 3000 μm, more preferably 400 μm to 2500 μm, and even more preferably 500 μm to 2000 μm. For example, Lc may have lengths of approximately 500 μm, 1000 μm, 1500 μm, 2000 μm, 2500 μm, or 3000 μm.

[0078] The straight channel is either perpendicular to the direction of extension of the corrugated channel or forms a constant angle with respect to the direction of the wave of the corrugated channel. For example, the angle may be 45° or less, preferably 9° to 27°.

[0079] In a single-layer channel embodiment, it is preferable that the straight channel is perpendicular to the direction of extension of the corrugated channel. In the case of a multi-layer channel, the angle may be related to various factors.

[0080] For example, if the widths of two adjacent layers of channels are not equal, the size of the angle between them may depend on the length (Lc) of the straight channel, the width of the two-layer channel, the difference in width between the two layers, and their alignment (e.g., alignment of inner or outer edges). In some embodiments, the straight channel with the widest width among the multilayer channels is always perpendicular to the direction of extension of the corrugated channel.

[0081] For example, if the widths of the channels in two adjacent layers are equal, the magnitude of the angle between them may depend on the length (Lc) of the straight channel, the width of the channel, and the degree to which the channels in the two adjacent layers are misaligned. In some embodiments, the angle between the direction of extension of the straight channel and the corrugated channel in two channels of equal width is equal, but their directions are opposite.

[0082] In order to generate turbulence, it is preferable to adjust the arrangement of the flow channels in such a way that, in at least one flow channel, the angle between the two straight flow channels causes them to be drawn inward into the semicircular or semielliptical flow channel between them (resembling a horseshoe shape).

[0083] Within the aforementioned mixed unit, there may be n semicircular or semielliptic rings, whether single-layered or multi-layered. n can be any integer other than 1, preferably 6 or greater, for example 6, 7, 8, 9, 10 or greater, for example 10-15, 15-20.

[0084] The mixed fluid, after passing through the mixing unit, flows out from the outlet of the outlet section. In a preferred embodiment, the outlet section further includes a flow path section connecting the end of the mixing section to the outlet. The design and orientation of the flow path in this section can be determined as needed and there are no special requirements. In a specific embodiment, the flow path between the end of the mixing section and the outlet is a straight flow path.

[0085] In embodiments of multilayer microstructured mixing units, it should be noted that the cross-section of the mixing section is not necessarily rectangular. In particular, in the case of a multilayer microstructured mixing unit that includes layers with different flow path widths, the cross-section of the entire mixing section is not rectangular. However, other parts of the mixing section, such as the confluence and outlet sections that have direct fluid communication with the mixing section, do not have the effect of creating a break, and therefore do not become multilayered, resulting in a rectangular cross-section. Consequently, when the mixing section is connected to the flow paths of the confluence and outlet sections, which have rectangular cross-sections, a transition section exists, in which the flow path width of one or multiple layers gradually changes, and eventually the multilayered structure disappears, and the connection becomes possible because the confluence or outlet section has the same rectangular cross-section at the connection point with the mixing section.

[0086] In specific embodiments, the multilayer microstructured mixed unit of the present invention has the size and structure shown in any of D-A1, D-B1, D-B2, D-B2-3, D-B2-5, D-B2-7, D-B3, D-B4, D-B5, D-B6, D-B7, D-C1, T-B1 in Table 1 of Examples, preferably D-A1, D-B1, D-B2, D-B2-3, D-B2-5, D-B2-7, D-B3, D-B4, D-B5, D-B6, D-B7, D-C1, T-B1, and preferably D-B1 and T-B1.

[0087] In a specific embodiment, the SML microstructure mixing unit of the present invention has the D-B1 structure shown in Figure 1. The structure of the D-B1 mixing unit will be described in detail below. The D-B1 SML microstructure mixing unit includes a fluid-communicated inlet, confluence, multilayer mixing section, and outlet. The multilayer mixing section includes two layers of corrugated channels, each layer of the corrugated channels containing 10 semicircular rings, with each semicircular ring having a Do of 2000 μm and a Di of 1000 μm. Any two adjacent semicircular rings have opposite curvature directions and are connected by a straight channel, with an Lc of 1000 μm. The channels in the semicircular rings of the two-layer corrugated channels are always parallel and each has a constant width and height. The width of the narrow layer is 250 μm, the width of the wide layer is 500 μm, and the height of both the narrow and wide layers is 125 μm. The inlet section includes two inlets, and these inlets are in fluid communication with the confluence section, so that different fluids flowing in from the inlets merge at the confluence section. The straight flow path of the inlet section and the confluence section form a Y shape.

[0088] In a specific embodiment, the SML microstructure mixing unit of the present invention has the structure of T-B1. The installation of the inlet, confluence, and outlet of the T-B1 mixing unit is the same as that of D-B1. The structure of the mixing section of the T-B1 mixing unit will be described in detail below. The SML microstructure mixing multilayer section of T-B1 includes three layers of corrugated channels, and its cross-section is shown in Figure 5D. Each of the three layers of corrugated channels contains 10 semicircular rings, with Do being 2000 μm and Di being 1000 μm. Any two adjacent semicircular rings have opposite curvature directions and are connected by a straight channel, with Lc being 1000 μm. The channels in the semicircular sections of the three-layer corrugated channels are always parallel and each has a constant width and height, with the upper and lower layers being narrow (narrow layers) and the middle layer being wide (wide layer). The width of the narrow layer is 250 μm, the width of the wide layer is 500 μm, and the height of both the narrow and wide layers is 100 μm. The inlet section includes two inlets, and these inlets are in fluid communication with the confluence section, so that different fluids flowing in from the inlets merge at the confluence section. The straight flow path of the inlet section and the confluence section form a Y shape.

[0089] (Multi-stage SML mixing device) The present invention also relates to a mixing apparatus comprising the waveform microstructure mixing unit, and more particularly to a waveform multilayer (SML) microstructure mixing unit.

[0090] The SML microstructure mixing unit of the present invention is less prone to clogging and is therefore suitable for scale-up. Accordingly, in a preferred embodiment, the mixing apparatus of the present invention comprises multiple SML microstructure mixing units, also known as a "multistage parallel mixing apparatus." In a preferred embodiment, the multiple SML microstructure mixing units are connected via parallelization. Through this parallelization, the apparatus of the present invention can achieve greater throughput.

[0091] In a preferred embodiment, a multistage parallel mixing apparatus has m parallel SML microstructure mixing units that share a single inlet, which allows the fluid flowing in from the same inlet to flow into m channels. Each channel is in fluid communication with a junction or mixing section of an SML microstructure mixing unit. For example, after the fluid flows in from at least two inlets, the channels that pass through the branching structures contained in each inlet are divided into m branched channels, which are then mixed in the independent junction or mixing sections of each SML mixing unit. The branching structures can ultimately be divided into m branched channels via one or more branching points.

[0092] Similarly, the m outlet ends of the m mixing units are in fluid communication with the outlet. The m parallel SML microstructure mixing units share one outlet. The outlet has a branching structure, which, via one or more branching points, can ultimately merge the fluids discharged from the m mixing units into a single flow path and establish fluid communication with the outlet.

[0093] In one embodiment, the number m of parallelized units is an even number, preferably 2 to the power of n. In this way, multiple branch structures formed by branching points that diverge in two or more directions can be designed, and since each branch is symmetrical, accuracy of pressure division is ensured. To ensure accuracy of pressure division, it is preferable that the parallelized units are identical, that is, have the same size and structure, and are arranged symmetrically and evenly within the device. When m is an even number, preferably 2 to the power of n, two independent mixing units can be paired in a mirror-symmetric manner to facilitate combination.

[0094] Examples of arrangement methods are shown in Figures 3 and 4. This embodiment of the present invention will be explained using the four-stage parallel mixing unit in Figure 3. As shown in Figure 3, the first layer is the inlet layer (Figure 3A), which includes two total fluid inlets (left and center) and one total fluid outlet (right). The two inlets are used for the inflow of two fluids to be mixed, and the one outlet is used for the outflow of the mixed fluid. The second layer is the flow division layer (Figure 3B), which includes straight flow channels in the fluid communication sections with the two inlets. Subsequently, the straight flow channels divide into two symmetrical branched flow channels, so that the two fluids to be mixed flow in from the two inlets and then divide into two separate flows in the flow division layer before flowing into the third layer. The third layer is the mixing layer (Figure 3C). Each inflow channel divides into two again in the third layer, passes through symmetrical non-linear flow channels, and then merges with other fluids. After passing through the merging section, it enters the multi-layer waveform mixing unit and is mixed. The mixed fluid, after mixing, passes through an outlet channel with two branching points, merging the two into one each time, and finally converging at a single point before flowing out through an outlet that connects to the second and first layers.

[0095] As can be seen from Figures 3C and 3D, the multi-stage SML mixing unit consists of two sets of SML mixing units, each set containing two mirror-symmetric mixing units, exhibiting a lantern-like structure as shown in Figure 3. Since each set of the lantern-like structure has an entrance surrounded by a flow channel, flow separation between the sets of lantern-like structures must be carried out in a separate layer, i.e., a flow separation layer. Those skilled in the art will understand that, as long as the waveform flow channel of the present invention is included, the number of layers of material that the final mixing apparatus is composed of is not important to the present invention.

[0096] In one embodiment of the present invention, the device of the present invention is a device having four parallelized channels as shown in Figure 3, or a device having sixteen parallelized channels as shown in Figure 4.

[0097] The confluence and inlet sections, including the branching structure, may be on different planes from the mixing section and / or outlet section. The plane in which the confluence and inlet sections are located is called the "flow separation layer," and it exerts a flow separation effect on the confluence fluid. The plane formed by the mixing section and outlet section is called the "mixing layer," and it exerts a flow mixing and outflow effect.

[0098] In a specific embodiment, the SML mixing device is a microfluidic chip. The mixing device, such as a microfluidic chip, may include, or be connected to, other elements such as syringes, pumps, heaters, detectors, and other flow rate control elements.

[0099] (In-line dilution) In one embodiment of the present invention, an inlet for in-line dilution is optionally further included downstream of the SML mixing unit within the apparatus.

[0100] Using Figure 19 as an example, the technical means of in-line dilution are described. Specifically, the outlet of the SML mixing unit, preferably located close to the mixing section within the outlet, further includes a diluent inlet or diluent channel for delivering a diluent solvent such as PBS or another suitable buffer or carrier, so that the concentration of each component in the fluid flowing through the mixing section is diluted before it flows out of the outlet. For example, the proportion of the original organic solvent, such as ethanol, in the solution is reduced. A diluent inlet and / or diluent channel are preferred because if the ethanol concentration is too high, the stability of the nanoparticles as a product may decrease.

[0101] The mixing method in the in-line dilution is not particularly limited. The in-line dilution can improve the stability of the nanoparticles.

[0102] The rate at which the diluent flows from the diluent inlet can be adjusted according to the desired dilution ratio. When fabricating self-assembling nanoparticles, the dilution ratio can be any value from 1 to 40 times.

[0103] The diluent used for dilution may be a commonly used diluent in this field, such as physiological saline or PBS.

[0104] The diluent flow path may include other components, such as a flow rate control device like a pump, or it may be connected to other components, such as a device for introducing fluid like a syringe.

[0105] (Materials for the mixed unit) The mixed units of the present invention can be made from materials having specific mechanical properties and biocompatibility, for example. Specific examples include polymer materials, metallic materials, or inorganic nonmetallic materials.

[0106] Metal materials that can be used in the manufacture of the mixed unit of the present invention may include, for example, stainless steel, aluminum alloy, magnesium alloy, and titanium alloy. 304 stainless steel, 316L stainless steel, and Hastelloy are preferred.

[0107] Polymer materials that can be used to manufacture the mixed unit of the present invention may include, for example, polyether ether ketone (PEEK), acrylic (PMMA), polydimethylsiloxane (PDMS), polyamide-imide (PAI), polychlorotrifluoroethylene (PCTFE), polytetrafluoroethylene (PTFE), polycarbonate (PC), polypropylene (PP), polyphenylene sulfide (PPS), cyclic olefin copolymer (COC), and cyclic olefin polymer (COP). Preferably, polyether ether ketone (PEEK), polyester (PET / PETP), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), and polydimethylsiloxane (PDMS) are used.

[0108] Inorganic nonmetallic materials that can be used in the manufacture of the mixing unit of the present invention may be, for example, glass, ceramics, etc.

[0109] (Manufacturing of mixed units) The flow channels in the mixing unit of the present invention can be manufactured by any method known in the art or to be developed in the future, as long as the structure described in the present invention can be realized. For example, machining such as precision CNC milling can be used.

[0110] When manufacturing mixed units using polymer materials, processing can also be done using thermoforming (e.g., injection molding) or etching (e.g., SU-8 adhesive). In the case of multilayer mixed units, each layer can be manufactured individually and then sealed together. Common sealing processes such as hot press sealing, laser sealing, and film sealing can be used to seal between different layers.

[0111] In other embodiments, the packaging can also be done directly using metal or polymer materials.

[0112] (application) In one embodiment, the mixing unit and mixing apparatus of the present invention can be used to produce the following four types of nanoparticles. Specifically, different fluids used to produce nanoparticles, such as a fluid containing a carrier material and a fluid containing a load, are added from the inlet of the mixing unit or mixing apparatus of the present invention and mixed by the mixing section of the present invention.

[0113] In a preferred embodiment, the nanoparticles complete self-assembly within the mixing section of the mixing unit and mixing apparatus of the present invention.

[0114] The mixing unit or mixing apparatus of the present invention can produce nanoparticles having a relatively uniform size through efficient mixing. In embodiments of the present invention, nanoparticles produced by the mixing unit of the present invention, in particular LNPs using therapeutic molecules such as nucleic acid molecules as the carrier material and lipids as the support material, have a DPI of 0.3 or less, preferably 0.2 or less.

[0115] (1) Lipid nanoparticles The mixing unit and apparatus of the present invention are particularly suitable for the production of lipid nanoparticles (LNPs) carrying a delivery target. The delivery target may be a nucleic acid molecule including plasmids, ceDNA, mRNA, siRNA, microRNA, sgRNA, etc., and may also be a small molecule or a protein. When used for carrying nucleic acid material, the nucleic acid molecule can be of any size from 20 bp to 15 kb. In a preferred embodiment, the mixing unit and apparatus of the present invention are used to produce nucleic acid-carrying LNPs. In a preferred embodiment, the LNP is an LNP containing an ionizable lipid.

[0116] Lipid nanoparticles are generally composed of four types of lipids: (a) ionizable lipids or cationic lipids, (b) neutral lipids (or non-cationic lipids), (c) PEG-bound lipids, and (d) components that provide membrane integrity. In a specific embodiment of the present invention, the four types of lipids are dissolved in an ethanol phase, and the mixture enters the mixing unit or mixing apparatus of the present invention through at least one inlet. Suitable components as lipid components of (a) to (d) above for producing lipid nanoparticles are listed below.

[0117] (a) Ionizable lipids or cationic lipids Examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), 3-(N-(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), or N-(1,2-dimyristiloxypropane-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide (DMRIE).

[0118] Examples of ionizable lipids include DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, cKK-E12, SM-102, and ALC-0315.

[0119] Other suitable ionizable lipids reported in the relevant technical field are also considered.

[0120] (b) Neutral lipids (or noncationic lipids) Examples of noncationic lipids include distearoyl-sn-glycerophosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoylphosphatidylglycerol (DPPG), dioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), and palmitoylphosphatidylethanolamine. Luamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidyl-ethanolamine (DSPE), monomethyl-phosphatidylethanolamine (16-O-monomethylPE, etc.), dimethyl-phosphatidylethanolamine (16-O-dimethylPE, etc.), 18-1- Lance PE, 1-stearoyl-2-oleoyl-phosphatidiethanolamine (SOPE), hydrogenated soybean phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diylcoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglycerol Examples include, but are not limited to, cerol (POPG), dierydoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalosporin, cardiolipin, phosphaticaside, cerebroside, dicetylphosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It should be understood that other diacyl phosphatidylcholine and diacyl phosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably C. 10 ~C 24 acyl groups derived from fatty acids having a carbon chain, such as lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0121] Other examples of non-cationic lipids suitable for use in lipid nanoparticles include, for example, stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glyceryl ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethoxylated fatty acid amide, dioctadecyldimethylammonium bromide, ceramides, sphingomyelin, and other non-phospholipids. In some embodiments, the non-cationic lipid is a phospholipid. In some embodiments, the non-cationic lipid is selected from the group consisting of DSPC, DPPC, DMPC, DOPC, POPC, DOPE, and SM.

[0122] In some preferred embodiments, the non-cationic lipid is selected from DPSC, DPSC, or DOPE.

[0123] (c) PEG-conjugated lipid The lipid particles further contain bound lipids, which can be selected from the group consisting of, for example, PEG-diacylglycerol (DAG) (1-(monomethoxy-polyethylene glycol)-2,3-dimethoxyglycerol (PEG-DMG)), PEG-distearoylphosphatidylethanolamine (PEG-DSPE), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), PEG-PE, PEG-diacylglycerol succinate (PEGS-DAG) (4-O-(2′,3′-bis(tetradecanoyloxy)propyl-1-O-(w-methoxy(polyethoxy)ethyl) succinate, etc. (PEG-S-DMG)), PEG-dioxypropylcarbam,N-(carbonyl-methoxypolyethylene glycol 2000)-1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine sodium salt.

[0124] (d) Components that provide film integrity Examples of components that provide membrane integrity include sterols and cholesterol.

[0125] (2) Polymer nanoparticles As polymer nanoparticles, the materials used are similar to those in liposome nanoparticles. Examples of carriers include PLGA nanoparticles and PLA nanoparticles. Generally speaking, polymer nanoparticles include polymers and / or amphiphilic polymers.

[0126] Non-limiting examples of hydrophobic polymers include polylactic acid (PLA), polypropylene oxide, poly(lactic acid-coglycolic acid) (PLGA), poly(ε-caprolactone), poly(ethylene), polybutadiene, polyethylene glycol, polymethacrylate, polyvinyl butyl ether, polystyrene, polycyclopentadienylmethylnorbornene, polyethylene, polyisobutylene, and polysiloxane. As another example, the hydrophobic polymer may be any of the following polymers: methyl acrylate, ethyl acrylate, propyl acrylate, n-butyl acrylate, isobutyl acrylate, 2-ethyl acrylate, tert-butyl acrylate, methacrylic acid (e.g., ethyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, ethyl methacrylate, n-butyl methacrylate, isobutyl methacrylate, etc.), propylene nitrile, methacrylonitrile, vinyl ester (e.g., vinyl acetate, trans vinyl ester, vinyl propionate, vinylformamide, vinylacetamide, vinylpyridine, vinylimidazole), aminoalkyl ester (e.g., aminoalkyl acrylate, aminoalkyl methacrylate, aminoalkyl(meth)acrylamide), styrene ester, lactic acid ester.

[0127] Amphoteric polymers include poly(ethylene glycol)-poly(D,L-lactic acid) copolymer (PLA-PEG), PLGA-PEG, polystyrene-polyoxyethylene copolymer, polybutyl acrylate-polyacrylic acid copolymer, or polybutyl methacrylate-polyethylene oxide copolymer. Other examples of amphoteric copolymers are described in Patent Document 3 (which is incorporated herein by reference in its entirety). Other examples of amphoteric polymers (e.g., amphoteric copolymers) are known in the art.

[0128] (3) Self-assembly of lipid-polymer hybrid nanoparticles Lipid-polymer hybrid nanoparticle materials include the lipid nanoparticles described in (1) and the polymer nanoparticles described in (2) above. For the selection of these two components, please refer to the respective sections above.

[0129] (4) Self-assembly of liposomes The mixing unit and apparatus of the present invention can also be used for the self-assembly of liposomes (or similar vesicles). The target of delivery may be small molecules (e.g., poorly soluble cytotoxic drugs such as paclitaxel and camptothecin) or biological macromolecules (protein drugs).

[0130] Liposomes typically consist of phospholipids (such as the neutral lipids, cationic lipids, and ionizable lipids mentioned above) and components that provide membrane integrity. Special types of liposomes may also contain other components, such as long-cycle liposomal lipids including PEG-bound lipids.

[0131] (Flow velocity and TFR) In the production of nanoparticles, the two fluids to be mixed are typically an ethanol phase and an aqueous phase, with a volume ratio of 1:3. Similarly, to achieve a 1:3 volume ratio, the velocity ratio of the two fluids entering the inlet must also be 1:3. In other words, the inlet velocity of the ethanol phase is 25% TFR, while the inlet velocity of the aqueous phase is 75% TFR. Following the same principle, the inlet velocities of the different fluids to be mixed can be adjusted according to the desired volume ratio.

[0132] The inventors have found that different flow path designs require different TFRs. The various SML mixing units of the present invention are particularly suitable for achieving excellent mixing effects at low flow rates, i.e., TFRs of 1 mL / min to 4 mL / min.

[0133] In a preferred embodiment, the present invention provides a self-assembly method for producing nanoparticles, the method comprising the step of mixing a first fluid containing a carrier material (e.g., one or more lipids) and a second fluid containing a load (e.g., nucleic acids such as DNA or RNA) using the corrugated multilayer microstructure mixing unit of the present invention. In this method, a TFR of 1 mL / min to 12 mL / min, preferably 1 mL / min to 6 mL / min, more preferably 1 mL / min to 5 mL / min, and even more preferably 1 mL / min to 4 mL / min is used. For example, when using D-B4, D-B5, D-B6, or D-B7 mixing units, a higher TFR, for example 4 mL / min to 12 mL / min, can be used, for example, using the corresponding TFRs listed in Table 3. When using preferred D-B1, D-B2, D-B2-7, or T-B1 mixing units, a lower TFR, for example 1 mL / min to 4 mL / min, can be used, and these mixing units can obtain ideal products at a low TFR of 1 mL / min.

[0134] This invention also relates to the following items:

[0135] 1. A multilayer corrugated (SML) microstructure mixing unit comprising a fluid-communicated inlet, a confluence, a multilayer mixing section, and an outlet, Each layer of the multilayer mixing section is a waveform channel, and each waveform channel of the layer contains n semicircular or semielliptical rings, where any two adjacent semicircular or semielliptical rings have opposite curvature directions and are connected by a straight channel. The projected shapes of two adjacent layers in the multilayer mixing section always overlap at least partially on the flow path of the entire mixing section, but do not completely overlap. The inlet section includes at least two inlets, which are in fluid communication with the confluence section, so that different fluids flowing in from the inlets merge at the confluence section.

[0136] 2. The mixing section includes two, three, four, or five waveform channels, as described in item 1, for the SML microstructure mixing unit.

[0137] 3. The SML microstructure mixing unit according to item 1 or 2, wherein each layer of waveform channels has a certain width, and the widths of the waveform channels in each layer may be the same or different from each other, and it is preferable that at least two layers of waveform channels have different widths.

[0138] 4. The SML microstructure mixing unit described in any one of items 1 to 3, wherein the flow channels of each layer on the semi-circular or semi-elliptical ring portion of the mixing section are always parallel.

[0139] 5. The outer edges of the flow channels in each layer on the semi-circular or semi-elliptical ring portion of the mixing section always overlap, as described in item 4, for the SML microstructure mixing unit.

[0140] 6. The SML microstructure mixing unit according to any one of items 1 to 5, wherein (a) the mixing section includes two layers of waveform channels, and the cross-section of the waveform channels on the semi-annular or semi-elliptical annular section is L-shaped, or (b) the mixing section includes three layers of waveform channels, and the cross-section of the waveform channels on the semi-annular or semi-elliptical annular section is T-shaped, rotated by 90°.

[0141] 7. An SML microstructured mixing unit according to any one of items 1 to 6, wherein the outer diameter of each semicircular ring in the corrugated channel of each layer, or the outer axis length Do of each elliptical ring in the direction of the corrugation, is 3 to 5 times the width of the channel, and / or the inner diameter of each semicircular ring, or the inner axis length Di of each elliptical ring in the direction of the corrugation, is 1 to 3 times the width of the channel.

[0142] 8. The width W of the overall projected shape of the 8.1 or more layered waveform channel is 200 μm to 3000 μm, as described in any one of items 1 to 7 of the SML microstructure mixing unit.

[0143] 9.1 The total height H of the waveform channel with 1 or more layers is 100 μm to 750 μm, as described in any one of items 1 to 8 of the SML microstructure mixing unit.

[0144] 10. The SML microstructure mixing unit according to any one of items 1 to 9, wherein the semicircular or semielliptical rings with opposite curvature directions are preferably connected by a straight channel, and the length Lc of the straight channel is 0.5W to 4W, preferably 1W to 3W, more preferably 1W to 2W.

[0145] 11. An SML microstructure mixing unit according to any one of items 1 to 10, wherein n is an integer of 6 or greater, preferably an integer of any integer from 7 to 15.

[0146] 12. An SML microstructure mixing unit according to any one of items 1 to 11, comprising two or three layers of corrugated channels, wherein each layer of corrugated channels comprises six to ten semicircular channels, and the length Lc of the straight channels between each semicircular channel is greater than or equal to the inner diameter Di of the projected shape of the semicircular channel.

[0147] 13. The microstructure mixing unit according to item 12, wherein at least two layers in the waveform channel have different widths, and the width of the narrower layer is 25% to 75%, preferably about 50%, of the width of the wider layer.

[0148] 14. The microstructure mixing unit according to item 13, wherein the narrow layer always overlaps with the outer edge of the wider layer throughout the entire semicircular channel, preferably, all layers always overlap at their outer edges throughout the entire semicircular channel.

[0149] 15. A microstructure mixing unit as described in item 13, having the structure and size of D-B1 or T-B1 as described in Table 1.

[0150] 16. A microstructure mixing apparatus comprising m microstructure mixing units described in any one of items 1 to 15 arranged in parallel, wherein the m microstructure mixing units are in fluid communication through an inlet, a confluence and / or an outlet, where m is an integer greater than 1.

[0151] 17. The microstructure mixing apparatus according to item 16, wherein m is an even number, preferably a power of 2.

[0152] 18. A microfluidic chip comprising, optionally, other elements selected from the group consisting of syringes, pumps, heaters and detectors, as described in item 16 or 17.

[0153] 19. A method for mixing different fluids, comprising the steps of mixing a first fluid and a second fluid using a microstructure mixing unit as described in any one of items 1 to 15 or a microstructure mixing apparatus as described in any one of items 16 to 18, and introducing the first fluid into a first inlet and introducing the second fluid into a second inlet.

[0154] 20. A method for producing nanoparticles by self-assembly, comprising mixing a first fluid and a second fluid using a microstructure mixing unit described in any one of items 1 to 15 or a microstructure mixing apparatus described in any one of items 16 to 18, introducing the first fluid into a first inlet and introducing the second fluid into a second inlet, wherein the first fluid is a fluid containing a nanoparticle carrier material and the second fluid is a fluid containing nanoparticle carriers.

[0155] 21. The method according to item 20, wherein the nanoparticles are lipid nanoparticles, polymer nanoparticles, lipid-polymer hybrid nanoparticles, or liposomes.

[0156] 22. The method according to item 21, wherein the nanoparticles are lipid nanoparticles, the first fluid is a fluid containing one or more types of lipids, and the second fluid is a fluid containing nucleic acids such as DNA or RNA. [Examples]

[0157] The multilayer waveform microstructure mixing unit and a fluid mixing apparatus equipped therewith of the present invention will be described through the following embodiments.

[0158] (Design of the mixing unit) The parameters of the mixing units for specific examples and comparative examples of the present invention are shown in Table 1 below.

[0159] For the sake of explanation and comparison, width and height are normalized and shown separately. Specifically, assuming 1W = 1000 μm and 1H = 500 μm, the width and height of the flow channels in each layer of the mixing section are expressed as multiples or fractions of these values.

[0160] In Table 1, Y1, Y2, and T1 are simple conventional Y-shaped and T-shaped channels, while S1 to S4 are single-layer corrugated microstructure mixing units. The cross-sections of these seven channels are rectangular throughout the entire mixing section, specifically square. The structure of each mixing unit is also shown in the schematic configuration diagrams in Figures 1 and 5 to 8.

[0161] Table 1. Chip parameters used in the examples and their relationships (In the table, 1W = 500um, 1H = 250um, 1Lc = 1000um, 1Do = 2000um, 1Di = 1000um) TIFF0007897621000001.tif220170TIFF0007897621000002.tif58170

[0162] Preparation of LNP samples To test the effectiveness of the microfluidic chip of the present invention, LNP samples are prepared by mixing them using the various flow channels described in Table 1. Specifically, an aqueous phase (a citrate buffer for anionic long-chain natural polymer molecules, the molecular structure of which is similar to mRNA with a molecular weight of 15,000 Da; hereinafter referred to as "mRNA-like") and an ethanol phase (a lipid mixture solution) are mixed in a volume ratio of 3:1 at different flow rates to prepare LNP samples.

[0163] For the ethanol phase, ethanol stock solutions of SM-102 (8-[(2-hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino]-octanoic acid, 1-octyrnonyl ester), DSPC (distearoylphosphatidylcholine), Cholesterol, and DMG-PEG (distearoylphosphatidylethanolamine-polyethylene glycol) are prepared according to the concentrations shown in Table 2, and then uniformly mixed in a constant volume ratio to obtain the lipid material working solution as the ethanol phase. The concentrations and molar ratios of the various components in the lipid material working solution are shown in Table 2. Similarly, a citrate buffer of mRNA-like substances at a concentration of 0.1 mg / mL is prepared according to the concentrations shown in Table 2. The prepared ethanol phase and aqueous phase are injected into a mixer (prepared with a syringe pump) in a volume ratio of 1:3 (100 μL:300 μL).

[0164] Table 2. LNP Recipes JPEG0007897621000003.jpg70170

[0165] Measurement of particle size and PD After mixing using the microfluidic chip of the present invention, the resulting mixture is diluted with 400 μL of PBS and placed on a dialysis plate for 2 hours of ice bath dialysis. After dialysis, the particle size and PDI of the obtained particles are measured using a nanoparticle size analyzer (BeNano 90 Zeta).

[0166] In the following examples, the particle size is always the volume-based particle size. This is because the inventors found that the volume-based particle size is the closest to the particle size measured by a transmission electron microscope (TEM) for the LNP sample under test, and can more accurately reflect the size of the nanoparticles.

[0167] In the embodiments of the present invention, a PDI of 0.2 or less was considered good or ideal, a PDI greater than 0.2 and less than 0.3 was considered acceptable, and a PDI of 0.3 or more was considered undesirable.

[0168] In the embodiments of the present invention, a particle size of less than 100 nm (volume-based particle size) is considered ideal.

[0169] (Example 1: Comparison of mixed units with different structural shapes) In this embodiment, microfluidic chips were prepared using mixing units numbered Y1, T1, S1, S2, and D-B1 in Table 1. Y1 is shown in Figure 8, T1 in Figure 7, and S1 in Figure 6. The Lc of S2 was half that of S1, and the other parameters were the same. D-B1 is shown in Figure 5B.

[0170] In these five types of mixing units, the straight channel at the inlet and the straight channel at the merging point of T1 are perpendicular. The other four types all have a constant angle. Furthermore, while Y1, T1, S1, and S2 use conventional rectangular cross-sections as in the prior art, D-B1 is the two-layer waveform mixing unit of the present invention.

[0171] The test method was as described in "LNP Preparation" above. Different flow rates were tested. Specifically, total flow rates of 1 mL / min and 6 mL / min were tested with five types of mixing units. An additional 4 mL / min was tested with the only two-layer waveform mixing unit, D-B1. Figure 10 shows the statistical results of PDI and volume-based particle size for self-assembling nanoparticles fabricated using tips containing these mixing units.

[0172] Figure 10 shows that, at a total flow rate of 1 mL / min, there was no significant difference in particle size and PDI between the single-layer waveform mixing unit S1 and S2. In the case of the single-layer waveform mixing unit, Lc varied within the range of 0.5 to 1 mm and did not significantly affect PDI. At a flow rate of 1 mL / min, both S1 and S2 demonstrated excellent mixing effects.

[0173] At a total flow rate of 6 mL / min, compared to tip S2, the particle size of nanoparticles produced with tip S1 (which has a shorter Lc) was slightly larger, and the PDI was also slightly larger. This suggests that when using a single-layer waveform mixing unit, when the Lc decreases from 1 mm in S1 to 0.5 mm in S2, the increased flow rate (from 1 mL / min to 6 mL / min) may have resulted in excessive mixing and potential aggregation.

[0174] Therefore, when considering shortening Lc in the structure of the present invention, especially when it is less than 1 mm, it is preferable to maintain the total flow rate as is or to lower the total flow rate in order to ensure particle quality, for example, by maintaining the total flow rate at about 1 mL / min.

[0175] On the other hand, when designing the same waveform, the effects of mixing units with different numbers of layers were compared. Compared to single-layer S1, double-layer D-B1 obtained a lower particle size at 1 mL / min. When the rate was increased to 6 mL / min, double-layer D-B1 had the same particle size as single-layer S1.

[0176] Considering particle dispersion and aggregation, the two-layer D-B1 also showed an increase in PDI as the total flow rate increased from 1 mL / min to 6 mL / min, but at a total flow rate of 4 mL / min, the PDI of D-B1 was at a good level of 0.2.

[0177] In short, as can be seen from Figure 10, each tip has a different optimal flow rate. Y1 is suitable for 6 mL / min, T1 is suitable for 6 mL / min, S1 and S2 have large particle size and low PDI at 1 mL / min, and small particle size and high PDI at 6 mL / min, and neither flow rate was ideal. For these tips, D-B1 had good particle size and PDI at 1 mL / min and 4 mL / min, especially at 1 mL / min.

[0178] (Example 2: Proportional expansion of a microstructure mixing unit) In this embodiment, the mixing units numbered Y2, S3, and D-B4 in Table 1 were used as chips. These three types of chips were manufactured by proportionally scaling up the sizes of Y1, S1, and D-B2 by 200%. Proportional scaling means that the length, width, and height were all enlarged by a specified ratio. The angles between the inlet and junction sections of these three types of chips are the same, but the difference is that the mixing section of Y2 is straight, S3 is a single-layer waveform mixing unit, and D-B4 is a double-layer waveform mixing unit.

[0179] The test method is as described above. Fabrication of LNPs As described in [reference], different flow rates were tested. Specifically, six total flow rates of 1, 6, 12, 20, 24, and 30 mL / min were tested for three types of mixing units. The 4 mL / min rate, which showed good results in Example 1, was also tested for two types of waveform mixing units. Figure 11 shows the statistical results of PDI and volume-based particle size for self-assembling nanoparticles produced using tips containing these mixing units.

[0180] Figure 11 shows that after proportional expansion to 200% at each test flow velocity, the particles obtained from mixing units Y2, S3, and D-B4 uniformly showed a decrease in particle size with increasing flow velocity.

[0181] Simultaneously, it was found that increasing the flow rate to 20 mL / min increased the PDI of each group compared to the previous flow rate (although the PDI was still less than 0.3). This may be related to the vigorous mixing that occurs after the flow rate exceeds 20 mL / min, causing aggregation. Particularly at high flow rates, the PDI of D-B4 was greater than that of Y2 and S3, suggesting that the two-phase mixing process occurred more vigorously in the D-B4 structure than in the other two structures.

[0182] Specifically, at low flow rates of 1, 4, and 6 mL / min, and medium flow rates of 6 and 12 mL / min, the particle size obtained with the 200% enlarged waveform double-layer structure D-B4 was smaller than the particle size of the 200% enlarged waveform single-layer structure S3. At the low flow rates mentioned above, the advantages of mixing with the waveform double-layer structure are clear. Furthermore, as the total flow rate increased to 20 mL / min, it was suggested that the difference in particle size between the 200% enlarged waveform single-layer structure S3 and the 200% enlarged waveform double-layer structure D-B4 decreased.

[0183] The results above suggest that, after increasing the size, the mixing advantage due to the difference in the number of layers decreased as the total flow velocity increased.

[0184] Of these three structures, D-B4 mixes well at 6 mL / min and can produce small, uniform particles, indicating that it requires a lower flow rate than the other two tips and is suitable for lower working flow rates. S3 is suitable for working flow rates of 6 mL / min, 12 mL / min, 20 mL / min, and 24 mL / min, while Y2 is suitable for working flow rates of 12 mL / min and 20 mL / min.

[0185] In short, after proportional expansion, both the single-layer and double-layer microstructure mixing units of the present invention exhibit good mixing effects. The wave-shaped microstructure mixing unit of the present invention allows the length and width of the flow channel to be varied within a certain range, enabling the production of nanoparticles with smaller and more uniform particle sizes.

[0186] (Example 3: Comparison of different Lc values) In this embodiment, mixed units numbered D-B1 and D-B2, and D-B4 and D-B5 in Table 1 were used as chips. D-B4 and D-B5 are proportionally scaled versions of the structures of D-B2 and D-B1, respectively. The Lc of D-B1 was twice that of D-B2, and the Lc of D-B5 was twice that of D-B4.

[0187] The test method is as described above. Fabrication of LNPsAs described in [reference], different flow rates were tested in the comparison between D-B1 and D-B2. Specifically, total flow rates of 1 mL / min, 4 mL / min, and 6 mL / min were tested. Figure 13 shows the PD of self-assembling nanoparticles and the statistical results of the mixing unit produced by the chips containing these mixing units.

[0188] As can be seen from Figure 13, at flow rates of 4 mL / min and 6 mL / min, the PDI of D-B1 was significantly smaller than that of D-B2, while the particle size was similar. This indicates that, for a tip with a channel of size 0.5 W × 0.5 H, a longer Lc allowed for better control of the PDI. This may be because a shorter Lc results in more vigorous mixing, leading to collisions and aggregation of some nanoparticles.

[0189] The test method is as described above. Fabrication of LNPs As described in [reference], different flow rates were tested in the comparison between D-B4 and D-B5. Specifically, seven total flow rates of 1, 4, 6, 12, 20, 24, and 30 mL / min were tested with the two types of mixing units. Figure 12 shows the statistical results of the PDI and volume-based particle size of self-assembling nanoparticles produced using tips containing these mixing units.

[0190] Figure 12 shows that, compared to group D-B4, the volume-based particle size of group D-B5 increased at each velocity. Of these, at flow rates of 12, 24, and 30 mL / min, the volume-based particle size of group D-B5 was close to that of group D-B4. This suggests that after proportionally scaling the system, the increase in Lc reduced the intensity of local mixing, potentially leading to an increase in particle size.

[0191] At the same time, this embodiment suggests that in a proportionally scaled system, when the total flow rate is 1 to 30 mL / min, the observation results for group 2 show that the particle size decreases with increasing flow rate and the PDI increases with increasing flow rate. When the flow rate exceeds 12 mL / min, the PDI for group 2 exceeds 0.2, and the increasing trend of PDI with increasing flow rate becomes more pronounced. Notably, when the flow rate is equal to 12 mL / min, the particle sizes of D-B4 and D-B5 were the same, but the PDI of D-B5 was clearly smaller than that of D-B4, resulting in LNPs with ideal particle size and PDI.

[0192] This example suggests that by increasing the length of Lc, the PDI of the fabricated LNP can be controlled more effectively, allowing for the acquisition of LNPs with ideal particle size and PDI over a wider range of fabrication flow rates.

[0193] (Example 4: Comparison of different numbers of semicircles) In this embodiment, microfluidic chips were fabricated using mixing units numbered D-B2-3, D-B2-5, D-B2-7, and D-B2 in Table 1. All four of these mixing units are two-layer waveform mixing units, the only difference being the number of semicircular rings, or curves, included in the waveform portion.

[0194] The test method is as described above. Fabrication of LNPs As described in [reference], different flow rates were tested. Specifically, four types of mixing units were tested with total flow rates of 1 mL / min, 4 mL / min, and 6 mL / min. Figure 20 shows the statistical results of the PDI and volume-based particle size of self-assembling nanoparticles produced using tips containing these mixing units.

[0195] As can be seen from Figure 20, both D-B2 and D-B2-7 are suitable for 1 mL / min, but D-B2-3 and D-B2-5 were difficult to produce small-sized, uniform LNPs at these flow rates. The suitable flow rates for D-B2 were the same as in the other examples. The results in Figure 20 also show that the number of semicircular rings in the waveform mixing unit should not be too few, preferably at least 6, and more preferably at least 7.

[0196] (Example 6: Comparison of single-layer and multi-layer waveform microstructure mixed units) In this embodiment, the inventors fabricated microfluidic chips using mixing units numbered S1, D-A1, D-B1, D-C1, and T-B1 in Table 1, with each chip containing only one mixing unit. The chips used the same design as in Figure 1, except that the mixing section was different.

[0197] Lipid nanoparticles were fabricated using these chips, and parameters such as PDI and volume-based particle size of the obtained lipid nanoparticles were measured.

[0198] These five types of mixing units are all waveform microstructure mixing units. Except for the height of each layer in T-B1, the width, Do, Di, Lc, and other major parameters of each type of mixing unit are basically the same, and the main difference lies in the number of layers. The mixing section of S1 is a single-layer waveform channel, the mixing sections of D-A1, D-B1, and D-C1 are two-layer waveform channels, and the mixing section of T-B1 is a three-layer waveform channel. Schematic diagrams and cross-sectional diagrams of D-A1, D-B1, D-C1, and T-B1 are shown in Figures 5A to 5D, respectively, with the left side being a top perspective view and the right side being a schematic diagram of the corresponding cross-sectional shape.

[0199] As shown in Figure 5, D-A1, D-B1, and D-C1 all have two-layered channels. The inner edge of the narrow channel layer of D-A1 overlaps with the inner edge of the wide channel layer of the semi-ring portion, and the inner edge of the narrow channel layer of D-B1 overlaps with the outer edge of the semi-ring portion. If the length and shape of the wide channel layer are the same and the width of the narrow channel layer is also the same, the narrow channel layer of D-B1 is longer than the narrow channel layer of D-A1. Unlike D-A1 and D-B1, D-C1 has two-layered channels of the same width, and the widths of the two-layered channels on the semi-ring portion are offset, forming a cross-section similar to the Z shape of Tetris.

[0200] the above" Fabrication of LNPs As described in [the relevant section], we tested different flow rates of 1, 4, and 6 mL / min. Figure 9 shows the PDI and volume-based particle size statistics for self-assembling nanoparticles produced using tips containing these mixing units.

[0201] As can be seen from Figure 9, at a total flow rate of 1 mL / min, the average particle size obtained with a single-layer tip (S1) is larger than the average particle size of the double-layer (D-A1, D-B1, D-C1) and triple-layer (T-B1) tips, while the average particle size obtained with the double-layer (D-A1, D-B1, D-C1) tips is smaller than the average particle size of the triple-layer (T-B1) tip. The particle size between the double-layer tips is D-C1 > D-B1 > D-A1, and the PDI is D-A1 > D-C1 > D-B1. At a low flow rate of 1 mL / min, the double-layer structure performed better than the triple-layer and single-layer structures. Among the double-layer structures, the D-B1 structure, where the narrower part of the flow path is on the outside, performed better than the other double-layer structures, exhibiting superior particle size and dispersibility. This is the same as the result in Example 1. Furthermore, the D-A1 is not very desirable because it was difficult to control the PDI to 0.2 or less at each flow velocity.

[0202] Based on the results above, it can be seen that of these five structures, D-B1 was suitable for an operating flow rate of 4 mL / min, and T-B1 was suitable for an operating flow rate of 1 mL / min. The LNPs obtained under these conditions have a small particle size and uniform dispersibility.

[0203] (Example 7: Number of layers in the microstructure mixing unit) In this embodiment, chips numbered S2 and D-B2, S3 and D-B4, and S4 and D-B7 were used. S2 and D-B2 have the same shape, and are single-layered and double-layered, respectively. S3 and D-B4 have the same shape, and are single-layered and double-layered, respectively. S4 and D-B7 have the same shape, and are single-layered and double-layered, respectively.

[0204] The test method was as described in "Preparation of LNPs," and different total flow velocities were tested. The statistical results for the tested flow velocities, PDI, and volume-based particle size are shown in Figure 14, where A represents the results for S2 and D-B2, B represents the results for S3 and D-B4, and C represents the results for S4 and D-B7.

[0205] Figure 14A shows that particle size decreases with increasing flow rate, while PDI increases with increasing flow rate. Comparing the two types of mixing units, the LNPs produced with the two-layer D-B2 unit have smaller particle sizes. The single-layer S2 unit produced large particle sizes and low PDI at 1 mL / min, and small particle sizes and high PDI at 6 mL / min, indicating that neither flow rate was suitable. The two-layer D-B2 unit was able to produce LNPs with small size and good uniformity at 1 mL / min, but the PDI was too high at flow rates of 4 mL / min and 6 mL / min.

[0206] Figure 14B shows that particle size decreased with increasing flow rate, with single-layer PDI decreasing first and then increasing, while double-layer PDI increased after the flow rate reached a certain value. Furthermore, at the same flow rate, LNP particle size produced by the double-layer D-B4 tip was generally smaller, indicating a better mixing effect with the double-layer tip. However, when mixing with D-B4 after the flow rate exceeded 12 mL / min, aggregation may have occurred, potentially increasing the PDI. However, at a flow rate of 30 mL / min, the PDI of S3 increased only slightly. S3 showed good mixing effects at flow rates of 6 mL / min, 12 mL / min, 20 mL / min, and 24 mL / min, while D-B4 showed the best mixing effect at 6 mL / min.

[0207] Figure 14C shows that particle size decreases with increasing flow rate, PDI decreases first, and then increases after the flow rate reaches 12 mL / min. Comparing single-layer and double-layer structures, the particle size of double-layer D-B7 is smaller than that of single-layer, and the PDI of D-B7 is initially smaller than that of S4, and after the flow rate reaches 12 mL / min, the PDI is larger than that of S4. This is thought to be because the aqueous and ethanol phases are mixed more uniformly in the double-layer tip, resulting in a lower optimal flow rate. When S4 is at 12 mL / min and D-B7 is at 4 mL / min, the objective of producing LNPs with small particle size and good uniformity can be achieved.

[0208] In short, the two-layer structure, due to the effect of vigorous mixing, may yield nanoparticles with smaller particle sizes at lower flow rates. At the same time, the two-layer structure can yield nanoparticles with relatively smaller particle sizes and PDIs within a given flow rate range compared to the single-layer structure.

[0209] (Example 8: Enlargement of the microstructure mixing unit) In this embodiment, two groups of proportionally expanded and contracted mixing units were compared. Specifically, the mixing units numbered D-B3, D-B2, D-B4, and D-B6 in Table 1 were used as one group, and D-B1, D-B5, and D-B7 were used as the other group. Chips were fabricated and compared within each group. In the second group, as mentioned above, the difference between D-B1 and D-B2 was that the former had a longer Lc. Within each group, the difference between each mixing unit is a multiple of the proportional expansion or contraction of the flow path size.

[0210] The test method was as described in "Preparation of LNPs," and total flow rates of 1, 4, 6, 12, 20, 24, and 30 mL / min were used. Statistical results for PDI and volume-based particle size are shown in Figure 15. Figure 15A shows the results for D-B3, D-B2, D-B4, and D-B6, and Figure 15B shows the results for D-B1, D-B5, and D-B7.

[0211] Figure 15A shows that the internal pressure of the small channel was too high, making it unsuitable for high flow rates. In D-B3, the channel narrowed, and at low flow rates the particle size was small. However, the PDI was very large, and at a flow rate of 6 mL / min, the result was the opposite, indicating that it was difficult to find a suitable flow rate for small channels in order to produce small-sized, uniform LNPs. In D-B2, a normal channel, small-sized, uniform LNPs were obtained at a flow rate of 1 mL / min. It is presumed that the small channel D-B3 was suitable for even lower flow rates, such as less than 1 mL / min. Notably, D-B6 could not obtain LNPs with a particle size of less than 100 nm even at a flow rate of 30 mL / min, indicating that the double structure lost the desired mixing effect when the channel was expanded to a certain size.

[0212] As can be seen from Figure 15B, the D-B1, D-B5, and D-B7 channels continued to increase in size, and as the flow velocity increased, the particle size of the produced LNPs continued to decrease, which was basically the same trend as in Figure 15A. However, at the same flow velocity, the particle size was D-B1 <D-B5<D-B7となった。

[0213] At 1 mL / min, the PDI of LNPs prepared with the three tips was very low. As the flow rate increased, the PDI of D-B1 continued to increase, indicating that D-B1 was suitable for the low flow rate of 1 mL / min in the test, but not for the high flow rates. The PDI of D-B5 and D-B7 increased only when the flow rate exceeded 12 mL / min. As can be seen from the particle size results, the uniformity of LNPs prepared with D-B5 and D-B7 at 12 mL / min was good, and they were suited to higher flow rates compared to D-B1.

[0214] From the results of Examples 1 to 8, the various mixing units in Table 1 can obtain an optimal flow velocity range with acceptable DPI and particle size, which are specifically shown in Table 3 below.

[0215] Table 3. Optimal flow rates for different mixing units JPEG0007897621000004.jpg198170JPEG0007897621000005.jpg32170

[0216] (Example 9: Observe the mixing effect using a colored solution) The mixing process of the ethanol phase and the aqueous phase was simulated using an ethanol solution containing phenol red and a phosphate buffer solution containing trypan blue. The difference in color of the solutions before and after mixing reflects the mixing effect. Before mixing, the ethanol phase is yellow and the aqueous phase is blue. After mixing, the liquid changes to red, and the darker the red color of the liquid after mixing, the more complete the mixing is (as shown in Figure 23, the original photograph is red, and the darker the color, the darker the red).

[0217] A comparison was made between Y1, a single-layer waveform channel S1, and the multi-layer waveform channel D-B1 of the present invention. The results are shown in Figure 23A. In Figure 23A, it can be seen that after passing through the mixing section over the same distance, the mixing effect of the three channels is best in D-B1 and worst in Y1, according to the fluid color. From the figure, it can be seen that the two fluids in D-B1 are mixed first, resulting in a darker color. It was also observed that the fluid flow was more stable and less turbulent (undisturbed) in the two-layer waveform channel, while more turbulence occurred in the single-layer waveform channel.

[0218] This test demonstrates the superiority and stability of the two-layer SML channel of the present invention in terms of mixing effect.

[0219] (Example 10: Mixing apparatus equipped with multiple parallelized multilayer waveform mixing units) A 4-stage parallelized multilayer waveform microstructure mixing unit, as shown in Figure 3, and a 16-stage parallelized unit, as shown in Figure 4, were fabricated. The mixing effect was reflected using the colored solution from Example 9.

[0220] As shown in Figure 23B, the mixed fluid in the outlet channel of the 16-stage SML mixer changes to a darker color (the original photo was red), indicating that the two introduced fluids have been completely mixed.

[0221] (Example 11: Comparison with a non-waveform channel chip) In this embodiment, a microfluidic chip was fabricated using the size and design of the mixing units numbered D-B1 in Table 1, the only difference being the use of nine semi-rings, hence it is called D-B1(9C). For comparison, a conventional non-corrugated channel chip was used, specifically the NxGen microfluidic chip (Precision NanoSystems Co., Canada) mounted on an Ignite model microfluidic device. This chip features a channel consisting of four rings as disclosed in the aforementioned Patent Document 2. The structures of the two types of mixing units are shown in Figure 21. As shown in Figure 21B, the mixing unit of the NxGen microfluidic chip has multiple continuous annular structures in the mixing section and includes more channel branches.

[0222] To test the effectiveness of the microfluidic chip of the present invention compared to prior art, GFP mRNA-laden LNP samples were mixed and prepared using the various flow channels listed in the table below. Specifically, LNP samples were prepared by mixing an aqueous solution of GFP mRNA (citrate buffer, pH=4) and an ethanol phase (lipid-operated solution) in a volume ratio of 3:1 at different flow rates.

[0223] For the ethanol phase, stock ethanol solutions of SM-102, DSPC (distearoylphosphatidylcholine), Cholesterol, and DMG-PEG (distearoylphosphatidylethanolamine-polyethylene glycol) were prepared according to the concentrations shown in Table 4, and uniformly mixed in a constant volume ratio to obtain a lipid material working solution as the ethanol phase. The concentrations and molar ratios of the various components in the lipid material working solution are shown in Table 4. Similarly, a 0.1 mg / mL GFP mRNA aqueous solution (citrate buffer, pH=4) was also prepared according to the concentrations shown in Table 4. The prepared ethanol phase and aqueous phase were injected into a mixer D-B1 (9C) or NxGen microfluidic tip in a volume ratio of 1:3 (100 μL:300 μL).

[0224] Table 4. mGFP LNP recipes JPEG0007897621000006.jpg60170

[0225] We tested different flow rates. Specifically, we tested total flow rates of 1 mL / min, 2 mL / min, 4 mL / min, and 12 mL / min using two types of tips. Figure 22 shows the particle size and PDI statistics for GFP mRNA-loaded LNP samples.

[0226] As can be seen from Figure 22, both chips showed a tendency for particle size to decrease with increasing flow rate. At flow rates of 4 mL / min and 12 mL / min, the LNP particle size produced by both chips was approximately 80 nm. At flow rates of 1 mL / min and 2 mL / min, the LNP particle size obtained by the D-B1(9C) chip was smaller than that of the NxGen microfluidic chip. At the same time, the PDI of the LNPs produced by both chips was controlled to 0.2 or less at all tested flow rates. This result indicates that, compared to conventional techniques, the microstructure mixing unit of the present invention was able to produce nanoparticles with smaller particle sizes at lower flow rates.

Claims

1. A multilayer corrugated (SML) microstructure mixing unit comprising a fluid-communicated inlet, a confluence, a multilayer mixing section, and an outlet, Each layer of the multilayer mixing section is a waveform channel, and each waveform channel of the layer contains n semicircular or semielliptical rings, any two adjacent semicircular or semielliptical rings having opposite curvature directions and connected by a straight channel. Each of the waveform channels in the aforementioned layers has a certain width, and at least two of the waveform channels have different widths. The projected shapes of two adjacent layers in the multilayer mixing section always overlap at least partially on the flow path of the entire mixing section, but do not completely overlap. The flow paths of each layer on the semicircular or semielliptical ring portion of the mixing section are always parallel. The outer edges of the flow channels in each layer on the semicircular or semielliptical ring portion of the mixing section always overlap. The inlet section includes at least two inlets, which are in fluid communication with the confluence section so that different fluids flowing in from the inlets merge at the confluence section. SML microstructure mixing unit.

2. The SML microstructure mixing unit according to claim 1, wherein the mixing section includes two, three, four, or five waveform channels.

3. The SML microstructure mixing unit according to claim 1, wherein each of the waveform channels in the respective layers has a certain width, and the widths of the waveform channels in each layer are the same as those of the other.

4. (a) The mixing section includes two layers of waveform channels, the cross-sections of the two layers of waveform channels on the semi-circular or semi-elliptical ring-shaped section are L-shaped, or (b) The mixing section includes three layers of waveform channels, the cross-sections of the three layers of waveform channels on the semi-circular or semi-elliptical ring-shaped section are T-shaped, rotated by 90°, according to claim 1.

5. The SML microstructure mixing unit according to claim 1, wherein the outer diameter of each semicircular ring or the outer axis length Do of each elliptical ring in the waveform extension direction in each layer of the waveform channel is 3 to 5 times the width of the channel, and / or the inner diameter of each semicircular ring or the inner axis length Di of each elliptical ring in the waveform extension direction is 1 to 3 times the width of the channel.

6. The SML microstructure mixing unit according to claim 1, wherein the length Lc of the straight channel is 0.5W to 4W.

7. The SML microstructure mixing unit according to claim 1, wherein n is an integer of 5 or more.

8. The SML microstructure mixing unit according to claim 1, comprising two or three layers of corrugated channels, wherein each layer of corrugated channels comprises five to ten semicircular channels, and the length Lc of the straight channels between each semicircular channel is greater than or equal to the inner diameter Di of the projected shape of the semicircular channel.

9. The SML microstructure mixing unit according to claim 1, wherein at least two layers in the waveform channel have different widths, and the width of the narrower layer is 25% to 75% of the width of the wider layer, preferably about 50%.

10. A microstructure mixing apparatus comprising m parallel-arranged SML microstructure mixing units according to any one of claims 1 to 9, wherein the m microstructure mixing units share one inlet and one outlet, The aforementioned inlet allows the fluid flowing in from the same inlet to flow into m flow channels, and each flow channel is in fluid communication with the aforementioned confluence or mixing section of the SML microstructure mixing unit. The outlet section has a branching structure, and ultimately the fluids flowing out from the m mixing sections are merged into a single flow path, and the fluids are connected to the outlet via one or more branching points. A microstructure mixing apparatus in which m is an integer greater than 1.

11. The microstructure mixing apparatus according to claim 10, wherein m is an even number, preferably a power of 2.

12. A method for mixing different fluids, comprising the step of mixing a first fluid and a second fluid using a microstructure mixing unit according to any one of claims 1 to 9, wherein the first fluid is allowed to flow into a first inlet, the second fluid into a second inlet, the fluids are allowed to merge at the confluence, and the fluids are allowed to mix at the mixing section.

13. A method for mixing different fluids, comprising the step of mixing a first fluid and a second fluid using the microstructure mixing apparatus described in claim 10, wherein the first fluid is allowed to flow into a first inlet, the second fluid into a second inlet, the fluids are allowed to merge at the confluence, and the fluids are allowed to mix at the mixing section.

14. A method for producing nanoparticles by self-assembly, comprising: using a microstructure mixing unit according to any one of claims 1 to 9, mixing a first fluid and a second fluid; introducing the first fluid into a first inlet; introducing the second fluid into a second inlet; wherein the first fluid is a fluid containing a nanoparticle carrier material; and the second fluid is a fluid containing a nanoparticle carrier.

15. A method for producing nanoparticles by self-assembly, comprising: using the microstructure mixing apparatus described in claim 13, mixing a first fluid and a second fluid; introducing the first fluid into a first inlet; introducing the second fluid into a second inlet; wherein the first fluid is a fluid containing a nanoparticle carrier material; and the second fluid is a fluid containing a nanoparticle carrier.

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