Corrugated microstructure mixing unit and uses thereof

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 TO RELATED APPLICATIONS) This application claims priority to Chinese Patent Application No. 202210119774.0, entitled "Wavy Microstructure Mixing Unit and Use Thereof," filed on February 8, 2022.

[0002] The present invention relates to the field of microfluidics, in particular to a multi-layer corrugated microstructure mixing unit and a mixing device having the mixing unit. The present invention also relates to the use of the mixing unit and the mixing device in the production of nanoparticles, such as nucleic acid molecule-loaded lipid nanoparticles. The mixing unit and the mixing device of the present invention are particularly suitable for the production of self-assembling lipid nanoparticles as drugs. [Background technology]

[0003] Microfluidics technology has been used to fabricate nano-sized delivery carriers, especially drug delivery carriers such as LNPs, polymeric 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 used in the production of nucleic acid drugs and gene therapy. As an innovative delivery technology, LNP technology solves the problem of nucleic acid delivery and makes it possible to realize mRNA vaccines. As a typical organic nanoparticle, the LNP delivery system has the advantages of strong permeability, high nucleic acid loading capacity, low toxicity, good long-term stability, and good biocompatibility compared to viral delivery systems.

[0005] In the manufacturing process of nanoparticles such as LNPs, an ethanol phase is mixed with an aqueous phase, and the rapid mixing process causes supersaturation of lipid or polymer molecules, promoting the self-assembly of nanoparticles to form nano-sized particles.

[0006] During this process, rapid mixing of the two solutions 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, ultrasound, etc.

[0007] Because the nanoparticles are formed by self-assembly, these manufacturing methods are considered bottom-up methods.Compared with traditional top-down methods, the main advantages of the rapid mixing process are enhanced control over physicochemical properties, improved encapsulation efficiency, and improved feasibility of process scale-up.

[0008] Although the 3D structures of different microstructured mixers are different, 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 soluble phases and rapidly increase the polarity of the solvent environment of materials such as lipids or polymers, thus forming lipid or polymer nanoparticles.

[0009] For example, in the preparation of lipid nanoparticles (LNPs) used for mRNA delivery, the mixing process typically involves rapid mixing of an ethanol phase containing hydrophobic lipids with a buffered aqueous phase containing mRNA in a microfluidic chip or a T-shaped mixer. This process realizes the formation of LNPs through hydrophobic interactions, while simultaneously completing the encapsulation of mRNA through electrostatic interactions.

[0010] In a typical process for making mRNA-loaded lipid nanoparticles, four lipid materials (ionizable lipids, distearoylphosphatidylcholine (DSPC), cholesterol, and PEGylated lipids) are dissolved in an ethanol solution, while 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 the mRNA. The lipids become insoluble when exposed to the aqueous buffer, while the ionizable lipids become ionized and positively charged. The positive charges of the ionizable lipids then drive the negative charges of the ionizable lipids and the mRNA phosphate backbone, resulting in electrostatic interactions. This allows the lipid materials to complete self-assembly in a supersaturated state, forming mRNA-loaded lipid nanoparticles.

[0011] The key to the mixing technique in LNP fabrication is to form turbulent flow, i.e., chaotic flow, when mixing the ethanol phase and the aqueous phase. When the flow rate is low or the Reynolds number is low, the solution in the fluid channel generally forms laminar flow, and the mixing at this time belongs to diffusive mixing, which is 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 the aqueous phase contact and mix in laminar flow, a phospholipid bilayer is formed at the interface, and eventually a structure similar to liposomes or multilamellar vesicles is formed, which affects the particle yield and particle uniformity. Under turbulent flow, lipid materials can quickly become supersaturated and self-assemble to form a uniform solid structure. When the Reynolds number (Re) or flow rate is high, turbulence improves the mixing efficiency and shortens the mixing time, and the shorter mixing time reduces the mass transfer effect. The mass transfer effect increases lipid aggregation and the heterogeneity of particle morphology. In LNP preparation, the mixing channel has a low mixing velocity and Reynolds number, and turbulence is difficult to form under such conditions.The self-assembly process of LNPs is based on the fact that when an ethanol phase is diluted into an aqueous phase, the polarity of the solvent increases, resulting in the precipitation of lipids.

[0012] Patent Document 1 discloses a flow 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, and it may be difficult to use at the industrial level.

[0013] Hirota et al. first reported the use of T-tube mixing in the fabrication of lipid-based drug delivery systems in 1999. T-tube mixing offers an alternative to macroscopic mixing methods for generating DNA-lipid complexes. As the name suggests, a T-tube is a three-sided T-shaped tube with inflow from both ends of the horizontal axis of the "T" and outflow from the vertical end of the "T". Compared to macroscopic mixing methods (e.g., vortexing and dropwise addition), the T-junction geometry mixer provides a relatively controlled mixing environment, allowing for reproducible generation of nanoparticles. The collision of the two input fluids at the T-junction causes rapid mixing, forming a turbulent or chaotic flow in the outflowing fluid.

[0014] T-tube mixing is one of the methods recommended for large-scale LNP production by mRNA vaccine companies. However, in the early stages of drug development, the use of the T-tube mixing method for small-scale laboratory-scale production is very limited. The main reason for its limited use is that the T-shaped structure is relatively simple, so a high flow rate is required to ensure effective mixing. In general, a high flow rate corresponds to a larger amount of material used, and the material flows through the device faster. However, for nucleic acid drugs, the high cost of raw materials and the small amount required for the product make it unsuitable for a situation where a large dose meets a high flow rate. For example, if the flow rate is 60 mL / min, and the shortest production time is only 20 seconds, at least about 20 mL of feed liquid is required. Taking mRNA as an example, such an amount would include mg-level RNA molecules, but studies such as cytology may only require μg-level RNA, which is costly and wasteful.

[0015] In 2002, Stroock et al. found that adding a herringbone structure to the flow channel improves the mixing effect at low Reynolds numbers, ensuring fast mixing in the millisecond timescale at lower flow rates. Pieter Cullis team used a staggered herringbone mixer (SHM) to produce LNPs through chaotic convective mixing. This technology was subsequently commercialized by Precision Nanosystems. This method was developed to enhance control over the mixing process and reduce mixing time. Although SHMs are widely used in laboratories, such mixers are difficult to fabricate and have certain performance limitations. SHM designs are difficult to scale up and process, limiting their clinical translation and large-scale application.

[0016] The patent literature discloses a diverging mixer utilizing Dean vortexing that includes multiple rings arranged in series.

[0017] It remains desirable to design improved microfluidic mixing devices to improve mixing efficiency, reduce production costs, and be widely applicable to drug production in the pharmaceutical field, especially for the production of self-assembled nanoparticles. [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 Summary of the Invention [Problem to be solved by the invention]

[0019] Simple curved or spiral designs require very high flow velocities for rapid mixing of the fluids, which can induce the formation of secondary Dean vortices, turning the fluid into a chaotic convective state. This situation usually corresponds to very high Reynolds numbers, which is not suitable for many practical scenarios.

[0020] In the multi-layer wavy microstructure mixing unit of the present invention, each layer of the flow channel is designed to have a geometric shape that alternately bends in different directions, and multiple layers of such flow channels are combined to generate more flow and help induce chaotic convection. Therefore, the wavy microstructure mixing unit of the present invention can induce stronger chaotic convection in the production of self-assembled lipid or polymer nanoparticles, allowing two fluids to surround and effectively wrap each other, so that the interface between the fluids is exponentially increased and the fluids are quickly mixed.

[0021] Specifically, the corrugated microstructure of the present invention is a corrugated multi-layer microstructure, each layer having the same or different width, and the projections between two adjacent layers on the entire flow path do not completely overlap, but always overlap. It is also conceivable to provide one or more flow-restricting ribs along a flow path having an original rectangular cross section, so that at least a part of the cross section of the flow path forms one or more recesses with respect to the original rectangular cross section. In particular, the cross section of the curved part of the corrugated structure is no longer rectangular. This design can further promote the generation of vortexes and improve the mixing effect. Based on these designs, the invention of has been completed. [Means for solving the problem]

[0022] In a first aspect, the present invention provides a sinuous multilayer (SML) microstructure mixing unit comprising two or more layers of corrugated channels, where the projections between two adjacent layers on the entire channel always overlap, but do not completely overlap. Preferably, the projections between two adjacent layers always overlap only partially, with semicircular or semi-elliptical rings of the corrugations.

[0023] In a second aspect, the present invention further provides a microfluidics device comprising a corrugated microstructure mixing unit or a corrugated multi-layer microstructure mixing unit according to the first aspect, in which the corrugated microstructure mixing units or the corrugated multi-layer microstructure mixing units are combined in parallel and / or in series.

[0024] In a third aspect, the present invention relates to a method of making self-assembling nanoparticles using the microstructured mixing unit of the first aspect or the apparatus of the second aspect. In a specific embodiment, the self-assembling nanoparticles are nucleic acid-loaded lipid nanoparticles. Effect of the Invention

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

[0026] The microstructured mixing units of the present invention can induce turbulence in the liquid during mixing at lower mixing speeds (e.g., total flow rates) and Reynolds numbers (Re), increasing the intensity of mixing. The multi-layer corrugated channels with different widths or staggered shapes repeatedly change the relative direction of fluid flow between different layers, causing the main rotation center of the Dean vortices to move between the mixing units.

[0027] The combination of the corrugated microstructured mixing unit in the device of the present invention can provide precise liquid partial pressure. By using a curved or spiral shape design, the microstructured mixing unit of the present invention provides a way to enhance convective transport in microchannels through simple geometric design.

[0028] The corrugated microstructure mixing unit of the present invention can maintain excellent mixing effect even when scaled up proportionally (e.g., when the cross-sectional area and length of the flow path are scaled up proportionally) or when the scale is increased (e.g., by connecting multiple mixing units in parallel), making the applicable scenarios of the mixing unit more diverse.

[0029] The corrugated microstructure mixing unit of the present invention and the microfluidic chip comprising said unit are particularly suitable for the production of nanoparticles, in particular for the self-assembly of nanoparticles loaded with drug molecules, for example the self-assembly of lipid nanoparticles loaded with nucleic acids.

[0030] The corrugated multi-layer microstructure mixing unit of the present invention can achieve millisecond-level mixing and form nanoparticles, with an effect similar to that of the herringbone SHM mixer. This effect is achieved without the need for complex 3D surface structures such as the ridge structure of the staggered herringbone mixer. In view of the fact that complex 3D surface structures can induce high local shear forces, the flow channels formed by the unit of the present invention are more suitable for production processes involving biopolymers (especially nucleic acid molecules). At the same time, due to the absence of complex 3D surface structures, such mixing devices (such as chips) are easy to clean and reusable. [Brief description of the drawings]

[0031] [Figure 1] Schematic diagram of the corrugated microstructure mixing unit D-B1. The two small drawings on the left side are respectively a top view of a repeating unit of corrugated channels in the mixing section (upper small drawing; includes two semicircular annular channels and two short straight channels), and a cross-sectional view of this channel (lower small drawing). [Diagram 2] FIG. 3D schematic diagram of the corrugated microstructure mixing unit D-B1. [Figure 3A-3E] Figure 1 shows the mixing device with four parallel corrugated multilayer microstructure mixing units: (A) Schematic diagram of the inlet layer as the first layer, (B) Schematic diagram of the dividing layer as the second layer, (C) Schematic diagram of the mixing layer as the third layer, (D) Perspective view of all layers merged, (E) Photograph of the actual device. [Figure 4A-4D] Figure 1 shows a mixing device with 16 parallel corrugated multilayer microstructure mixing units: (A) Schematic diagram of the inlet layer as the first layer, (B) Schematic diagram of the dividing layer as the second layer, (C) Schematic diagram of the mixing layer as the third layer, (D) Perspective view of all layers merged. [Fig. 4E-4F] The figure shows a mixing device in which 16 corrugated multilayer microstructure mixing units are arranged in parallel. (E-F) Photographs of the actual device. [Figure 5A-5B] The top view and cross-sectional view of the repeating unit in the mixing section of four wavy multilayer microstructure mixing units D-A1, D-B1, D-C1 and T-B1 of the present invention. (A) D-A1 is a two-layer structure in which the inner edge of the narrow channel layer on the semicircular ring always overlaps with the inner edge of the wide channel layer, and (B) D-B1 is a two-layer structure in which the outer edge of the narrow channel layer on the semicircular ring always overlaps with the outer edge of the wide channel layer. The figures show the dimensions of each layer, where W is width, H is height, Lc is the length of the straight channel, Do is the outer diameter of the semicircular ring in the projected shape, and Di is the inner diameter of the semicircular ring in the projected shape. [Fig. 5C-5D] Top view and cross-sectional view of repeating units in the mixing section of four corrugated multi-layer 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, in which the width of two-layer channels on the semicircular ring part is shifted to form a cross section similar to the Z-shape of Tetris. (D) Three-layer structure T-B1. The figure shows the dimensions of each layer, W: width, H: height, Lc: length of straight channel, Do: outer diameter of semicircular ring in projected shape, Di: inner diameter of semicircular ring in projected shape. [Figure 6A-6B] Schematic diagram of the corrugated single-layer microstructured mixing units S1 (A) and S2 (B). The small drawing at the top left shows the top view of a repeating unit of the corrugated channel of the mixing section (left; contains two semicircular annular and two short straight channels) and a cross-section of this channel (right). [Figure 6C-6D] Schematic diagrams of the corrugated single-layer microstructured mixing units S3 (C) and S4 (D). The small drawings at the top left show a top view of a repeating unit of the corrugated channel in the mixing section (left; contains two semicircular annular and two short straight channels) and a cross-section of this channel (right). [Figure 7] A schematic diagram of a T-shaped mixing unit T1. The small drawing on the left shows a cross-sectional view of the flow path of the mixing section. [Figure 8A-8B] Schematic diagram of Y-shaped mixing units Y1 (A) and Y2 (B). The small drawing on the left shows a cross-sectional view of the flow path of the mixing section. [Figure 9] 1 shows histograms of particle size (particle size based on volume) and PDI of particles obtained by mixing using the corrugated microstructure mixing units S1, D-A1, D-B1, D-C1, and T-B1. [Figure 10] 1 shows histograms of particle size (volume-based particle size) and PDI of particles obtained by the corrugated microstructure mixing units Y1, T1, S1, S2, and D-B1. [Figure 11] 1 shows histograms of particle size (volume-based particle size) and PDI of particles obtained by the corrugated microstructure mixing units Y2, S3, and D-B4. [Figure 12] 13 is a histogram of particle size (particle size based on volume) and PDI of particles obtained by the wavy microstructure mixing units D-B4 and D-B5. [Figure 13] 1 is a histogram of particle size (volume-based particle size) and PDI of particles obtained by the wavy microstructure mixing units D-B1, D-B2, S1, and S2. [Figure 14] Figure A shows a histogram of particle size (particle size by volume) and PDI of particles obtained using the wavy microstructure mixing unit S2 and D-B2. Figure B shows a histogram of particle size (particle size by volume) and PDI of particles obtained using the wavy microstructure mixing unit S3 and D-B2. Figure C shows a histogram of particle size (particle size by volume) and PDI of particles obtained using the wavy microstructure mixing unit S4 and D-B7. [Figure 15] Figure A shows the particle size (particle size by volume) and PDI histograms of particles obtained using the wavy microstructure mixing units D-B2, D-B3, D-B4, and D-B6, and Figure B shows the particle size (particle size by volume) and PDI histograms of particles obtained using the wavy microstructure mixing units D-B1, D-B5, and D-B7. [Figure 16] 1A-F are schematic diagrams of a three-layer corrugated microstructure mixing unit; [Figure 17] 1A and 1B are schematic diagrams of a three-layer corrugated microstructure mixing unit according to different embodiments. [Figure 18] FIG. 1 is a schematic diagram of a two-layer mixing unit with a T-shaped mixing section. [Figure 19] FIG. 1 is a schematic diagram of a dual-phase mixing unit with an in-line dilution channel at the outlet. [Figure 20]FIG. 13 shows the results of Example 5 comparing the histograms of PDI and particle size (volume-based particle size) of nanoparticles produced by two-layer structure mixing units having different numbers of semicircular rings. [Figure 21] FIG. 13 is a schematic diagram of the mixing unit of the present invention (A) in Example 10 and the annular mixing unit of the prior art (B). [Figure 22] FIG. 13 shows the results of measuring the size and DPI of RNA-loaded self-assembling LNPs produced by the mixing unit of the present invention in Example 10 and the circular mixing unit in the prior art. [Figure 23A-23B] These are actual photographs showing the mixing effect due to the different colors of the two fluids before and after mixing when an ethanol solution containing phenol red and a phosphate buffer solution containing trypan blue are mixed using the Y-1, S-1, and D-B1 mixing units (A) and a 16-stage parallel mixer (B). [Figure 24] FIG. 1 is a schematic diagram of the turbulent flow direction of a two-phase mixing unit such as D-B1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0032] <Detailed Description of the Invention> The terms of this application are defined below. Undefined terms have the same meaning as understood by one of ordinary skill in the art.

[0033] (definition) A "microstructured mixing unit" in the context of the present invention refers to a device for mixing fluids having sub-millimeter sized structures in one dimension. In particular, said microstructured mixing unit is part of a microfluidic chip or is a microfluidic chip itself.

[0034] "Channel" or "flow passage" have the same meaning in the context of the present invention and refer to a cavity in a microfluidic chip through which fluids can pass, typically an elongated passage. Channels in prior art microfluidic chips generally have a rectangular cross section. Channels of the present invention preferably do not have a rectangular cross section over their entire length. Such channels of the present invention can be considered to consist of multiple layers of channels with rectangular cross section arranged one above the other. Thus, the reference to a "multi-layer channel" is only to more clearly describe the structure of the mixing unit of the present invention, and does not imply that the fluids between these channels are physically isolated from each other to form multiple independent channels. In fact, the "multi-layer channel" ultimately forms a single fluid channel.

[0035] A "wavy" flow channel refers to a flow channel that is composed of multiple circular arcs connected from end to end and curved in opposite directions, so that the entire flow channel is wavy. In a specific embodiment of the present invention, the circular arcs are semicircular or semielliptical rings. Since the bending directions of two adjacent semicircular or semielliptical rings are opposite, two consecutive semicircular or semielliptical rings can be considered as a "repeated structure" or a "repeated unit" if each semicircular or semielliptical ring is the same size. The direction of the wave motion of the "wave" is similar to the direction of wave vibration in physics. The direction of the extension of the "wave" is similar to the direction of wave propagation in physics. Therefore, the wave motion direction of the wave and the extension direction of the wave are two perpendicular directions.

[0036] The term "cross section" refers to a cross section perpendicular to the direction of liquid flow in a channel.

[0037] "Projected shape" refers to the shape of the microfluidic chip or the corrugated microstructure mixing unit when placed horizontally as a whole. "Projected shape" overlap refers to a complete overlap in size or shape, and does not include the case where one projected shape is smaller and fits within the other projected shape.

[0038] "Fluid communication" or "fluidly communicable" refers to a connection between structures, components, or elements, respectively, such that fluid can flow from one to the other.

[0039] "Parallelization" refers to two elements or components connected head-to-head or tail-to-tail. In the context of the present invention, "parallelization" generally refers to the parallelization of two or more mixing units, i.e., the mixing units are connected via a junction at their inlet ends or share one junction, and the outlet ends are connected via the same outlet or share one outlet.

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

[0041] "Dean vortex" refers to the phenomenon in which, when a fluid flows through a curved channel, the fluid moving toward the center of the channel is pushed outward (caused by the no-slip boundary condition) by the centripetal force and the higher velocity of the fluid at that point. The action of these forces causes the fluid to rotate perpendicular to the channel.

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

[0043] "Nanoparticle" refers to a particulate material having a diameter of submicron size, usually less than 100 nm. In the context of the present invention, nanoparticles generally refer to nanocarriers loaded with molecules having pharma- ceutically active ingredients.

[0044] "LNP" is an abbreviation for lipid nanoparticle.

[0045] "Carrier material" in the context of this application refers to the main material forming the nanoparticles, which is generally a biocompatible component and is capable of forming nanoparticles by itself. In the present invention, the "carrier material" generally does not have the desired pharmaceutical efficacy, e.g. therapeutic efficacy.

[0046] "Payload", in the context of this application, refers to a substance having a pharmaceutical benefit, eg, a therapeutic benefit, that can be delivered by the carrier material.

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

[0048] "Particle size" in the context of this specification refers to the diameter of a spherical particulate material. The particle size of submicron-sized particles is usually measured by dynamic light scattering (DLS). DLS estimates the particle size distribution of particles by irradiating submicron-sized particles with laser light to cause scattering and analyzing the fluctuation change of the scattered light. The measurement results include volume-based particle size, intensity-based particle size, and number-based particle size, as well as an average particle size calculated based on these three parameters. Unless otherwise specified, when referring to "particle size" in the context of the present invention, it refers to the volume-based particle size measured by dynamic light scattering.

[0049] "PDI" is an abbreviation for Poly dispersity index, which is a parameter for the width of the particle size distribution of nanoparticles. The larger this index is, the wider the distribution is, and the higher the polydispersity is. The smaller this index is, the narrower the distribution is, and the better the uniformity is.

[0050] (SML Microstructure Mixing Unit) To achieve nanoparticle self-assembly, turbulent flow is usually required. At low Reynolds numbers, the typical flow characteristics are laminar rather than turbulent. Therefore, for microstructured mixing units (such as microfluidic chips) used for nanoparticle self-assembly, the flow rate is accelerated to increase the Reynolds number and the formation of turbulence, or a special flow design is used to achieve turbulence at low flow rates / Reynolds numbers.

[0051] The present invention uses a corrugated channel consisting of alternating semicircular or semi-elliptical rings curved in different directions, and has multiple layers of such corrugated channels in the mixing section to induce alternating flow and chaotic convection in the mixer, thereby promoting the mixing of components in the fluid (Figure 24). By using a curved or spiral shape design, a simple geometric design can promote the convective transport in the microchannel and generate Dean vortices during the mixing process, improving the mixing effect.

[0052] The present invention provides a corrugated multi-layer microstructure mixing unit, the mixing unit comprising an inlet section, a junction section, a multi-layer mixing section, and an outlet section, which are fluidly connected to each other, each layer of the multi-layer mixing section is a corrugated channel, the corrugated channel includes n semicircular or semi-elliptical rings, and the curvature directions of any two adjacent semicircular or semi-elliptical rings are opposite to each other and are connected to each other by a straight channel. The projected shapes of two adjacent layers of the multi-layer mixing section always overlap at least partially, but not completely, on the flow path of the entire mixing section. The inlet section includes at least two inlets, which are fluidly connected to the junction section, so that different fluids flowing from the inlets are joined at the junction section.

[0053] The inlet portion is used to allow different fluids to be mixed to flow in, and thus includes at least two inlets, for example, two inlets, for example, when preparing nucleic acid-loaded liposome nanoparticles, at least one inlet is an inlet for the lipid-containing fluid to flow in, and at least one other inlet is an inlet for the nucleic acid-containing fluid to flow in.

[0054] After the different fluids enter through the inlets, they preferably flow through a channel section before joining together. Thus, in a preferred embodiment, the inlet section includes a channel section between each inlet and the joining section. In a preferred embodiment, the channel length between the inlet section and the joining section is at least 2000 μm. In a specific embodiment, the channel between the inlet section and the joining section is a straight channel.

[0055] The different liquids flowing in from different inlets are merged at the junction after passing through the inlet section. The length of the junction can be arbitrary. The length and shape of the junction can be adjusted according to the needs of chip manufacturing and flow channel design.

[0056] In some embodiments, the mixing unit of the present invention does not include a junction, in which case the fluids entering from the different inlets join directly in the mixing section.

[0057] Fluids flowing from different inlets can be joined at the same location in the junction. In this case, taking the two inlet scheme as an example, the inlet and junction can form a T-shaped or Y-shaped structure. In this case, the junction is the vertical part of the T or Y, and the inlet channel is the two branches at the top. In a preferred embodiment, the included angle α between the straight channel of the inlet and the straight channel of the junction is greater than or equal to 90° and less than 180°.

[0058] After passing through the junction, the different fluids are roughly mixed. The fluid mixture then enters the mixing section, where the special structure of the mixing section creates turbulence, resulting in thorough mixing and promoting the formation of desired products, such as self-assembled nanoparticles.

[0059] The straight channels of the junction are perpendicular to or coincident with the direction of the undulations of the corrugated channels of the mixing section. In a specific embodiment, the straight channels of the junction are perpendicular to or coincident with the extension direction of the corrugated channels of the mixing section.

[0060] There are many implementations of the multi-layer mixing unit, but the main purpose is to create a more complex channel structure than the conventional simple rectangular channel, so as to generate more turbulence under the same conditions. Therefore, the corrugated multi-layer microstructure mixing unit can produce a better mixing effect. Specifically, the fluid can become turbulent even when the flow velocity and Reynolds number are not high.

[0061] For example, the multi-layered mixed unit may include 2 to 20 layers. Considering the manufacturing process and cost, it is preferable that the number of layers does not exceed 5 layers, for example, 2 layers, 3 layers, 4 layers, 5 layers. However, considering the future development of the manufacturing process, it is possible to provide more layers.

[0062] The width of the channels of each layer of the multi-layer 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. Incomplete overlap of projected shapes can be achieved by 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 width of the widest layer, such as 1 / 5, 1 / 4, 1 / 3, 1 / 2, or 2 / 3, 3 / 4, 2 / 5, 3 / 5, 4 / 5, etc., of the width.

[0064] The height of the channels in each layer in a multi-layer mixing unit may be the same or different, hi a specific embodiment, the height of each layer is the same.

[0065] In a preferred embodiment, in the corrugated multi-layer microstructure mixing unit, the semicircular annular or semi-elliptical annular channels of each layer are always parallel. In other words, each semicircular annular channel of each layer can form a concentric circle, or each semi-elliptical annular channel of each layer can form a concentric and same-direction ellipse.

[0066] In light of this, when the widths of two adjacent layers are different, it is preferable that the outer edges of the flow channels of the semicircular rings or semi-elliptical rings of each layer always overlap. Similarly, when the widths of two adjacent layers are different, the orientation of the flow channels can be adjusted by connecting the straight lines of the two semicircular rings or semi-elliptical rings, and the above arrangement can be realized for each semicircular ring or semi-elliptical ring. Specifically, when the widths of each layer are different, the straight lines of the layers with different widths form different angles with respect to the extension direction of the corrugated structure. In such an arrangement, the cross section of the multilayer mixing section in the semicircular ring or semi-elliptical ring portion can be, for example, L-shaped (two layers), and can be, for example, three layers B, three layers C, and three layers F shown in FIG. 16.

[0067] In another embodiment, when the widths of two adjacent layers are different, the inner edges of the channels of the semicircular or semi-elliptical rings of each layer always overlap. In such an arrangement, the cross section of the multi-layer mixing section in the semicircular or semi-elliptical ring can be, for example, L-shaped (two layers), or can be, for example, three layers A shown in FIG.

[0068] In another embodiment, when the widths of two adjacent layers are different, for a mixing unit with at least three layers, there are two adjacent layers whose outer edges of the channels of the semicircular ring or semi-elliptical ring always overlap, and there are also two adjacent layers whose inner edges of the channels of the semicircular ring or semi-elliptical ring always overlap. In such an arrangement, the cross section of the multi-layer mixing section in the semicircular ring or semi-elliptical ring may be, for example, the cross section of three layers C, three layers E shown in Figure 16 and the cross section of four layers B in Figure 17.

[0069] When the width of each layer is the same or when the width of two adjacent layers is the same, the projected shapes of the two adjacent layers are different at the same time, and the flow paths in the semicircular ring or semicircular ring are always parallel, so that the widths of two adjacent layers of the same width can be shifted in the semicircular ring or semielliptical ring. Therefore, the direction of the flow path is adjusted by connecting the straight lines of the two semicircular rings or semielliptical rings. In one embodiment, when the widths of the two adjacent layers are the same and the widths are shifted in the semicircular ring or semielliptical ring, the one on the outside of each semicircular ring or semielliptical ring (i.e., the one with the larger inner and outer diameters of the ring) is always on the outside, and the one on the inside is always on the inside. In such an arrangement, the cross section of the multi-layer mixing section in the semicircular ring or semielliptical ring may be, for example, FIG. 5C (two layers), FIG. 16, three layers D, and FIG. 17, four layers A.

[0070] In various configurations, the preferred technical means is that the flow paths of the semicircular or semi-elliptical rings are always parallel, and the outer edges of the flow paths of the semicircular or semi-elliptical rings of each layer (such as each layer 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 corrugated channel of the mixing section in the multi-layer mixing unit is represented as W, and the height is represented as H. Preferably, the height of the channel of the mixing section is always constant. In the case of the multi-layer mixing unit, the width and height of each layer can be represented separately, for example, the width and height of the narrow layer can be represented as W and H, respectively. narrow and H narrow and the width of the wide layer is W wide and H wide When only the width W is stated without specifying which layer it is, W is measured based on the projected shape of the flow channel in the semicircular or semi-elliptical ring. When only the height H is stated without specifying which layer it is, it is the overall width of the stacked multi-layer flow channels.

[0072] In a preferred embodiment, the width (W) of the corrugated channel portion is any value between 200 μm and 3000 μm, preferably between 250 μm and 2000 μm, more preferably between 300 μm and 1500 μm, even more preferably between 400 μm and 1000 μm, for example on the order 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 corrugated channel portion is any value between 100 μm and 1500 μm, preferably between 125 μm and 1000 μm, more preferably between 150 μm and 750 μm, even more preferably between 200 μm and 500 μm, for example on the order 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 corrugated flow passage portion is 0.01 to 1 mm 2 Preferably, the range is between 0.04 and 0.8 mm. 2 , more preferably 0.06 to 0.5 mm 2 , and even more preferably 0.08 to 0.4 mm 2 It is.

[0075] In a preferred embodiment, the width of each layer of the multi-layer mixing section is smaller than or equal to the width of the flow path of the junction and outlet of the multi-layer microstructure mixing unit and the connection of the mixing unit. In a preferred embodiment, the width of the widest layer of the multi-layer mixing section is equal to the width of the flow path of the junction and outlet of the multi-layer microstructure mixing section and the connection of 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 extension direction of the corrugation is represented as Do, and the inner diameter of each semicircular ring or the inner axis length of each elliptical ring in the extension direction of the corrugation is represented as Di. Since Do and Di are the inner diameter and the outer diameter, respectively, Do is equal to Di plus W by 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, for example about 4 times, that of W, and the length of Di may be about 1 to 3 times, for example about 2 times, that of W. In the case of a multi-layered mixing unit, the possible values ​​of Do and Di can be described for each layer. When only Do and Di are described without specifying which layer they are, the values ​​of Do and Di are measured based on the projected shape of the flow channel of the semicircular ring or semi-elliptical ring.

[0077] In the mixing section, it is preferable that the semicircular or semielliptical rings having opposite curvature directions are connected by a straight flow path. The length of the straight flow path is represented by a parameter Lc. It should be noted that Lc means the straight distance between two semicircular or semielliptical rings in the wave direction of the corrugated flow path as shown in the drawings of the present invention, and does not take into account the included angle between this straight flow path and the wave direction of the corrugated flow path. For example, the length (Lc) of the straight flow path 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 flow path can be any value between 200 μm to 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, the Lc may have a length of about 500 μm, 1000 μm, 1500 μm, 2000 μm, 2500 μm, or 3000 μm.

[0078] The straight flow paths are perpendicular to the extension direction of the corrugated flow paths or form a certain angle with the direction of the wavy motion of the corrugated flow paths. For example, the included angle may be 45° or less, preferably 9° to 27°.

[0079] In a single layer channel embodiment, the straight channels are preferably perpendicular to the extension direction of the corrugated channels. In the case of multi-layer channels, the included angle may depend on a variety of factors.

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

[0081] For example, when the widths of the channels of two adjacent layers are equal, the magnitude of the included angle may depend on the length (Lc) of the straight channels, the width of the channels, and the degree of offset of the channels of the two adjacent layers. In some embodiments, the angles between the extension directions of the straight channels and the corrugated channels in two-layer channels of equal width are equal but opposite in direction.

[0082] To generate turbulent flow, it is preferred that the flow paths are arranged such that, in at least one flow path, the included angle causes the two straight flow paths to form a structure (resembling a horseshoe) that recedes inward relative to the semicircular or semi-elliptical annular flow path between them.

[0083] In the mixing unit, whether single-layer or multi-layer, there may be n semicircular or semielliptical rings, where n may be any integer other than 1, and is preferably 6 or more, for example, 6, 7, 8, 9, 10 or more, for example, 10 to 15, 15 to 20.

[0084] After passing through the mixing unit, the mixed fluid flows out of the outlet of the outlet section. In a preferred embodiment, the outlet section further includes a channel section for connecting the end of the mixing section and the outlet. The design and orientation of the channel of this section can be determined as needed and there is no special requirement. In a specific embodiment, the channel between the end of the mixing section and the outlet is a straight channel.

[0085] It should be noted that in the embodiment of the multi-layer microstructured mixing unit, only the cross section of the mixing section is not necessarily rectangular. In particular, in the case of a multi-layer microstructured mixing unit including layers with different channel widths, the cross section of the entire mixing section is not rectangular. However, other parts of the mixing section, such as the junction section and the outlet section, which are in direct fluid communication with the mixing section, do not have the effect of creating a cut, so that they do not have a multi-layer structure, and therefore have a rectangular cross section. Therefore, when the mixing section is connected to the channels of the junction section and the outlet section, which have a rectangular cross section, there is a transition section in which the channel width of one layer or multiple layers gradually changes, and finally the multi-layer structure disappears, and the connection point between the junction section or the outlet section and the mixing section has the same rectangular cross section, making it possible to connect.

[0086] In a specific embodiment, the multi-layer microstructure mixing 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 the embodiment, 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 structure of D-B1 shown in FIG. 1. The structure of the D-B1 mixing unit is described in detail below. The SML microstructure mixing unit of D-B1 includes an inlet section, a junction section, a multi-layer mixing section, and an outlet section that are fluidly connected. The multi-layer mixing section includes a two-layer corrugated channel, and each layer of the corrugated channel includes 10 semicircular rings, and each semicircular ring has Do of 2000 μm and Di of 1000 μm. The curvature directions of any two adjacent semicircular rings are opposite and connected by a straight channel, and Lc is 1000 μm. The channels in the semicircular ring section of the two-layer corrugated channel are always parallel and have a certain width and height, respectively. The width of the narrow layer is 250 μm, the width of the wide layer is 500 μm, and the heights of the narrow layer and the wide layer are both 125 μm. The inlet section includes two inlets, and the inlets are in fluid communication with a junction section such that different fluids flowing through the inlets join at the junction section. The straight flow paths of the inlet section and the junction 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 inlet, junction and outlet of the T-B1 mixing unit are installed similarly to D-B1. The structure of the mixing section of the T-B1 mixing unit is described in detail below. The SML microstructure mixing multi-layer mixing section of the T-B1 includes a three-layer corrugated channel, and the cross section is shown in FIG. 5D. Each of the three layers of the corrugated channel includes 10 semicircular rings with Do of 2000 μm and Di of 1000 μm. The curvature directions of any two adjacent semicircular rings are opposite, and they are connected by a straight channel, and Lc is 1000 μm. The channels in the semicircular ring section of the three-layer corrugated channel are always parallel, and each has a certain width and height, with the upper and lower layers being narrow (narrow layer) 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 heights of the narrow layer and the wide layer are both 100 μm. The inlet section includes two inlets, and the inlets are in fluid communication with a junction section such that different fluids flowing through the inlets join at the junction section. The straight flow paths of the inlet section and the junction section form a Y-shape.

[0089] (Multi-stage SML mixing device) The invention also relates to a mixing device comprising said corrugated microstructured mixing unit, in particular a corrugated multi-layer (SML) microstructured mixing unit.

[0090] The SML microstructure mixing unit of the present invention is suitable for scale-up because it is less susceptible to clogging. Thus, in a preferred embodiment, the mixing device of the present invention comprises a plurality of SML microstructure mixing units, also known as a "multi-stage parallelized mixing device". In a preferred embodiment, the plurality of SML microstructure mixing units are connected via parallelization. Through the parallelization, the device of the present invention can achieve a greater throughput.

[0091] In a preferred embodiment, m parallel SML microstructure mixing units of the multi-stage parallel mixer share one inlet, which allows fluids flowing from the same inlet into m channels. Each channel is in fluid communication with a junction or mixing section of the SML microstructure mixing unit. For example, after fluids flow in from at least two inlets, the channels pass through branching structures included in each inlet and are split into m branching channels, which are then mixed in independent junctions or mixing sections of each SML mixing unit. The branching structures can ultimately be split into m branching channels via one or more branch points.

[0092] Similarly, the m outlet ends of the m mixing sections are in fluid communication with an outlet section. The m parallel SML microstructured mixing units share one outlet section. The outlet section has a branching structure that can finally merge the fluids flowing out of the m mixing sections into one flow path through one or more branch points to be in fluid communication with the outlet.

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

[0094] Examples of the arrangement are shown in Fig. 3 and Fig. 4. This embodiment of the present invention is explained using the four-stage parallel mixing unit in Fig. 3. As shown in Fig. 3, the first layer is an inlet layer (Fig. 3A), which includes two fluid (general) inlets (left and center) and one fluid (general) outlet (right). The two inlets are used for the inflow of the two mixed fluids, respectively, and the one outlet is used for the outflow of the mixed fluid. The second layer is a flow-division layer (Fig. 3B), which includes straight flow paths in the parts fluidically communicating with the two inlets. Then, the straight flow paths are divided into two to form two symmetrical branching flow paths, so that the two mixed fluids flow in from the two inlets, and then are divided into two separate flows in the flow-division layer and flow into the third layer. The third layer is a mixing layer (Fig. 3C). Each input flow path is divided into two again in the third layer, and after passing through the left-right symmetrical non-linear flow paths, it merges with the other fluids. After passing through the merger section, it enters the multi-layered wave mixing unit and is mixed. After mixing, the mixed fluid passes through an outlet channel with two branching points, combining the two at each point, and finally merging into one point and flowing out from an outlet that is fluidly connected to the second layer and the first layer.

[0095] As can be seen from Figures 3C and 3D, the multi-stage SML mixing unit is composed of two sets of SML mixing units, each set containing two mirror-symmetric mixing units, and presents a lantern-like structure as shown in Figure 3. Since each set of lantern-like structures necessarily has an inlet surrounded by a flow channel, the diversion between the sets of lantern-like structures must be done in another layer, i.e., the diversion layer. Those skilled in the art will understand that it is not important to the present invention how many layers of material the final mixing device is composed of, as long as it contains the corrugated flow channels of the present invention.

[0096] In one embodiment of the present invention, the device of the present invention is a device having four channels parallelized as shown in FIG. 3, or a device having 16 channels connected in parallel as shown in FIG. 4.

[0097] The confluence section including the branch structure and the inlet section may be in a different plane from the mixing section and / or the outlet section. The plane where the confluence section and the inlet section are located is called the "diverging layer" and acts to divide the confluence fluid. The plane formed by the mixing section and the outlet section is called the "mixing layer" and acts to mix and flow out.

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

[0099] (In-line dilution) In one embodiment of the invention, the apparatus alternatively further includes an inlet for in-line dilution downstream of the SML mixing unit.

[0100] The technical means of in-line dilution will be described with reference to FIG. 19 as an example. Specifically, the outlet of the SML mixing unit further includes a diluent input channel or diluent inlet for delivering a dilution solvent such as PBS or other suitable buffer, carrier, etc., at a position close to the mixing section in the outlet, so that the concentration of each component in the fluid flowing through the mixing section is diluted before flowing out of the outlet. For example, the proportion of the original organic solvent such as ethanol in the solution is reduced. A too high ethanol concentration may reduce the stability of the nanoparticles as a product, so that a diluent input channel and / or diluent inlet are preferred.

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

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

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

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

[0105] (Mixed unit material) The mixing unit of the present invention can be made of, for example, materials with specific mechanical properties and biocompatibility, such as polymeric materials, metallic materials, or inorganic non-metallic materials.

[0106] Metallic materials that can be used to manufacture the mixing unit of the present invention can be, for example, stainless steel, aluminum alloy, magnesium alloy, titanium alloy, etc. 304 stainless steel, 316L stainless steel, Hastelloy are preferred.

[0107] Polymeric materials that can be used to manufacture the mixing unit of the present invention can be, for example, polyetheretherketone (PEEK), acrylic (PMMA), polydimethylsiloxane (PDMS), polyamideimide (PAI), polychlorotrifluoroethylene (PCTFE), polytetrafluoroethylene (PTFE), polycarbonate (PC), polypropylene (PP), polyphenylene sulfide (PPS), cyclic olefin copolymer (COC), cyclic olefin polymer (COP). Preferably, polyetheretherketone (PEEK), polyester (PET / PETP), cyclic olefin copolymer (COC), cyclic olefin polymer (COP), polydimethylsiloxane (PDMS) are used.

[0108] Inorganic non-metallic materials that can be used to manufacture the mixing unit of the present invention can be, for example, glass, ceramics, and the like.

[0109] (Mixing unit manufacturing) The flow channels in the mixing unit of the present invention can be manufactured by any method known in the art or 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 polymer materials are used to manufacture the mixed unit, they can also be processed using thermoforming (e.g. injection molding process) or etching process (e.g. SU-8 adhesive). For multi-layer mixed units, each layer can be manufactured separately and then sealed together. Common sealing processes such as hot press sealing, laser sealing, film sealing, etc. can be used to seal between different layers.

[0111] In other embodiments, it may be packaged directly with metal or polymeric materials.

[0112] (application) In one embodiment, the mixing unit and mixing device 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 payload, are added through the inlet of the mixing unit or mixing device of the present invention and mixed by the mixing part of the present invention.

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

[0114] The mixing unit or mixing device of the present invention can obtain nanoparticles with relatively uniform size through efficient mixing. In an embodiment of the present invention, the nanoparticles produced by the mixing unit of the present invention, especially LNPs with therapeutic molecules such as nucleic acid molecules as payloads and lipids as carrier materials, have a DPI of 0.3 or less, preferably 0.2 or less.

[0115] (1) Lipid nanoparticles The mixing unit and device of the present invention are particularly suitable for producing lipid nanoparticles (LNPs) loaded with a target for delivery. The target for delivery may be a nucleic acid molecule, including plasmid, ceDNA, mRNA, siRNA, microRNA, sgRNA, etc., and may also be a small molecule or a protein. When used to load a nucleic acid material, the nucleic acid molecule may be any size between 20 bp and 15 kb. In a preferred embodiment, the mixing unit and device of the present invention are used to produce LNPs loaded with nucleic acids. In a preferred embodiment, the LNPs are LNPs containing ionizable lipids.

[0116] Lipid nanoparticles are generally composed of four types of lipids: (a) ionizable or cationic lipids, (b) neutral lipids (or non-cationic lipids), (c) PEG-conjugated 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 device of the present invention through at least one inlet. The following are suitable components as the lipid components (a) to (d) for preparing lipid nanoparticles.

[0117] (a) Ionizable 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-dimyristyloxyprop-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, ALC-0315.

[0119] Other suitable ionizable lipids reported in the art are also contemplated.

[0120] (b) Neutral lipids (or non-cationic lipids) Examples of non-cationic lipids include distearoyl-sn-glycero-phosphoethanolamine, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoylphosphatidylethanolamine (PPE ... 16-O-dimethyl-1-propanolamine (DMPE), 18-O-dimethyl-1-propanolamine (16-O-dimethyl-1-propanolamine ... Lance PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), hydrogenated soy phosphatidylcholine (HSPC), egg phosphatidylcholine (EPC), dioleoyl phosphatidylserine (DOPS), sphingomyelin (SM), dimyristoyl phosphatidylcholine (DMPC), dimyristoyl phosphatidylglycerol (DMPG), distearoyl phosphatidylglycerol (DSPG), diercoyl phosphatidylcholine (DEPC), palmitoyl oleoyl phosphatidylglyc These include, but are not limited to, cerol (POPG), dielaidoyl-phosphatidylethanolamine (DEPE), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalosporin, cardiolipin, phosphatidic acid, cerebroside, dicetyl phosphate, lysophosphatidylcholine, dilinoleoylphosphatidylcholine, or mixtures thereof.It should be understood that other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids may also be used. The acyl groups in these lipids are preferably C. 10 ~C 24 The acyl group is derived from a fatty acid having a carbon chain, for example, lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl.

[0121] Other examples of non-cationic lipid suitable for use in lipid nanoparticles include non-phosphoryl lipids such as stearylamine, dodecylamine, hexadecylamine, acetyl palmitate, glycerol ricinoleate, hexadecyl stearate, isopropyl myristate, amphoteric acrylic polymers, triethanolamine-lauryl sulfate, alkyl-aryl sulfate polyethyloxylated fatty acid amides, dioctadecyldimethylammonium bromide, ceramide, sphingomyelin, etc. 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 may further comprise a conjugated lipid, which may 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)succinic acid, 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 membrane integrity Examples of components that provide membrane integrity include sterols and cholesterol.

[0125] (2) Polymer nanoparticles As polymeric nanoparticles, the loaded substances are similar to liposomal nanoparticles. Carriers include, for example, PLGA nanoparticles, PLA nanoparticles, etc. Generally speaking, polymeric nanoparticles include polymers and / or amphiphilic polymers.

[0126] Non-limiting examples of hydrophobic polymers include polylactic acid (PLA), polypropylene oxide, poly(lactic-co-glycolic acid) (PLGA), poly(ε-caprolactone), poly(ethylene), polybutadiene, polyethylene glycol, polymethacrylate, polyvinyl butyl ether, polystyrene, polycyclopentadienyl methyl norbornene, polyethylene, polyisobutylene, 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 esters (e.g., vinyl acetate, trans vinyl esters, vinyl propionate, vinyl formamide, vinyl acetamide, vinyl pyridine, vinyl imidazole), amino alkyl esters (e.g., amino alkyl acrylates, amino alkyl methacrylates, amino alkyl (meth) acrylamides), styrene esters, and lactic acid esters.

[0127] Amphoteric polymers include poly(ethylene glycol)-poly(D,L-lactic acid) copolymers (PLA-PEG), PLGA-PEG, polystyrene-polyoxyethylene copolymers, polybutyl acrylate-polyacrylic acid copolymers, or polybutyl methacrylate-polyethylene oxide copolymers. Other examples of amphiphilic copolymers are described in U.S. Patent No. 5,399,633, 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 The lipid-polymer hybrid nanoparticle material comprises the lipid nanoparticles described above in (1) and the polymeric nanoparticles described above in (2). For the selection of these two components, please refer to the respective sections above.

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

[0130] Liposomes are typically composed of phospholipids (such as the neutral, cationic, or ionizable lipids listed above) and components that provide membrane integrity. Special types of liposomes may also contain other components, such as long-circulating liposomal lipids, including PEG-conjugated lipids.

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

[0132] The inventors have found that different flow channel 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 between 1 mL / min and 4 mL / min.

[0133] In a preferred embodiment, the present invention provides a method for producing nanoparticles by self-assembly, the method comprising mixing a first fluid containing a carrier material (e.g., one or more lipids) with a second fluid containing a payload (e.g., nucleic acid such as DNA or RNA) using a corrugated multi-layer microstructured mixing unit of the present invention. The method uses 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, even more preferably 1 mL / min to 4 mL / min. For example, when using D-B4, D-B5, D-B6, D-B7 mixing units, a higher TFR can be used, for example a TFR of 4 mL / min to 12 mL / min, for example using the corresponding TFRs listed in Table 3. When using the preferred D-B1, D-B2, D-B2-7 or T-B1 mixing units, a lower TFR can be used, for example a TFR of 1 mL / min to 4 mL / min, these mixing units can obtain an ideal product with a TFR as low as 1 mL / min.

[0134] The present invention also relates to the following items:

[0135] 1. A multi-layer corrugated (SML) microstructured mixing unit, comprising an inlet section, a confluence section, a multi-layer mixing section, and an outlet section, the inlet section being in fluid communication with the confluence section; Each layer of the multi-layer mixing section is a corrugated flow channel, and the corrugated flow channel of each layer includes n semicircular rings or semi-elliptical rings, and the curvature directions of any two adjacent semicircular rings or semi-elliptical rings are opposite to each other and are connected by a straight flow channel; The projected shapes of two adjacent layers of the multi-layer mixing section always at least partially overlap each other on the flow path of the entire mixing section, but do not completely overlap each other; The inlet section includes at least two inlets, and the inlets are in fluid communication with a junction section such that different fluids flowing through the inlets are joined at the junction section.

[0136] 2. The SML microstructure mixing unit according to item 1, wherein the mixing section comprises two, three, four or five layers of the corrugated channels.

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

[0138] 4. The SML microstructured mixing unit according to any one of items 1 to 3, wherein the flow paths of each layer on the semicircular annulus or semi-elliptical annulus of the mixing section are always parallel.

[0139] 5. The SML microstructured mixing unit according to item 4, wherein the outer edges of the channels of each layer on the semicircular or semi-elliptical annular section of the mixing section always overlap.

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

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

[0142] 8. The SML microstructure mixing unit according to any one of items 1 to 7, wherein the width W of the overall projected shape of one or more layers of corrugated channels is 200 μm to 3000 μm.

[0143] 9. The SML microstructure mixing unit according to any one of items 1 to 8, wherein the total height H of the corrugated channels of one or more layers is 100 μm to 750 μm.

[0144] 10. The SML microstructure mixing unit according to any one of items 1 to 9, wherein the semicircular or semi-elliptical rings having opposite curvature directions are preferably connected by straight flow paths, and the length Lc of the straight flow path is 0.5W to 4W, preferably 1W to 3W, and more preferably 1W to 2W.

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

[0146] 12. The SML microstructure mixing unit according to any one of items 1 to 11, comprising two or three layers of corrugated channels, each of the layers of corrugated channels comprising 6 to 10 semicircular rings, and the length Lc of the straight channel between each of the semicircular ring channels is greater than or equal to the inner diameter Di of the projected shape of the semicircular ring channel.

[0147] 13. The microstructure mixing unit according to item 12, wherein at least two layers in the corrugated 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%.

[0148] 14. A microstructure mixing unit as described in item 13, wherein the narrow layer always overlaps the outer edge of the wide layer throughout the entire semi-annular channel, and preferably all layers always have overlapping outer edges throughout the entire semi-annular channel.

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

[0150] 16. A microstructure mixing device, comprising m microstructure mixing units according to any one of items 1 to 15 arranged in parallel, the m microstructure mixing units being in fluid communication with each other through an inlet section, a junction section and / or an outlet section, where m is an integer greater than 1.

[0151] 17. A microstructured mixer according to item 16, wherein m is an even number, preferably m is a power of two.

[0152] 18. The microstructured mixing device according to item 16 or 17, which is a microfluidic chip and optionally comprises other elements selected from the group consisting of a syringe, a pump, a heater and a detector.

[0153] 19. A method for mixing different fluids, comprising the steps of mixing a first fluid and a second fluid using a microstructured mixing unit according to any one of items 1 to 15 or a microstructured mixing device according to any one of items 16 to 18, causing the first fluid to flow into a first inlet and the second fluid to flow 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 device described in any one of items 16 to 18; flowing the first fluid into a first inlet and flowing the second fluid into a second inlet; the first fluid is a fluid containing a nanoparticle carrier material, and the second fluid is a fluid containing a nanoparticle payload.

[0155] 21. The method according to item 20, wherein the nanoparticle is a lipid nanoparticle, a polymer nanoparticle, a lipid-polymer hybrid nanoparticle or a liposome.

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

[0157] The multi-layer corrugated microstructure mixing unit and the fluid mixing device equipped therewith of the present invention will be described through the following embodiments.

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

[0159] For ease of explanation and comparison, the width and height are normalized and shown separately. Specifically, 1W=1000 μm, 1H=500 μm, and the width and height of the flow channel in each layer of the mixing section are expressed as a multiple or fraction of these.

[0160] In Table 1, Y1, Y2, and T1 are simple conventional Y-shaped and T-shaped channels, and S1 to S4 are single-layer corrugated microstructure mixing units, and the cross sections of these seven channels are all rectangular throughout the mixing section, specifically, square. The structure of each mixing unit is also shown in the schematic diagrams of 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) JPEG2025506436000002.jpg107170JPEG2025506436000003.jpg112170JPEG2025506436000004.jpg61170

[0162] Preparation of LNP samples In order to test the effect of the microfluidic chip of the present invention, LNP samples are mixed and prepared using various flow paths listed in Table 1. Specifically, an aqueous phase (a citrate buffer solution of anionic long-chain natural polymer molecules, the molecular structure of which is similar to mRNA with a molecular weight of 15,000 Da. Hereinafter, this will be referred to as "mRNA-like") and an ethanol phase (lipid mixture solution) are mixed at a volume ratio of 3:1 at different flow rates to prepare LNP samples.

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

[0164] Table 2. LNP recipes JPEG2025506436000005.jpg70170

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

[0166] The particle sizes in the following examples are all based on volume because the inventors found that the volume-based particle size is closest to the particle size measured by transmission electron microscopy (TEM) for the LNP samples tested and can more accurately reflect the size of the nanoparticles.

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

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

[0169] (Example 1: Comparison of Mixing Units with Different Shape Structures) In this example, microfluidic chips were prepared with 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 of that of S1, and 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 of T1 is perpendicular to the straight channel at the junction. The other four types all have a certain angle. In addition, Y1, T1, S1, and S2 use the conventional rectangular cross section in the prior art, while D-B1 is the two-layer corrugated mixing unit of the present invention.

[0171] The test method was as described above in "Preparation of LNPs". Different flow rates were tested. Specifically, total flow rates of 1 mL / min and 6 mL / min were tested for the five types of mixing units. An additional 4 mL / min was tested for the only two-layer corrugated mixing unit, D-B1. The statistical results of PDI, volumetric particle size, of the self-assembled nanoparticles produced by the chips containing these mixing units are shown in Figure 10.

[0172] From Figure 10, it can be seen that when the total flow rate was 1 mL / min, the single-layer corrugated mixer S1 had no significant difference in particle size and PDI compared with S2. For the single-layer corrugated mixer, Lc varied in the range of 0.5-1 mm and did not have a significant effect on PDI. At a flow rate of 1 mL / min, both S1 and S2 could achieve excellent mixing effects.

[0173] At a total flow rate of 6 mL / min, the particle size and PDI of nanoparticles produced by chip S2 with shorter Lc were slightly larger than those of chip S2 when compared with chip S2. This indicates that when using the single-layer corrugated mixing unit, when Lc decreased from 1 mm for S1 to 0.5 mm for S2, the increase in flow rate (from 1 mL / min to 6 mL / min) may have caused mixing to become too vigorous, resulting in aggregation.

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

[0175] Meanwhile, the effect of mixing units with different number of layers was compared when designing the same waveform. Compared with single layer S1, bilayer D-B1 obtained lower particle size at 1 mL / min. When the speed was increased to 6 mL / min, bilayer D-B1 had the same particle size compared with single layer S1.

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

[0177] In summary, as can be seen from Figure 10, the suitable flow rate for each chip is different, Y1 is suitable for 6 mL / min, T1 is suitable for 6 mL / min, S1 and S2 have large particle size and small PDI at 1 mL / min, and small particle size and large PDI at 6 mL / min, neither flow rate is ideal. In these chips, D-B1 has good particle size and PDI at 1 mL / min and 4 mL / min, especially at 1 mL / min.

[0178] Example 2: Proportional expansion of microstructured mixing units In this example, the mixing units numbered Y2, S3 and D-B4 in Table 1 were used as chips. These three chips were made by proportionally scaling up the size of Y1, S1 and D-B2 by 200%. Proportional scaling means that the length, width and height are all increased by a specified ratio. The angles between the inlet and the junction of these three chips are the same, but the difference is that the mixing section of Y2 is straight, S3 is a single-layer corrugated mixing unit, and D-B4 is a two-layer corrugated mixing unit.

[0179] The test method is as described above. Preparation of LNPs ". Different flow rates were tried. Specifically, six total flow rates of 1, 6, 12, 20, 24, and 30 mL / min were tried for the three types of mixing units. The flow rate of 4 mL / min, which had a good effect in Example 1, was also tried for the two types of waveform mixing units. The statistical results of the PDI and volumetric particle size of the self-assembling nanoparticles produced by the chips including these mixing units are shown in Figure 11.

[0180] From FIG. 11, it can be seen that after being scaled up by 200% at each test flow rate, the particles obtained from mixing units Y2, S3, and D-B4 uniformly exhibited a decrease in particle size with increasing flow rate.

[0181] At the same time, we also found that increasing the flow rate to 20 mL / min increased the PDI of each group compared with the previous flow rate (although the PDI was still less than 0.3). This could be related to the vigorous mixing after the flow rate exceeded 20 mL / min, which caused aggregation. Especially at high flow rates, the PDI of D-B4 was larger than those of Y2 and S3, suggesting that the two-phase mixing process occurred more violently in the structure D-B4 chip 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 200% expanded corrugated bilayer structure D-B4 was smaller than that of 200% expanded corrugated monolayer structure S3, suggesting that at the above low flow rates, the mixing advantage of corrugated bilayer structure is obvious, and when the total flow rate is increased to 20 mL / min, the difference in particle size obtained with 200% expanded corrugated monolayer structure S3 and 200% expanded corrugated bilayer structure D-B4 became smaller.

[0183] The above results suggest that after enlarging the size, the mixing advantage of different layer numbers decreased with increasing total flow rate.

[0184] Among these three structures, D-B4 can obtain small and uniform particles with good mixing at 6 mL / min, which indicates that it requires less flow rate than the other two chips and is suitable for low working flow rates. S3 is suitable for working flow rates of 6 mL / min, 12 mL / min, 20 mL / min, and 24 mL / min, and Y2 is suitable for working flow rates of 12 mL / min and 20 mL / min.

[0185] In short, after proportional expansion, the single-layer and double-layer microstructure mixing units of the present invention both show good mixing effect. The corrugated microstructure mixing unit of the present invention can change the length and width of the flow channel within a certain range, and can produce nanoparticles with smaller and more uniform particle size.

[0186] (Example 3: Comparison of different Lc) In this example, the 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 enlarged 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. Preparation of LNPs". In comparing D-B1 and D-B2, different flow rates were tried. Specifically, total flow rates of 1 mL / min, 4 mL / min, and 6 mL / min were tried. The statistical results of the PD and mixing units of the self-assembled nanoparticles produced by the chips containing these mixing units are shown in Figure 13.

[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 diameters were comparable. This indicates that for the chip with a channel size of 0.5W × 0.5H, a longer Lc could better control the PDI. This may be because a shorter Lc would result in more vigorous mixing, leading to collision and aggregation of some nanoparticles.

[0189] The test method is as described above. Preparation of LNPs ". In comparing D-B4 and D-B5, different flow rates were tried. Specifically, seven total flow rates, including 1, 4, 6, 12, 20, 24, and 30 mL / min, were tried with the two types of mixing units. The statistical results of PDI, volumetric particle size, of the self-assembled nanoparticles produced by the chips containing these mixing units are shown in Figure 12.

[0190] From Figure 12, we can see that the volumetric particle size of the D-B5 group is larger at each flow rate compared to the D-B4 group. Among them, the volumetric particle size of the D-B5 group is close to that of the D-B4 group at flow rates of 12, 24, and 30 mL / min. This suggests that after the proportional expansion of the system, the increase in Lc may have reduced the intensity of local mixing, leading to larger particle size.

[0191] At the same time, this example suggests that in the proportionally expanded system, when the total flow rate is 1-30 mL / min, the observation results of the two groups 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 of the two groups exceeds 0.2, and the tendency of the PDI to increase with increasing flow rate becomes obvious. It is noteworthy that when the flow rate is equal to 12 mL / min, the particle sizes of D-B4 and D-B5 are the same, but the PDI of D-B5 is obviously smaller than that of D-B4, and LNPs with ideal particle size and PDI can be obtained.

[0192] This example suggests that by increasing Lc, the PDI of the LNPs produced can be more appropriately controlled, and therefore LNPs with ideal particle size and PDI can be obtained over a wider range of production flow rates.

[0193] Example 4: Comparison of different numbers of semicircular rings In this example, microfluidic chips were fabricated with mixing units numbered D-B2-3, D-B2-5, D-B2-7, and D-B2 in Table 1. These four types of mixing units are all double-layer corrugated mixing units, and the only difference is the number of semicircular rings in the corrugated part, i.e., the number of bends.

[0194] The test method is as described above. Preparation of LNPs ". Different flow rates were tested. Specifically, total flow rates of 1 mL / min, 4 mL / min, and 6 mL / min were tested for the four types of mixing units. The statistical results of PDI, volumetric particle size, of the self-assembled nanoparticles produced by the chips containing these mixing units are shown in Figure 20.

[0195] As can be seen from Figure 20, D-B2 and D-B2-7 are both suitable for 1 mL / min, while D-B2-3 and D-B2-5 had difficulty in producing LNPs with small size and good uniformity under these flow rates. The results of suitable flow rates for D-B2 were the same as other examples. The results in Figure 20 also show that the number of semicircular rings in the waveform mixing unit should not be too small, preferably at least six, more preferably at least seven.

[0196] Example 6: Comparison of single layer and multi-layer corrugated microstructure mixing units In this example, we fabricated microfluidic chips with mixing units numbered S1, D-A1, D-B1, D-C1, and T-B1 in Table 1, and each chip contained only one mixing unit. The chips used a similar design to that in Figure 1, except for the mixing section.

[0197] These chips were used to produce lipid nanoparticles, and parameters such as PDI and volume-based particle size of the resulting lipid nanoparticles were measured.

[0198] All five types of mixing units are corrugated microstructure mixing units. Except for the height of each layer of T-B1, the main parameters such as width, Do, Di, Lc, etc. 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 corrugated channel, the mixing sections of D-A1, D-B1, and D-C1 are two-layer corrugated channels, and the mixing section of T-B1 is a three-layer corrugated channel. The schematic diagrams and cross-sectional views of D-A1, D-B1, D-C1, and T-B1 are shown in Figures 5A-D, respectively, where the left side is a top perspective view and the right side is a schematic diagram of the corresponding cross-sectional shape.

[0199] As shown in FIG. 5, D-A1, D-B1, and D-C1 all have a two-layer flow channel. The inner edge of the narrow flow channel layer of D-A1 overlaps with the inner edge of the wide flow channel layer of the semicircular ring, and the inner edge of the narrow flow channel layer of D-B1 overlaps with the outer edge of the semicircular ring. When the length and shape of the wide flow channel layer are the same and the width of the narrow flow channel layer is also the same, the narrow flow channel layer of D-B1 is longer than the narrow flow channel layer of D-A1. Unlike D-A1 and D-B1, D-C1 has two-layer flow channels of the same width, and the width of the two-layer flow channels on the semicircular ring is shifted to form a cross section similar to the Z-shape of Tetris.

[0200] the above" Preparation of LNPs The flow rates were as described in ", and different total flow rates of 1, 4, and 6 mL / min were tested. The statistical results of the PDI and volumetric particle size of the self-assembled nanoparticles produced by the chips containing these mixing units are shown in Figure 9.

[0201] As can be seen from FIG. 9, when the total flow rate is 1 mL / min, the average particle size obtained when the chip is a single-layer structure (S1) is larger than the average particle size of the two-layer structure (D-A1, D-B1, D-C1) and three-layer structure (T-B1), and the average particle size obtained with the two-layer structure (D-A1, D-B1, D-C1) is smaller than the average particle size of the three-layer structure (T-B1). The particle size between the two-layer structure chips 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 two-layer structure performed better than the three-layer structure and single-layer structure. Among the two-layer structures, the D-B1 structure, in which the narrow part of the flow path is on the outside, was better than the other two-layer structures, and had excellent particle size and dispersibility. This is the same as the result of Example 1. In addition, D-A1 is not very preferable because it was difficult to control the PDI to 0.2 or less at each flow rate.

[0202] The results above show that, among 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 small particle sizes and uniform dispersibility.

[0203] Example 7: Number of layers of microstructure mixed unit In this embodiment, chips 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 rates were tested. The statistical results of the tested flow rates, PDI, and volumetric particle size are shown in Figure 14, where A is the result of S2 and D-B2, B is the result of S3 and D-B4, and C is the result of S4 and D-B7.

[0205] From Figure 14A, it can be seen that the particle size decreases with increasing flow rate, and the PDI increases with increasing flow rate. Comparing the two types of mixing units, the particle size of LNPs produced by bilayer D-B2 is small. Single layer S2 produced large particle size and small PDI at 1 mL / min, and small particle size and large PDI at 6 mL / min flow rate, indicating that neither flow rate was suitable. Bilayer D-B2 could produce LNPs with small size and good uniformity at 1 mL / min, but the PDI at 4 mL / min and 6 mL / min flow rates was too large.

[0206] From Figure 14B, it can be seen that the particle size decreased with the increase of flow rate, the PDI of the single layer decreased first and then increased, and the PDI of the double layer increased after the flow rate reached a certain value. Also, at the same flow rate, the LNP particle size produced by the double-layer D-B4 chip was generally smaller, which indicates that the mixing effect of the double-layer chip was better. However, when mixing with D-B4 after the flow rate exceeded 12 mL / min, aggregation may have occurred, which increased the PDI. However, when the flow rate was 30 mL / min, the PDI of S3 only increased slightly. S3 has good mixing effect at flow rates of 6 mL / min, 12 mL / min, 20 mL / min, and 24 mL / min, and D-B4 has the best mixing effect at 6 mL / min.

[0207] From Figure 14C, we can see that the particle size decreases with the increase of flow rate, and the PDI decreases first, and after the flow rate reaches 12 mL / min, the PDI increases. Comparing the monolayer and bilayer, the particle size of bilayer D-B7 is smaller than monolayer, and the PDI of D-B7 is smaller than S4 at first, and after the flow rate reaches 12 mL / min, the PDI is larger than S4. This is because the aqueous phase and the ethanol phase are more uniformly mixed in the bilayer chip, and the suitable flow rate is smaller. At 12 mL / min for S4 and 4 mL / min for D-B7, the goal of preparing LNPs with small particle size and good uniformity can be achieved.

[0208] In summary, the bilayer structure may be able to produce smaller nanoparticles at lower flow rates due to the effect of vigorous mixing. At the same time, the bilayer structure can produce nanoparticles with relatively smaller particle sizes and PDIs within a certain flow rate range than the monolayer structure.

[0209] Example 8: Enlargement of the microstructure mixing unit In this example, two groups of proportionally scaled mixing units were compared. Specifically, 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 another group, and chips were fabricated and compared within the groups. In the two groups, the difference between D-B1 and D-B2 was that the former had a longer Lc, as mentioned above. Within each group, the difference between each mixing unit was a multiple of the proportional scale of the channel size.

[0210] The test method was as described in "Preparation of LNPs", and the flow rates were 1, 4, 6, 12, 20, 24, and 30 mL / min. The statistical results of PDI and volumetric 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] From Fig. 15A, it can be seen that the internal pressure of the small channel was too high and was not suitable for high flow rates. D-B3 had a narrow channel and small particle size at low flow rates, but the results were reversed when the PDI was very large and the flow rate was 6 mL / min, indicating that it was difficult to find a suitable flow rate for the small channel to produce LNPs with good uniformity at small sizes. The normal channel D-B2 produced LNPs with good uniformity at a flow rate of 1 mL / min. It is speculated that the small channel D-B3 was suitable for lower flow rates, such as flow rates below 1 mL / min. It is noteworthy that D-B6 could not obtain LNPs with particle sizes below 100 nm even at a flow rate of 30 mL / min, indicating that the dual structure lost the desired mixing effect when the channel was enlarged to a certain size.

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

[0213] At 1 mL / min, the PDI of LNPs prepared by the three chips was very small. With the increase of flow rate, the PDI of D-B1 continued to increase, indicating that D-B1 was suitable for the low 1 mL / min flow rate among the tested flow rates, but not for the high flow rates. The PDI of D-B5 and D-B7 increased only when the flow rate was above 12 mL / min. As can be seen from the particle size results, the uniformity of LNPs prepared by D-B5 and D-B7 at 12 mL / min was both good, and the suitable flow rate was larger compared to D-B1.

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

[0215] Table 3. Optimal flow rates for different mixing units JPEG2025506436000006.jpg198170JPEG2025506436000007.jpg32170

[0216] Example 9: Observing the mixing effect using colored solutions 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 mixing effect is reflected by the difference in the color of the solutions before and after mixing. The ethanol phase before mixing is yellow, and the aqueous phase is blue. The liquid after mixing turns red, and the deeper the red of the liquid after mixing, the more completely it is mixed (as shown in Figure 23, the original photo is red, and the deeper the color, the deeper the red).

[0217] Y1, single-layer corrugated channel S1, and multi-layer corrugated channel D-B1 of the present invention were compared. The results are shown in Figure 23A. In Figure 23A, it can be seen that after passing through the mixing section at the same distance, the mixing effect of the three channels is the best in D-B1 and the worst in Y1 according to the color of the fluid. It can be seen from the figure that the two fluids in D-B1 are mixed first and have a darker color. It was also observed that in the channel with two-layer corrugated structure, the flow of the fluid is more stable and less disturbed ((undisturbed)), while in the single-layer corrugated channel, more disturbance occurs.

[0218] This test demonstrates the mixing advantage and stability of the two-layer SML channel of the present invention.

[0219] (Example 10: Mixing device with multiple parallel multi-layer waveform mixing units) A 4-stage parallel multi-layer corrugated microstructure mixing unit as shown in Figure 3 and a 16-stage parallel multi-layer corrugated microstructure mixing unit as shown in Figure 4 were fabricated. The coloring solution of Example 9 was used to reflect the mixing effect.

[0220] As shown in Figure 23B, the mixed fluid in the outlet channel of the 16-stage SML mixer turned a darker color (red in the original photo), indicating that the two input fluids were completely mixed.

[0221] (Example 11: Comparison with a non-wavy channel chip) In this example, a microfluidic chip was fabricated using the size and design of the mixing unit numbered D-B1 in Table 1, with the only difference being the use of nine semicircular rings, hence called D-B1 (9C). A non-corrugated channel chip of the prior art was used for comparison, specifically an NxGen microfluidic chip (Precision NanoSystems co., Inc., Canada) mounted on an Ignite model microfluidics device. This chip has 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 FIG. 21. As shown in FIG. 21B, the mixing unit of the NxGen microfluidic chip has multiple continuous ring structures in the mixing section and contains more channel branches.

[0222] To test the effect of comparing the microfluidic chip of the present invention with the conventional technology, GFP mRNA-loaded LNP samples were mixed and prepared using various flow paths listed in the following table. Specifically, the aqueous solution of GFP mRNA (citrate buffer, pH=4) and the ethanol phase (lipid working solution) were mixed at a volume ratio of 3:1 at different flow rates to prepare LNP samples.

[0223] For the ethanol phase, ethanol stock solutions of SM-102, DSPC (DSPC (distearoylphosphatidylcholine), Cholesterol, and DMG-PEG (distearoylphosphatidylethanolamine-polyethylene glycol) were prepared according to the concentrations shown in Table 4, and mixed uniformly at a certain volume ratio to obtain a lipid material working solution as an ethanol phase. The concentrations and molar ratios of various components in the lipid material working solution are shown in Table 4. Similarly, a 0.1 mg / mL GFP mRNA-specific 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 chip at a volume ratio of 1:3 (100 μL: 300 μL).

[0224] Table 4. mGFP LNP recipe JPEG2025506436000008.jpg60170

[0225] Different flow rates were tested. Specifically, total flow rates of 1 mL / min, 2 mL / min, 4 mL / min, and 12 mL / min were tested for the two types of chips. The statistical results of particle size and PDI of the GFP mRNA-loaded LNP sample are shown in Figure 22.

[0226] As can be seen from FIG. 22, both chips show a tendency that the particle size decreased with increasing flow rate. At flow rates of 4 mL / min and 12 mL / min, the LNP particle size produced by both chips was about 80 nm. At 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 LNP produced by both chips was controlled below 0.2 at all flow rates tested. This result indicates that compared with the conventional technology, the microstructured mixing unit of the present invention can obtain nanoparticles with smaller particle size at a lower flow rate.

Claims

1. A multi-layer corrugated (SML) microstructured mixing unit, comprising an inlet section, a confluence section, a multi-layer mixing section, and an outlet section, the inlet section and the confluence section being in fluid communication with each other; Each layer of the multi-layer mixing section is a corrugated flow channel, and the corrugated flow channel of each layer includes n semicircular rings or semi-elliptical rings, and the curvature directions of any two adjacent semicircular rings or semi-elliptical rings are opposite to each other and are connected by a straight flow channel; The projected shapes of two adjacent layers of the multi-layer mixing section always at least partially overlap each other on the flow path of the entire mixing section, but do not completely overlap each other; The inlet section includes at least two inlets, the inlets being in fluid communication with the junction section such that different fluids flowing through the inlets are joined at the junction section. SML Microstructure Mixing Unit.

2. 10. The SML microstructured mixing unit of claim 1, wherein the mixing section comprises two, three, four or five layers of the corrugated channels.

3. The SML microstructure mixing unit according to claim 1 or 2, wherein the corrugated channels of each layer each have a constant width, and the widths of the corrugated channels of each layer may be the same or different from each other, and it is preferable that the corrugated channels of at least two layers have different widths.

4. The SML microstructured mixing unit according to any one of claims 1 to 3, wherein the flow paths of each layer on the semicircular or semi-elliptical annular portion of the mixing section are always parallel.

5. 5. The SML microstructured mixing unit of claim 4, wherein the outer edges of the flow channels of each layer on the semicircular or semi-elliptical annulus of the mixing section always overlap.

6. 6. The SML microstructure mixing unit according to any one of claims 1 to 5, wherein (a) the mixing section comprises two layers of corrugated channels, and a cross section of the corrugated channels on the semicircular or semi-elliptical annular section is L-shaped; or (b) the mixing section comprises three layers of corrugated channels, and a cross section of the corrugated channels on the semicircular or semi-elliptical annular section is T-shaped rotated by 90 degrees.

7. The SML microstructure mixing unit according to any one of claims 1 to 6, wherein the outer diameter of each of the semicircular rings in the corrugated flow channel of each layer or the outer axis length Do of each of the elliptical rings in the extension direction of the corrugation is 3 to 5 times the width of the flow channel, and / or the inner diameter of each of the semicircular rings or the inner axis length Di of each of the elliptical rings in the extension direction of the corrugation is 1 to 3 times the width of the flow channel.

8. The SML microstructure mixing unit according to any one of claims 1 to 7, wherein each of the semicircular or semi-elliptical rings with opposite curvature directions is preferably connected by a straight flow path, and the length Lc of the straight flow path is 0.5W to 4W, preferably 1W to 3W, and more preferably 1W to 2W.

9. The SML microstructure mixing unit according to any one of claims 1 to 8, wherein n is an integer equal to or greater than 6, preferably an integer between 7 and 15.

10. The SML microstructure mixing unit according to any one of claims 1 to 9, comprising two or three layers of corrugated channels, each of the layers of corrugated channels comprising 6 to 10 of the semicircular rings, and the length Lc of the straight channel between each of the semicircular ring channels is greater than or equal to the inner diameter Di of the projected shape of the semicircular ring channel.

11. 11. The microstructure mixing unit according to any one of claims 1 to 10, wherein at least two layers in the corrugated channels have different widths, the width of the narrower layer being 25% to 75%, preferably around 50%, of the width of the wider layer.

12. A microstructure mixing apparatus, comprising m microstructure mixing units according to any one of claims 1 to 11 arranged in parallel, the m microstructure mixing units being in fluid communication with each other through an inlet section, a junction section and / or an outlet section, where m is an integer greater than 1.

13. 13. The microstructure mixer of claim 12, wherein m is an even number, preferably m is a power of two.

14. A method for mixing different fluids, comprising the steps of mixing a first fluid and a second fluid using a microstructured mixing unit according to any one of claims 1 to 11 or a microstructured mixing device according to claim 12 or 13, causing the first fluid to flow into a first inlet and the second fluid to flow into a second inlet.

15. A method for producing nanoparticles by self-assembly, comprising: mixing a first fluid and a second fluid using a microstructure mixing unit according to any one of claims 1 to 11 or a microstructure mixing device according to claim 12 or 13; flowing the first fluid into a first inlet and flowing the second fluid into a second inlet; the first fluid being a fluid containing a nanoparticle carrier material and the second fluid being a fluid containing a nanoparticle payload.

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