Micro-channel system, micro-fluidic chip, nano-particles and preparation method of nano-particles

Abstract

The invention discloses a micro-channel system, a micro-fluidic chip, nano-particles and a preparation method of the nano-particles. The micro-channel system is arranged on a substrate of the micro-fluidic chip and comprises a liquid inlet channel, a front-end main channel, a main mixing unit and a tail-end main channel which are communicated in sequence, the liquid inlet channel comprises N1 liquid inlet flow channels which are connected in parallel, and the tail ends of the N1 liquid inlet flow channels are converged through a convergence point and then are communicated with the front end of the front-end main channel; the main mixing unit comprises N2 annular micro-channel structures which are connected in series; each annular micro-channel structure comprises a main sub-channel and a secondary sub-channel, and the front ends and the tail ends of the main sub-channel and the secondary sub-channel are respectively connected to form an annular shape; and the width ratio of the main sub-channel to the secondary sub-channel is (1-6): 1. The nano-particles prepared by the micro-fluidic chip have high uniformity, small particle size and high encapsulation efficiency.

Description

Technical Field

[0001] The present invention relates to a microfluidic system, a microfluidic chip, nanoparticles and a method for preparing the same. Background Art

[0002] Microfluidic technology refers to the technology of precisely controlling the flow of fluids in microchannels (micrometers or millimeters) with different geometric shapes and different sizes, which has shown great application potential in disciplines such as medicine, physics, materials, machinery, chemistry, and energy.

[0003] The mixing of multiple liquids and the construction of micro / nanoparticles are common operation processes in current fields such as chemical engineering, materials, and biomedicine. Traditional liquid mixing and particle construction usually have defects such as poor homogeneity of mixing conditions or reaction conditions, low mass transfer and heat transfer efficiency, strong pollution, and low yield. However, through the use of a microfluidic chip, microfluidic technology can achieve uniform and rapid mixing of fluids, provide a stable and excellent self-assembly or nucleation environment for the generation of particles, and ensure batch-to-batch reproducibility. In addition, the preparation of particles using microfluidic technology also has many advantages such as simple preparation, short time consumption, less reagent consumption, environmental friendliness, and small space occupation of equipment.

[0004] The above technical advantages of microfluidic technology make it possible to construct cost-effective monodisperse nanoparticles. With the help of microchannels or microfluidic chips with different structures, many technical bottlenecks in drug delivery have been solved. For example, the stable encapsulation and delivery of nucleic acid drugs, such as ONPATTRO (2018, Alnylam Pharmaceuticals, Inc., siRNA-LNP), COMIRNATY (2020, Pfizer-BioNTech, mRNA-LNP), and SPIKEVAX (2020, Moderna, mRNA-LNP), all benefit from microfluidic technology.

[0005] Currently, however, the microfluidic chips used for drug delivery systems such as lipid nanoparticles are still relatively limited, and various microchannels and chips have certain defects. For example, the herringbone micromixer (SHM) based on obstacle design has limited flow rate, is prone to blockage, and has a small batch size, while the impingement jet mixer (IJM) is not suitable for low flow rates and has poor mixing effects at low flow rates.

[0006] In order to further overcome some of the problems existing in the above microfluidic chips or microchannels, it is necessary to design a microfluidic chip that can be highly efficient and rapid and is easy to transfer from the laboratory to industrialization, while realizing the construction of lipid nanoparticles within a wider flow rate range. Summary of the Invention

[0007] In order to solve the problems of poor mixing effect, poor uniformity of nanoparticles obtained, and large particle size existing in the prior art of microfluidic chips, the present invention provides a microchannel system, a microfluidic chip, nanoparticles and a preparation method thereof. The nanoparticles obtained by using the microfluidic chip can achieve high mixing efficiency and high encapsulation rate, and the obtained nanoparticles have small particle size and good uniformity.

[0008] The present invention mainly adopts the following technical solutions to solve the above technical problems:

[0009] The present invention provides a microchannel system, which is arranged on the substrate of the microfluidic chip. The microchannel system includes a liquid inlet channel (IFC), a front-end main channel (MC1), a main mixing unit (MMU) and a terminal main channel (MC2) that are connected in sequence;

[0010] The liquid inlet channel includes N1 parallel liquid inlet channels, where N1≥2; a liquid inlet (inlet) is provided at the front end of each liquid inlet channel, and the ends of the N1 liquid inlet channels are joined together through a convergence point (MP) and then communicated with the front end of the front-end main channel;

[0011] The main mixing unit includes N2 series-connected annular microchannel structures, where N2≥1; each annular microchannel structure includes a main sub-channel and a secondary sub-channel. The front ends and the ends of the main sub-channel and the secondary sub-channel are respectively connected to form an annular shape. The connection point at the front end is called a splitting point (SP), and the connection point at the end is called a combination point (CP); the splitting point of the first annular microchannel structure is communicated with the end of the front-end main channel, the combination point of the first annular microchannel structure is the splitting point of the second annular microchannel structure, and so on. The combination point of the last annular microchannel structure is communicated with the front end of the terminal main channel; the width ratio of the main sub-channel to the secondary sub-channel is (1-6):1;

[0012] The terminal main channel includes a liquid outlet channel and a liquid outlet (outlet) provided at the end of the liquid outlet channel.

[0013] In the present invention, the mixing principle of the fluid is symmetric / asymmetric splitting and recombination. When the width ratio of the main sub-channel to the secondary sub-channel is 1, it is symmetric splitting and recombination. At this time, the fluid undergoes continuous symmetric splitting, extrusion, and recombination, improving the mixing efficiency of the fluid. When the width ratio of the main sub-channel to the secondary sub-channel is not 1, it is asymmetric splitting and recombination. At this time, compared with symmetric splitting and recombination, the fluid undergoes continuous asymmetric splitting, extrusion, and recombination, further improving the mixing efficiency of the fluid. An increase in the width ratio of the main sub-channel to the secondary sub-channel will cause higher-intensity asymmetric splitting, which can further improve the mixing efficiency of the fluid. However, if the width ratio of the main sub-channel to the secondary sub-channel is too high, such as higher than 6:1, it may cause too large a pressure difference in each liquid inlet channel, resulting in situations such as backflow and countercurrent, which is not conducive to improving the mixing efficiency and the formation of uniform particles.

[0014] In the present invention, when the material flows through each channel, especially during the process of the main mixing unit, the material continuously splits, extrudes, and recombines. Substances between the material fluids and in the material fluids will undergo mutual diffusion and exchange, thereby promoting the homogeneity of each phase and meeting the requirements of thorough mixing and self-assembly of monodisperse particles in each phase.

[0015] In some preferred embodiments, the liquid inlet channel includes 2 parallel liquid inlet flow channels, and the liquid inlet flow channels form an angle at the confluence point. The size of the angle is preferably 0° - 180°, more preferably 45° - 180°, and even more preferably 60° - 180°, for example 90°. The size of this angle affects the shear force and contact area of the material entering the liquid inlet flow channels, thereby improving the mixing efficiency.

[0016] In some other preferred embodiments, the liquid inlet channel includes more than 3 parallel liquid inlet flow channels, and adjacent two liquid inlet flow channels form an angle at the confluence point, and the sizes of each angle are equal.

[0017] In the present invention, the width of the front-end main channel is preferably less than or equal to the minimum value of the width of the liquid inlet flow channels.

[0018] Among them, the ratio of the minimum value of the width of the liquid inlet flow channels to the width of the front-end main channel is preferably (1 - 5):1, more preferably (1 - 3):1, and even more preferably (2 - 3):1. The meaning of the minimum value of the width of the liquid inlet flow channels is that when the number of liquid inlet flow channels is greater than or equal to 2 and the widths of each liquid inlet flow channel are different, the smallest width among the multiple liquid inlet flow channels with different widths is the minimum value of the width of the liquid inlet flow channels.

[0019] In the present invention, the width of the liquid inlet flow channels is preferably 10 - 1000 μm, more preferably 50 - 500 μm, and even more preferably 100 - 300 μm.

[0020] In the present invention, the depth of the liquid inlet flow channel is preferably 10 - 500 μm, more preferably 50 - 400 μm, and even more preferably 100 - 300 μm. Herein, the meaning of the depth is the depth that the liquid inlet flow channel extends from the substrate surface into the substrate interior.

[0021] In the present invention, the length of the liquid inlet flow channel is preferably 10 - 35 mm, more preferably 15 - 30 mm, and even more preferably 18 - 25 mm.

[0022] In the present invention, the width of the front-end main channel is preferably 10 - 1000 μm, more preferably 50 - 500 μm, and even more preferably 100 - 300 μm.

[0023] In the present invention, the depth of the front-end main channel is preferably 10 - 500 μm, more preferably 50 - 400 μm, and even more preferably 100 - 300 μm. Herein, the meaning of the depth is the depth that the front-end main channel extends from the substrate surface into the substrate interior.

[0024] In the present invention, the length of the front-end main channel is preferably 100 - 2000 μm, more preferably 200 - 1000 μm, and even more preferably 400 - 800 μm.

[0025] In the present invention, the annular microchannel structure can be symmetric or asymmetric.

[0026] In the present invention, the main sub-channel and the secondary sub-channel are, for example, two complementary semi-circular arcs. The use of an arc design for the main sub-channel and the secondary sub-channel can avoid the generation of flow dead zones in the mixing channel, and compared with the herringbone staggered SHM microfluidic chip, the fluid flow is smoother and more continuous.

[0027] In the present invention, the width ratio of the main sub-channel to the secondary sub-channel is preferably (1 - 4):1, more preferably (1.5 - 3):1, and for example, 2:1.

[0028] In the present invention, the width of the main sub-channel is preferably 10 - 1000 μm, more preferably 20 - 500 μm, and even more preferably 50 - 300 μm.

[0029] In the present invention, the width of the secondary sub-channel is preferably 10 - 1000 μm, more preferably 20 - 500 μm, and even more preferably 50 - 300 μm.

[0030] In the present invention, the depth of the main sub-channel is preferably 10 - 500 μm, more preferably 50 - 400 μm, and even more preferably 100 - 300 μm. Herein, the meaning of the depth is the depth that the main sub-channel extends from the substrate surface into the substrate interior.

[0031] In the present invention, the depth of the secondary sub-channel is preferably 10 - 500 μm, more preferably 50 - 400 μm, and even more preferably 100 - 300 μm. Herein, the meaning of the depth is the depth that the secondary sub-channel extends from the substrate surface into the substrate interior.

[0032] In the present invention, the curvature radius ratio of the main sub-channel to the secondary sub-channel is preferably (0.8 - 2):1, more preferably (0.9 - 1.5):1, and even more preferably (1 - 1.3):1. This curvature radius ratio is beneficial to the symmetric or asymmetric splitting, extrusion, and recombination of the fluid, improving the mixing efficiency of the fluid. An increase in the curvature radius ratio will cause a higher-intensity asymmetric splitting, which can further improve the mixing efficiency of the fluid. However, if the curvature radius ratio is too high, it may cause too large a pressure difference in each liquid inlet channel, resulting in situations such as backflow and countercurrent, which is not conducive to improving the mixing efficiency and the formation of uniform particles.

[0033] In the present invention, the curvature radius of the main sub-channel is preferably 100 - 1000 μm, more preferably 200 - 800 μm, and even more preferably 400 - 700 μm.

[0034] In the present invention, the curvature radius of the secondary sub-channel is preferably 100 - 1000 μm, more preferably 200 - 800 μm, and even more preferably 400 - 700 μm.

[0035] Herein, the curvature radii of the main sub-channels of each of the annular micro-channel structures may be the same or different; the curvature radii of the secondary sub-channels of each of the annular micro-channel structures may be the same or different.

[0036] In the present invention, N2 is preferably 1 - 100, more preferably 2 - 30, and even more preferably 3 - 10, such as 4, 5, or 6.

[0037] In the present invention, the width of the terminal main channel is preferably 10 - 1000 μm, more preferably 50 - 500 μm, and even more preferably 100 - 300 μm.

[0038] In the present invention, the depth of the terminal main channel is preferably 10 - 500 μm, more preferably 50 - 400 μm, and even more preferably 100 - 300 μm. Herein, the meaning of the depth is the depth that the terminal main channel extends from the substrate surface into the substrate interior.

[0039] In the present invention, the length of the terminal main channel is preferably 0.01 - 10 cm, more preferably 0.1 - 8 cm, and even more preferably 1 - 5 cm.

[0040] In the present invention, the shape of the liquid outlet channel is preferably linear or curved. More preferably, the liquid outlet channel includes a linear front section and an arc-shaped rear section. Among them, the angle between the tangent of the outer edge of the arc-shaped rear section and the extension line of the linear front section is preferably 0° - 90°, more preferably 10° - 80°, and even more preferably 20° - 60°; the ratio of the length of the linear front section to the arc length of the arc-shaped rear section is preferably (0.1 - 5):1, more preferably (0.3 - 3):1, and even more preferably (0.5 - 2):1.

[0041] The present invention also provides a microfluidic chip, which includes a cover plate and a substrate, and the above-mentioned microchannel system is arranged on the substrate.

[0042] The applicable characteristics of the microfluidic chip of the present invention for rapid, thorough mixing of different fluids and self-assembly of monodisperse particles enable it to be used in various industries and for various purposes, such as compound synthesis, fine chemicals, drug delivery, analysis and detection, polymer materials, etc.

[0043] In the present invention, the materials of the cover plate and the substrate can be conventional in the art, for example, one or more of metal, silicon, quartz, ceramic, glass, Hastelloy, and polymer.

[0044] Among them, the metal is, for example, stainless steel.

[0045] Among them, the polymer is, for example, polymethyl methacrylate (PMMA), polycarbonate (PC), polystyrene (PS), polypropylene (PP), polyethylene (PE), polyamide (PI), polydimethylsiloxane (PDMS), polyethylene terephthalate (PET), polyvinyl chloride (PVC), photoresist, polyester (TPE), polyethylene glycol diacrylate (PEGDA), perfluorinated compound, polyurethane (PU), cycloolefin copolymer (COC), or cycloolefin polymer (COP).

[0046] Among them, the photoresist is, for example, SU-8 photoresist.

[0047] Among them, the perfluorinated compound is, for example, one or more of polyperfluoroethylene propylene (PFEP), perfluoroalkoxy (PFA), and perfluoropolyether (PFPE).

[0048] In the present invention, the number of the microchannel systems can be one or more.

[0049] In some preferred embodiments, the number of the microchannel systems is more than two, and the microchannel systems adopt a staggered design. This design can achieve effective utilization of space, is conducive to reducing the cost of the microfluidic chip, and improving the utilization rate.

[0050] In the present invention, during the actual application process, multiple microfluidic chips can be integrated and used in series and / or parallel according to the usage and batch requirements.

[0051] The present invention also provides a method for preparing nanoparticles, which uses the above-mentioned microfluidic chip and includes the following steps: at least two materials including a first material and a second material are respectively introduced into the liquid inlet channel through different liquid inlets, and react through the front-end main channel, the main mixing unit, and the end main channel in sequence to obtain nanoparticles.

[0052] In the present invention, the first material preferably includes one or more of lipid materials, lipid-like materials, and high molecular polymers.

[0053] Among them, the high molecular polymer is preferably a natural polymer with a specific charge, a synthetic polymer with a specific charge, or a semi-synthetic polymer with a specific charge. Here, the meaning of having a specific charge is carrying a positive charge or a negative charge, and the quantity of carrying a positive charge or a negative charge is not particularly limited in the present invention. A high molecular compound formed by covalent bonding of multiple small molecule units in nature, the meaning of the natural polymer in the present invention is a high molecular compound directly extracted from nature, the semi-synthetic polymer means a polymer formed by processing a natural polymer as a substrate, and the synthetic polymer is a high molecular compound obtained by artificial synthesis.

[0054] In the present invention, the first material preferably further includes a solvent, and the solvent is preferably an alcohol, such as ethanol.

[0055] In some preferred embodiments, the first material includes a lipid material and / or a lipid-like material, and the concentration of the total lipid in the first material is preferably 1-50 mM, more preferably 5-30 mM. Here, the definition of the concentration of the total lipid is the total molar concentration of the lipid material and / or the lipid-like material in the first material.

[0056] In the present invention, the second material preferably includes an active ingredient.

[0057] Among them, the active ingredient can be a small molecule compound and / or a biological macromolecule compound. The molecular weight of the small molecule compound is below 1000, and the molecular weight of the biological macromolecule compound is higher than 10000.

[0058] Among them, the biomacromolecular compound is, for example, one or more of antisense nucleic acid (ASO), small interfering RNA (siRNA), messenger RNA (mRNA), DNA, microRNA (miRNA), aptamer, ribozyme, antibody nucleic acid conjugate (ARC), and CRISPR-Cas9 antibody.

[0059] In the present invention, the second material preferably further includes a buffer solution.

[0060] Among them, the buffer solution is, for example, one or more of citrate buffer, sodium acetate buffer, and malic acid buffer.

[0061] Among them, the pH value of the second material is preferably 4 - 6.

[0062] In the present invention, the flow rate ratio of the first material to the second material is preferably 1:(1 - 10), more preferably 1:(2 - 7), such as 1:3, 1:4, 1:5, or 1:6.

[0063] In the present invention, the sum of the flow rates of the first material and the second material is preferably 0.001 - 300 mL / min, more preferably 0.01 - 150 mL / min, still more preferably 1 - 100 mL / min, preferably 1 - 20 mL / min, further preferably 4 - 20 mL / min, such as 6 mL / min, 8 mL / min, 9 mL / min, 12 mL / min, or 16 mL / min. Among them, the meaning of the sum of the flow rates is the addition of the flow rate of the first material and the flow rate of the second material. Among them, the sum of the flow rates of the first material and the second material is the tolerable total flow rate range of the microfluidic chip.

[0064] In the present invention, the nanoparticles are preferably liposomes, microspheres, vesicles, lipid nanoparticles, polymer nanoparticles, or microcapsules.

[0065] In the present invention, when there are two or more materials, each material can be independently introduced into multiple parallel inlet channels.

[0066] In some specific embodiments of the present invention, the number of inlet channels through which the first material is introduced is less than or equal to the number of inlet channels through which the second material is introduced.

[0067] In some specific embodiments of the present invention, the nanoparticles are lipid nanoparticles, the first material includes a lipid material and a lipidoid material, the lipid material is an ionizable lipid molecule, a co-lipid molecule, and cholesterol, the lipidoid material is a polymer-conjugated lipid molecule; the second material includes an active ingredient.

[0068] Among them, the ionizable lipid molecule means a lipid that can exhibit a cationic polar head under specific conditions; the co-lipid molecule means a natural or synthetic neutral lipid; the polymer-conjugated lipid molecule means a molecule containing a lipid moiety and a polymer moiety.

[0069] In the above embodiments, in the first material, the molar ratio of the ionizable lipid molecule, the co-lipid molecule, the cholesterol, and the polymer-conjugated lipid molecule is preferably (5 - 60):(5 - 60):(5 - 50):(1 - 10), more preferably (40 - 60):(5 - 20):(30 - 50):(0.5 - 5), and even more preferably (45 - 55):(8 - 12):(35 - 45):(1 - 2).

[0070] In the above embodiments, in the first material, the ionizable lipid molecule is, for example, methyl 4-(N,N-dimethylamino)butyrate (dilinoleoyl) (DLin-MC3-DMA); the co-lipid molecule is, for example, distearoylphosphatidylcholine (DSPC); the polymer-conjugated lipid molecule is, for example, 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol 2000 (DMG-PEG2000).

[0071] Among them, the molar ratio of DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 is, for example, 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6.

[0072] In the above embodiments, in the second material, the active ingredient includes eGFP-mRNA or siRNA. When the active ingredient is eGFP-mRNA, the mass ratio of the ionizable lipid molecule to the eGFP-mRNA is preferably (1 - 100):1, more preferably (2 - 50):1, and even more preferably (5 - 40):1, for example, 20:1. When the active ingredient is siRNA, the mass ratio of the ionizable lipid molecule to the siRNA is preferably (1 - 100):1, more preferably (2 - 50):1, and even more preferably (5 - 40):1.

[0073] In the present invention, those skilled in the art know that after the reaction through the terminal main channel and discharged through the liquid outlet, generally, it also includes steps of removing the solvent and filtration.

[0074] In the present invention, preferably, the material is introduced through a driving pump. For example, the material is pumped into through a precision driving pump through the liquid inlet.

[0075] In the present invention, the at least two materials can be miscible or immiscible.

[0076] In certain specific embodiments of the present invention, there is provided a method for preparing lipid nanoparticles, comprising the following steps:

[0077] (1) Dissolving an ionizable lipid molecule, a helper lipid molecule, cholesterol, and a polyethylene glycolylated lipid molecule in ethanol as a first material, and dissolving siRNA in a citrate buffer as a second material;

[0078] (2) Feeding the above-mentioned first material and second material respectively through two liquid inlets into different liquid inlet channels, and successively reacting through a front-end main channel, a main mixing unit, and a terminal main channel, and obtaining a lipid nanoparticle solution after the reaction, which is discharged from the liquid outlet;

[0079] (3) Removing ethanol from the above-mentioned lipid nanoparticle solution by dialysis or ultrafiltration, and making up the volume with a buffer solution, and the buffer solution can be, for example, phosphate buffer solution (PBS);

[0080] (4) Filtering the solution after volume adjustment through a sterile filter, and namely obtaining lipid nanoparticles, and the precision of the sterile filter can be, for example, 0.22 μm.

[0081] The present invention also provides a nanoparticle, which is prepared by the above method for preparing nanoparticles.

[0082] In the present invention, the nanoparticle is preferably a lipid nanoparticle, and the lipid nanoparticle can stably encapsulate and deliver an active ingredient or a therapeutic agent (such as a nucleic acid).

[0083] In the present invention, the particle size of the lipid nanoparticle is preferably 20 - 500 nm, more preferably 50 - 300 nm, still more preferably 70 - 150 nm, for example, 73 nm, 74 nm, 77 nm, 79 nm, 87 nm, 104 nm, 129 nm or 130 nm.

[0084] In the present invention, the PDI of the lipid nanoparticle is preferably 0.01 - 0.3, more preferably 0.02 - 0.2, still more preferably 0.03 - 0.15, for example, 0.04, 0.07, 0.08, 0.12 or 0.13. The PDI is the polydispersity index.

[0085] In the present invention, the nanoparticles comprise an active ingredient, and the encapsulation efficiency of the nanoparticles is preferably 80%-98%, more preferably 80%-98%, and even more preferably 90%-95%, such as 90.2%, 91.3%, 91.7%, 92.2%, 92.6%, 92.7% or 92.8%. Among them, the nanoparticles comprise a carrier and an active ingredient, and the active ingredient can be distributed in the cavity formed by the carrier wrapping, in the carrier and on the surface of the carrier. The meaning of the encapsulation efficiency is the mass percentage of the active ingredient encapsulated in the cavity formed by the carrier in the total active ingredient in the nanoparticles.

[0086] In the present invention, the nanoparticles can be used as the main component of a pharmaceutical composition for the treatment and prevention of various diseases. When the nanoparticles are used as the main component of the pharmaceutical composition, the pharmaceutical composition generally further comprises pharmaceutically acceptable excipients.

[0087] Among them, according to the Pharmacopoeia of the People's Republic of China (2020 Edition), the term "pharmaceutically acceptable excipients" generally refers to excipients and additives used in the production of drugs and the preparation of prescriptions, and all substances contained in pharmaceutical preparations except the active ingredient.

[0088] On the basis of conforming to the common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present invention.

[0089] The reagents and raw materials used in the present invention are all commercially available.

[0090] The positive and progressive effects of the present invention are as follows:

[0091] The microfluidic chip containing a microchannel system of the present invention is designed based on the principle of splitting-recombination. Flow rate driving and Dean vortices effectively promote the uniform and rapid mixing of various fluids and the self-assembly of particles. Compared with the impinging jet mixer IJM, it is more conducive to the formation of monodisperse particles at low flow rates, and thus has high applicability in both the early pilot product research stage and the later batch production stage of nanoparticles.

[0092] The nanoparticles prepared by using the microfluidic chip of the present invention have high uniformity, small particle size and high encapsulation efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0093] Figure 1 It is a schematic diagram of the microchannel system of Example 1.

[0094] Figure 2 It is a schematic diagram of the microchannel system of Example 2.

[0095] Figure 3 It is a schematic diagram of the microchannel system of Example 3.

[0096] Figure 4 Schematic diagram of the microchannel system of Example 4.

[0097] Figure 5 Schematic diagram of the main mixing unit in the microchannel system of Example 4.

[0098] Figure 6 Schematic diagram of the microchannel system of Example 5.

[0099] Figure 7 Schematic diagram of the main mixing unit in the microchannel system of Example 5.

[0100] Figure 8 Schematic diagram of the microchannel system of Example 6.

[0101] Figure 9 Schematic diagram of the main mixing unit in the microchannel system of Example 6.

[0102] Figure 10 Effect data graph of the mixing index of Examples 7 - 35.

[0103] Figure 11 Effect data graph of the mixing index of Examples 37 - 66.

[0104] Figure 12 Effect data graph of the mixing index of Examples 67 - 96.

[0105] Figure 13 Effect data graph of the particle size and PDI of Examples 97 - 104.

[0106] Figure 14 Effect data graph of the particle size and PDI of Examples 105 - 110.

[0107] Figure 15 Transmission electron microscope image of Example 1 magnified 30,000 times.

[0108] Figure 16 Transmission electron microscope image of Example 1 magnified 80,000 times.

[0109] Figure 17 Cryo - scanning electron microscope image (one) of Example 1 magnified 92,000 times.

[0110] Figure 18 Cryo - scanning electron microscope image (two) of Example 1 magnified 92,000 times.

[0111] Figure 19 Schematic diagram of a microfluidic chip with a staggered design for two microchannel systems.

[0112] The reference numerals are as follows:

[0113] 1 - Liquid inlet; 2 - Liquid inlet channel; 3 - Confluence point; 4 - Front-end main channel; 5 - Main mixing unit; 501 - Main sub-channel; 502 - Secondary sub-channel; 503 - Splitting point; 504 - Combining point; 505 - Merging point of splitting point and combining point; 6 - Terminal main channel; 7 - Liquid outlet. Detailed implementation mode

[0114] The present invention will be further described below by way of examples, but the present invention is not limited to the scope of the described examples. For the experimental methods without specific conditions in the following examples, they are carried out according to conventional methods and conditions, or selected according to the product instructions.

[0115] In the following examples and comparative examples of the present invention, the manufacturer of DLin-MC3-DMA is Lipoid GmbH; the manufacturer of DSPC is Lipoid GmbH; the manufacturer of cholesterol is Shanghai Avectro Pharmaceutical Technology Co., Ltd.; the manufacturer of DMG-PEG2000 is Lipoid GmbH; the manufacturer of eGFP-mRNA is CanSino Biologics Inc. The above DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 are all for injection grade.

[0116] Examples 1 - 3

[0117] Examples 1 - 3 were prepared with the following preparation method to prepare lipid nanoparticles:

[0118] (1) Mix DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 50:10:38.5:1.5 and dissolve them in absolute ethanol as the first material, and dissolve eGFP-mRNA in a citric acid buffer solution with a pH value of 4.0 as the second material; wherein, the mass ratio of DLin-MC3-DMA to eGFP-mRNA is 20:1;

[0119] (2) Inject the above first material and second material into the liquid inlet channel through different liquid inlets by an injection pump at a flow rate ratio of 1:3 and a total flow rate of 6 mL / min for the sum of the flow rates of the first material and the second material; react through the front-end main channel, the main mixing unit, and the terminal main channel in sequence to obtain a lipid nanoparticle solution;

[0120] (3) Dialyze the above lipid nanoparticle solution overnight in PBS and filter it with a 0.22 μm microporous membrane to obtain lipid nanoparticles.

[0121] The microchannel system in the microfluidic chip used in Example 1 is as Figure 1As shown, the number of liquid inlets and liquid inlet channels is 2, and the two liquid inlet channels are arranged at an angle of 60°; the microchannel system in the microfluidic chip used in Example 2 is as shown in Figure 2 As shown, the number of liquid inlets and liquid inlet channels is 2, and the two liquid inlet channels are arranged at an angle of 90°; the microchannel system in the microfluidic chip used in Example 3 is as shown in Figure 3 As shown, the number of liquid inlets and liquid inlet channels is 2, and the two liquid inlet channels are arranged at an angle of 180°. In the microchannel systems of the microfluidic chips used in Examples 1-3, the width of the main sub-channels is 300 μm, the depth of the main sub-channels is 200 μm, the depth of the secondary sub-channels is 200 μm, the curvature radius ratio of the main sub-channels to the secondary sub-channels is 1:1, and N2 is 6.

[0122] Figure 15 、 16 is the transmission electron micrograph of the lipid nanoparticles prepared in Example 1. Figure 17 、 18 is the cryo-scanning electron micrograph of the lipid nanoparticles prepared in Example 1. Among them, the instrument used for the transmission electron microscopy test is JEM-1400 of JEOL Ltd. (Japan), and the instrument used for the cryo-scanning electron microscopy test is FEI Talos F200C electron microscope, and the test conditions are 200 KV and the magnification is 92000.

[0123] The size and PDI of the lipid nanoparticles were determined by dynamic light scattering; the encapsulation efficiency was determined using the Quant it Ribogreen RNA quantification kit; the measured results are listed in Table 1 below.

[0124] Table 1

[0125]

[0126] From the results in the above table, it can be seen that the mixing efficiency of Examples 1-3 can reach more than 99%, and the average particle size of the prepared lipid nanoparticles is between 60-70 nm; PDI reflects the particle size distribution width of the prepared particles, and its range is between 0-1. The smaller the value, the more uniform the particle size. According to the above table, the PDI of the lipid nanoparticles in Examples 1-3 is close to 0, indicating that the particles in Examples 1-3 have good uniformity; the encapsulation efficiency of Examples 1-3 is all as high as more than 91%.

[0127] Examples 4-6 and Comparative Examples 1 and 2

[0128] Lipid nanoparticles were prepared in Examples 4-6 using the following preparation method:

[0129] (1) Mix DLin-MC3-DMA, DSPC, cholesterol, and DMG-PEG2000 in a molar ratio of 46.3:9.4:42.7:1.6 and dissolve them in absolute ethanol as the first material, and dissolve eGFP-mRNA in a citric acid buffer solution with a pH value of 4.0 as the second material; wherein, the mass ratio of DLin-MC3-DMA to eGFP-mRNA is 20:1;

[0130] (2) Inject the above-mentioned first material and second material into the liquid inlet channel through different liquid inlet ports by an injection pump at a flow rate ratio of 1:6 and a total flow rate of 12 mL / min for the sum of the flow rates of the first material and the second material; react successively through the front-end main channel, the main mixing unit, and the end main channel to obtain a lipid nanoparticle solution;

[0131] (3) Dialyze the above-mentioned lipid nanoparticle solution overnight in PBS and filter it with a 0.22 μm microporous membrane to obtain lipid nanoparticles.

[0132] The microchannel system in the microfluidic chip used in Example 4 is as Figure 4 shown, the number of its liquid inlet and liquid inlet channels is 2, the width ratio of its main sub-channel to its secondary sub-channel is 1:1, and the main mixing unit is as Figure 5 shown; the microchannel system in the microfluidic chip used in Example 5 is as Figure 6 shown, the number of its liquid inlet and liquid inlet channels is 2, the width ratio of its main sub-channel to its secondary sub-channel is 2:1; the main mixing unit is as Figure 7 shown; the microchannel system in the microfluidic chip used in Example 6 is as Figure 8 shown, the number of its liquid inlet and liquid inlet channels is 2, the width ratio of its main sub-channel to its secondary sub-channel is 3:1; the main mixing unit is as Figure 9 shown. In the microchannel systems of the microfluidic chips used in Examples 4-6, the width of the main sub-channel is 300 μm, the depth of the main sub-channel is 200 μm, the depth of the secondary sub-channel is 200 μm, the curvature radius ratio of the main sub-channel to the secondary sub-channel is 1:1, and N2 is 6.

[0133] In Comparative Example 1, the width ratio of the main sub-channel to the secondary sub-channel is 0.5:1, and the other conditions are the same as those in Example 4.

[0134] In Comparative Example 2, the width ratio of the main sub-channel to the secondary sub-channel is 7:1, and the other conditions are the same as those in Example 4.

[0135] The size and PDI of lipid nanoparticles were determined by dynamic light scattering; the encapsulation efficiency was determined using the Quant it Ribogreen RNA Quantification Kit; the above-mentioned tests were performed on Examples 4-6 and Comparative Examples 1 and 2, and the measured results are listed in Table 2.

[0136] Table 2

[0137]

[0138] From the results in the above table, it can be seen that the mixing efficiency of Examples 4-6 can reach more than 89%, and the average particle size of the prepared lipid nanoparticles is between 60-104 nm; the PDI of the lipid nanoparticles in Examples 4-6 is close to 0, indicating good particle uniformity in Examples 4-6; the encapsulation efficiency of Examples 1-3 is all above 92%. Compared with the angle between the two liquid inlet channels, the width ratio of the main sub-channel and the secondary sub-channel has a greater impact on the technical effect. According to the above results, when the width ratio of the main sub-channel and the secondary sub-channel increases from 1:1 to 2:1 or 3:1, the mixing efficiency increases significantly and the particle size decreases significantly.

[0139] Compared with Example 4, the width ratio of the main sub-channel and the secondary sub-channel in Comparative Example 1 is too small, resulting in a decrease in both the mixing efficiency and the encapsulation efficiency in Comparative Example 1, an increase in PDI, and uneven particle size.

[0140] Compared with Example 4, the width ratio of the main sub-channel and the secondary sub-channel in Comparative Example 2 is too large, resulting in a significant decrease in the particle size and encapsulation efficiency in Comparative Example 2, an increase in PDI, and uneven particle size.

[0141] Examples 7-36

[0142] The preparation method used in Examples 7-36 is the same as that in Example 4 except for the sum of the flow rates of the first material and the second material. The sum of the flow rates of the first material and the second material in Examples 7-35 is listed in Table 3 below.

[0143] The microfluidic chip used in Examples 7-36 is the same as that in Example 4 except for the number of annular microchannel structures. The number of annular microchannel structures in Examples 7-35 is listed in Figure 10 and Table 3 below, where Figure 10 the mixing index on the ordinate is the mixing efficiency in Table 3.

[0144] Table 3

[0145]

[0146]

[0147] Examples 37-66

[0148] Examples 37 - 66 were prepared in the same manner as Example 5, except that the sum of the flow rates of the first and second materials was different. The sum of the flow rates of the first and second materials in Examples 37 - 66 is listed in Table 3 below.

[0149] The microfluidic chips used in Examples 37 - 66 were the same as those in Example 5, except for the number of circular microchannel structures. The number of circular microchannel structures in Examples 37 - 66 is listed in Figure 11 and Table 4 below, where Figure 11 the mixing index on the vertical axis in is the mixing efficiency in Table 4.

[0150] Table 4

[0151]

[0152]

[0153] Examples 67 - 96

[0154] Examples 67 - 96 were prepared in the same manner as Example 6, except that the sum of the flow rates of the first and second materials was different. The sum of the flow rates of the first and second materials in Examples 67 - 96 is listed in Table 3 below.

[0155] The microfluidic chips used in Examples 67 - 96 were the same as those in Example 6, except for the number of circular microchannel structures. The number of circular microchannel structures in Examples 67 - 96 is listed in Figure 12 and Table 5 below, where Figure 12 the mixing index on the vertical axis in is the mixing efficiency in Table 5.

[0156] Table 5

[0157]

[0158]

[0159] According to the results in Tables 3 - 5 above, it can be seen that there is a correlation between the number of circular microchannel structures and the mixing efficiency. In the early stage, the mixing efficiency increases with the increase in the number of cycles, but when the mixing efficiency reaches 90%, it tends to be stable, and at this time, the increase in the number of cycles has no significant improvement on the mixing efficiency.

[0160] Examples 97 - 104

[0161] Examples 97 - 104 were prepared in the same manner as Example 1, except that the flow rate ratio of the first and second materials was different. The flow rate ratio of the first and second materials in Examples 97 - 104 is listed in Table 6 below. The particle size and PDI are as Figure 13and Table 6.

[0162] The microfluidic chip used in Examples 97 - 104 is the same as that in Example 1.

[0163] Table 6

[0164]

[0165] As can be seen from Table 6 above, after fixing the number of fixed annular microchannel structures and the sum of the flow rates of the first material and the second material, different flow rate ratios of the first material and the second material have an impact on the particle size and PDI of lipid nanoparticles. When the flow rate ratio is 1:(2 - 7), the particle size and PDI are smaller.

[0166] Examples 105 - 110

[0167] The preparation methods used in Examples 105 - 110 are the same as those in Example 1 except for the sum of the flow rates of the first material and the second material. The sum of the flow rates of the first material and the second material in Examples 105 - 110 is listed in Table 7 below, and the particle size and PDI are as Figure 14 and Table 7.

[0168] The microfluidic chip used in Examples 105 - 110 is the same as that in Example 1.

[0169] Table 7

[0170]

[0171] As can be seen from Table 7 above, after fixing the number of fixed annular microchannel structures and the flow rate ratio of the first material and the second material, different sums of the flow rates of the first material and the second material have an impact on the particle size and PDI of lipid nanoparticles. When the flow rate is in the range of 1 - 20 mL / min, lipid nanoparticles with a particle size below 200 nm can be prepared. When the flow rate is in the range of 4 - 20 mL / min, the particle size < 100 nm and PDI < 0.15.

[0172] Example 111

[0173] The microchannel system in the microfluidic chip used in Example 111 is as Figure 19 shown, where the number of microchannel systems is 2, and the microchannel systems adopt a staggered design.

Claims

1. A microfluidic channel system, characterized in that, The microchannel system is disposed on the substrate of the microfluidic chip. The microchannel system includes a liquid inlet channel, a front-end main channel, a main mixing unit, and a terminal main channel that are connected in sequence; The liquid inlet channel includes N1 parallel liquid inlet channels, where N1≥2; a liquid inlet is provided at the front end of each liquid inlet channel, and the ends of the N1 liquid inlet channels are joined at a confluence point and then connected to the front end of the front-end main channel; The main mixing unit includes N2 series-connected annular microchannel structures, where N2≥1; each annular microchannel structure includes a main sub-channel and a secondary sub-channel. The front and end of the main sub-channel and the secondary sub-channel are respectively connected to form a ring. The connection point at the front is called the splitting point, and the connection point at the end is called the combining point; the splitting point of the first annular microchannel structure is connected to the end of the front-end main channel, the combining point of the first annular microchannel structure is the splitting point of the second annular microchannel structure, and so on. The combining point of the last annular microchannel structure is connected to the front end of the terminal main channel; the width ratio of the main sub-channel to the secondary sub-channel is (1-6):1; The terminal main channel includes a liquid outlet channel and a liquid outlet provided at the end of the liquid outlet channel.

2. The microfluidic channel system according to claim 1, characterized in that, The width ratio of the main sub-channel to the secondary sub-channel is (1-4):1, preferably (1.5-3):1, for example 2:1; And / or, the width of the main sub-channel is 10-1000μm, preferably 20-500μm, more preferably 50-300μm; And / or, the width of the secondary sub-channel is 10-1000μm, preferably 20-500μm, more preferably 50-300μm; And / or, the depth of the main sub-channel is 10-500μm, preferably 50-400μm, more preferably 100-300μm; And / or, the depth of the secondary sub-channel is 10-500μm, preferably 50-400μm, more preferably 100-300μm; And / or, the curvature radius ratio of the main sub-channel to the secondary sub-channel is (0.8-2):1, preferably (0.9-1.5):1, more preferably (1-1.3):1; And / or, the curvature radius of the main sub-channel is 100-1000μm, preferably 200-800μm , more preferably 400 - 700 μm; And / or, the curvature radius of the secondary sub-channel is 100-1000μm, preferably 200-800μm, more preferably 400-700μm; And / or, N2 is 1-100, preferably 2-30, more preferably 3-10, for example 4, 5 or 6.

3. The microfluidic channel system according to claim 1, characterized in that, The liquid inlet channel includes 2 parallel liquid inlet channels. The liquid inlet channels form an angle at the confluence point. The size of the angle is 0°-180°, preferably 45°-180°, more preferably 60°-180°, for example 90°; And / or, the width of the front main channel is less than or equal to the minimum value of the width of the liquid inlet channel; wherein, the ratio of the minimum value of the width of the liquid inlet channel to the width of the front main channel is preferably (1 - 5):1, more preferably (1 - 3):1, and still more preferably (2 - 3):1; And / or, the width of the liquid inlet channel is 10 - 1000 μm, preferably 50 - 500 μm, more preferably 100 - 300 μm; And / or, the depth of the liquid inlet channel is 10 - 500 μm, preferably 50 - 400 μm, more preferably 100 - 300 μm; And / or, the length of the liquid inlet channel is 10 - 35 mm, preferably 15 - 30 mm, more preferably 18 - 25 mm; And / or, the width of the front main channel is 10 - 1000 μm, preferably 50 - 500 μm, more preferably 100 - 300 μm; And / or, the depth of the front main channel is 10 - 500 μm, preferably 50 - 400 μm, more preferably 100 - 300 μm; And / or, the length of the front main channel is 100 - 2000 μm, preferably 200 - 1000 μm, more preferably 400 - 800 μm.

4. The microfluidic channel system according to claim 1, characterized in that, The width of the end main channel is 10 - 1000 μm, preferably 50 - 500 μm, more preferably 100 - 300 μm; And / or, the depth of the end main channel is 10 - 500 μm, preferably 50 - 400 μm, more preferably 100 - 300 μm; And / or, the length of the end main channel is 0.01 - 10 cm, preferably 0.1 - 8 cm, more preferably 1 - 5 cm; And / or, the shape of the liquid outlet channel is linear or curved; preferably, the liquid outlet channel includes a linear front section and an arc-shaped rear section, wherein the angle between the tangent of the outer edge of the arc-shaped rear section and the extension line of the linear front section is preferably 0° - 90°, more preferably 10° - 80°, and still more preferably 20° - 60°; the ratio of the length of the linear front section to the arc length of the arc-shaped rear section is preferably (0.1 - 5):1, more preferably (0.3 - 3):1, and still more preferably (0.5 - 2):1; And / or, the main sub-channel and the secondary sub-channel are two complementary semi-circles.

5. A microfluidic chip, characterized in that, It includes a cover plate and a substrate, and the microchannel system as described in any one of claims 1 - 4 is provided on the substrate.

6. A method for preparing nanoparticles, characterized in that, It uses the microfluidic chip as described in claim 5, and it includes the following steps: respectively introducing at least two materials including a first material and a second material through different liquid inlet ports into the liquid inlet channel, and sequentially reacting through the front main channel, the main mixing unit, and the end main channel to obtain nanoparticles.

7. The method for preparing nanoparticles according to claim 6, characterized in that, The first material includes one or more of lipid materials, lipid-like materials, and high molecular polymers; Wherein, the high molecular polymer is preferably a natural polymer with a specific charge, a synthetic polymer with a specific charge, or a semi-synthetic polymer with a specific charge; Among them, the first material preferably further includes a solvent, and the solvent is preferably an alcohol, such as ethanol; Among them, when the first material includes a lipid material and / or a lipid-like material, the concentration of the total lipid in the first material is preferably 1-50 mM, more preferably 5-30 mM; And / or, the second material includes an active ingredient, and the active ingredient is preferably a small molecule compound and / or a biopolymer compound; the biopolymer compound is, for example, one or more of antisense nucleic acid, small interfering RNA, messenger RNA, DNA, microRNA, aptamer, ribozyme, antibody-nucleic acid conjugate drug, and CRISPR-Cas9 antibody; among them, the second material preferably further includes a buffer solution, and the buffer solution is, for example, one or more of citrate buffer solution, sodium acetate buffer solution, and malic acid buffer solution; among them, the pH value of the second material is preferably 4-6; And / or, the flow rate ratio of the first material to the second material is 1:(1-10), preferably 1:(2-7), such as 1:3, 1:4, 1:5, or 1:6; And / or, the sum of the flow rates of the first material and the second material is 0.001-300 mL / min, preferably 0.01-150 mL / min, more preferably 1-100 mL / min, further more preferably 1-20 mL / min, preferably 4-20 mL / min, such as 6 mL / min, 8 mL / min, 9 mL / min, 12 mL / min, or 16 mL / min; And / or, the nanoparticles are liposomes, microspheres, vesicles, lipid nanoparticles, polymer nanoparticles, or microcapsules.

8. The method for preparing nanoparticles according to claim 7, wherein, The nanoparticles are lipid nanoparticles, the first material includes a lipid material and a lipid-like material, the lipid material is an ionizable lipid molecule, a co-lipid molecule, and cholesterol, and the lipid-like material is a polymer-conjugated lipid molecule; the second material includes an active ingredient; Preferably, in the first material, the molar ratio of the ionizable lipid molecule, the co-lipid molecule, the cholesterol, and the polymer-conjugated lipid molecule is (5-60):(5-60):(5-50):(1-10), preferably (40-60):(5-20):(30-50):(0.5-5), more preferably (45-55):(8-12):(35-45):(1-2); Preferably, in the first material, the ionizable lipid molecule is DLin-MC3-DMA, the co-lipid molecule is DSPC, and the polymer-conjugated lipid molecule is DMG-PEG2000. Among them, the molar ratio of DLin-MC3-DMA:DSPC:cholesterol:DMG-PEG2000 is, for example, 50:10:38.5:1.5 or 46.3:9.4:42.7:1.6; Preferably, in the second material, the active ingredient includes eGFP-mRNA or siRNA; when the active ingredient is eGFP-mRNA, the mass ratio of the ionizable lipid molecule to the eGFP-mRNA is preferably (1-100):1, more preferably (2-50):1, still more preferably (5-40):1, such as 20:1; when the active ingredient is siRNA, the mass ratio of the ionizable lipid molecule to the siRNA is preferably (1-100):1, more preferably (2-50):1, still more preferably (5-40):

1.

9. A nanoparticle, wherein, It is prepared by using the preparation method of the nanoparticles according to any one of claims 6-8.

10. The nanoparticle according to claim 9, wherein, The nanoparticles are lipid nanoparticles; And / or, the particle size of the nanoparticles is 20-500 nm, preferably 50-300 nm, more preferably 70-150 nm, such as 73 nm, 74 nm, 77 nm, 79 nm, 87 nm, 104 nm, 129 nm or 130 nm; And / or, the PDI of the nanoparticles is 0.01-0.3, preferably 0.02-0.2, more preferably 0.03-0.15, such as 0.04, 0.07, 0.08, 0.12 or 0.13; And / or, the nanoparticles include an active ingredient, and the encapsulation rate of the nanoparticles is 80%-98%, preferably 80%-98%, more preferably 90%-95%, such as 90.2%, 91.3%, 91.7%, 92.2%, 92.6%, 92.7% or 92.8%.

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