Microfluidic chip, microfluidic chip assembly and method for preparing delivery nanoparticles

Abstract

The present invention provides a microfluidic chip, a microfluidic chip assembly and a method for preparing delivery nanoparticles, comprising a chip body, a fluid flow channel and a mixing unit constructed in the chip body, the mixing unit comprising an inlet channel, the inlet channel is used to input other components other than the first component, the inlet channel is between the first inlet and the nanoparticle collection port and one end is connected to the fluid flow channel, and the other end is the second inlet, the mixing unit also includes a first obstacle structure constructed on the inner wall of the channel, when the first component is input at the first inlet, the first obstacle structure causes the fluid in the fluid flow channel to form a vortex on the side of the first obstacle structure close to the nanoparticle collection port by the Karman vortex street effect, and the fluid flowing out of the inlet channel is in the vortex formed by the first obstacle structure. The present invention effectively improves the encapsulation rate and reduces the empty package rate, and the size of the generated nanoparticles is controllable and uniform with strong repeatability.

Description

Technical Field

[0001] The present invention belongs to the field of microfluidics, and in particular relates to a microfluidics chip, a microfluidics chip component and a method for preparing delivery nanoparticles. Background Art

[0002] In recent years, RNA vaccines (COVID-19 mRNA, tumor mRNA vaccines, circRNA vaccines), small nucleic acid drugs (siRNA, ASO, miRNA) or other molecular drugs have received great attention, and the FDA has also issued new guidelines to encourage innovation in related gene therapy, including nucleic acid or other molecular drugs. Gene therapy uses nucleic acid or other molecular drugs as functional molecules to perform specific biological treatments for a variety of diseases (such as cancer, heart disease, cystic fibrosis, hemophilia, diabetes and AIDS, etc.).

[0003] Nanoscale delivery vectors are one of the core technical barriers of RNA vaccines, small nucleic acid drugs or other molecular drugs. Delivery vectors are responsible for protecting and allowing nucleic acid drugs or other molecular drug components to be effectively absorbed by the body through various body cell barriers, transported to the target site, and released at the right time to complete gene therapy and reduce toxic side effects. Non-viral nucleic acid or other molecular drug nano-delivery systems are mainly designed for liposomes, polymers, peptides and inorganic compounds, and their smaller toxic side effects make them more advantageous than viral vectors. At present, the main methods for preparing delivery nanoparticles are high-pressure homogenization, nanoprecipitation, material self-assembly, in-situ synthesis / polymerization, etc. However, the nanoparticles prepared by these methods have uneven structures, wide particle size distribution, complex synthesis steps and large batch differences, and the amount of reaction substrates and reagents required for synthesis is large, which is easy to cause waste of raw materials, which greatly limits its application in the preparation of delivery materials. In contrast, the use of microfluidic mixing technology to prepare nanoparticles has obvious advantages. This method is relatively simple and fast, the conditions are controllable, and it is easy to achieve production scale-up.

[0004] The channel size of the existing microfluidic chip is usually at the micro-nano level (without a protruding structure in the channel). The Reynolds number (Re) of the micro-nano scale fluid is small, usually less than 2300, the viscosity of the fluid is dominant, and the flow is in a laminar state. At this time, the turbulent mixing between the traditional two liquids will not be produced, that is, the liquids are not easy to mix. At present, the method of enhancing the mixing effect is mainly to hedge, stretch, and fold the fluid to increase the contact area between the fluids. Although the above mixing method is simple, the mixing uniformity is poor, the encapsulation rate is low, the particle size difference is large, and the empty package rate is high. In addition, the microfluidic chip currently for the synthesis of nucleic acid or other molecular drug delivery nanoparticles is basically designed in the form of nucleic acid or other molecular drug / aqueous phase-delivery material / organic phase two inlets, focusing on increasing the mixing effect of the two phases to achieve the purpose of high encapsulation rate. However, the components of nucleic acid or other molecular drug delivery materials are often more than one, and the order and proportion of contact between the components have a great influence on the physical properties and delivery efficiency of the delivery material. Summary of the invention

[0005] Therefore, the technical problem to be solved by the present invention is to provide a microfluidic chip, a microfluidic chip component and a method for preparing delivery nanoparticles, which can overcome the shortcomings of the prior art microfluidic chips that use a mixing method of hedging, stretching, and folding fluids to increase the contact area between fluids, such as poor mixing uniformity, low encapsulation rate, large particle size differences, and high empty package rate.

[0006] In order to solve the above problems, the present invention provides a microfluidic chip for generating molecular drug lipid delivery nanoparticles. The microfluidic chip includes a chip body, a fluid flow channel and at least one mixing unit are constructed in the chip body, the fluid flow channel has a first inlet for inputting a first component and a nanoparticle collection port, the mixing unit includes an inlet channel, the inlet channel is used to input other components other than the first component, the inlet channel is between the first inlet and the nanoparticle collection port and one end is connected to the fluid flow channel, and the other end is a second inlet, the mixing unit also includes a first obstacle structure constructed on the inner wall of the fluid flow channel between the first inlet and the inlet channel, when the first component is input at the first inlet, the first obstacle structure causes the fluid in the fluid flow channel to form a vortex on the side of the first obstacle structure close to the nanoparticle collection port due to the Karman vortex street effect, and the fluid flowing out of the inlet channel is in the vortex formed by the first obstacle structure.

[0007] In some embodiments, the first obstacle structure is a protrusion extending toward the inner side of the fluid flow channel, and along the flow direction of the fluid in the fluid flow channel, the extension length of the protrusion first increases and then decreases; and / or, the Reynolds number when the first component passes from the side of the first obstacle structure close to the first inlet to the side of the first obstacle structure close to the nanoparticle collection port is 90 to 200.

[0008] In some embodiments, the flow cross-section of the fluid flow channel is circular, and on a central axis surface of the fluid flow channel, the first obstacle structure is semicircular.

[0009] In some embodiments, the fluid flow channel extends in a straight line, the flow cross-section of the confluence channel is circular and extends in a straight line, the fluid flow channel between the confluence channel and the nanoparticle collection port is a first channel segment, an angle α1 is formed between the fluid flow channel and the first channel segment, 35°≤α1≤165°, and the α1 is positively correlated with the radius of the vortex; and / or, the diameter of the fluid flow channel is d, the maximum extension length of the first obstacle structure is s, d / 2≤s≤3d / 4.

[0010] In some embodiments, the flow mixing unit further includes a second obstacle structure constructed on an inner wall of the fluid flow channel between the inlet channel and the nanoparticle collecting port.

[0011] In some embodiments, the first obstacle structure and the second obstacle structure are located at opposite sides of the fluid flow channel; and / or the first obstacle structure and the second obstacle structure have the same shape and size.

[0012] In some embodiments, the flow mixing units have at least two, and at least two of the flow mixing units are arranged in sequence along the length extension direction of the fluid flow channel; and / or the fluid flow channel has a plurality of parallel and spaced ones, and the first inlets respectively provided in the fluid flow channels are combined into the same total inlet, the nanoparticle collection ports respectively provided in the fluid flow channels are combined into the same total collection port, and the second inlets of the flow mixing units respectively provided in the fluid flow channels for inputting the same component are combined into the same total inlet.

[0013] The present invention also provides a microfluidic chip assembly, which is formed by assembling the above-mentioned microfluidic chips in an upper and lower stack, wherein the first inlets of each of the upper and lower adjacent microfluidic chips are plugged and connected to each other, and the nanoparticle collection ports of each of the upper and lower adjacent microfluidic chips are plugged and connected to each other. When at least two of the mixing flow units are included, the second inlets of the mixing flow units of each of the upper and lower adjacent microfluidic chips are plugged and connected to each other.

[0014] The present invention also provides a method for preparing delivery nanoparticles, which is carried out using the above-mentioned microfluidic chip or microfluidic chip assembly, and the preparation method comprises:

[0015] Dissolving each nano-encapsulated component in a corresponding organic phase solution to form each encapsulated solution, and dissolving the component to be encapsulated in an aqueous phase solution to form a solution to be encapsulated;

[0016] The mixing unit closest to the first inlet is the first mixing unit, the solution to be encapsulated is connected to the second inlet of the first mixing unit, and each of the encapsulating solutions is connected to the first inlet and the second inlets of the remaining mixing units respectively along the direction from the first inlet to the nanoparticle collection port according to the order of each nano-encapsulating component relative to the component to be encapsulated in the formed delivery nanoparticle from inside to outside;

[0017] The to-be-wrapped solution and each wrapping solution are input into the fluid flow channel.

[0018] In some embodiments, the input speed of the coating solution input into the first inlet is controlled so that the Reynolds number Re of the coating solution when it flows to the first obstacle structure in the first flow mixing unit close to the nanoparticle collecting port is 90-200.

[0019] In some embodiments, the mixing unit closest to the nanoparticle collection port is a second mixing unit, each nanoparticle encapsulation component includes a dispersant, and the encapsulation solution formed by the dispersant is connected to the second inlet of the second mixing unit.

[0020] In some embodiments, the component to be encapsulated includes nucleic acid, the nano-encapsulation component further includes cationic lipid, cholesterol, phospholipid material DSPC, and the dispersant includes PEG2000.

[0021] In some embodiments, the concentration range of the nano-encapsulation component in each of the encapsulation solutions is 0.01 to 20 mM; and / or the solution to be encapsulated contains a buffer solution, and the buffer solution concentration is 0.01 to 1 μg / μL; and / or the input speed range of each of the encapsulation solutions and the solution to be encapsulated is 0.02 to 20 mL / min.

[0022] The present invention provides a microfluidic chip, a microfluidic chip assembly and a method for preparing delivery nanoparticles. A first obstacle structure is set on the inner wall of a fluid flow channel. When a first component fluid passes over the first obstacle structure, a velocity gradient is generated, boundary layer separation occurs, and a vortex is formed on the downstream side of the first obstacle structure. Nucleic acids or other molecular drug components (i.e., other components) are directly merged into the aforementioned vortex on the downstream side of the first obstacle structure. The two components complete wrapped mixing to obtain more complete electrostatic or ionic force bonding, which effectively improves the encapsulation rate and reduces the empty package rate. The size of the generated nanoparticles is controllable and uniform, and the repeatability is strong. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the overall structure of the microfluidic chip according to an embodiment of the present invention;

[0024] Figure 2 Schematic diagram of a local structure in which the first obstacle structure and the second obstacle structure in the microfluidic chip of an embodiment of the present invention have a semicircular cross section on the central axis plane of the fluid flow channel, and arrows in the figure indicate the flow directions of the corresponding component fluids;

[0025] Figure 3 This is a schematic diagram of a local structure in which the first obstacle structure and the second obstacle structure in the microfluidic chip of an embodiment of the present invention have a semi-elliptical cross-section on the central axis plane of the fluid flow channel, and the arrows in the figure indicate the flow directions of the corresponding component fluids.

[0026] Figure 4 This is a schematic diagram of a local structure in which the first obstacle structure and the second obstacle structure in the microfluidic chip of an embodiment of the present invention have isosceles trapezoidal cross-sections on the central axis plane of the fluid flow channel, and arrows in the figure indicate the flow directions of the corresponding component fluids.

[0027] Figure 5 This is a schematic diagram of a local structure in which the first obstacle structure and the second obstacle structure in the microfluidic chip of an embodiment of the present invention have isosceles triangle cross-sections on the central axis plane of the fluid flow channel, and arrows in the figure indicate the flow directions of the corresponding component fluids.

[0028] Figure 6 It is a schematic diagram of a microfluidic chip according to an embodiment of the present invention being connected to an external connection catheter in a specific application.

[0029] Figure 7This is a schematic diagram of the Karman vortex street effect on the fluid when the first obstacle structure of the present invention is semicircular, and the arrows in the figure indicate the flow directions of the corresponding component fluids.

[0030] Figure 8 The figure is a schematic diagram of the process of delivering nanoparticles formed by the microfluidic chip in the embodiment of the present invention in practical application.

[0031] Fig. 9 FIG. 4 is a schematic diagram of the structure of a microfluidic chip in another embodiment of the present invention.

[0032] Fig.10 Schematic diagram of the structure of a microfluidic chip assembly in an embodiment of the present invention.

[0033] The reference numerals are:

[0034] 1. Chip body; 2. Fluid flow channel; 21. First inlet; 22. Nanoparticle collection port; 3. Confluence channel; 30. Mixing flow unit; 31. Second inlet; 32. First obstacle structure; 33. Second obstacle structure; 41. Total inlet; 42. Total collection port; 43. Total confluence port. DETAILED DESCRIPTION

[0035] See also Figures 1 to 10 As shown, according to an embodiment of the present invention, a microfluidic chip is provided for generating molecular drug lipid delivery nanoparticles, wherein the aforementioned molecular drug lipid delivery nanoparticles are specifically nucleic acid drug lipid delivery nanoparticles and other molecular drug lipid delivery nanoparticles, the microfluidic chip comprises a chip body 1, wherein the chip body 1 is provided with a fluid flow channel 2 and at least one flow mixing unit 30, wherein the fluid flow channel 2 has a first inlet 21 for inputting a first component and a nanoparticle collecting port 22, wherein the flow mixing unit 30 comprises an inlet channel 3, wherein the inlet channel 3 is used for inputting other components other than the first component, wherein the inlet channel 3 is located between the first inlet 21 and the nanoparticle collecting port 22, wherein one end of the inlet channel 3 is connected to the fluid flow channel 2, and the other end is a second inlet 31, wherein the flow mixing unit 30 further comprises A first obstacle structure 32 is constructed on the inner wall of the fluid flow channel 2 between the first inlet 21 and the confluence channel 3. When the first component is input into the first inlet 21, the first obstacle structure 32 causes the fluid in the fluid flow channel 2 to form a vortex (also called a vortex) by the Karman vortex street effect on the side of the first obstacle structure 32 close to the nanoparticle collection port 22, and the fluid flowing out of the confluence channel 3 is in the vortex formed by the first obstacle structure 32. The aforementioned first component can be, for example, one of the encapsulated components or the components to be encapsulated, and the other components other than the first component can be at least the other of the encapsulated components or the components to be encapsulated, so as to achieve effective mixing and encapsulation of at least two components after flowing through the aforementioned first obstacle structure 32, thereby generating molecular drug lipid delivery nanoparticles.

[0036] In this technical solution, by setting the first obstacle structure 32 on the inner wall of the fluid flow channel 2, the first component (i.e., the encapsulated component) fluid generates a velocity gradient and boundary layer separation when passing over the first obstacle structure 32, and a vortex is formed on the downstream side of the first obstacle structure 32. The nucleic acid or other molecular drug components (i.e., other components) directly merge into the aforementioned vortex area on the downstream side of the first obstacle structure 32. The two components complete the encapsulation mixing to obtain a more sufficient electrostatic or ionic force combination, which effectively improves the encapsulation rate and reduces the empty package rate. The size of the generated nanoparticles is controllable, uniform, and highly repeatable. In some cases, the aforementioned first component may also be the component to be encapsulated, i.e., the aforementioned nucleic acid or other molecular drug component, and the other components are encapsulated components.

[0037] The material of the chip body 1 can specifically be one of polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, polystyrene (PS), silicon material, stainless steel, polyetheretherketone (PEEK), polytetrafluoroethylene (PTEE), polyethylene (PE), fluorinated ethylene propylene (FEP), ethylene tetrafluoroethylene (ETFE), polyethylene (PP), or a combination of two or more thereof.

[0038] The fluid flow cross-sectional shapes of the aforementioned inlet channel 3 and the fluid flow channel 2 can be various in theory, any one or combination of circular, semicircular, elliptical, trapezoidal, rectangular, triangular, polygonal, etc. In order to prevent the reduction of the fluid resistance along the way, the various shapes should be designed as arcs at the corners. In a specific embodiment, for example, when the shape is rectangular, the width of the rectangle ranges from 10 to 1000 μm, the depth ranges from 10 to 1000 μm, and the corresponding first inlet 21, nanoparticle collection port 22 and second inlet 31 have an inner diameter range of 10 to 1500 μm. In a preferred embodiment, the flow cross section of the fluid flow channel 2 (i.e., the fluid flow cross section) is circular, and the flow cross section of the inlet channel 3 is also preferably circular. The circular aforementioned channel is more convenient in terms of manufacturing and formation.

[0039] In some embodiments, on a central axis surface of the fluid flow channel 2, the cross-sectional shape of the first obstacle structure 32 can be various, for example Figure 3 The semi-ellipse shown in Figure 4 The isosceles trapezoid shown in Figure 5The triangle, rectangle, wing shape and other polygons shown in the figure, preferably in some embodiments, the first obstacle structure 32 is a protrusion extending toward the inner side of the fluid flow channel 2, and along the flow direction of the fluid in the fluid flow channel 2, the extension length of the protrusion first increases and then decreases, which effectively guides the flow of the fluid on the upstream side, and can more effectively form a vortex on the downstream side. In a preferred embodiment, the cross-sectional shape of the first obstacle structure 32 is semicircular. On the one hand, the use of a semicircular shape is conducive to reducing the difficulty of constructing the obstacle structure in the fluid flow channel 2, making it easier to process. More importantly, the use of a semicircular cross-section can form a secondary vortex on the downstream side of the obstacle structure. The formation of a secondary vortex can make the mixing of the two components that intersect here more complete, and the wrapping effect of the wrapped component on the component to be wrapped is better. See specifically. Figure 7 As shown, the first component (i.e., fluid u1 in the figure) flows into the fluid flow channel 2 from the upstream side of the first obstacle structure 32, and enters the downstream side of the first obstacle structure 32 after crossing the flow channel formed between the first obstacle structure 32 (from the direction of position a1-position a2-position a3) and the inner wall of the fluid flow channel 2. In this process, the first component forms a boundary layer under the action of the first obstacle structure 32. The existence of the boundary layer causes at least part of the fluid of the first component to deflect toward the direction a4 when it reaches the downstream side, forming a primary vortex (i.e., roughly the area at b in the figure). At the same time, other components (i.e., fluid u2 in the figure) converge from the confluence channel 3 roughly toward the downstream side of the first obstacle structure 32 (from the direction of position a4-position a3), and flow back under the disturbance of the fluid. The primary vortex formed by the first component further forms a secondary vortex (i.e., roughly the area at c in the figure), thereby further improving the mixing and encapsulation effect of the two components at this point, further improving the encapsulation rate, and reducing the empty package rate. Further, according to the Reynolds number calculation formula Re=ρvd / μ, ρ is the density (kg / m 3 ), v is the average flow velocity, i.e., the average flow velocity when crossing the first obstacle structure 32 (m / s), d is the diameter of the channel (i.e., the aforementioned fluid flow channel 2) (m), and μ is the liquid viscosity coefficient (s / m 2 ), in a specific embodiment, ρ is taken as ethanol density 0.789 g / cm at 20°C 3, μ is taken as 1.17×10^-3Pa·s, the average velocity of the fluid flowing through this place is taken as the ratio of volume velocity to cross-sectional area 2m / s, d is taken as the equivalent diameter of 4 times the hydraulic radius (100μm), and Re is equal to 134.87. According to relevant research: when the laminar flow velocity is small, water particles with different flow rates are distributed in layers and do not mix with each other, which has a poor effect on liquid mixing; when the turbulent flow velocity is large or changes, the water flow in the tube is full of vortices, and the flow of water particles with different flow rates mixes with each other, which has a better effect on liquid mixing; at the same time, combined with the Karman vortex street principle, when Re≈40, the viscous fluid flows around When passing through the cylinder, boundary layer separation occurs, and a pair of symmetrical vortices with opposite rotation directions are generated behind the cylinder; when Re = 40-70, the position of the symmetrical vortex is unstable, and the wake has periodic oscillations; when Re = 90-200, the vortices on the back flow surface are constantly produced and separated alternately, and form a glue map arrangement in the wake vortex area, with opposite rotation directions, regular (that is, vortex formation periodic law) and stable vortex structure, which is conducive to the effective mixing of the two components, and when Re is greater than 200, the vortex begins to fall off randomly, and the vortex formation period is irregular. As the Reynolds number increases, the randomness of vortex shedding also increases. That is, the Reynolds number when the first component passes from the side of the first obstacle structure 32 close to the first inlet 21 to the side of the first obstacle structure 32 close to the nanoparticle collection port 22 is 90-200, and the vortex shedding has a periodic law, so that the first component and the component entering the inlet channel 3 are more effectively mixed and wrapped here, and the size of the formed inclusions (also called wrapped particles, nanoparticles) is more uniform.

[0040] In some embodiments, the fluid flow channel 2 extends in a straight line, the confluence channel 3 also extends in a straight line, and each flow channel extends in a straight line, without a curved structure, dead space, or dead angle, which facilitates channel processing, is not easy to form blockage of the flow channel, and is easy to clean. The microfluidics can be reused, reducing production costs. Figure 1 and Figure 2 As shown, the fluid flow channel 2 between the inlet channel 3 and the nanoparticle collection port 22 is the first channel section, and an angle α1 is formed between the fluid flow channel 2 and the first channel section, 35°≤α1≤165°, and α1 is positively correlated with the radius of the vortex, that is, when the radius of the formed vortex is large, α1 can be designed to be relatively large, and when the radius of the formed vortex is small, α1 can be designed to be relatively small, so as to ensure that the different components are fully mixed and wrapped. In a specific embodiment, α1 = 45°.

[0041] In a specific embodiment, the diameter of the fluid flow channel 2 is d, the maximum extension length of the first obstacle structure 32 is s, d / 2≤s≤3d / 4, specifically, when the cross-sectional shape of the first obstacle structure 32 is semicircular, the aforementioned s is also the radius of the semicircle.

[0042] See Figure 2 As shown, the flow mixing unit 30 also includes a second obstacle structure 33 constructed on the inner wall of the fluid flow channel 2 between the inlet channel 3 and the nanoparticle collection port 22, which can further enhance the mixing and encapsulation of the components mixed before the obstacle structure, and further improve the size uniformity of the formed encapsulated particles. In a more preferred embodiment, the first obstacle structure 32 and the second obstacle structure 33 are respectively located on opposite sides of the fluid flow channel 2, that is, the rotation directions of the vortices formed after the fluid passes through the first obstacle structure 32 and the second obstacle structure 33 are opposite, and the reverse rotation is conducive to further improving the mixing and encapsulation effect. In order to further simplify the manufacturing difficulty, the first obstacle structure 32 and the second obstacle structure 33 have the same shape and size.

[0043] In some embodiments, there are at least two mixing units 30, and the specific number of mixing units 30 can be 2 to 10. At least two mixing units 30 are sequentially arranged along the length extension direction of the fluid flow channel 2, and the number thereof varies according to the structure of the synthetic product and the number of the encapsulated components. Multiple confluence channels 3 introduce components in a distributed manner to achieve precise control of the injection order and injection ratio of multiple components, for example Figure 1 The case with two mixing units 30 is shown in FIG. Figure 6 The figure shows a case where there are four mixing units 30. Specifically, the number of mixing units 30 can be set according to the specific packaging layer process of the drug delivery nanoparticles. In the present invention, a plurality of inlet channels 3 are set on the fluid flow channel 2 extending in a straight line, and the injection sequence and even the injection ratio of the multiple components can be precisely controlled by inputting different packages or components to be packaged into each inlet channel 3, so that the generated drug delivery nanoparticles are of higher quality. In a specific embodiment, the flow cross-section of the fluid flow channel 2 and the inlet channel 3 is circular with a diameter of 100 μm and the total pipe length L of the fluid flow channel 2 (i.e. Figure 6 The minimum straight line distance between the first inlet 21 and the nanoparticle collecting port 22 is 35m, the first inlet 21, the second inlet 31 and the nanoparticle collecting port 22 are circular holes with a diameter of 100 μm, Figure 6Also shown is the minimum straight-line spacing l1 between the first obstacle structure 32 and the second obstacle structure 33 in each mixing unit 30, l1 is preferably 100 μm, the minimum straight-line spacing l0 between the first obstacle structure 32 of the first mixing unit and the first inlet 21, l0 is preferably 10 mm, and the minimum straight-line spacing l2 between the second obstacle structure 33 of the previous mixing unit and the first obstacle structure 32 of the next mixing unit in two adjacent mixing units 30, l2 is preferably 500 μm, s=40 μm (that is, the diameter of the first obstacle structure 32 is 80 μm), so that the wrapping effect of each mixing unit on its own components can be guaranteed. In addition, the inclination angles of the confluence channels 3 in each mixing unit 30 can be equal, that is, unified as the aforementioned α1, α1=α2=α3=α4, that is, in other cases, depending on the different components to be mixed, α1, α2, α3, and α4 in the figure can be different, and the first obstacle structure 32 and the second obstacle structure 33 respectively possessed by each mixing unit 30 are preferably the same in shape and size.

[0044] See also Fig. 9 As shown, in another specific embodiment, the fluid flow channel 2 has a plurality of parallel and spaced first inlets 21 respectively provided in each fluid flow channel 2 are summarized in the same total inlet 41, the nanoparticle collecting ports 22 respectively provided in each fluid flow channel 2 are summarized in the same total collecting port 42, and the second inlets 31 of the mixing flow units 30 respectively provided in each fluid flow channel 2 for inputting the same component are summarized in the same total inlet 43, that is, the first inlets 21 are connected in parallel with each other, the nanoparticle collecting ports 22 are connected in parallel with each other, and the second inlets 31 of the same component are connected in parallel with each other, thereby realizing the synchronous quantitative input of multiple components with a simple structure, greatly improving the throughput of the microfluidic chip and improving the production efficiency.

[0045] According to the embodiments of the present invention, see Fig.10 As shown, a microfluidic chip assembly is also provided, which is formed by stacking and assembling the above-mentioned microfluidic chips, wherein the first inlets 21 respectively provided by the upper and lower adjacent microfluidic chips are plugged and connected to each other, and the nanoparticle collection ports 22 respectively provided by the upper and lower adjacent microfluidic chips are plugged and connected to each other. When at least two mixing flow units 30 are included, the second inlets 31 of the mixing flow units 30 for inputting the same component of the upper and lower adjacent microfluidic chips are plugged and connected to each other, so that a high-throughput nanoparticle production device can be formed by assembling and building the upper and lower microfluidic chips, which is conducive to expanding production. The minimum spacing between the fluid flow channels 2 respectively provided in the two upper and lower adjacent microfluidic chips is w1, and the depth of each fluid flow channel 2 in the upper and lower directions is w2, w1≥w2, and when the cross-section of the fluid flow channel 2 is circular, w2 is also the aforementioned d, so that the deformation of the fluid flow channel 2 caused by the high input pressure of various components can be prevented, and a smaller amplification effect is achieved.

[0046] According to an embodiment of the present invention, a method for preparing delivery nanoparticles is also provided, which is performed using the above-mentioned microfluidic chip or microfluidic chip assembly, and the preparation method comprises:

[0047] Dissolving each nano-encapsulated component in a corresponding organic phase solution to form each encapsulated solution, and dissolving the component to be encapsulated in an aqueous phase solution to form a solution to be encapsulated;

[0048] The mixing unit 30 closest to the first inlet 21 is the first mixing unit, which connects the solution to be encapsulated with the second inlet 31 of the first mixing unit, and connects each encapsulation solution with the first inlet 21 and the second inlets 31 of the remaining mixing units 30 along the direction from the first inlet 21 to the nanoparticle collection port 22 according to the order of each nano-encapsulation component from the inside to the outside in the formed delivery nanoparticle relative to the component to be encapsulated;

[0049] The solution to be coated and each coating solution are input into the fluid flow channel 2 .

[0050] This technical solution can utilize the distributed structure between the inlet channel 3 and the fluid flow channel 2 to introduce various components in a distributed manner, thereby achieving precise control of the injection sequence and injection ratio of multiple components.

[0051] The input speed of the encapsulated solution input into the first inlet 21 is controlled so that the Reynolds number Re of the encapsulated solution when it flows to the first obstacle structure 32 in the first mixed flow unit close to the nanoparticle collecting port 22 is 90-200, and the Reynolds number of the encapsulated solution when it passes from the side of the first obstacle structure 32 close to the first inlet 21 to the side of the first obstacle structure 32 close to the nanoparticle collecting port 22 is 90-200, and the vortex shedding has a periodic pattern, so that the encapsulated solution and the components (such as nucleic acid molecules) introduced into the inlet channel 3 are more effectively mixed and encapsulated here, and the size of the formed encapsulated objects is more uniform.

[0052] In some embodiments, the mixing unit 30 closest to the nanoparticle collection port 22 is a second mixing unit, each nanoparticle encapsulation component includes a dispersant, and the encapsulation solution formed by the dispersant is connected to the second inlet 31 of the second mixing unit. Figure 6 As shown in the example, the first mixing unit is the leftmost mixing unit, which is used to input the mRNA aqueous solution, and the second mixing unit is the rightmost mixing unit, which is used to input the dispersant (specifically, PEG lipid) organic phase (such as ethanol) solution. The components to be encapsulated include nucleic acids, and the nano-encapsulation components also include cationic lipids, cholesterol, and phospholipid material DSPC, and the dispersant includes PEG2000.

[0053] The concentration range of the nano-encapsulation components in each encapsulation solution is 0.01-20 mM to ensure the quality of the nanoparticles formed after the mixed encapsulation; and / or, the solution to be encapsulated contains a buffer solution with a concentration of 0.01-1 μg / μL. In a specific embodiment, when the component in the solution to be encapsulated is mRNA, the aforementioned buffer solution can specifically use sodium citrate buffer; in a specific embodiment, the input speed range of each encapsulation solution and the solution to be encapsulated is 0.02-20 mL / min. The aforementioned nucleic acid or other molecular drug delivery nanoparticles include but are not limited to liposomes, micelles, inorganic nanoparticles and polymer nanoparticles.

[0054] Specifically, when preparing nucleic acid or other molecular drug delivery nanoparticles, the specific operation steps are as follows:

[0055] 1) dissolving the encapsulated components in an organic phase solution and dissolving the nucleic acid or other molecular drugs in an aqueous phase solution;

[0056] 2) Using a syringe to absorb the aqueous phase solution and the organic phase solution, and then connecting the syringe interface to the first inlet 21 (or the main inlet 41) and each second inlet 31 (or the main inlet 43) of the microfluidic chip respectively;

[0057] 3) setting the syringe advancement speed according to the aqueous phase flow rate and the organic phase flow rate respectively, and starting the syringe pump system (existing technology) to prepare the product, wherein the microfluidic flow rate can be achieved by adjusting the flow rate of each input fluid and the ratio of each flow rate, and the fluid velocity range is 0.02 to 20 mL / min.

[0058] See also Figure 5 and Figure 8 As shown, the different components of mRNA / citrate buffer are introduced here, and the two-phase encapsulation is quickly mixed, and the negatively charged mRNA and the positively charged cationic lipids achieve efficient electrostatic binding. Other components such as cholesterol, PEG2000, PVA, etc. can also be further mixed one by one or mixed with the mixture components to form the desired nucleic acid drug lipid nanoencapsulation (i.e., nanoparticles).

[0059] In a specific embodiment, Figure 5As shown, the first inlet 21 is connected to the SM102 / ethanol solution with a concentration of 8mM, the second inlet 31 of the first mixed flow unit is connected to the mRNA / sodium citrate buffer solution with a concentration of 0.072μg / μL, the mixed flow unit adjacent to the first mixed flow unit is defined as the third mixed flow unit, the second inlet 31 of the third mixed flow unit is connected to the cholesterol / ethanol solution with a concentration of 6mM, the mixed flow unit between the third mixed flow unit and the second mixed flow unit is the fourth mixed flow unit, the second inlet 31 of the fourth mixed flow unit is connected to the DSPC / ethanol solution with a concentration of 2mM, and the second inlet 31 of the second mixed flow unit is connected to the PEG2000 / ethanol solution with a concentration of 0.24mM. After all solutions are prepared, they are filtered using a 0.22μm filter membrane, and then they are sucked into syringes respectively, connected to catheters and installed on injection pumps (not shown in the figure). Set the mRNA / citric acid buffer to 6000 μL / min and the lipid / ethanol solution to 2000 μL / min, run the device, and start collecting the liquid flowing out of the nanoparticle collection port 22 after the output liquid flow rate stabilizes. The collected liquid is diluted with 30 times the volume of PBS to obtain mRNA-lipid nanoparticles with small and uniform particle size. The aforementioned catheter can specifically use non-toxic and harmless connecting hoses such as polyethylene, polypropylene plastic, polyvinyl chloride, and polyolefin thermoplastic elastomer (TPE).

[0060] See also Figure 8 As shown, Figure 8 The synthesis process of mRNA-lipid nanoparticles is schematically shown. The cationic lipid (SM102) and the nucleic acid drug (mRNA) first come into contact and combine under the action of electrostatic force, and the hydrophobic end of the cationic lipid faces outward to form a vesicle that encapsulates the nucleic acid drug; then cholesterol and the phospholipid material DSPC are introduced successively, and are in full contact with the nucleic acid or other molecular drugs / cationic lipid vesicles; finally, PEG2000 is introduced, and its hydrophobic end contacts the hydrophobic end of SM102, cholesterol, and DCSP, and the hydrophilic end faces outward to form the final product, mRNA-lipid nanoparticles.

[0061] It is easy for those skilled in the art to understand that, under the premise of no conflict, the above-mentioned advantageous methods can be freely combined and superimposed.

[0062] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention shall be included in the protection scope of the present invention. The above are only preferred embodiments of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and variations can be made without departing from the technical principles of the present invention, and these improvements and variations should also be regarded as the protection scope of the present invention.

Claims

1. A method for generating molecular drug lipid delivery nanoparticles, wherein the molecular drug lipid delivery nanoparticles are generated through a fluid flow channel (2) and a flow mixing unit (30), wherein the fluid flow channel (2) has a first inlet (21) for inputting a first component and a nanoparticle collection port (22), wherein the flow mixing unit (30) comprises an inlet channel (3), wherein the inlet channel (3) is used to input other components other than the first component, wherein the inlet channel (3) is located between the first inlet (21) and the nanoparticle collection port (22) and one end of the inlet channel (3) is connected to the fluid flow channel (2), and the other end is a second inlet (31), wherein the flow mixing unit (30) further comprises a first obstacle structure (31) constructed on the inner wall of the fluid flow channel (2) between the first inlet (21) and the inlet channel (3) 2), when the first component is input into the first inlet (21), the first obstacle structure (32) causes the fluid in the fluid flow channel (2) to form a primary vortex on the side of the first obstacle structure (32) close to the nanoparticle collection port (22) by the Karman vortex street effect, and the fluid flowing out of the confluence channel (3) is in the primary vortex formed by the first obstacle structure (32); the first obstacle structure (32) and the confluence channel (3) are on the same side of the fluid flow channel (2), and at least part of the fluid in the fluid flow channel (2) forms a backflow under the disturbance of the fluid flowing out of the confluence channel (3), so that the primary vortex further forms a secondary vortex; The fluid flow channel (2) has a plurality of parallel and spaced-apartmental inlets, wherein the first inlets (21) respectively possessed by each fluid flow channel (2) are integrated into a common main inlet (41), the nanoparticle collection ports (22) respectively possessed by each fluid flow channel (2) are integrated into a common main collection port (42), and the second inlets (31) respectively possessed by each fluid flow channel (2) for inputting a mixing flow unit (30) of the same component are integrated into a common main inlet (43), that is, the first inlets (21) are connected in parallel to each other, the nanoparticle collection ports (22) are connected in parallel to each other, and the second inlets (31) of the same component are connected in parallel to each other.

2. The method according to claim 1, characterized in that The first obstacle structure (32) is a protrusion extending toward the inner side of the fluid flow channel (2), and along the flow direction of the fluid in the fluid flow channel (2), the extension length of the protrusion first increases and then decreases.

3. The method according to claim 1, characterized in that The Reynolds number when the first component passes from the side of the first obstacle structure (32) close to the first inlet (21) to the side of the first obstacle structure (32) close to the nanoparticle collection port (22) is 90-200.

4. The method according to claim 2, characterized in that: The flow cross-section of the fluid flow channel (2) is circular, and on a central axis surface of the fluid flow channel (2), the first obstacle structure (32) is semicircular.

5. The method according to claim 4, characterized in that The diameter of the fluid flow channel (2) is d, the maximum extension length of the first obstacle structure (32) is s, and d / 2≤s≤3d / 4.

6. The method according to claim 1, characterized in that The flow mixing unit (30) further comprises a second obstacle structure (33) constructed on the inner wall of the fluid flow channel (2) between the inlet channel (3) and the nanoparticle collection port (22); the inlet channel (3) is located on the fluid flow channel (2) between the first obstacle structure (32) and the second obstacle structure (33); and the first obstacle structure (32) and the second obstacle structure (33) are arranged adjacent to each other and are respectively located on both sides of the inlet channel (3).

7. The method according to claim 6, characterized in that The first obstacle structure (32) and the second obstacle structure (33) have the same shape and size.

8. The method according to claim 1, characterized in that There are at least two flow mixing units (30), and the at least two flow mixing units (30) are arranged in sequence along the length extension direction of the fluid flow channel (2).

Images