CN116037236A - Microfluidic chip, microfluidic chip assembly and preparation method of delivery nanoparticles - Google Patents
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Abstract
The invention provides a microfluidic chip, a microfluidic chip assembly and a preparation method for delivering nano particles, which comprise a chip body, wherein a fluid flow channel and a mixed flow unit are constructed in the chip body, the mixed flow unit comprises an inlet channel, the inlet channel is used for inputting other components except a first component, the inlet channel is positioned between a first inlet and a nano particle collecting port, one end of the inlet channel is communicated with the fluid flow channel, the other end of the inlet channel is a second inlet, the mixed flow unit further comprises a first barrier structure constructed on the inner wall of the channel, when the first component is input into the first inlet, the first barrier structure enables fluid in the fluid flow channel to form a karman vortex street effect on one side of the first barrier structure, which is close to the nano particle collecting port, to form a vortex, and fluid flowing out of the inlet channel is positioned in the vortex formed by the first barrier structure. The invention effectively improves the encapsulation efficiency, reduces the empty encapsulation efficiency, and the generated nano particles have controllable and uniform size and strong repeatability.
Description
Technical Field
The invention belongs to the technical field of microfluidics, and particularly relates to a microfluidic chip, a microfluidic chip assembly and a preparation method of delivery nanoparticles.
Background
In recent years, RNA vaccines (new coronamrnas, 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 released new guidelines to encourage innovation of related gene therapies including nucleic acids or other molecular drugs. Gene therapy uses nucleic acids or other molecular drugs as functional molecules for specific biological therapy against a variety of diseases (e.g., cancer, heart disease, cystic fibrosis, hemophilia, diabetes, AIDS, etc.).
The delivery carrier based on nano level is one of the core technical barriers of RNA vaccine, small nucleic acid medicine or other molecular medicine, and is responsible for protecting and allowing nucleic acid medicine or other molecular medicine components to be effectively absorbed by the body, transported to target sites, released at proper time, and completed gene therapy and reduced toxic and side effects. Non-viral nucleic acid or other molecular drug nano-delivery systems are designed primarily for liposomes, polymers, polypeptides and inorganic compounds, with less toxic side effects making them more advantageous than viral vectors. The existing preparation methods for delivering nano particles mainly comprise a high-pressure homogenization method, a nano precipitation method, a material self-assembly method, an in-situ synthesis/polymerization method and the like, however, the nano particles prepared by the methods are nonuniform in structure, wide in particle size distribution, complex in synthesis steps and large in batch-to-batch difference, and the raw materials are easy to waste due to large reaction substrates and large reagent amounts required by synthesis, so that the application of the nano particles in preparing the delivering materials is limited to a great extent. In contrast, the microfluidic mixing technology is adopted to prepare the nano particles, and the method is relatively simple, convenient and rapid, has controllable conditions and is easy to realize production amplification.
The size of the conventional microfluidic chip channel is usually in the micro-nano level (under the condition that no protruding structure exists in the channel), the Reynolds number (Re) of the micro-nano fluid is smaller, usually smaller than 2300, the viscous force of the fluid is dominant, the flow is in a laminar flow state, and at the moment, turbulent mixing between the conventional two liquids cannot be generated, namely, the liquids are not easy to mix. At present, the mode of enhancing the mixing effect is mainly to increase the contact area between fluids by opposite-impact, stretching and folding fluids, and the mixing mode is simple, but has poor mixing uniformity, lower encapsulation efficiency, larger particle size difference and higher blank inclusion rate. In addition, the microfluidic chip for nucleic acid or other molecular drug delivery nanoparticle synthesis is designed in a manner of two inlets of nucleic acid or other molecular drug/water phase-delivery material/organic phase, and the aim of achieving high encapsulation efficiency is focused on increasing the mixing effect of the two phases. However, the components of nucleic acid or other molecular drug delivery materials are often more than one, and the order and ratio of the contact between the components has a great influence on the physical properties and delivery efficiency of the delivery materials.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a microfluidic chip, a microfluidic chip assembly and a preparation method for delivering nano particles, which can overcome the defects of poor mixing uniformity, low encapsulation efficiency, large particle size difference, high blank rate and the like in a mixing mode that the contact area between fluids is increased by using opposite-flow, stretching and folding fluids in the microfluidic chip in the prior art.
In order to solve the above problems, the present invention provides a microfluidic chip for generating molecular drug lipid delivery nanoparticles, the microfluidic chip comprising a chip body, in which a fluid flow channel and at least one mixing flow unit are configured, the fluid flow channel has a first inlet for inputting a first component, a nanoparticle collection port, the mixing flow unit comprises an inlet channel for inputting other components except the first component, the inlet channel is located between the first inlet and the nanoparticle collection port, one end of the inlet channel is communicated with the fluid flow channel, and the other end of the inlet channel is a second inlet, the mixing flow unit further comprises a first barrier structure configured on an inner wall of the fluid flow channel between the first inlet and the inlet channel, when the first inlet inputs the first component, the first barrier structure forms a karman vortex effect on one side of the first barrier structure, which is close to the nanoparticle collection port, and the fluid in the fluid flow channel forms a vortex effect on one side of the first barrier structure, which forms a vortex in the first vortex structure.
In some embodiments, the first barrier is a protrusion extending toward an inner side of the fluid flow channel, an extension length of the protrusion increasing and then decreasing along a flow direction of the fluid within the fluid flow channel; and/or the Reynolds number of the first component when the first component passes from one side of the first barrier structure close to the first inlet to one side of the first barrier structure close to the nanoparticle collection port is 90-200.
In some embodiments, the flow passage has a circular flow cross-section, and the first barrier structure is semicircular in shape on a central axial plane of the flow passage.
In some embodiments, the fluid flow channel extends along a straight line, the through-flow section of the converging channel is circular and extends along a straight line, the fluid flow channel between the converging channel and the nanoparticle collection port is a first channel section, and an included angle alpha is formed between the fluid flow channel and the first channel section 1 ，35°≤α 1 Not more than 165 DEG, and the alpha 1 is positively correlated with the radius of the vortex; and/or the diameter of the fluid flow channel is d, and the maximum extension length of the first barrier structure is s, and d/2 is less than or equal to s is less than or equal to 3d/4.
In some embodiments, the flow mixing unit further comprises a second barrier structure configured on a channel inner wall of the fluid flow channel between the sink channel and the nanoparticle collection port.
In some embodiments, the first barrier structure and the second barrier structure are on opposite sides of the fluid flow channel, respectively; and/or the first barrier structure and the second barrier structure are the same in shape and size.
In some embodiments, the mixed flow unit has at least two, and at least two of the mixed flow units are sequentially disposed along a length extension direction of the fluid flow channel; and/or the fluid flow channels are provided with a plurality of parallel intervals, the first inlets of the fluid flow channels are respectively integrated into the same total inlet, the nanoparticle collecting ports of the fluid flow channels are respectively integrated into the same total collecting port, and the second inlets of the mixing units for inputting the same component of the fluid flow channels are respectively integrated into the same total integrating port.
The invention also provides a microfluidic chip assembly, which is formed by stacking and assembling the microfluidic chips up and down, wherein the first inlets respectively arranged on the microfluidic chips adjacent up and down are mutually connected in an inserting way, the nanoparticle collecting ports respectively arranged on the microfluidic chips adjacent up and down are mutually connected in an inserting way, and when the microfluidic chip assembly comprises at least two mixing units, the second inlets of the mixing units for inputting the same component are mutually connected in an inserting way.
The invention also provides a preparation method of the delivery nano-particles, which is carried out by adopting the micro-fluidic chip or the micro-fluidic chip assembly, and comprises the following steps: respectively dissolving each nano coating component in a corresponding organic phase solution to form each coating solution, and dissolving the component to be coated in an aqueous phase solution to form a solution to be coated; the flow mixing unit closest to the first inlet is a first flow mixing unit, the solution to be wrapped is communicated with the second inlet of the first flow mixing unit, and each wrapping solution is respectively communicated with the first inlet and the second inlet of the rest flow mixing units along the direction from the first inlet to the nanoparticle collecting port according to the sequence of each nanometer wrapping component relative to the component to be wrapped in the formed delivery nanoparticle from inside to outside;
inputting the solution to be wrapped and each wrapping solution into the fluid flow channel.
In some embodiments, the input speed of the wrapping solution input by the first inlet is controlled so that the reynolds number Re of the wrapping solution flowing to the side of the first barrier structure in the first mixed flow unit near the nanoparticle collection port is 90 to 200.
In some embodiments, the mixed flow unit nearest to the nanoparticle collection port is a second mixed flow unit, each nanoparticle encapsulation composition includes a dispersant, and the encapsulation solution formed by the dispersant communicates with the second inlet provided by the second mixed flow unit.
In some embodiments, the component to be encapsulated comprises a nucleic acid, the nanocapsule composition further comprises a cationic lipid, cholesterol, phospholipid material DSPC, and the dispersing agent comprises PEG2000.
In some embodiments, the concentration of the nano-encapsulation component in each of the encapsulation solutions ranges from 0.01 mm to 20mm; and/or the solution to be wrapped contains buffer solution, and the concentration of the buffer solution is 0.01-1 mug/mu L; and/or the input speed range of each wrapping solution and the solution to be wrapped is 0.02-20 mL/min.
According to the microfluidic chip, the microfluidic chip assembly and the preparation method of the delivery nano particles, the first barrier structure is arranged on the inner wall of the fluid flow channel, the first component fluid generates a speed gradient when crossing the first barrier structure, boundary layer separation occurs, a vortex is formed on the downstream side of the first barrier structure, nucleic acid or other molecular medicine components (namely other components) directly enter the vortex on the downstream side of the first barrier structure, the two components are mixed in a wrapping mode to obtain more sufficient electrostatic or ionic force combination, the encapsulation rate is effectively improved, the blank rate is reduced, and the generated nano particles are controllable in size, uniform and high in repeatability.
Drawings
Fig. 1 is a schematic general structural diagram of a microfluidic chip according to an embodiment of the present invention.
Fig. 2 is a schematic diagram of a partial structure of a microfluidic chip according to an embodiment of the present invention, in which a first barrier structure and a second barrier structure are semicircular in cross section on a central axis surface of a fluid flow channel, and arrows in the figure show flow directions of corresponding component fluids.
Fig. 3 is a schematic diagram of a partial structure of a microfluidic chip according to an embodiment of the present invention, in which a first barrier structure and a second barrier structure are semi-elliptical in cross section on a central axis surface of a fluid flow channel, and arrows in the figure show flow directions of corresponding component fluids.
Fig. 4 is a schematic diagram of a partial structure of a first barrier structure and a second barrier structure of a microfluidic chip according to an embodiment of the present invention, in which the cross section of the first barrier structure and the second barrier structure on a central axis surface of a fluid flow channel is isosceles trapezoid, and arrows in the figure show flow directions of corresponding component fluids.
Fig. 5 is a schematic diagram of a partial structure of a micro-fluidic chip according to an embodiment of the present invention, in which a cross section of a first barrier structure and a cross section of a second barrier structure on a central axis surface of a fluid flow channel are isosceles triangles, and arrows in the figure show flow directions of corresponding component fluids.
Fig. 6 is a schematic diagram of a microfluidic chip according to an embodiment of the present invention connected to an external connection conduit in a specific application.
Fig. 7 is a schematic view showing the karman vortex street effect generated on the fluid when the first barrier structure is semicircular, and the arrows in the figure show the flowing directions of the corresponding component fluids.
Fig. 8 is a schematic process of delivering nanoparticles for the formation of microfluidic chips in practical applications in an embodiment of the present invention.
Fig. 9 is a schematic structural diagram of a microfluidic chip according to another embodiment of the present invention.
Fig. 10 is a schematic structural diagram of a microfluidic chip assembly according to an embodiment of the present invention.
The reference numerals are expressed as: 1. a chip body; 2. a fluid flow channel; 21. a first inlet; 22. a nanoparticle collection port; 3. an afflux channel; 30. a mixed flow unit; 31. a second inlet; 32. a first barrier structure; 33. a second barrier structure; 41. a main inlet; 42. a total collection port; 43. and a summary portal.
Description of the embodiments
Referring to fig. 1 to 10 in combination, according to an embodiment of the present invention, there is provided a microfluidic chip, for generating molecular drug lipid delivery nanoparticles, in particular nucleic acid drug lipid delivery nanoparticles and other molecular drug lipid delivery nanoparticles, the microfluidic chip comprises a chip body 1, a fluid flow channel 2 and at least one mixing flow unit 30 are configured in the chip body 1, the fluid flow channel 2 has a first inlet 21 for inputting a first component, a nanoparticle collection port 22, the mixing flow unit 30 comprises an inlet channel 3 for inputting other components than the first component, the inlet channel 3 is positioned between the first inlet 21 and the nanoparticle collection port 22 and is in communication with the fluid flow channel 2 at one end and with a second inlet 31 at the other end, the mixing flow unit 30 further comprises a first barrier structure 32 configured on the channel inner wall of the fluid flow channel 2 between the first inlet 21 and the inlet channel 3, when the first component is input into the first inlet 21, the first barrier structure 32 forms a karman vortex street effect on the side of the first barrier structure 32 near the nanoparticle collection port 22 to form a vortex (also referred to as a vortex), the fluid flowing out of the inlet channel 3 is in the vortex formed by the first barrier structure 32, and the first component can be, for example, one of a package component or a component to be packaged, while the other components than the first component are at least the other of the encapsulation component or the component to be encapsulated, to achieve efficient mixing and encapsulation of at least two components after flowing through the aforementioned first barrier structure 32, thereby generating molecular drug lipid delivery nanoparticles.
According to the technical scheme, the first barrier structure 32 is arranged on the inner wall of the fluid flow channel 2, the first component (namely, the wrapping component) fluid generates a speed gradient and generates boundary layer separation when passing through the first barrier structure 32, a vortex is formed at the downstream side of the first barrier structure 32, nucleic acid or other molecular medicine components (namely, other components) directly enter the vortex area at the downstream side of the first barrier structure 32, the wrapping type mixing of the two components is completed to obtain more sufficient electrostatic or ionic force combination, the encapsulation rate is effectively improved, the blank packing rate is reduced, and the generated nano particles are controllable in size, uniform and strong in repeatability. In some cases, the first component may also be the component to be encapsulated, i.e. the nucleic acid or other molecular drug component described above, while the other component is the encapsulation component.
The material of the chip body 1 may specifically be one or a combination of two or more of Polydimethylsiloxane (PDMS), polymethyl methacrylate (PMMA), glass, polystyrene (PS), a silicon material, stainless steel, polyetheretherketone (PEEK), polytetrafluoroethylene (PTEE), polyethylene (PE), fluorinated Ethylene Propylene (FEP), ethylene Tetrafluoroethylene (ETFE), and polyethylene (PP).
The cross-sectional shape of the inlet channel 3 and the fluid flow channel 2 may be any one or a combination of a plurality of circular, semicircular, elliptical, trapezoidal, rectangular, triangular, and polygonal shapes, and in order to prevent the reduction of the resistance of the fluid along the way, the various shapes should be designed to be arc-shaped at the corner, and in a specific embodiment, for example, rectangular, the rectangular shape has a width ranging from 10 μm to 1000 μm and a depth ranging from 10 μm to 1000 μm, and the corresponding inner diameters of the first inlet 21, the nanoparticle collection port 22, and the second inlet 31 range from 10 μm 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, which is more convenient in terms of manufacturing and forming.
In some embodiments, in one of the fluid flow channels 2The cross-sectional shape of the first barrier 32 on the central axis surface may be varied, such as a semi-oval shape as shown in fig. 3, an isosceles trapezoid shape as shown in fig. 4, a triangle shape as shown in fig. 5, a rectangle shape, a wing shape, and other polygons, and preferably in some embodiments, the first barrier 32 is a protrusion extending toward the inside of the fluid flow channel 2, and the extending length of the protrusion increases and decreases in the flow direction of the fluid in the fluid flow channel 2, so that the fluid flow is effectively guided on the upstream side, and a vortex can be more effectively formed on the downstream side. In a preferred embodiment, the cross-sectional shape of the first barrier structure 32 is semicircular, which is advantageous in reducing the difficulty of the construction of the barrier structure in the fluid flow channel 2, making the processing easier, and more importantly, the semicircular cross-section can form a secondary vortex on the downstream side of the barrier structure, so that the two components meeting with each other can be mixed more fully by forming the secondary vortex, and the wrapping effect of the wrapping component on the component to be wrapped is better. Referring specifically to FIG. 7, a first component (i.e., u in the figure 1 Fluid) flows into the fluid flow channel 2 from the upstream side of the first barrier 32 and passes over the flow channel formed between the first barrier 32 (from the position a 1-the position a 2-the position a 3) and the inner wall of the fluid flow channel 2 and then enters the downstream side of the first barrier 32, in which process the first component forms a boundary layer under the influence of the first barrier 32, the presence of which boundary layer causes at least part of the fluid to deflect in the direction a4 to form a primary vortex (i.e. approximately the region b in the figure) while the other components (i.e. u in the figure) 2 Fluid) flows back from the inflow channel 3 toward the downstream side of the first barrier 32 (from the position a4 to the position a 3), and the primary vortex formed by the first component forms a secondary vortex (i.e., the region c in the figure), so that the mixing and wrapping effect of the two components can be further improved, the encapsulation efficiency can be further improved, and the blank rate can be reduced. Further, according to the reynolds number calculation formula re=ρvd/μ, ρ is a liquid (fluid passing over the first barrier structure 32I.e. first component) density (kg/m 3 ) V is the average flow rate, i.e. the average flow rate (m/s) over the first barrier 32, d is the diameter (m) of the channel (i.e. the aforementioned fluid flow channel 2), μ is the liquid viscosity coefficient (s/m 2 ) In a specific embodiment, ρ is 0.789g/cm of ethanol density at 20deg.C 3 Mu is 1.17X10-3 Pa.s, the average flow rate of fluid flowing therethrough is the ratio of volume flow rate to cross-sectional area is 2 m/s, d is the equivalent diameter is 4 times the hydraulic radius (100 μm), re is equal to 134.87, and according to the related study: when the laminar flow velocity is smaller, water particles with different flow velocities are distributed in a layered manner and are not mixed with each other, so that the liquid mixing effect is poor; when the turbulent flow velocity is large or changed, the water flow in the pipe is full of vortex, and the water particles with different flow velocities are mixed and mixed with each other to flow, so that the liquid mixing effect is good; simultaneously, by combining with the karman vortex street principle, when Re is approximately equal to 40, viscous fluid bypasses the cylinder to generate boundary layer separation, and a pair of symmetrical vortices with opposite rotation directions are generated behind the cylinder; re=40-70 symmetrical vortex positions are unstable, and wake flow has periodic oscillation; when re=90-200, the vortex on the back flow surface is continuously and alternately produced and separated, a glue pattern arrangement is formed in the wake vortex area, the rotation directions are opposite, a regular (namely, the vortex forms a periodic rule) and stable vortex structure is beneficial to effectively mixing the two components, when Re is greater than 200, the vortex starts to fall off randomly, the vortex forms a periodic irregularity, and the randomness of vortex falling off is increased along with the increase of the Reynolds number. That is, the reynolds number of the first component passing over from the side of the first barrier structure 32 near the first inlet 21 to the side of the first barrier structure 32 near the nanoparticle collection port 22 is 90 to 200, and vortex shedding is periodic, so that the first component and the component entering at the inlet channel 3 form more effective mixing and wrapping at the position, and the sizes of the formed wrappers (also called wrapping particles and nanoparticles) are more uniform.
In some embodiments, the fluid flow channels 2 extend along a straight line, the inlet channels 3 extend along a straight line, and each flow channel extends along a straight line, so that there is no curved structure, no dead space or dead angle, and the channels are easy to process and the flow channels are not easy to formThe blocking of the microfluidic device is easy to clean, the microfluidic device can be reused, and the production cost is reduced. Referring to fig. 1 and 2 in combination, the fluid flow channel 2 between the inlet channel 3 and the nanoparticle collection port 22 is a first channel segment, and an included angle α is formed between the fluid flow channel 2 and the first channel segment 1 ，35°≤α 1 Less than or equal to 165 DEG and alpha 1 Positively correlated with the radius of the vortex, i.e. alpha when the radius of the vortex formed is large 1 Can be designed to be relatively large, and alpha when the radius of the vortex formed is small 1 Can be designed to be relatively small, so that the full mixing and wrapping of the different component ingredients can be ensured. In a specific embodiment, α 1 =45°。
In a specific embodiment, the fluid flow channel 2 has a diameter d and the first barrier 32 has a maximum extension of s, d/2. Ltoreq.s.ltoreq.3d/4, and in particular, when the first barrier 32 has a semicircular cross-sectional shape, s is the radius of the semicircle.
Referring specifically to fig. 2, the mixed flow unit 30 further includes a second barrier structure 33 configured 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 packing of the components mixed before the barrier structure and further enhance the size uniformity of the formed packed particles. In a more preferred embodiment, the first barrier 32 and the second barrier 33 are located on opposite sides of the fluid flow channel 2, respectively, i.e. the opposite direction of the vortex formed by the fluid after passing over the first barrier 32 and the second barrier 33, and the opposite direction of rotation is beneficial to further improving the mixing and packaging effect. To further simplify the manufacturing difficulty, the first barrier structure 32 and the second barrier structure 33 are identical in shape and size.
In some embodiments, the mixing units 30 have at least two, the number of specific mixing units 30 may be 2-10, at least two mixing units 30 are sequentially arranged along the length extending direction of the fluid flow channel 2, the number of the mixing units is changed according to the structure of the synthesized product and the number of the wrapping components, and the components are introduced in a distributed manner into the plurality of the converging channels 3 to realizeThe precise control of the injection sequence and the injection proportion of the multicomponent is shown in fig. 1, for example, in the case of two mixed flow units 30, while in fig. 6, in the case of four mixed flow units 30, specifically, the design number of the mixed flow units 30 can be set according to the coating layering process of specific drug delivery nanoparticles, while the invention is provided with a plurality of inlet channels 3 on the fluid flow channel 2 extending in a straight line substantially, so that the precise control of the injection sequence and even the injection proportion of the multicomponent can be realized by inputting different coatings or components to be coated into each inlet channel 3, and the quality of the generated drug delivery nanoparticles is higher. In a specific embodiment, the flow cross-section of the fluid flow channel 2 and the inlet channel 3 is circular and has a diameter of 100 μm, and the total path length L (i.e. the minimum linear distance between the first inlet 21 and the nanoparticle collection port 22 in FIG. 6) of the fluid flow channel 2 is 35m, the first inlet 21, the second inlet 31 and the nanoparticle collection port 22 are circular holes having a diameter of 100 μm, and the minimum linear distance L between the first barrier structure 32 and the second barrier structure 33 in each of the mixing units 30 is shown in FIG. 6 1 ，l 1 Preferably 100 μm, the minimum linear distance l between the first obstruction 32 of the first flow mixing unit and the first inlet 21 0 ，l 0 Preferably 10mm, the minimum linear distance l between the second barrier structure 33 of the preceding mixed flow unit and the first barrier structure 32 of the following mixed flow unit of the two adjacent mixed flow units 30 2 ，l 2 Preferably 500 μm, s=40 μm (i.e., the diameter of the first barrier structure 32 is 80 μm), so that the wrapping effect of each mixed flow unit on the respective components can be ensured. In addition, the inclination angles of the converging passages 3 in the respective mixing units 30 may be equal, that is, uniform to the above-mentioned α 1 ，α 1 =α 2 =α 3 =α 4 I.e. in other cases alpha in the figure depending on the components to be mixed 1 、α 2 、α 3 、α 4 The first barrier structure 32 and the second barrier structure 33 of each mixed flow unit 30 may be different from each other, and are preferably identical in shape and size.
Referring to fig. 9, in another specific embodiment, the fluid flow channels 2 have a plurality of parallel spaced apart first inlets 21 respectively provided in each fluid flow channel 2 are summarized in a same total inlet 41, the nanoparticle collecting ports 22 respectively provided in each fluid flow channel 2 are summarized in a same total collecting port 42, the second inlets 31 respectively provided in each fluid flow channel 2 for inputting the mixing units 30 of the same component are summarized in a same total collecting port 43, that is, each first inlet 21 is connected in parallel, the nanoparticle collecting ports 22 are connected in parallel, and each second inlet 31 of the same component is connected in parallel, so that the synchronous quantitative input of a plurality of components is realized, the structure is simple, and the flux of the microfluidic chip is greatly improved, and the production efficiency is improved.
According to an embodiment of the present invention, referring specifically to fig. 10, there is further provided a microfluidic chip assembly formed by stacking the above-mentioned microfluidic chips up and down, wherein the first inlets 21 respectively provided in the upper and lower adjacent microfluidic chips are connected to each other by plugging, the nanoparticle collecting ports 22 respectively provided in the upper and lower adjacent microfluidic chips are connected to each other by plugging, and when at least two mixing units 30 are included, the second inlets 31 of the mixing units 30 for inputting the same component are connected to each other by plugging, so that a high-flux nanoparticle production device can be formed by assembling the upper and lower microfluidic chips, which is beneficial to expansion of production. The minimum distance between the fluid flow channels 2 respectively arranged in the two micro-fluidic chips adjacent to each other is w 1 The depth of each fluid flow channel 2 in the up-down direction is w 2 ，w 1 ≥w 2 When the cross section of the fluid flow channel 2 is circular, w 2 I.e. d as described above, this can prevent deformation of the fluid flow path 2 caused when the input pressure of the various components is high, with less amplification effect.
According to an embodiment of the present invention, there is also provided a method for preparing a delivery nanoparticle, which is performed using the microfluidic chip or the microfluidic chip assembly described above, the method comprising: respectively dissolving each nano coating component in a corresponding organic phase solution to form each coating solution, and dissolving the component to be coated in an aqueous phase solution to form a solution to be coated; the flow mixing unit 30 nearest to the first inlet 21 is a first flow mixing unit, the solution to be wrapped is communicated with the second inlet 31 of the first flow mixing unit, and each wrapping solution is respectively communicated with the first inlet 21 and the second inlet 31 of the rest of the flow mixing units 30 along the direction from the first inlet 21 to the nanoparticle collecting port 22 according to the sequence of each nanoparticle wrapping component from inside to outside in the formed delivery nanoparticle relative to the to-be-wrapped component; the solution to be packed and each packing solution are fed into the fluid flow channel 2.
According to the technical scheme, each component can be introduced in a distributed mode by utilizing a distributed structure between the inlet channel 3 and the fluid flow channel 2, and the accurate regulation and control of the sampling sequence and the sampling proportion of multiple components can be realized.
The input speed of the wrapping solution input by the first inlet 21 is controlled so that the Reynolds number Re of the wrapping solution flowing to the side of the first barrier structure 32 close to the nanoparticle collection port 22 in the first mixed flow unit is 90-200, the Reynolds number of the wrapping solution flowing from the side of the first barrier structure 32 close to the first inlet 21 to the side of the first barrier structure 32 close to the nanoparticle collection port 22 is 90-200, vortex shedding is regular, so that the wrapping solution and components (such as nucleic acid molecules) collected at the collecting channel 3 can be mixed and wrapped more effectively, and the size of the formed wrapping object is more uniform.
In some embodiments, the mixed flow unit 30 closest to the nanoparticle collection port 22 is a second mixed flow unit, each nanoparticle encapsulation composition includes a dispersant, and the encapsulation solution formed by the dispersant communicates with a second inlet 31 provided in the second mixed flow unit. Specifically, as shown in fig. 6, the first mixing unit is the leftmost mixing unit for inputting the mRNA aqueous solution, and the second mixing unit is the rightmost mixing unit for inputting the dispersant (specifically, for example, PEG lipid) organic phase (for example, ethanol) solution. The component to be coated comprises nucleic acid, the nano-coating component also comprises cationic lipid, cholesterol and phospholipid material DSPC, and the dispersing agent comprises PEG2000.
The concentration range of the nano coating components in each coating solution is 0.01-20 mM, so that the quality of the nano particles formed after mixing and coating is ensured; and/or, the solution to be wrapped contains buffer solution, the concentration of the buffer solution is 0.01-1 mug/mu L, and in a specific embodiment, when the component in the solution to be wrapped is mRNA, the buffer solution can be sodium citrate buffer solution; in one embodiment, the input speed of each wrapping solution and the solution to be wrapped is in the range of 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 polymeric nanoparticles.
Specifically, when nucleic acid or other molecular drug delivery nanoparticles are prepared, the specific operation steps are as follows:
1) Dissolving the coating composition in an organic phase solution, and dissolving nucleic acids or other molecular drugs in an aqueous phase solution;
2) Sucking the aqueous phase solution and the organic phase solution by using a syringe, and connecting the syringe interfaces with the first inlet 21 (or the total inlet 41) and the second inlets 31 (or the total inlet 43) of the microfluidic chip respectively;
3) The method comprises the steps of respectively setting the propulsion speed of an injector according to the flow speed of an aqueous phase and the flow speed of an organic phase, and starting an injection pump system (in the prior art) to prepare a product, wherein the microfluidic flow speed can be realized by adjusting the flow speed of each input fluid to the flow rate ratio of each input fluid, and the fluid speed ranges from 0.02 mL/min to 20mL/min.
Referring to fig. 5 and 8, the mRNA/citric acid buffer solution of different components is combined, two-phase coated and rapidly mixed, and the mRNA with negative charges and the positive charged cationic lipid are combined in a high-efficiency static manner, so that other components such as cholesterol, PEG2000, PVA and the like can be further mixed one by one or with the mixture components to form the nucleic acid drug lipid nano-coating (i.e. nano-particles) of the intended purpose.
In a specific embodiment, as shown in FIG. 5, the first inlet 21 is connected to SM 102/ethanol solution with a concentration of 8mM, the second inlet 31 of the first mixed flow unit is connected to mRNA/sodium citrate buffer solution with a concentration of 0.072. Mu.g/. Mu.L, the mixed flow unit next to the first mixed flow unit is defined as a third mixed flow unit, the second inlet 31 of the third mixed flow unit is connected to 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 a fourth mixed flow unit, the second inlet 31 of the fourth mixed flow unit is connected to DSPC/ethanol solution with a concentration of 2mM, and the second inlet 31 of the second mixed flow unit is connected to PEG 2000/ethanol solution with a concentration of 0.24 mM. All solutions were prepared and filtered using a 0.22 μm filter and then separately aspirated into syringes, connected to tubing and mounted on syringe pumps (not shown). Setting 6000 mu L/min of mRNA/citric acid buffer solution, 2000 mu L/min of lipid/ethanol solution, operating equipment, starting to collect the effluent liquid from the nanoparticle collecting port 22 after the flow rate of output liquid is stable, and diluting the collected liquid by using 30 times of PBS (phosphate buffer solution), so that mRNA-lipid nanoparticles with small and uniform particle size can be obtained. The catheter can be a nontoxic and harmless connecting hose such as polyethylene, polypropylene plastic, polyvinyl chloride, polyolefin thermoplastic elastomer (TPE) and the like.
Referring further to fig. 8, fig. 8 illustrates the mRNA-lipid nanoparticle synthesis process, wherein the cationic lipid (SM 102) is first contacted with the nucleic acid drug (mRNA) to bind under electrostatic force, and the hydrophobic end of the cationic lipid faces outwards to form vesicles encapsulating the nucleic acid drug; cholesterol and phospholipid material DSPC are then introduced sequentially into sufficient contact with nucleic acid or other molecular drug/cationic lipid vesicles; finally PEG2000 was introduced with its hydrophobic end in contact with the hydrophobic end of SM102, cholesterol, DCSP and the hydrophilic end facing outwards forming the final product mRNA-lipid nanoparticle.
It will be readily appreciated by those skilled in the art that the above advantageous ways can be freely combined and superimposed without conflict.
The foregoing description of the preferred embodiments of the invention is not intended to be limiting, but rather is intended to cover all modifications, equivalents, and alternatives falling within the spirit and principles of the invention. The foregoing is merely a preferred embodiment of the present invention, and it should be noted that it will be apparent to those skilled in the art that modifications and variations can be made without departing from the technical principles of the present invention, and these modifications and variations should also be regarded as the scope of the invention.
Claims (13)
1. Microfluidic chip for generating molecular drug lipid delivery nanoparticles, comprising a chip body (1), the chip body (1) being internally configured with a fluid flow channel (2) and at least one mixing flow unit (30), the fluid flow channel (2) having a first inlet (21) for inputting a first component, a nanoparticle collection port (22), the mixing flow unit (30) comprising an inlet channel (3) for inputting a further component than the first component, the inlet channel (3) being located between the first inlet (21) and the nanoparticle collection port (22) and being in communication with the fluid flow channel (2) at one end and with a second inlet (31) at the other end, the mixing flow unit (30) further comprising a first barrier structure (32) configured on the inner wall of the fluid flow channel (2) between the first inlet (21) and the inlet channel (3), whereby the first barrier structure (32) forms a vortex effect in the fluid flow channel (2) when the first inlet (21) is inputted, the first barrier structure forms a vortex effect in the first side of the fluid flow channel (2), the fluid flowing out of the inlet channel (3) is located within the vortex formed by the first barrier (32).
2. The microfluidic chip according to claim 1, wherein the first barrier structure (32) is a protrusion extending towards the inner side of the fluid flow channel (2), the extension length of the protrusion increasing and decreasing in the flow direction of the fluid within the fluid flow channel (2); and/or the Reynolds number of the first component when the first component passes from one side of the first barrier structure (32) close to the first inlet (21) to one side of the first barrier structure (32) close to the nanoparticle collection port (22) is 90-200.
3. The microfluidic chip according to claim 2, wherein the through-flow cross-section of the fluid flow channel (2) is circular, and the first barrier structure (32) is semicircular on a central axial plane of the fluid flow channel (2).
4. A microfluidic chip according to claim 3, wherein the fluid flow channel (2) extends in a straight line, the through-flow cross-section of the inlet channel (3) is circular and extends in a straight line, the fluid flow channel (2) between the inlet channel (3) and the nanoparticle collection port (22) is a first channel section, and an angle α is formed between the fluid flow channel (2) and the first channel section 1 ，35°≤α 1 Not more than 165 DEG, and said alpha 1 Positively correlated to the radius of the vortex; and/or the diameter of the fluid flow channel (2) is d, and the maximum extension length of the first barrier structure (32) is s, and d/2 is less than or equal to s is less than or equal to 3d/4.
5. The microfluidic chip according to claim 1, wherein the flow mixing unit (30) further comprises a second barrier structure (33) configured on a channel inner wall of the fluid flow channel (2) between the inlet channel (3) and the nanoparticle collection port (22).
6. The microfluidic chip according to claim 5, wherein the first barrier structure (32) and the second barrier structure (33) are on opposite sides of the fluid flow channel (2), respectively; and/or the first barrier structure (32) and the second barrier structure (33) are the same in shape and size.
7. The microfluidic chip according to claim 1, wherein the mixed flow units (30) have at least two, at least two of the mixed flow units (30) being disposed in sequence along a length extension direction of the fluid flow channel (2); and/or the fluid flow channels (2) have a plurality of parallel intervals, the first inlets (21) of the fluid flow channels (2) are integrated into the same total inlet (41), the nanoparticle collection ports (22) of the fluid flow channels (2) are integrated into the same total collection port (42), and the second inlets (31) of the mixing units (30) of the fluid flow channels (2) for inputting the same component are integrated into the same total inlet (43).
8. A microfluidic chip assembly, characterized in that the microfluidic chip assembly is formed by stacking and assembling the microfluidic chips according to any one of claims 1 to 7, wherein the first inlets (21) respectively provided by the microfluidic chips adjacent to each other are mutually connected in a plugging manner, the nanoparticle collecting ports (22) respectively provided by the microfluidic chips adjacent to each other are mutually connected in a plugging manner, and when at least two mixing units (30) are included, the microfluidic chips adjacent to each other are used for inputting the same component, and the second inlets (31) of the mixing units (30) are mutually connected in a plugging manner.
9. A method of preparing a delivery nanoparticle using the microfluidic chip of claim 7 or the microfluidic chip assembly of claim 8, the method comprising:
respectively dissolving each nano coating component in a corresponding organic phase solution to form each coating solution, and dissolving the component to be coated in an aqueous phase solution to form a solution to be coated;
the flow mixing unit (30) nearest to the first inlet (21) is a first flow mixing unit, the solution to be wrapped is communicated with the second inlet (31) of the first flow mixing unit, and each wrapping solution is respectively communicated with the first inlet (21) and the second inlet (31) of the rest flow mixing units (30) along the direction from the first inlet (21) to the nanoparticle collecting port (22) according to the sequence of each nanoparticle wrapping component relative to the component to be wrapped in the formed delivery nanoparticles from inside to outside;
the solution to be wrapped is fed into the fluid flow channel (2).
10. The delivery nanoparticle preparation method according to claim 9, wherein the input speed of the encapsulation solution input by the first inlet (21) is controlled such that the reynolds number Re of the encapsulation solution flowing to the side of the first barrier structure (32) in the first mixed flow unit near the nanoparticle collection port (22) is 90 to 200.
11. The method of preparing delivery nanoparticles according to claim 9, characterized in that said mixed flow unit (30) closest to said nanoparticle collection port (22) is a second mixed flow unit, each nanoparticle encapsulation composition comprising a dispersing agent, said encapsulation solution formed by said dispersing agent being in communication with said second inlet (31) provided by said second mixed flow unit.
12. The method of claim 11, wherein the component to be encapsulated comprises a nucleic acid, the nano-encapsulation component further comprises a cationic lipid, cholesterol, phospholipid material DSPC, and the dispersing agent comprises PEG2000.
13. The method of claim 9, wherein the concentration of the nano-encapsulation component in each of the encapsulation solutions is in the range of 0.01-20 mm; and/or the solution to be wrapped contains buffer solution, and the concentration of the buffer solution is 0.01-1 mug/mu L; and/or the input speed range of each wrapping solution and the solution to be wrapped is 0.02-20 mL/min.
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| CN116618103A (en) | 2023-08-22 |
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