阅读说明：本技术 一种用于粒子生成的混合器 (Mixer for particle generation ) 是由 黄雷 张育坚 李航文 于 2020-12-08 设计创作，主要内容包括：本发明公开了一种用于粒子生成的混合器,所述混合器创新了管道设置,使每个混合单元同时包括直线型混合路径和曲线型混合路径,独创了由六个半圆形混合单元构成的混合管道,并且所有管道宽度一致,可以提高混合效率,尽可能降低流阻,又能提高混合效果,不易为异物堵塞,性能更稳定,特别适用于纳米粒子的生产。本发明还公开了采用该混合器制得的微流混合芯片盒,其进液口与出液口均垂直于于芯片侧壁设置,使注射时能够防止气泡进入,同时也避免了人工手工操作驱赶注射器头部的气泡,造成的昂贵样本液体的浪费,还能多个微流混合芯片盒并行高通量使用,使用便捷,易于推广,实现了纳米粒子的高质量和高效率生产。(The invention discloses a mixer for generating particles, which innovatively sets pipelines, enables each mixing unit to simultaneously comprise a linear mixing path and a curved mixing path, originally creates a mixing pipeline consisting of six semicircular mixing units, ensures that all the pipelines have the same width, can improve the mixing efficiency, reduces the flow resistance as far as possible, can improve the mixing effect, is not easy to be blocked by foreign matters, has more stable performance, and is particularly suitable for the production of nano particles. The invention also discloses a microflow mixing chip box prepared by the mixer, wherein the liquid inlet and the liquid outlet are arranged vertical to the side wall of the chip, so that bubbles can be prevented from entering during injection, simultaneously the waste of expensive sample liquid caused by driving the bubbles at the head of the injector by manual operation is avoided, a plurality of microflow mixing chip boxes can be used in parallel at high flux, the use is convenient and fast, the popularization is easy, and the high-quality and high-efficiency production of nano particles is realized.)

1. A mixer for particle generation, the mixer comprising a first mixing unit, wherein the first mixing unit comprises a first channel comprising a straight channel and a second channel comprising a curved channel.

2. The mixer of claim 1, wherein the first passageway includes a first inlet and a first outlet, the second passageway includes a second inlet and a second outlet, and the first inlet and the second inlet are in fluid communication; the first outlet and the second outlet are in fluid communication.

3. The mixer of claim 1, wherein the mixing unit further comprises a first merging region, the merging region communicating with the first inlet of the first passage and the second inlet of the second passage to effect the splitting of the fluid.

4. The mixer of claim 3, wherein the mixing unit further comprises a second junction region, the second junction region being in communication with the first outlet of the first passage and the second outlet of the second passage to achieve the junction of the fluids.

5. The mixer of claim 1, wherein the curvilinear passage of the second passage comprises a semi-circular arcuate passage.

6. The mixer of claim 1 wherein the second passageway further comprises a straight initial segment passageway, the initial segment being upstream of the curved passageway.

7. The mixer of claim 6 wherein the length of the initial section passage is less than or equal to 1/3 times the length of the second passage.

8. The mixer of claim 6 wherein the initial passageway is at an acute angle of less than 90 degrees to the first passageway.

9. The mixer of claim 3, further comprising a premixing passage in communication with the first junction region, the premixing region effecting mixing of two different fluids.

10. The mixer of claim 9, further comprising a passage for delivering a first fluid and a passage for delivering a second fluid, the first delivery passage and the second delivery passage being in fluid communication with the premixing passage.

11. The mixer of claim 2, further comprising a second mixing element comprising a third channel and a fourth channel, wherein the third channel comprises a curvilinear channel and the fourth channel comprises a linear channel.

12. The mixer of claim 11, wherein the third passageway includes a third inlet and the fourth passageway includes a fourth inlet.

13. The mixer of claim 12, wherein the inlet of the fourth channel is adjacent to the outlet of the second channel of the first mixing unit, or the inlet of the fourth channel is on the same side of the channel as the outlet of the second channel of the first mixing unit, or the third inlet of the third channel is positioned opposite to the outlet of the first channel of the first mixing unit.

14. The mixer of claim 12 wherein the fourth passageway is disposed at an obtuse angle of greater than 90 degrees to the first passageway.

15. The mixer of claim 12, wherein the third channel further comprises a linear initial channel upstream of the curvilinear channel, the initial channel being a partial extension of the first linear channel.

16. The mixer of claim 12, including a third confluence region, the flow at the third confluence region partially entering the third passage and partially entering the second passage.

17. The mixer of claim 1, further comprising a second mixing unit comprising a third channel and a fourth channel, wherein the third channel comprises a curved channel and the fourth channel comprises a straight channel, the third channel is located on the same side of the mixing unit as the first channel, and the fourth channel is located on the same side of the mixing unit as the second channel.

18. The mixer of claim 1, wherein the mixer further comprises a second mixing element, wherein the first mixing element is located upstream of the second mixing element, the second mixing element comprising a third channel and a fourth channel, wherein the third channel comprises a curvilinear channel and the fourth channel comprises a rectilinear channel; and by taking the fourth channel as a reference, the curved channel of the first mixing unit and the curved channel of the second mixing unit are respectively positioned at two sides of the fourth channel.

19. A mixer according to any of claims 1-18, wherein all channels are of the same width or depth.

20. A mixer according to any of claims 1-19, wherein the cross-section of said channels is rectangular.

21. A mixer for nanoparticle generation, the mixer comprising n mixing units, wherein each of the mixing units comprises a first channel and a second channel, the first channel comprises a linear channel, the second channel comprises a curvilinear channel, the first channel has a first inlet and a second outlet, the second channel has a third inlet and a fourth outlet, the first inlet and the third inlet are in fluid communication, and wherein n is a natural integer from 1 to 6.

22. A mixer for nanoparticle generation, the mixer comprising a first mixing unit, wherein the first mixing unit comprises a first channel for receiving a first fluid and a second channel for receiving a second fluid, wherein the flow path of the first fluid in the first channel is smaller than the flow path of the second fluid in the second channel.

23. A mixer for nanoparticle generation, the mixer comprising a first mixing unit, wherein the first mixing unit comprises a first channel for receiving a first fluid and a second channel for receiving a second fluid, wherein the length of the first channel is less than the length of the second channel.

24. A mixer for nanoparticles, comprising N +1 mixing units, wherein the nth mixing unit comprises an a-th linear channel and an a + 1-th curvilinear channel, the a-th linear channel comprises an a-th fluid inlet and an a-th fluid outlet, the a + 1-th curvilinear channel comprises an a + 1-th inflow inlet and an a + 1-th fluid outlet, wherein N is a natural integer equal to or greater than 1, and a is a natural number greater than or equal to 1.

25. The mixer of claim 24, wherein the fluid inlets of the a-th linear channel and the a + 1-th curved channel comprise a-th intersection region, and the fluid in the intersection region is divided; alternatively, the fluid outlet of the a-th linear channel and the fluid outlet of the a + 1-th curved channel comprise a + 1-th intersection region, so that the fluids from the two channels are mixed or converged.

26. The mixer of claim 24, wherein the (N + 1) th mixing unit comprises a +2 th linear channel and a +3 th curved channel, the a +2 th linear channel comprises a +2 th fluid inlet and a +2 th fluid outlet, and the a +3 th curved channel comprises a +3 th fluid inlet and a +3 th fluid outlet.

27. The mixer of claim 26 wherein the a-th fluid outlet is disposed in face-to-face relation with the a + 3-th fluid inlet.

28. The mixer of claim 26, wherein the a +1 th fluid outlet is disposed adjacent to the a +2 th fluid inlet or on the same side of the passageway.

29. The mixer of claim 26, comprising a straight channel upstream of the curved channel, the channel comprising a curved channel fluid inlet.

30. The mixer according to any one of claims 21-29 wherein the mixer comprises a pre-premix channel for flowing fluid into the first and second channels, the pre-premix channel being upstream of the first and second channels, or an a-th linear channel and an a + 1-th curvilinear channel, wherein a is 1.

31. The mixer of claim 30 wherein the pre-premix passage includes a mixing fluid of the first fluid and the second fluid.

32. The mixer of claim 31, wherein the first fluid comprises a nucleic acid and the second fluid comprises a polymer.

33. The mixer of claim 31, wherein the first fluid comprises a nucleic acid and the second fluid comprises a lipid component.

34. The mixer of claim 31, wherein the first fluid comprises particles of nucleic acid and polymer and the second fluid comprises lipid components.

35. A method of producing microparticles, the method comprising providing a mixer according to any one of claims 1 to 34, passing a fluid from the premixing passage into the first mixing unit, wherein a portion of the fluid is passed into the first passage of the first mixing unit and another portion of the fluid is passed into the second passage of the first mixing unit.

36. The method of claim 35, flowing the premixed fluid through a first inlet of a first passage in fluid communication with the first junction region and a second inlet of a second passage.

37. The method of claim 30, wherein the fluids passing through the first and second channels of the first mixing unit are combined at a second combining region.

38. The method of claim 37, passing fluid from the first junction region through inlets of third and fourth channels of the second mixing unit in communication with a third junction region into the third and fourth channels, respectively.

39. The method of claim 36, wherein flowing the fluid in the first mixing unit is accomplished by externally applying pressure to the channel.

40. The method of claim 37, wherein the first fluid and the second fluid are premixed in the premixing passage.

41. A method of preparing microparticles, the method comprising providing a mixed fluid, passing a portion of the fluid through a first channel and another portion of the fluid remaining through a second channel, wherein the fluid path for the fluid through the first channel is smaller than the fluid path for the fluid through the second channel.

42. The method of claim 41, wherein the fluid comprises one or more of a nucleic acid material, a polymer material, or a lipid material.

43. The method of claim 41, wherein the first channel comprises a straight channel and the second channel comprises a curved channel.

44. The method of claim 41, wherein a premix passage is provided, the premix passage being upstream of the first passage and the second passage, wherein the first fluid and the second fluid mix into the mixing fluid in the premix passage.

Technical Field

The invention relates to the field of micro-fluidic control, in particular to a mixer for particle generation.

Background

The micron and nanometer materials have wide application in the fields of chemical industry, electronics, medicine, biology and the like, the traditional micron nano particle synthesis usually adopts a chemical stirring synthesis mode, and the size and the shape of the particle can be controlled by various factors such as a reducing agent, a surfactant, the volume of a reaction vessel, the stirring efficiency, the reaction time and the like. The mixing of the reaction liquid is the most critical factor for the synthesis of the nano particles, and in the traditional synthesis device, a liquid stirring and mixing mode is generally adopted, and although the stirring and mixing mode is mature, the mixing efficiency and the mixing uniformity are difficult to quantitatively or accurately control, and the requirement for producing high-quality nano particles cannot be met.

The microfluidic technology is a new cross-scientific technology, has been applied to various fields such as chemistry, chemical engineering, biology, physics and the like, and comprises the aspects of organic synthesis, inorganic particle synthesis, biological materials, drug synthesis and the like, and is characterized by being capable of accurately controlling micro-fluid and having the advantages of miniaturization, multiple functions, easy integration and the like. In the aspect of micro-nano particle synthesis, the micro-fluidic technology adopted to replace the traditional synthesis mode has become a development trend in basic research and industrial application at present.

The performance of the microfluidic mixer is the key core of the microfluidic synthesis technology, and determines the quality and efficiency of nanoparticle generation. The micro-flow mixer is generally divided into an active micro-mixer and a passive micro-mixer, wherein the active micro-mixer realizes effective mixing by utilizing a moving part or external energy, and has a complex structure and difficult integration; the passive micro mixer does not need any external energy source, changes the flow field of fluid by changing the geometric shape of the micro channel, further realizes the effective mixing of fluid working media, has simple and convenient manufacture and few supporting facilities, and is widely developed.

Ansari et al propose staggered chevron micromixers, the main idea of this design is to increase the contact area between the 2 fluids by creating a lateral flow; meneaud and the like carry out researches on experiments and numerical simulation of the zigzag micro-channel, and vortex is formed by utilizing a turning region of the micro-channel, so that the mixing efficiency is improved; liu et al have studied three-dimensional serpentine channel micromixers, square waveform micromixers and straight channel micromixers; the effect of Ansari et al on the geometry of a three-dimensional serpentine channel with repeating L-shaped circulation units on fluid flow and mixing; mouza and the like further improve a micro mixer with an arc channel by using the principle of separation and recombination, utilize fluid in split sub-channels with uniform width to generate balanced collision, and simultaneously induce Dean vortex by using the arc sub-channels to enhance mixing; on the basis, Ansari and the like carry out numerical simulation and experimental research on the mixing characteristics of the in-plane asymmetric circular channel separation recombination micro-mixer, and aim at the problems in the Mouza and the like research, unbalanced collision generated by asymmetric sub-channels of the micro-mixer is utilized to enhance mixing.

However, when the existing microfluidic mixing chip is used for preparing the nano particles, the mixing effect is not high, and even the blockage is easy to occur, so that the quality of the prepared nano particles is not stable enough.

In addition, under current traditional technical condition, the feed liquor port of miniflow mixing chip box sets up the lower surface at the chip usually, and the feed liquor port becomes perpendicular setting with the lower surface of chip, uses the vertical upwards injection sample liquid of syringe during the use, because the blank region of syringe head can cause the bubble to remain in the syringe this moment, though the bubble of syringe head can be driven in advance to manual operation, makes the liquid sample full of whole syringe, nevertheless can cause the waste to the liquid sample that the price is expensive like this. Meanwhile, the liquid inlet port and the lower surface of the chip are arranged vertically in a T shape, so that a plurality of microflow mixing chip boxes cannot be superposed, and parallel high-flux use of the microflow mixing chip boxes is difficult to realize.

Disclosure of Invention

In order to solve the above problems, the present invention provides a mixer for parallel high-throughput nanoparticle generation and a microfluidic mixing cartridge including the mixer, which can greatly improve mixing efficiency by an inventive structural design, effectively reduce waste of expensive sample liquid, and enable parallel high-throughput use of a plurality of microfluidic mixing cartridges, thereby achieving high-quality and high-efficiency production of nanoparticles.

In one aspect, the invention provides a mixer comprising a first mixing unit comprising a first channel comprising a rectilinear or substantially rectilinear channel and a second channel comprising a curvilinear or substantially curvilinear channel.

In some forms, the first channel and the second channel form a mixing unit.

In some aspects, the first channel includes a channel inlet and a channel outlet, and the second channel also includes a channel inlet and a channel outlet. In some forms the inlet of the first channel and the inlet of the second channel are in communication, thereby allowing fluid to flow into the first channel at the inlet of the first channel. In some forms the fluid is allowed to flow into the second channel at an inlet of the second channel. In some approaches, the fluid enters the first and second channels at a first intersection of the first and second channels, respectively. In some approaches, the fluid passing through the first channel and the fluid passing through the second channel mix or merge at the outlets of the first channel and the second channel. In some embodiments, the fluids are mixed or merged at the exit of the first and second passages into the second merge region.

In some forms, the first channel and the second channel are each connected end-to-end to provide fluid communication.

In some embodiments, the first channel and the second channel are connected end to end, respectively, meaning that the head and the head of the first channel and the second channel are connected together, and the tail are connected together.

Further, the first channel comprises a first inlet and a first outlet, the second channel comprises a second inlet and a second outlet, and the first inlet and the second inlet are in fluid communication; the first outlet and the second outlet are in fluid communication.

Further, the mixing unit further comprises a first merging region, and the merging region is communicated with the first inlet of the first channel and the second inlet of the second channel, so that the flow division is realized.

Further, the mixing unit further comprises a second merging region, and the second merging region is communicated with the first outlet of the first channel and the second outlet of the second channel, so that the fluids are merged.

Further, the curvilinear channel of the second channel comprises a semi-circular arc channel.

In the foregoing manner, the second path includes a straight initial path upstream of the arcuate or curvilinear path. In some embodiments, the initial channel is at an acute angle to the linear first channel. In some forms the initial segment length is less than or equal to 1/3 of the second channel length.

Further, the included angle between the initial channel and the first channel is an acute angle smaller than 90 degrees.

Further, the mixer also includes a premixing passage in communication with the first junction region, the premixing region effecting mixing of two different fluids.

Further, the mixer also includes a passage for delivering a first fluid and a passage for delivering a second fluid, the first and second delivery passages being in fluid communication with the premixing passage.

Further, the mixer also comprises a second mixing unit, wherein the second mixing unit comprises a third channel and a fourth channel, the third channel comprises a curved channel, and the fourth channel comprises a linear channel.

Further, the third channel includes a third inlet and the fourth channel includes a fourth inlet.

Further, the inlet of the fourth channel is adjacent to the outlet of the second channel of the first mixing unit, or the inlet of the fourth channel and the outlet of the second channel of the first mixing unit are located on the same side of the channel, or the third inlet of the third channel and the outlet of the first channel of the first mixing unit are arranged in a face-to-face manner.

Further, the fourth channel and the first channel are arranged at an obtuse angle larger than 90 degrees.

Further, the third channel further comprises a linear initial channel located upstream of the curved channel, and the initial channel is a partial extension of the first linear channel.

Further, the fluid at the third confluence region partially enters the third channel and partially enters the second channel.

Further, the mixer also comprises a second mixing unit, the second mixing unit comprises a third channel and a fourth channel, the third channel comprises a curved channel, the fourth channel comprises a linear channel, the third channel and the first channel are positioned on the same side of the mixing unit, and the fourth channel and the second channel are positioned on the other same side of the mixing unit.

Further, the mixer further comprises a second mixing unit, wherein the first mixing unit is positioned upstream of the second mixing unit, the second mixing unit comprises a third channel and a fourth channel, the third channel comprises a curved channel, and the fourth channel comprises a linear channel; and by taking the fourth channel as a reference, the curved channel of the first mixing unit and the curved channel of the second mixing unit are respectively positioned at two sides of the fourth channel.

In all the above-mentioned ways, the width or height of the channels is equal, or the cross-section of the channels is the same.

In some forms, the present invention provides mixers in which the channels are rectangular in cross-section and the length and width of all channel cross-sections are uniform.

In another aspect, the present invention provides a mixer for nanoparticle generation, the mixer comprising n mixing units, wherein each of the mixing units comprises a first channel and a second channel, the first channel comprises a linear channel, the second channel comprises a curvilinear channel, the first channel has a first inlet and a second outlet, the second channel has a third inlet and a fourth outlet, the first inlet and the third inlet are in fluid communication, and n is a natural integer from 1 to 6.

In yet another aspect, the present invention provides a mixer for nanoparticle generation, the mixer comprising a first mixing unit, wherein the first mixing unit comprises a first channel for receiving a first fluid and a second channel for receiving a second fluid, wherein the flow path of the first fluid in the first channel is smaller than the flow path of the second fluid in the second channel.

In yet another aspect, the present invention provides a mixer for nanoparticle generation, the mixer comprising a first mixing unit, wherein the first mixing unit comprises a first channel for receiving a first fluid and a second channel for receiving a second fluid, wherein the length of the first channel is less than the length of the second channel.

In some embodiments, the first channel has a first fluid inlet and a first fluid outlet, the second channel has a second fluid inlet and a second fluid outlet, and the first fluid inlet and the second fluid inlet are in fluid communication.

In some embodiments, a mixing channel is included upstream of the first junction region, the mixing channel connecting the first fluid inlet and the second fluid inlet, such that fluid flows partially into the first channel and partially into the second channel at the first junction region.

In still another aspect, the present invention provides a nanoparticle mixer, comprising N +1 mixing units, wherein the nth mixing unit comprises an a-th linear channel and an a + 1-th curvilinear channel, the a-th linear channel comprises an a-th fluid inlet and an a-th fluid outlet, and the a + 1-th curvilinear channel comprises an a + 1-th inflow inlet and an a + 1-th fluid outlet, wherein N is a natural integer equal to or greater than 1, and a is a natural number greater than or equal to 1.

Furthermore, the fluid inlet of the a-th linear channel and the fluid inlet of the a + 1-th curved channel comprise an a-th intersection area, so that the fluid in the intersection area is divided; alternatively, the fluid outlet of the a-th linear channel and the fluid outlet of the a + 1-th curved channel comprise a + 1-th intersection region, so that the fluids from the two channels are mixed or converged.

Further, the N +1 th mixing unit comprises a +2 th linear channel and a +3 th curved channel, the a +2 th linear channel comprises an a +2 th fluid inlet and an a +2 th fluid outlet, and the a +3 th curved channel comprises an a +3 th fluid inlet and an a +3 th fluid outlet.

Further, the a-th fluid outlet is arranged opposite to the a + 3-th fluid inlet.

Further, the a +1 th fluid outlet is disposed adjacent to the a +2 th fluid inlet, or on the same side of the channel.

Further, a straight channel is included upstream of the curved channel, and the channel includes a fluid inlet of the curved channel.

Further, the mixer comprises a pre-premixing channel for flowing fluid into the first channel and the second channel, wherein the pre-premixing channel is positioned at the upstream of the first channel and the second channel, or an a-th linear channel and an a + 1-th curvilinear channel, wherein a is 1.

Further, the pre-premix passage includes a mixed fluid of a first fluid and a second fluid.

Further wherein the first fluid comprises a nucleic acid and the second fluid comprises a polymer.

Further wherein the first fluid comprises nucleic acids and the second fluid comprises lipid components.

Further, the first fluid comprises particles formed by nucleic acid and polymer, and the second fluid comprises lipid components.

In some embodiments, two channels are included upstream of the mixing channel for introducing two liquids or fluids, the two channels having a junction where the two fluids meet and flow into the mixing channel to form a mixed fluid.

In some aspects, connecting upstream of the mixing channel comprises a first entry channel and a second entry channel that meet at an inlet of the mixing channel. In some embodiments, the first inlet channel is configured to receive a first fluid and the second channel is configured to receive a second fluid, the first fluid and the second fluid being mixed at an inlet of the mixing channel to form a mixed fluid and flowing into the first mixing channel.

In the foregoing manner, the second channel includes an arc-shaped channel, and the first channel is a linear channel.

In some embodiments, the mixer further comprises a second mixing unit comprising a third channel and a fourth channel, the third channel comprising a curved channel and the fourth channel comprising a straight channel. In some embodiments, the inlet of the curved channel in the second mixing unit is in the same straight line or substantially the same straight line position as the straight line channel of the first mixing unit. In some forms the inlet of the linear channel in the second mixing unit is at an acute angle to the outlet of the linear channel. In some embodiments, the second mixing unit further comprises a linear initial channel, the initial channel being located upstream of the curved channel. In some embodiments, the initial channel is collinear or substantially collinear with the linear channel of the first mixing unit. In some embodiments, the linear initial channel and the fourth channel have an acute included angle.

In another mode, when the fluid is, for example, to the second mixing unit, the fluid enters the third and fourth channels, wherein the path through which the fluid flows in the third channel is larger than the path through which the fluid flows in the fourth channel.

In some embodiments, the mixer further comprises a third mixing unit and a fourth mixing unit, the third mixing unit has the same structure as the first mixing unit, the fourth mixing unit has the same structure as the second mixing unit, and the third mixing unit and the fourth mixing unit are distributed in the same way as the first mixing unit and the second mixing unit.

In some approaches, the third channel of the second mixing unit includes a third inlet and a third outlet; the fourth channel includes a fourth inlet and a fourth outlet. In some forms the outlet of the first channel faces the inlet of the third channel. In some forms, the inlet of the fourth channel is adjacent to the inlet of the second channel, the latter being adjacent to each other. In some forms the inlet of the fourth channel is located adjacent to the inlet of the second channel on the same side of the channel. In some approaches, the inlet of the third passageway and the inlet of the fourth passageway communicate with the third intersection region. In some approaches, the third intersection is located downstream of the second intersection.

In some aspects, the present disclosure provides a mixer comprising N mixing units, where N is a natural integer greater than 1; n can be a natural number such as 1,2,3,4,5,6,7,8 and the like. In some embodiments, N is 2, N is 3, N is 4, N is 5, N is 6, N is 7, or any other natural integer. In some embodiments, N is a natural even number. In some modes, when N is a natural even number, the mixing units are connected end to end; the two adjacent mixing units are a first mixing unit and a second mixing unit, the second channel of the first mixing unit is positioned on the right side of the first channel, and the second channel of the second mixing unit is positioned on the left side of the first channel. In some embodiments, the first channel of the first mixing unit and the first channel of the second mixing unit are linear channels, and the second channel of the first mixing unit and the second channel of the second mixing unit comprise curvilinear channels. Alternatively, in some approaches, the length of the first channel of the first mixing unit is less than the length of the second channel.

In some aspects, the present invention provides a mixer comprising a first mixing unit, the mixing unit comprising two mixing channels, the two mixing channels forming a "D" shape. In some embodiments, the channel includes an inlet end and an outlet end, and the inlet end of the fluid to be mixed is divided into two channels. After passing through the latter two channels, the flows merge at one end of the outgoing flow. In some embodiments, the mixer includes a second mixing unit, the mixing unit including two mixing channels, the two mixing channels forming a "D" shape, wherein the second mixing unit is arranged in an opposite direction to the first mixing unit. In some embodiments, the curved channels of the first mixing unit are oppositely oriented from the curved channels of the second mixing unit. In some forms, the first mixing unit and the second mixing unit have an angle therebetween, and the angle may be an obtuse angle or an acute angle.

In some aspects, the mixer comprises N mixing units, where N is a natural integer greater than 1; n may be a natural number such as 1,2,3,4,5,6,7, or 8. In some embodiments, N is 2, N is 3, N is 4, N is 5, N is 6, N is 7, or any other natural integer, where each mixing unit includes two mixing channels forming a "D" shaped channel mixing unit.

In some forms, the mixer includes a passage for discharging the particles or granules, the passage being located downstream of the mixing unit.

The invention carries out structural creative design and improvement on the basis of the existing separation and recombination type mixing pipeline, ensures that one channel is linear, simultaneously adopts a semicircular arc with an innovative structure on the other path, has consistent width of all channels, reduces flow resistance as far as possible, is not easy to block foreign matters, and greatly improves the mixing effect.

In another aspect, the present invention provides a microfluidic mixing cartridge, which includes the above-mentioned mixer structure, and is provided with a liquid inlet, a liquid outlet, a liquid inlet pipeline, and a liquid outlet pipeline, where the two liquid inlets are used for respectively inputting different liquids or fluids.

The liquid inlet and the liquid outlet are vertical to the side wall of the chip; the liquid inlet pipeline is connected with the liquid inlet and the mixer, the liquid outlet pipeline is connected with the liquid outlet and the mixer, and the packaging box is arranged outside the chip.

Furthermore, the liquid inlet has two or more than two, and liquid inlet and liquid outlet are located the both ends of chip respectively.

When the two liquid inlets are provided, the two liquid inlets are respectively a first liquid inlet and a second liquid inlet, the solution from the first liquid inlet is called a first solution, and the solution from the second liquid inlet is called a second solution.

The liquid inlet pipeline I is connected with the liquid inlet I, the liquid inlet pipeline II is connected with the liquid inlet II, and the liquid inlet pipeline I and the liquid inlet pipeline II are connected with a top channel of the mixer together; the liquid outlet pipeline is connected with the bottom channel of the mixer, and the other end of the liquid outlet pipeline is connected with a liquid outlet.

Further, the liquid inlet and the liquid inlet pipeline are located on the same plane, and the liquid outlet pipeline are located on the same plane.

Furthermore, the liquid inlet pipeline, the liquid outlet pipeline and the chip are all basically located on the same plane.

Of course, the liquid inlet pipe, the liquid outlet pipe and the chip are basically located on the same plane, so that the volume is reduced, the manufacturing is convenient, in some modes, the liquid inlet pipe, the liquid outlet pipe and the chip can be respectively located on different planes, or any two or more of the liquid inlet, the liquid outlet pipe and the chip can be located on the same plane, and the situations are also within the protection scope of the invention.

Compared with the prior art, the microfluidic chip box provided by the invention has the advantages that the liquid inlet and the liquid outlet are perpendicular to the side wall of the chip, when the microfluidic chip box is used, the injector is used for vertically downwards injecting, the chip and the injector are positioned on the same plane, after the injector extracts a liquid sample, the injector is vertically downwards placed, bubbles naturally float to the top end inside the injector, then the injector is vertically downwards inserted into the liquid inlet of the chip, and then the liquid inside the injector is completely injected into the liquid inlet.

In another aspect, the invention further provides a microfluidic hybrid chip cartridge for parallel high-throughput nanoparticle generation, wherein the chip cartridge is formed by stacking a plurality of microfluidic hybrid chip cartridges in parallel.

Because inlet, liquid outlet and chip are in the coplanar, only need inject from the chip side during the application of sample, make miniflow mixes chip box can a plurality of superpositions to realize parallel high flux and use, make the miniflow that is used for parallel high flux nano particles to generate and mix chip box.

In yet another aspect, the present invention provides a method of making microparticles, the method comprising: providing a mixer as described above, letting the fluid from the premixing passage enter the first mixing unit, wherein a part of the fluid is let into the first passage of the first mixing unit and another part of the fluid is let into the second passage of the first mixing unit.

In some forms, the two passageways have substantially identically located converging inlets and substantially identically located converging outlets. In some approaches, the inlet includes an inlet into the first channel and an inlet into the second channel. In some forms, the outlet includes an outlet that exits the first channel and an outlet that exits the second channel.

Further, the premixed fluid is allowed to flow in through the first inlet of the first passage in fluid communication with the first junction region and the second inlet of the second passage.

Further, the fluids passing through the first and second passages of the first mixing unit are caused to join at a second joining region.

Further, the fluid from the first confluence region is let into the third and fourth passages through inlets of the third and fourth passages of the second mixing unit, which communicate with the third confluence region, respectively.

Further, allowing the fluid to flow in the first mixing unit is achieved by externally applying pressure to the channel.

Further, the first fluid and the second fluid are premixed in the premixing passage.

In yet another aspect, the invention provides a method of preparing microparticles, the method comprising providing a mixed fluid, passing a portion of the fluid through a first channel, and passing the remaining other portion of the fluid through a second channel, wherein the fluid path for the fluid through the first channel is smaller than the fluid path for the fluid through the second channel.

Further, the fluid comprises one or more of nucleic acid substances, polymer substances or lipid component substances.

Further, the first channel comprises a linear channel, and the second channel comprises a curvilinear channel.

Further, a premix passage is provided, said premix passage being located upstream of the first passage and the second passage, in which premix passage the first fluid and the second fluid are mixed into said mixing fluid.

In some forms, the fluid is admitted to the first and second passages through the junction inlets, respectively, and then the fluid is discharged through the junction outlets. In some forms, the fluid is allowed to flow through a path that is smaller in the first channel than in the second channel.

In some aspects, the fluid flows in a linear path in the first channel and the fluid flows in a curvilinear path in the second channel.

In some forms the mixer includes a second mixing element arranged in the same way as the first mixing element but at an angle to the first mixing element. In some aspects, the mixer may include a structure of first and second mixing units arranged repeatedly, and the repetition may be repeated three or more times.

In some forms, the fluid is mixed by a premixing passage upstream of the mixing unit before entering the mixing unit. In some embodiments, two channels are included upstream of the mixing channel, the two channels each receiving a different fluid, the two different fluids flowing into the mixing channel to mix to form a mixed fluid. In some embodiments, the mixed fluid flows into the junction inlet of the mixing unit, then into the first and second passages, and out through the junction outlet, and the mixed fluid after the outlet enters the next mixing unit.

In some embodiments, one of the two different fluids comprises a nucleic acid material and the other fluid comprises a polymer, a polypeptide, or the other fluid comprises a lipid component. Alternatively, one of the two different fluids comprises polymer particles formed in combination with nucleic acid species, or the other fluid comprises a lipid component.

The mixer and the microflow mixing chip box for parallel high-throughput nano particle generation provided by the invention have the following beneficial effects:

1. the pipeline arrangement of the innovative mixer enables each mixing unit to simultaneously comprise a linear mixing path and an arc mixing path, and all the pipelines are consistent in width, so that the flow resistance is reduced as far as possible, and the mixing effect can be improved.

2. The mixing pipeline which is originally created and consists of six semicircular mixing units can improve the mixing efficiency, has smaller flow resistance, is not easy to block by foreign matters, has more stable performance and is particularly suitable for the production of nano particles.

3. The liquid inlet and the liquid outlet are perpendicular to the side wall of the chip, so that bubbles can be prevented from entering during injection, and the problem that the bubbles at the head of the injector are driven by manual operation to cause waste of expensive sample liquid is avoided.

4. Because inlet, liquid outlet and chip are in the coplanar, only need inject from the chip side during the application of sample, make miniflow mixes chip box can a plurality of superpositions to realize parallel high throughput and use, can be used to parallel high throughput and generate the nano particle.

5. Convenient and efficient, and easy to popularize.

Detailed Description

The invention provides a microfluidic hybrid chip cartridge and a mixer thereof configured for the preparation of nanoparticles for scientific research or therapeutic applications. The system can be used to produce a wide variety of nanoparticles, including but not limited to polymer and lipid nanoparticles, which carry various loads. The system provides a simple workflow that can be used to produce a sterile product in certain embodiments.

Micro-flow mixing chip box

Microfluidic mixing chip cartridges are a hot spot in the current development of microfluidic analytical systems, and provide a convenient platform for combining two or more microfluidic streams in microfluidic mixers.

The microfluidic hybrid chip cartridge uses a chip as an operation platform, simultaneously uses analytical chemistry as a basis, uses a micro-electromechanical processing technology as a support, uses a micro-pipeline network as a structural characteristic, and uses life science as a main application object at present. The device is characterized in that the effective structure (channels, reaction chambers and other functional components) for containing the fluid is in a micron scale at least in one latitude, and due to the micron-scale structure, the fluid displays and generates special performance different from a macro scale in the structure, so that the device has the characteristics of controllable liquid flow, extremely less consumed samples and reagents and the like.

In some aspects, the present disclosure provides apparatus for the preparation of nanoparticles that enable simple, rapid and reproducible laboratory-scale nanoparticle preparation (0.5-20mL), which uses microfluidic hybrid chip cartridges for applications primarily in basic research, particle characterization, substance screening, and in vitro and in vivo studies, among others. The microfluidic hybrid chip cartridge used in the present disclosure has the advantage of precise control of environmental factors during fabrication, and microfluidic has the further advantage of allowing seamless amplification via parallelization. The disclosed embodiments are configured to mix the first solution and the second solution via a microfluidic mixer. Many methods for this mixing process are known. Compatible microfluidic mixing methods and devices are disclosed in: (1) U.S. patent application No.13/464690, a continuation of PCT/CA2010/001766 filed 11/4/2010, which claims the benefits of USSN 61/280510 filed 11/4/2009; (2) U.S. patent application No.14/353,460, a continuation of PCT/CA2012/000991 filed 10/25/2012, which claims the benefits of USSN 61/551366 filed 10/25/2011; (3) PCT/US2014/029116 filed 3/14/2014 (published as WO2014172045 at 10/23/2014) which requires the benefits of USSN 61/798495 filed 03/15/2013; (4) PCT/US2014/041865 filed 7/25/2014 (published as WO2015013596 at 1/29/2015) which requires the benefits of USSN 61/858973 filed 07/26/2013; (5) PCT/US2014/060961, which requires the benefits of USSN 61/891,758 filed by 10/16/2013; and (6) U.S. provisional patent application No.62/120179 filed on 24/2/2015, which is incorporated by reference in its entirety.

The microfluidic hybrid chip cartridge manufactured at present eliminates the user assembly step by integrating the accessory and the microfluidic into one cartridge, can have higher operating pressure and minimize internal volume, and can also provide a pre-sterilized microfluidic hybrid chip cartridge having a sterile fluid path. Microfluidic hybrid chip cartridges may include disposable and non-disposable, features of disposable cartridges that may reduce the risk of cross-contamination and reduce experimental time by eliminating the need for washing.

In some embodiments, the microfluidic mixing cartridge is disposable. The term "disposable" as used herein refers to a component that is relatively inexpensive relative to the products (e.g., nano-drugs) produced by microfluidic chip cartridges. In addition, disposable microfluidic hybrid chip cartridges have a limited useful life, such as being suitable for only a single use, as described below. Disposable materials broadly include plastics, magnets (e.g., inorganic materials) and metals.

In some embodiments, the microfluidic hybrid chip cartridge is configured for single use. In this regard, the construction of microfluidic hybrid chip cartridges results in low manufacturing costs and thus allows the user to dispose of the cartridge after use. In certain embodiments, the cassette characteristics change after a single use, thus negating or eliminating the possibility of further use of the cassette. For example, with a sterile cassette, after a single use, the cassette is no longer sterile and therefore cannot be reused as a sterile cassette. In addition, the single-use cartridge eliminates the risk of cross-contamination between mixes. In this regard, the single-use microfluidic mixing cartridge contains a completely unused (fluid-untouched) fluid path from the inlet connector to the outlet.

The sources of the solutions mixed in the microfluidic mixing cartridge include syringes and pumps. By configuring the inlet connector to mate with a connector connected to a solution source, the microfluidic mixing cartridge can be compatible with any solution source.

The microfluidic chip box comprises a microfluidic chip and a packaging box outside the chip, and a microfluidic structure is arranged in the chip, wherein the most important is a mixer.

The microflow mixing chip box provided by the invention is provided with a chip with an innovative structural design, a mixer with an innovative structural design is arranged in the chip, a liquid inlet, a liquid outlet, a liquid inlet pipeline, a liquid outlet pipeline and a mixer are arranged on the chip, and the liquid inlet and the liquid outlet are vertical to the side wall of the chip; the liquid inlet pipeline is connected with the liquid inlet and the mixer, the liquid outlet pipeline is connected with the liquid outlet and the mixer, and the packaging box is arranged outside the chip.

Furthermore, the liquid inlet has two or more than two, and liquid inlet and liquid outlet are located the both ends of chip respectively.

When the number of the liquid inlets is two, the two liquid inlets are respectively a first liquid inlet and a second liquid inlet, the solution from the first liquid inlet is called a first solution, and the solution from the second liquid inlet is called a second solution.

The liquid inlet pipeline I is connected with the liquid inlet I, the liquid inlet pipeline II is connected with the liquid inlet II, and the liquid inlet pipeline I and the liquid inlet pipeline II are connected with a top channel of the mixer together; the liquid outlet pipeline is connected with the bottom channel of the mixer, and the other end of the liquid outlet pipeline is connected with a liquid outlet.

Further, the liquid inlet and the liquid inlet pipeline are located on the same plane, and the liquid outlet pipeline are located on the same plane.

Furthermore, the liquid inlet pipeline, the liquid outlet pipeline and the chip are all basically located on the same plane.

Of course, the liquid inlet pipe, the liquid outlet pipe and the chip are basically located on the same plane only for reducing the volume and facilitating the manufacture and use, and in some modes, the liquid inlet pipe, the liquid outlet pipe and the chip can be respectively located on different planes, or any two or more of the liquid inlet, the liquid outlet pipe and the chip can be located on the same plane, and the situations are also within the protection scope of the invention.

Compared with the prior art, the microfluidic chip box provided by the invention has the advantages that the liquid inlet and the liquid outlet are perpendicular to the side wall of the chip, when the microfluidic chip box is used, the injector is used for vertically downwards injecting, the chip and the injector are positioned on the same plane, after the injector extracts a liquid sample, the injector is vertically downwards placed, bubbles naturally float to the top end inside the injector, then the injector is vertically downwards inserted into the liquid inlet of the chip, and then the liquid inside the injector is completely injected into the liquid inlet.

Microfluidic hybrid chip

In some embodiments, the microfluidic mixing chip comprises a first portion and a second portion, the first portion or the second portion comprising a first liquid inlet, a second liquid inlet, and a liquid outlet, or the first portion and the second portion being joined together to form a first liquid inlet, a second liquid inlet, and a liquid outlet, wherein the first portion and the second portion are joined together to enclose the mixer between the first portion and the second portion. Such as the configurations shown in fig. 1 and 2, the mixing chip configuration includes a first portion 30 and a second portion 20, and a mixer 22 containing microfluidic channels, the mixer 22 being sealed together by the first portion and the second portion, or the mixer configuration being disposed in a cartridge sealed together by the first portion and the second portion. The mixing device contains mixing elements which are in communication with microfluidic channels, for example, a base plate 102 of the mixer has microfluidic channels, and a cover plate 101 of the mixer covers the base plate 102 to form sealed microfluidic channels. In some embodiments, the mixer unit comprises a plurality of mixing units, and these mixing units generally comprise two channels that simultaneously allow the fluid to flow, by dividing the flow, converging the flow, then dividing the flow, and finally forming the desired particles. As will be described in detail later. In order to let the fluid enter the channel, there is typically an inlet into the channel, for example as shown in fig. 1 and 2, in the mixer there is a first inlet 12 and a second inlet 312, through which the different fluids are let into the channel, through the mixer the two fluids are mixed, finally obtaining particles, which are expelled out of the mixer through the outlet 313. Therefore, the lower plate 20 and the upper plate 30 are also provided with a structure of holes communicating with the inlet and the outlet, respectively, into which the fluid flows. For sealing, sealing gaskets 203,201, 202 may be provided between the channel and the inlet and outlet ports, respectively, to ensure sealing performance. Thus, the upper plate and the lower plate are combined together to form the chip case 100.

In some embodiments herein, the first portion of the chip may be referred to as a connection portion and the second portion may be referred to as a top plate. Some embodiments may require the use of additional components such as screws and plates to complete the coupling between the first and second portions of the chip. In one embodiment, the second portion functions to apply a clamping force to the assembly. In one embodiment, the second part contains a layer or structure to evenly distribute the clamping force on the mixer.

In some approaches, the first and second portions of the chip are secured together by one or more fasteners. In some approaches, one or more fasteners are removable. Exemplary removable fasteners are screws, nuts and bolts, clips, bands and pins. In still other approaches, one or more of the fasteners are non-removable. In such embodiments, the fastener may be a nail or a rivet. In additional embodiments, the fastener may be incorporated into a structure as a chip. In such embodiments, one portion may contain a pin or tab while the second portion has a recess, cut-out or other structure to receive the fastener.

In still other embodiments, the first portion and the second portion are joined together. In the described embodiment, the two parts are inseparable once coupled. In one embodiment, the first and second parts are bonded together with an adhesive. In one embodiment, the first portion and the second portion are joined together with a weld. Representative suitable welding methods include laser welding, ultrasonic welding, and solvent welding.

In still other embodiments, the chip further comprises a gasket configured to form separate fluid-tight seals between the mixer and the first inlet, second inlet, and outlet. There are also ways in which a flange or other feature integrated into the chip may be used to form the desired seal. The chip is internally provided with a microfluidic structure, wherein the microfluidic structure comprises a mixer for mixing two or more than two fluids.

Microfluidic structure

Microfluidic structures refer to systems or devices for manipulating (e.g., flowing, mixing, etc.) fluid samples that include channels on at least one micrometer scale (i.e., a dimension less than 1 mm). The microfluidic structure of the present invention includes a mixer, a liquid inlet pipe, a liquid outlet pipe, etc. in the microfluidic mixing chip, for example, in fig. 2, the microfluidic channel on the substrate includes two channels, namely, a liquid inlet channel 14 and a liquid outlet channel 314, both of which have respective liquid inlets 12 and 312, and the first fluid and the second fluid to be mixed are allowed to flow into the mixing unit through the liquid inlets 14 and 314 for mixing. After entering the first channel 14, the first fluid passes through the first preparation channel 103 and then enters the intersection 105; the second fluid enters the second channel 314, passes through the second preliminary channel 104, and then enters the intersection 105. The first and second fluids after first meeting in the meeting area 105 enter together the meeting channel 106 and then the mixing unit in the mixer. The fluid obtained after mixing by the mixer flows out through the liquid outlet channel 303.

Mixing unit in a mixer

The mixer is a "microfluidic element" in a microfluidic mixing cartridge, and is one of the key parts of a microfluidic structure, configured to perform functions beyond simple flowing solutions, such as mixing, heating, filtering, reaction, etc. The microfluidic component described in the present invention is a microfluidic mixer configured for mixing a first solution and a second solution in a chip structure to provide a mixed solution to form a particulate component. The mixed solution as described herein is not a pure mixed fluid or solution, but generally includes or dissolves, suspends, etc. substances such as nucleic acids, proteins, polypeptides, polymers, lipid components, etc. Typically two solutions of different composition are mixed, for example one solution containing the nucleic acid material and the other solution containing the polymer, and when the two solutions are mixed, the nucleic acid material and the polymer form a particulate material which is then mixed a number of times and then filtered or centrifuged to separate the particulate material. Such particulate material may be suspended in a solution and then mixed again with a solution containing a lipid component to coat the particulate material with the lipid component, thereby forming a particulate material. As will be described in detail below.

In some aspects, the present disclosure provides a mixer comprising a mixing unit comprising a first channel and a second channel, the first channel being linear and the second channel being curvilinear. For example, as shown in fig. 4, the mixer includes two channels, a first channel 702 and a second channel 701, wherein the length of the first channel is less than the length of the second channel. Thus, the fluid enters the first channel and the second channel, and the path through which the fluid flows in the first channel is smaller than the path through which the fluid flows in the second channel. In some ways, it will be appreciated that the fluid flows in the first channel for a shorter time than in the second channel if at the same pressure. In some embodiments, each of the two channels has an inlet and an outlet, e.g., a first channel 702 includes inlet 107 and outlet 113, and a second channel 702 includes inlet 108 and outlet 112. In some embodiments, the inlet 107 of the first channel and the inlet 108 of the second channel have a first region of intersection 900 at which fluid enters the first channel and the second channel for flow, respectively. In some embodiments, the first passage includes the exit port 113, the second passage includes the exit port 112, and a junction area 901 is also included between the exit ports, such as the second junction area 901, where the fluid from the first passage and the fluid from the second passage mix or join together.

Of course, the fluid meeting and remixing in the meeting area 901 may enter both the next mixing unit. Of course, fluid from the second junction area may flow into the third junction area 902, such that at the third junction area, the mixed fluid re-enters the second mixing unit to flow or flow in the third and fourth passages of the second mixing unit, respectively. By "junction area" is herein understood the area where the inlet and outlet of the channel are connected or the area of connection where a splitting of the fluid or a merging or remixing of the fluid is achieved. For example, at the inlet of both channels or at the outlet of both channels, may be considered a junction region where there is a splitting and/or pooling of the liquid. For example, there may be a split flow of liquid in first junction region 900, flowing into the first and second channels of the first mixing unit, respectively, and then a collection of liquid in second junction region 901. In the same way, the second mixing unit also has a third channel and a fourth channel, an inlet of the third channel and an inlet of the fourth channel, an outlet of the third channel and an outlet of the fourth channel, and a third intersection region 902 is also provided at the inlet of the third channel and the inlet of the fourth channel, and the mixed liquid is divided in this region. Likewise, at the outlet of the three channels and at the outlet of the fourth channel, there is a fourth junction region 903, where the mixing, pooling or junction of the fluids of the two channels is achieved. Similarly, in this manner, a plurality of mixing units may be provided in series, with a plurality of intersection regions, to achieve a first splitting, a first pooling, then a second splitting, a second pooling, a third splitting and a fourth pooling. This has N × 2 intersection regions, for example if N is 1, there are two intersection regions, N ═ 4, 8 intersection regions, N ═ 3, 6 intersection regions, and N ═ 6, 12 intersection regions. In some embodiments, there are two channels between each intersection, wherein one channel is linear and the other channel is curvilinear, or the length of one channel is less than the length of the other channel, or the flow path of the fluid in one channel is less than the flow path in the other channel. As shown in fig. 3, when there are 6 mixing units, the intersection region 900,902,904,906,908,910 realizes the diversion of the liquid or the diversion of the mixed liquid, and the intersection region 901,903,905,907,909,911 realizes the intersection, intersection or mixing of the liquids.

In some embodiments, the first channel 702 is a straight channel and the second channel 701 is a curved channel, but the two channels have the same location of the area of intersection. By "co-located junction region" is meant that the inlets and outlets of the two channels are substantially at the same location, and not offset by a substantial distance, it is also understood that the inlets of the two channels are at the same location, and that the liquid at the junction region is allowed to enter both the first channel 702 and the second channel 701 substantially simultaneously, e.g., the inlet port 107 of the first channel and the inlet port 108 of the second channel are both in fluid communication with the junction region, where the liquid from the junction region 900 flows into the first channel and the second channel, respectively. The inflow into the first and second channels, respectively, occurs almost simultaneously. Thus, the fluid can show different flow characteristics in different channels, such as the length of the flow path of the fluid, the difference of the flow resistance, the difference of the flow speed of the fluid, the easiness of the fluidity and the like. For example, the flow path in the first channel may be shorter than the flow path in the second channel, or the flow resistance of the fluid flowing in the first channel may be made smaller than the flow resistance of the fluid flowing in the second channel. Therefore, to achieve the flow characteristics of the fluid in the different channels, the first channel 702 may be a straight channel and the second channel may include the curved channel 111, such that the fluid has different flow characteristics in the two channels. In some embodiments, the second channel upstream of the curved channel 111 comprises a straight initial channel 110 which communicates with the loading port 108 or the straight initial channel 110 has the loading port 108. In this arrangement, the flow or mixture of flows from the junction region 900 flows partially into the straight channels 702 and partially into the curved channels 701, but with the initially straight channels connected to the curved channels, the flow from the junction region can be made to enter each channel at substantially the same flow rate, but substantial differentiation of the flow rate characteristics is largely represented by the curved channel portions. This will not cause fluid blockage or congestion in the junction area, and is particularly effective for particularly viscous fluids. As shown in fig. 4, arrows 109 and 123 show a flow route of a fluid in the straight channel 702, and arrows 110 and 111 show a flow of the fluid in the second channel 701. At the exit of the two channels 701,702, there is also included a junction region 901 where the solids from the two channels join and mix together. And then to the next mixing unit.

In some forms, the mixer further includes a second mixing unit connected to the first mixing unit, the second mixing unit having the same physical structure as the first mixing unit but being connected to the first mixing unit in a different manner or at a different angle. The second mixing unit comprises a third channel 117 comprising curved channels and a fourth channel 116 being a straight channel. Similarly, the third channel has a third fluid inlet 115 and a third fluid outlet 118, the fourth channel also has a fourth fluid inlet 114 and a fourth fluid outlet 119, the inlets of the two channels are connected to a junction region 902, and the outlets of the two channels are connected to a junction region 903. In some approaches, for convenience of illustration, intersection region 902 may be referred to as a third intersection region, intersection region 903 may be referred to as a fourth intersection region, intersection region 900 may be referred to as a first intersection region, and intersection region 901 may be referred to as a second intersection region. In some embodiments, the initial channel 117 of the second mixing unit is located on the same straight line as the straight line channel 702 of the first mixing unit, and it is understood that the initial channel 117 in the third channel 118 of the second mixing unit is an extension of the straight line channel 702 in the straight line direction. In some approaches, the inlet 114 of the straight-line channel of the second cell is on the same side of the channel as the outlet 112 of the curved channel of the first channel. Alternatively, in some arrangements, the inlet 114 of the straight-line channel of the second cell is disposed or arranged adjacent to the outlet 112 of the curved channel of the first channel. In some embodiments, the inlet 115 of the curved passage of the second mixing unit and the outlet 113 of the straight passage of the first mixing unit are positioned on the same straight line, or are disposed in a face-to-face relationship.

In some embodiments, the straight-line channel of each mixing unit is angled at an acute angle less than 90 °, e.g., 85 °,70 °,75 °,60 °, 65 °,50 °, 55 °,45 °,40 °, 30 °,35 °,20 °,25 °, or 10 °, with respect to the angle between the initial channel of another curved channel.

Fig. 4 is a schematic diagram showing a positional relationship between two identical mixing units. Fig. 3 is a schematic diagram of an arrangement structure of 6 mixing units of the same structure. As can be seen from fig. 4 and 3, the arrangement is regular, and as described in fig. 4, the combination of the first mixing unit and the second mixing unit, and the third mixing unit arranged below the second mixing unit, are connected in the same or substantially the same way as the second mixing unit is connected to the first mixing unit. Specifically, as shown in fig. 3, the third mixing unit is located downstream of the second mixing unit, which is located downstream of the first mixing unit. The third mixing unit comprises a straight channel 210 and a curved channel 211, and upstream of the curved channel a straight channel 212 is connected, which straight channel can be seen as an extended channel of the straight channel 116 of the second mixing unit, and similarly comprises a junction area 904, which enables a diversion of the flow from the second mixing unit, and a junction area 905, which enables a junction or mixing from the third mixing unit. In this view, if the straight channel 116 of the second mixing unit is taken as a reference, the curved channel 701 of the first mixing unit is located to the right of the straight channel, and the curved channel 118 of the second mixing unit is located to the left of the straight channel 116. Alternatively, the straight channel 702 of the first mixing unit and the straight channel of the third mixing unit are relatively parallel and at an angle to the straight channel 116 of the second mixing unit, which may be greater than 90 °, e.g., 95 °,98 °, 100 °, 105 °, 110 °, 115 °, 120 °, 125 °, 130 °, 135 °, 140 °, 145 °, 160 °, 170 °, etc.

Two different fluids are continuously shunted, mixed, shunted, mixed and shunted in the mixing unit, so that the preparation of the nano particles is realized. This similar microfluidic droplet preparation technique is similar in that two fluids converge at a junction to form a water-in-oil or oil-in-water droplet by shear force of the fluids. The invention produces nanoparticles or particles which may also be liposome-encapsulated nucleic acid material or liposome-encapsulated core structures which are similar structures formed by nucleic acids and polymers. Such materials may be core structured materials and shell structured materials as described in chinese patent, application No. 201880001680.5. All of the embodiments in this application are to be considered as specific embodiments of a part of this invention.

This regular connection, as can be seen from fig. 3, includes that the arrangement of the curved channel of the first mixing unit relative to the curved channel of the second mixing unit and the connection between the mixing units are self-configured. Fig. 3 is only one preferred way to enable the preparation of nanoparticles.

It is meant here that all repeating mixed units are of the same structure but are connected in a regular arrangement, but are not limited to other arrangements.

The structural arrangement of the mixing unit comprises the following arrangement, wherein the mixing unit comprises two channels, one channel is in a linear shape, the other channel is in a curved shape, the radian of the curve and the relative relation between the curve and the linear channel are included, so that the external shape of the integrally formed unit also comprises the changes of the area size and the shape of the inlet intersection area and the outlet intersection area of the two channels, the depth and the width of the channels or the size of the cross-street area of the channels and the like, and one of the factors is changed to be considered as different mixing units. If a plurality of mixing regions exist, it is preferable that each mixing region has the same structure and only different arrangements and combinations, but it is also possible that each mixing region has a different structure. For example, referring to fig. 4, two mixing units with the same structure in fig. 4 have the same structure, but different connection modes or combination modes. Of course, it is also possible to use the same connection for the mixing units of different configurations. For example, the first mixing unit is the same as the one illustrated in fig. 4, but the second mixing unit may have a different configuration than the first mixing unit, such as one or more of the characteristics of the length, width, depth, cross-sectional area, size of the inlet, size of the outlet, curvature or degree of curvature of the curved portion of the curved channel, length with the initial straight channel, etc. being different from that of the first mixing unit.

In some embodiments, the first channel and the second channel of the first mixing unit are connected end to end, respectively, meaning that the head and the head of the first channel and the head of the second channel are connected together, and the tail are connected together. The connection is not a connected relation, but can be connected together through different junction areas, so that the liquid diversion at the head part and the collection or the confluence at the tail part are realized.

Therefore, the present invention provides a mixer comprising N mixing units, each mixing unit comprising a linear channel and a curved channel, each mixing unit comprising a linear fluid inlet and a fluid outlet, an inflow inlet and a fluid outlet of the curved channel, wherein N is a natural integer equal to or greater than 1. In some embodiments, the fluid inlet of the linear channel and the solid inlet of the curved channel communicate with the intersection region to split the fluid in the intersection region, and the fluid outlet of the linear channel and the solid outlet of the curved channel communicate with the intersection region to mix or converge or merge the fluid from the two channels.

Therefore, the invention provides a mixer, which comprises N +1 mixing units, wherein the Nth mixing unit comprises an a-th linear channel and an a + 1-th curvilinear channel, the a-th linear channel comprises an a-th fluid inlet and an a-th fluid outlet, and the a + 1-th curvilinear channel comprises an a + 1-th inflow inlet and an a + 1-th fluid outlet, wherein N is a natural integer equal to or greater than 1, and a is a natural number greater than or equal to 1. Alternatively, the invention provides a mixer, which comprises N +1 mixing units, wherein the Nth mixing unit comprises an a-th linear channel and an a + 1-th curvilinear channel, the a-th linear channel comprises an a-th fluid inlet and an a-th fluid outlet, and the a + 1-th curvilinear channel comprises an a + 1-th inflow inlet and an a + 1-th fluid outlet, wherein N is a natural integer equal to or greater than 1, and a is a natural number greater than or equal to 1. In some embodiments, the a-th linear channel has a length less than the a + 1-th curvilinear channel; alternatively, the path through which the fluid flows in the a-th linear channel is smaller than the fluid path force of the fluid in the a + 1-th curved channel.

In some embodiments, the fluid inlet of the a-th linear channel and the stereoscopic inlet of the curved channel include an a-th intersection region to split the fluid in the intersection region, and the fluid outlet of the linear channel and the stereoscopic outlet of the curved channel include an a + 1-th intersection region to mix or converge or merge the fluid from the two channels.

In some embodiments, the (N + 1) th mixing unit also includes an a +2 th linear channel and an a +3 th curved channel, the a +2 th linear channel includes an a +2 th fluid inlet and an a +2 th fluid outlet, and the a +3 th curved channel includes an a +3 th fluid inlet and an a +3 th fluid outlet. In some embodiments, the fluid inlet of the a +2 linear channel and the fluid inlet of the a +3 curved channel include an a +2 junction region to split the fluid in the junction region, and the fluid outlet of the a +2 linear channel and the stereo outlet of the a +3 curved channel include an a +3 junction region to mix or converge or join the fluid from the two channels.

In some embodiments, the a-th fluid outlet is disposed opposite the a + 3-th fluid inlet. In some embodiments, the a +1 fluid outlet is disposed adjacent to the a +2 fluid inlet, or on the same side of the channel.

The mixer composed of a plurality of mixing units which are communicated with each other provided by the invention can be called as a separation and recombination type. The separation and recombination type refers to that each mixing unit is provided with more than two channels (such as a first channel and a second channel), each mixing unit is firstly divided into more than two channels which are connected in parallel, and then the channels are recombined into one channel. The recombination of the channels can at least achieve a short convergence in the junction region, which means that the length of the converged channels is short and basically the same concept as that of the convergence region, and the splitting and the convergence in the convergence region can occur almost simultaneously or at intervals. As shown in fig. 4, for example, the merging region 901 is a region where the outlet flows of the two mixing units of the first mixing unit are mixed or merged, and then, another merging region 902 is formed, in which the mixing solid is divided again into the channels of the second mixing unit. There is no strict boundary division between the merging region 901 and the merging region 902, which is only illustrated by way of example for convenience of description, and of course, the two merging regions may also be collectively referred to as a merging region to achieve mixing and splitting of the fluid, and such mixing and splitting may or may not occur simultaneously.

Furthermore, the curved channel is formed by combining a semi-circular arc shape or arc shapes with different circle centers. Of course, the linear channels are shown relative to the curved channels, where linear channels are only linear in general and are not necessarily the ones that require precision instrumentation to see. The term curve is also used in relation to a straight line.

In some ways, as shown in fig. 4 or 3, the two channels constituting the mixing unit constitute a shape resembling the letter "D", but of course, may also be combined into a shape resembling the letter "B", which may be a combination of the two letters "D", and the letter "B" includes two mixing units. Any other combination is also possible.

The research group finds that when the separation and recombination type mixing pipeline is used for generating the nano particles through a large number of experiments, the mixing efficiency of the separation and recombination type mixing pipeline is obviously higher than that of the mixing pipelines with other shapes, and the liquid in the pipeline is mixed and shunted for multiple times, and is mixed and shunted again, so that the mixing effect is higher; but because present separation heavy form mixing duct is mostly circular arc annular or fan-shaped etc. and the structure is more complicated, when being used for the nano particle to generate, the flow resistance is higher, blocks up easily, and the mixed effect still is not as good as one's mind. The invention carries out structural creative design and improvement on the basis of the existing separation and recombination type mixing pipeline, ensures that one path is linear and reduces the flow resistance as much as possible, and simultaneously improves the mixing effect greatly because the other path adopts a semi-circular arc shape.

In some modes, the semi-circular second channel has a linear initial section, and it can be seen that the second channel is not a regular semi-circular arc, but an approximate semi-circular arc through creative design, which can help to reduce flow resistance and improve mixing effect.

Further, the initial segment length is less than or equal to 1/3 of the second channel length. The straight initial segment must not be too long, which would affect the mixing effect, and therefore needs to be controlled within 1/3 of the length of the second channel. By "curved channel" is meant a channel comprising a curve in the second channel, which curve may be a curve of continuous curvature or a curve of constant curvature, such that the curve may be, for example, a serpentine curve, or the shape of some of the channels as shown in fig. 15. In fig. 15, the linear form is represented as a linear channel, and the curved form represents a curved channel. The curved channel may be a part of the second channel which is curved, but it is understood that the second channel itself is curved in whole or in whole relative to the straight channel. Correspondingly, a straight channel does not mean that all channels are straight, and some curved channels may be included in the straight channel, so as to make the length of the straight channel lower. In general, the inflow into the two channels is made to flow with different fluid flow characteristics, such as speed, length of the flow path, and volume per unit time.

Further, the mixer comprises two or more mixing units, and the mixing units are connected end to end; the two adjacent mixing units are a mixing unit A and a mixing unit B, the second channel of the mixing unit A is positioned on the right side of the first channel, and the second channel of the mixing unit B is positioned on the left side of the first channel.

The mixing units are connected end to end, namely the head of the mixing unit A is connected with the tail of the mixing unit B, and the mixing units are connected in series. But the semi-circular arc directions of the adjacent mixing units A and B are opposite, so that the passing paths of the fluid are ensured to be consistent in the separation and recombination processes, but the flowing directions are opposite, the generated turbulence and vortex are also opposite, and the impact force applied to the fluid is changed regularly, so that the fluid is mixed more uniformly and stably.

Further, all the channels are uniform in width. Because the width of the channel is consistent and the width of the channel is not obviously changed, the fluid is not easy to be blocked by foreign matters in the mixing process. In some forms the channels of the mixing unit are of the same size or cross-sectional area or cross-section, so that such a microsomal pathway can be more easily created, simply by changing the shape of the pathway. The depth, width, or cross-sectional area, etc. may be such that the two channels are uniform, but alternatives that do not preclude uniformity are not excluded.

Further, the first channel (straight channel) of each mixing unit is in straight communication with the initial segment of the second channel (including curved channel) of the next mixing unit. This further helps to reduce flow resistance and improve mixing.

Further, the cross section of the channel of the mixer provided by the invention is rectangular, and the length and the width of all the cross sections of the channel are kept consistent. The cross section of the channel of the mixer can be made into various shapes according to the requirement, such as round, semi-round, square, rectangle, triangle, trapezoid, etc., and the cross section of the channel of the mixer is preferably rectangular or square for convenient manufacture.

Further, the mixer comprises 6 mixing units, the 6 mixing units being connected in series in the same way as the first mixing unit and the second mixing unit. Further enhancement of the mixing effect can also be achieved by a series connection of more mixing units.

Multiple experiments prove that the mixing effect of the mixing pipeline containing 6 mixing units for preparing the nano particles can completely meet the requirement of the mixing effect required by the preparation of the nano particles. The present invention therefore preferably employs a mixer comprising 6 mixing units. This is merely a preferred solution and does not mean that a single mixing unit cannot be completed, and a flow path that can increase flow is also possible, and may include 2 or more than 2 mixing units connected in series.

Further, according to the needs of preparing products, a plurality of mixers can be connected in series to improve the mixing effect. Multiple mixers can also be connected in series or in parallel into a microfluidic mixing cartridge. In certain embodiments, this comprises a second mixer. Other inlet connections may also be added to support the function of additional mixers. In one embodiment, multiple mixers may be included in a chip.

There are also ways to include a third inlet connection and to include a second mixer to effect dilution of the mixed solution produced by the first mixer by mixing the diluted solution provided via the third inlet connector.

The mixer is incorporated entirely within the chip, which is meant to be that the mixer structure cannot be easily removed from the chip. For example, the mixer is integrally incorporated into the chip provided that the chip is openable with a tool (e.g., a screwdriver used to loosen a set screw) in order to expose the mixer structure. Additionally, the condition for the mixer to be integrated into the chip is that the chip is hermetically closed, so that the mixer can only be removed by breaking open the chip. In yet other ways, the mixer is integrally incorporated into the chip provided that the mixer is physically attached or part of the chip (e.g., the microfluidic mixing chip cartridge is of unitary construction or has been permanently attached with an adhesive, solvent soldering, or other technique). The above-described monolithic construction is not considered to be incorporated into a chip because the mixer is part of the chip that provides functions (e.g., structural support) in addition to microfluidic flow.

In yet another embodiment, integrally incorporated means that the microfluidic hybrid chip cartridge cannot be disassembled and reassembled. For example, the mixer cannot be removed from the chip and then replaced and sealed.

Cartridge material and construction

The chip and microfluidic structure are formed from materials capable of being formed into the desired shape and having the desired physical characteristics. The material of the microfluidic structure is capable of forming the desired micron-scale mixing channels and is capable of withstanding the pressures applied during mixing in the microfluidic structure. The material of the chip is sufficiently rigid that it will protect and support the microfluidic structures within the chip.

In one embodiment, the microfluidic structure and the chip are formed from different materials. In yet another embodiment, the microfluidic structure and the chip are formed from the same material. In yet another embodiment, the microfluidic structure and the chip are integrally formed.

In one embodiment, the chip is free of metal. In yet another embodiment, the chip may contain some metal, but at least 90% by weight of the chip is polymer. In one embodiment, the chip is free of metal. In yet another embodiment, the chip may contain some metal, but at least 99% by weight of the chip is polymer.

In one embodiment, the chip comprises a polymer selected from the group consisting of polypropylene, polycarbonate, COC, COP, polystyrene, nylon, acrylic polymers, HPDE, LPDE and other polyolefins.

In one embodiment, the chip does not include metal on the outer surface. The embodiments contemplate that magnets or other metal-containing elements may be present within the chip, but not on the outer surface.

In yet another embodiment, the first inlet connector and the second inlet connector are formed from a polymer. It is preferred in certain embodiments that the inlet connector is formed from a relatively soft polymeric material, particularly where a tapered or Luer connector is used. The softer polymer will improve minor manufacturing tolerances of the inlet and allow a fluid tight connection to be formed. More rigid polymers would not allow the fault tolerance features described above. In this regard, in one embodiment, the first inlet connector comprises a polymer having a young's modulus of 500MPa to 3500 MPa. In one embodiment, the first inlet connector comprises a polymer having a young's modulus of 2000MPa to 3000 MPa.

In one embodiment, the chip comprises a metal selected from the group consisting of aluminum and steel. As described above, in some embodiments, a small amount of metal can be incorporated into the chip.

In one embodiment, the microfluidic structure is not separable from the chip. In such embodiments, the microfluidic structure is connected (e.g., soldered or attached) to at least a portion of the carrier. In one embodiment, the microfluidic hybrid chip cartridge is of unitary construction, wherein the chip and the microfluidic structure are formed from the same material. In yet another embodiment, the microfluidic hybrid chip cartridge is made up of at least two parts (e.g., a connecting part and a top plate), wherein the microfluidic structure is incorporated into one of the two parts. That is, the microfluidic structure is attached (e.g., bonded or soldered) to a portion of the microfluidic hybrid chip cartridge that serves an additional function beyond providing a microfluidic element. In one embodiment, the microfluidic structure is connected to the top plate. In yet another embodiment, the microfluidic structure and the top plate are monolithic and formed from the same material. In yet another embodiment, the microfluidic structure is a unitary construction with one of the two portions.

In one embodiment, the chip surrounds the microfluidic structure. As used herein, the term "surrounding" means that the chip surrounds a majority of the surface area of the mixer. Most importantly, the chip facilitates liquid-tight sealing with the microfluidic structure and provides a rigid chamber that allows manipulation of the microfluidic mixing cartridge. In yet another embodiment, the chip completely surrounds the microfluidic structure, meaning that no surface area of the microfluidic structure is exposed outside the carrier.

In one embodiment, the first portion and the second portion are joined together to enclose the microfluidic structure.

In one embodiment, at least 90% by weight of the first part is polymer. In this embodiment, the first portion includes an inlet connector and an outlet opening.

In one embodiment, the first portion or the second portion comprises a microfluidic structure. In such embodiments, the microfluidic structure is connected to or integral with the first or second portion of the chip.

Fluid source

The fluid or solution reservoirs are selected to enable direct connection to the microfluidic mixing cartridge. In one embodiment, the fluid reservoir is a disposable syringe. In yet another embodiment, the fluid reservoir is a pre-filled syringe. Both the fluid and the reservoir can be sterile in order to produce sterile nanoparticles. The system contains a device through which fluid is caused to flow from the reservoir and through the cartridge at a specified flow rate. In one embodiment of the system, the fluid is flowed by pressurizing the reservoir, causing the first and second fluids to enter the cartridge (through the inlet into the microfluidic structure and its channels). Examples of pressurizing devices include, but are not limited to, linear actuators and inert gases. In one embodiment, each reservoir is pressurized independently. In one embodiment, two or more reservoirs are pressurized from the same source, while differential flow rates are achieved by varying the dimensions of the fluid channels. Differential flow ratios may be made possible by differential pressure drops across the fluid channels, differential channel impedances, or a combination thereof applied to the inlet stream. Differential channel impedance by varying channel height, width, length or surface characteristics can be used to achieve different flow rates. The fluid surface tension, viscosity, and other surface characteristics of the liquid stream in the one or more first streams and the one or more second streams may be used or considered to achieve different flow rates. The pressurization of the vessel may be controlled by a computer or microcontroller.

In certain embodiments, the system further comprises means for a full or partial system purge to minimize waste volume. Purging may be accomplished by flowing a gas or liquid through the junction and microfluidic structure after or during the preparation of the particles. Gases such as air, nitrogen, argon or others may be used. Liquids that may be used include water, aqueous buffers, ethanol, oil, or any other liquid.

Fixing mechanism

In one embodiment, the microfluidic mixing cartridge further comprises a securing mechanism configured to secure the microfluidic mixing cartridge to the support. In one embodiment, the holder is a device configured to arrange the microfluidic mixing cartridge relative to a fluid source (e.g., a syringe) and facilitate connection therebetween.

Sterile

Sterile cartridges are necessary for certain applications and provide the user with a convenient workflow to directly formulate sterile nanoparticles without the need for further filtration or processing. The above described workflow minimizes the loss of material associated with further sterilization steps. In one embodiment, the individual components of the cassette are sterilized prior to assembly. Representative sterilization methods include steam autoclave, dry heat, chemical sterilization (i.e., sodium hydroxide or ethylene oxide), gamma irradiation, gas, and combinations thereof. In certain embodiments, the microfluidic structure, inlet connector, outlet connector, and any other fluid contacting components are formed from materials compatible with gamma radiation and are sterilized by the means described. Materials compatible with gamma radiation are those that can be irradiated. For example, polycarbonates, cyclic olefin polymers, cyclic olefin copolymers, polypropylenes, and high density polyethylenes and low density polyethylenes. Materials that cannot be irradiated include polyamide, polytetrafluoroethylene and any metal. In yet another embodiment, the cartridge is sterilized after assembly.

In one embodiment, the cartridge is sterilizable. As used herein, the term "sterilizable" means that the cassette is formed from materials that are compatible with known sterilization methods, as described previously. In one embodiment, the cassette is sterilizable, in particular by gamma radiation. In yet another embodiment, the cartridge is formed from a polymer selected from the group consisting of polypropylene, polycarbonate, cyclic olefin polymer, cyclic olefin copolymer, high density polyethylene, low density polyethylene, and combinations thereof. In yet another embodiment, the cartridge does not comprise polyamide, polytetrafluoroethylene, or any metal.

In one embodiment, the microfluidic mixing cartridge is sterile.

In one embodiment, the microfluidic mixing cartridge includes a sterile fluid path from the first inlet connector and the second inlet connector, through the microfluidic structure, and to the outlet opening. The sterile fluid path described above allows mixing in a sterile environment. Since the inlet connector and the outlet opening are also sterile, sterile connections can easily be made possible.

In yet another aspect, a sterile package filled with sterile contents is provided. In one embodiment, the sterile package comprises a microfluidic cartridge according to any of the embodiments disclosed herein in a sterile state and sealed within the sterile package. The sterile package is defined by a housing containing sterile contents. The housing is a bag in one embodiment. By providing a microfluidic mixing chip cartridge that is sterile and sealed within sterile packaging, an end user can easily perform sterile microfluidic mixing with the cartridge: the sterile packaging is opened in a sterile environment and used for mixing without any preparation. Sterilization is not required for any sterile inlet connectors or fluid paths.

In one embodiment, the sterile package further comprises a first sterile syringe configured to couple with the first inlet connector of the microfluidic cartridge. In such embodiments, the sterile package is a kit comprising a microfluidic cartridge and a sterile syringe configured for use with the microfluidic cartridge. In one embodiment, the sterile package further comprises a first solution within the first sterile syringe.

In one embodiment, the first solution comprises nucleic acids in a first solvent. In yet another embodiment, the first solution is of a type configured to form lipid nanoparticles.

In one embodiment, the sterile package further comprises a second sterile syringe configured to couple with the second inlet connector of the microfluidic cartridge.

In one embodiment, the sterile package further comprises a second solution within a second sterile syringe.

In one embodiment, the second solution comprises the lipid particle-forming material in a second solvent. The above-described second solution can be combined with a first solution comprising nucleic acids in a first solvent to form a lipid nanoparticle solution via a microfluidic cartridge.

In one embodiment, the sterile package further comprises a sterile container configured to couple with the outlet opening of the microfluidic cartridge via an outlet opening connector.

In one embodiment, the sterile contents are disposable.

Nanoparticles

The nanoparticles of the present invention are homogeneous particles comprising more than one component substance (e.g., lipids, polymers, etc.) that are used to encapsulate a therapeutic substance and have a minimum dimension of less than 250 nanometers. Nanoparticles include, but are not limited to, lipid nanoparticles and polymer nanoparticles.

Lipid nanoparticles

In one embodiment, a lipid nanoparticle comprises: (a) a core; and (b) a shell surrounding the core, wherein the shell comprises a phospholipid. Of course, a nucleic acid substance encapsulated with a lipid may be used.

In one embodiment, the core comprises a lipid (e.g., a fatty acid triglyceride) and is a solid. In yet another embodiment, the core is a liquid (e.g., aqueous) and the particles are vesicles such as liposomes. In one embodiment, the shell surrounding the core is a single layer.

As noted above, in one embodiment, the lipid core comprises fatty acid triglycerides. Suitable fatty acid triglycerides include C8-C20 fatty acid triglycerides. In one embodiment, the fatty acid triglyceride is oleic acid triglyceride.

The lipid nanoparticle includes a shell comprising a phospholipid, the shell surrounding a core. Suitable phospholipids include diacyl phosphatidyl choline, diacyl phosphatidyl ethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebrosides. In one embodiment, the phospholipid is a C8-C20 fatty acid diacylphosphatidylcholine. A representative phospholipid is 1-palmitoyl-2-oleoyl phosphatidylcholine (POPC).

In certain embodiments, the ratio of phospholipids to fatty acid triglycerides is 20:80 (mol: mol) to 60:40 (mol: mol). Preferably, the triglyceride is present in a ratio of greater than 40% and less than 80%.

In certain embodiments, the nanoparticle further comprises a sterol. Representative sterols include cholesterol. In one embodiment, the ratio of phospholipid to cholesterol is 55:45 (mol: mol). In representative embodiments, the nanoparticles comprise 55-100% POPC and up to 10 mol% PEG-lipid.

In other embodiments, the lipid nanoparticle of the present disclosure may include one or more other lipids, including phosphoglycerides, representative examples of which include phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phosphatidic acid, palmitoyl oleoyl phosphatidylcholine, lysylphosphatidylcholine (lysophosphatidylethanolamine), lysophosphatidylethanolamine, dipalmitoyl phosphatidylcholine, dioleoylphosphatidylcholine, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking phosphorus such as sphingolipids and glycosphingolipids are useful. Triacylglycerols are also useful.

Representative nanoparticles of the present disclosure have a diameter of about 10 to about 100 nm. The lower diameter limit is about 10 to about 15 nm.

The size-limited lipid nanoparticles of the present disclosure can include one or more therapeutic and/or diagnostic agents. These agents are typically contained within the particle core. The nanoparticles of the present disclosure can include a wide variety of therapeutic and/or diagnostic agents.

Suitable therapeutic agents include chemotherapeutic agents (i.e., anti-neoplastic agents), anesthetics, beta-adrenergic blockers, antihypertensive agents, antidepressants, anticonvulsants, anti-emetics, antihistamines, antiarrhythmics, and antimalarials.

Representative antineoplastic agents include doxorubicin, daunorubicin, mitomycin, bleomycin, streptozocin, vinblastine, vincristine, mechlorethamine, hydrochloride, melphalan, cyclophosphamide, triethylenethiophosphoramide, carmustine, lomustine, semustine, fluorouracil, hydroxyurea, thioguanine, cytarabine, floxuridine, dacarbazine, cisplatin, procarbazine, vinorelbine, ciprofloxacin, norfloxacin, paclitaxel, docetaxel, etoposide, bexarotene, teniposide, tretinoin, isotretinoin, sirolimus, fulvestrant, valrubicin, vindesine, folinic acid, irinotecan, capecitabine, gemcitabine, mitoxantrone hydrochloride, oxaliplatin, doxorubicin, methotrexate, carboplatin, estramustine, and pharmaceutically acceptable salts thereof.

In yet another embodiment, the lipid nanoparticle is a nucleic acid-lipid nanoparticle.

Nucleic acid-lipid nanoparticles refer to lipid nanoparticles containing nucleic acids. The lipid nanoparticle includes one or more cationic lipids, one or more second lipids, and one or more nucleic acids.

The lipid nanoparticle includes a cationic lipid. Cationic lipids refer to lipids that are cationic or become cationic (protonated) as the pH decreases below the ionizable group pK of the lipid, but become progressively more neutral at higher pH values. At pH values below pK, the lipid is then able to bind to negatively charged nucleic acids (e.g. oligonucleotides). Cationic lipids include zwitterionic lipids that are positively charged when the pH is lowered.

Cationic lipids refer to any of a number of lipid species that carry a net positive charge at a selective pH, such as a physiological pH. The lipids include, but are not limited to, N-dioleyl-N, N-dimethylammonium chloride (DODAC); n- (2, 3-dioleyloxy) propyl) -N, N, N-trimethylammonium chloride (DOTMA); n, N-distearyl-N, N-dimethylammonium bromide (DDAB); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTAP); 3- (N ', N' -dimethylaminoethane) -carbamoyl) cholesterol (DC-Chol) and N- (1, 2-dimyristoyloxyprop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, there are many commercial formulations of cationic lipids that can be used in the present disclosure. These include, for example (commercially available cationic liposomes comprising DOTMA and 1, 2-dioleoyl-sn-3-phosphoethanolamine (DOPE), from GIBCO/BRL, Grand Island, NY); (commercially available cationic liposomes comprising N- (1- (2, 3-dioleyloxy) propyl) -N- (2- (spermidine carboxamido) ethyl) -N, N-dimethylammonium trifluoroacetate (DOSPA) and (DOPE) from GIBCO/BRL); and (commercially available cationic lipids, including dioctadecyl amidoglycyl carboxy spermine (DOGS) in ethanol, from Promega corp. The following lipids are cationic and have a positive charge at physiological pH: DODAP, DODMA, DMDMA, 1, 2-dioleyloxy-N, N-dimethylaminopropane (DLinDMA), 1, 2-diisolinolenyloxy-N, N-dimethylaminopropane (DLenDMA).

In one embodiment, the cationic lipid is an amino lipid. Suitable amino lipids for use in the disclosure include those described in WO2009/096558, which is incorporated herein by reference in its entirety. Representative amino lipids include l, 2-dioleylideneoxy-3- (dimethylamino) acetoxypropane (DLin-DAC), 1, 2-dioleylideneoxy-3-morpholinopropane (DLin-MA), l, 2-dioleylidene-3-dimethylaminopropane (DLInDAP), l, 2-dioleylidenethio-3-dimethylaminopropane (DLin-S-DMA), l-linoleyyl-2-linolenyloxy-3-dimethylaminopropane (DLin-2-DMAP), l, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA. Cl), l, 2-dioleyl-3-trimethylaminopropane chloride salt (DLin-Cl), l, 2-dioleylideneoxy-3- (N-methylpiperazino) propane (DLin-MPZ), 3- (N, N-dioleenylamino) -l, 2-propanediol (DLINAP), 3- (N, N-dioleenylamino) -l, 2-propanediol (DOAP), l, 2-dioleenyloxy-3- (2-N, N-dimethylamino) ethoxypropane (DLin-EG-DMA), and 2, 2-dioleylene-4-dimethylaminomethyl- [ l,3] -dioxolane (DLin-K-DMA).

Suitable amino lipids include those having the formula:

wherein R1 and R2 are the same or different and are independently optionally substituted C10-C24 alkyl, optionally substituted C10-C24 alkenyl, optionally substituted C10-C24 alkynyl, or optionally substituted C10-C24 acyl; r3 and R4 are the same or different and are independently optionally substituted C1-C6 alkyl, optionally substituted C2-C6 alkenyl, or optionally substituted C2-C6 alkynyl; or R3 and R4 may be linked to form an optionally substituted heterocyclic ring having 4 to 6 carbon atoms and 1 or 2 heteroatoms selected from nitrogen and oxygen; r5 is absent or present and when present is hydrogen or C1-C6 alkyl; m, n and p are the same or different and are independently 0 or 1, provided that m, n and p are not simultaneously 0; q is 0,1, 2,3 or 4; and Y and Z are the same or different and are independently O, S or NH.

In one embodiment, R1 and R2 are each linoleyl groups and the amino lipid is a dilinoleyl amino lipid. In one embodiment, the amino lipid is a dilinoleyl amino lipid.

Representative useful dilinoleyl amino lipids have the formula:

wherein n is 0,1, 2,3 or 4.

In one embodiment, the cationic lipid is DLin-K-DMA. In one embodiment, the cationic lipid is DLin-KC2-DMA (DLin-K-DMA described above, where n is 2).

In addition to those specifically described above, other suitable cationic lipids include cationic lipids that carry a net positive charge at about physiological pH: n, N-dioleyl-N, N-dimethylammonium chloride (DODAC); n- (2, 3-dioleyloxy) propyl-N, N-N-triethylammonium chloride (DOTMA); n, N-distearyl-N, N-dimethylammonium bromide (DDAB); n- (2, 3-dioleoyloxy) propyl) -N, N-trimethylammonium chloride (DOTAP); 1, 2-dioleyloxy-3-trimethylaminopropane chloride salt (DOTAP. Cl); 3 β - (N- (N ', N' -dimethylaminoethane) carbamoyl) cholesterol (DC-Chol), N- (1- (2, 3-dioleoyloxy) propyl) -N-2- (sperminoylaminoyl) ethyl) -N, N-dimethylammonium trifluoroacetate (DOSPA), dioctadecyl amidoglycyl carboxy spermine (DOGS), 1, 2-dioleoyl-3-dimethylammoniumpropane (DODAP), N, N-dimethyl-2, 3-dioleoyloxy) propylamine (DODMA), and N- (1, 2-dimyristoyloxy-prop-3-yl) -N, N-dimethyl-N-hydroxyethylammonium bromide (DMRIE). Additionally, many commercial formulations of cationic lipids can be used, such as LIPOFECTIN (including DOTMA and DOPE, available from GIBCO/BRL), and LIPOFECTIAMINE (including DOSPA and DOPE, available from GIBCO/BRL).

The cationic lipid is present in the lipid particle in an amount of about 30 to about 95 mole percent. In one embodiment, the cationic lipid is present in the lipid particle in an amount of about 30 to about 70 mole percent. In one embodiment, the cationic lipid is present in the lipid particle in an amount of about 40 to about 60 mole percent.

In one embodiment, the lipid particle comprises one or more cationic lipids and one or more nucleic acids.

In certain embodiments, the lipid nanoparticle comprises one or more second lipids. Suitable second lipids stabilize nanoparticle formation during formation.

Lipids refer to a class of organic compounds that are esters of fatty acids and are characterized by being insoluble in water but soluble in many organic solvents. Lipids generally fall into at least three categories: (1) "simple lipids" which include fats and oils and waxes; (2) "compound lipids" which include phospholipids and glycolipids; and (3) "derivatized lipids" such as steroids.

Suitable stabilizing lipids include neutral lipids and anionic lipids.

Neutral lipids refer to any of a number of lipid species that exist in uncharged or neutral zwitterionic forms at physiological pH. Representative neutral lipids include diacyl phosphatidyl choline, diacyl phosphatidyl ethanolamine, ceramide, sphingomyelin, dihydrosphingomyelin, cephalin, and cerebroside.

Exemplary lipids include, for example, Distearoylphosphatidylcholine (DSPC), Dioleoylphosphatidylcholine (DOPC), Dipalmitoylphosphatidylcholine (DPPC), Dioleoylphosphatidylglycerol (DOPG), Dipalmitoylphosphatidylglycerol (DPPG), Dioleoylphosphatidylethanolamine (DOPE), palmitoylphosphatidylcholine (POPC), palmitoylolethanolamine (POPE) and dioleoylphosphatidylethanolamine 4- (N-maleimidomethyl) -cyclohexane-1-carboxylate (DOPE-mal), Dipalmitoylphosphatidylethanolamine (DPPE), Dimyristoylphosphatidylethanolamine (DMPE), Distearoylphosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), and 1, 2-dioleoyl-sn-glycero-3-phosphoethanolamine (trans DOPE).

In one embodiment, the neutral lipid is 1, 2-distearoyl-sn-glycerol-3-phosphocholine (DSPC).

Anionic lipid refers to any lipid that is negatively charged at physiological pH. These lipids include phosphatidylglycerol, cardiolipin, diacylphosphatidylserine, diacylphosphatidic acid, N-dodecanoylphosphatidylethanolamine, N-succinylphosphatidylethanolamine, N-glutarylphosphatidylethanolamine, lysylphosphatidylglycerol, palmitoyloleoylphosphatidylglycerol (POPG), and other anionic modifying groups attached to neutral lipids.

Other suitable lipids include glycolipids (e.g. monosialoganglioside GM 1). Other suitable second lipids include sterols such as cholesterol.

In certain embodiments, the second lipid is a polyethylene glycol-lipid. Suitable polyethylene glycol-lipids include PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol. Representative polyethylene glycol-lipids include PEG-c-DOMG, PEG-c-DMA, and PEG-s-DMG. In one embodiment, the polyethylene glycol-lipid is N- [ (methoxypoly (ethylene glycol) 2000) carbamoyl ] -1, 2-dimyristoyloxypropyl-3-amine (PEG-c-DMA). In one embodiment, the polyethylene glycol-lipid is PEG-c-DOMG).

In certain embodiments, the second lipid is present in the lipid particle in an amount of about 0.5 to about 10 mole percent. In one embodiment, the second lipid is present in the lipid particle in an amount of about 1 to about 5 mole percent. In one embodiment, the second lipid is present in the lipid particle at about 1 mole percent.

The lipid nanoparticles disclosed herein can be used for systemic or local delivery of nucleic acids. As described herein, the nucleic acid is incorporated into the lipid particle during formation of the lipid particle.

Of course, the nanoparticle can also be a core-shell type particle, and if the nucleic acid is mixed with the polymer to form a core, and then the liposome is wrapped outside the core structure, the mixing can also be completed by the mixer of the present invention. The nucleic acid and the polymer may be formed into a microparticle structure by a mixer, and then the microparticle and the lipid component may be formed into a microparticle structure by a mixer. All core materials and shell materials of this so-called core-shell structure, such as that of patent application No. 201880001680.5, can be formed using the mixer of the present invention, and all of the core materials and shell-forming materials of this patent are embodiments of the present invention.

Nucleic acids

Nucleic acids include any oligonucleotide or polynucleotide. Fragments containing up to 50 nucleotides are generally referred to as oligonucleotides, while longer fragments are referred to as polynucleotides. In a particular embodiment, the oligonucleotide is 20-50 nucleotides in length. In the present invention, polynucleotides and oligonucleotides refer to polymers or oligomers of nucleotide or nucleoside monomers, which are composed of trona, sugars and intersugar (backbone) linkages. Polynucleotides and oligonucleotides also include polymers or oligomers comprising non-natural monomers or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over the native form because of enhanced cellular uptake characteristics and increased stability in the presence of nucleases. Oligonucleotides are defined as either deoxyribonucleotides or ribonucleotides. Deoxyribonucleotides consist of a 5-carbon sugar called deoxyribose, covalently linked to a phosphate at the 5 'and 3' carbons, forming an alternating, unbranched polymer. Ribooligonucleotides are composed of a similar repetitive structure in which the 5-carbon sugar is ribose. The nucleic acid present in the lipid particle according to the present disclosure includes any known form of nucleic acid. The nucleic acid used herein can be single-stranded DNA or RNA, or double-stranded DNA or RNA, or a DNA-RNA hybrid. Examples of double-stranded DNA include structural genes, genes including control and termination regions, and self-replicating systems such as viral or plasmid DNA. Examples of double-stranded RNA include siRNA and other RNA interfering agents. Single-stranded nucleic acids include antisense oligonucleotides, ribozymes, microRNAs, mRNAs, and triplex oligonucleotides.

In one embodiment, the polynucleic acid is an antisense oligonucleotide. In certain embodiments, the nucleic acid is an antisense nucleic acid, ribozyme, tRNA, snRNA, siRNA, shRNA, ncRNA, miRNA, mRNA, lncRNA, sgRNA, precondensed DNA, or aptamer.

Nucleic acids also refer to nucleotides, deoxynucleotides, modified nucleotides, modified deoxynucleotides, modified phosphate-sugar-backbone oligonucleotides, other nucleotides, nucleotide analogs, and combinations thereof, and can optionally be single-stranded, double-stranded, or contain a portion of both double-stranded and single-stranded sequence.

Nucleotides encompass the following terms defined below: nucleotide bases, nucleosides, nucleotide analogs, and nucleotides in general.

Nucleotide base refers to a substituted or unsubstituted parent aromatic monocyclic or polycyclic ring. In certain embodiments, the aromatic monocyclic or polycyclic ring contains at least one nitrogen atom. In certain embodiments, the nucleotide base is capable of forming Watson-Crick and/or Hoogsteen hydrogen bonds with an appropriately complementary nucleotide base. Exemplary nucleotide bases and analogs thereof include, but are not limited to, purines such as 2-aminopurine, 2, 6-diaminopurine, adenine (a), vinylidene adenine, N6-2-isopentenyl adenine (6iA), N6-2-isopentenyl-2-methylthioadenine (2ms6iA), N6-methyladenine, guanine (G), isoguanine, N2-dimethylguanine (dmG), 7-methylguanine (7mG), 2-thiopyrimidine, 6-thioguanine (6sG) hypoxanthine and O6-methylguanine; 7-deaza-purines such as 7-deaza-adenine (7-deaza-A) and 7-deaza-guanine (7-deaza-G); pyrimidines such as cytosine (C), 5-propynylcytosine, isocytosine, thymine (T), 4-thiothymine (4sT), 5, 6-dihydrothymine, O4-methylthymine, uracil (U), 4-thiouracil (4sU) and 5, 6-dihydrouracil (dihydrouracil; D); indoles such as nitroindole and 4-methylindole; pyrroles such as nitropyrrole; a nebularine; a base (Y); in certain embodiments, the nucleotide base is typically a nucleotide base. Additional exemplary nucleotide bases can be found in Fasman,1989, Practical Handbook of Biochemistry and Molecular Biology, pp.385-394, CRC Press, BocaRaton, Fla. Further examples of general bases can be found in, for example, Loakes, N.A.R.2001, vol 29: 2437-.

A nucleoside refers to a compound having a nucleotide base covalently linked to the C-1' carbon of a pentose sugar. In certain embodiments, the linkage is via a heteroaromatic ring nitrogen. Typical pentoses include, but are not limited to, those pentoses in which one OR more of the carbon atoms are each independently substituted with one OR more of the same OR different-R, -OR, -NRR, OR halogen groups, wherein each R is independently hydrogen, (C1-C6) alkyl OR (C5-C14) aryl. The pentose sugars may be saturated or unsaturated. Exemplary pentoses and their analogs include, but are not limited to, ribose, 2' -deoxyribose, 2' - (C1-C6) alkoxyribose, 2' - (C5-C14) aryloxyglycoribose, 2',3' -dideoxyribose, 2',3' -didehydroribose, 2' -deoxy-3 ' -haloribose, 2' -deoxy-3 ' -fluororibose, 2' -deoxy-3 ' -chlororibose, 2' -deoxy-3 ' -aminoribose, 2' -deoxy-3 ' - (C1-C6) alkylribose, 2' -deoxy-3 ' - (C1-C6) alkoxyribose, and 2' -deoxy-3 ' - (C5-C14) aryloxyglycoribose. See also, for example, 2' -O-methyl, 4' -. alpha. -anomeric nucleotides, 1' -. alpha. -anomeric nucleotides (Asseline (1991) nucleic acids Res.19:4067-74), 2' -4' -and 3' -4' -linked and other "locked" or "LNA" bicyclic sugar modifications (WO 98/22489; WO 98/39352; WO 99/14226). An "LNA" or "locked nucleic acid" is a conformationally locked DNA analog in which the ribose ring is constrained by a methylene group attached between the 2' -oxygen and the 3' -or 4' -carbon. The conformational constraints imposed by this linkage often increase the binding affinity of the complementary sequences and increase the thermal stability of the duplex.

The saccharides include modifications at the 2 '-or 3' -position such as methoxy, ethoxy, allyloxy, isopropoxy, butoxy, isobutoxy, methoxyethyl, alkoxy, phenoxy, azido, amino, alkylamino, fluoro, chloro and bromo. Nucleosides and nucleotides include the natural D-form isomer (D-form), as well as the L-form isomer (L-form) (Beigelman, U.S. Pat. No.6,251,666; Chu, U.S. Pat. No. 5,753,789; Shudo, EP 0540742; Garnesi (1993) Nucl. acids sRs.21: 4159-65; Fujimmori (1990) J.Amer. chem. Soc.112: 7435; Urata, (1993) nucleic acids Symposium Ser.No.29: 69-70). In the case of a purine as nucleobase, for example A or G, the ribose is attached to the N9-position of the nucleobase. In the case of a pyrimidine as the nucleobase, for example C, T or U, the pentose is attached to the N1-position of the nucleobase (Kornberg and Baker, (1992) DNA Replication,2nd Ed., Freeman, SanFrancisco, Calif.).

One or more of the pentose carbons of the nucleoside may be substituted with a phosphate ester. In certain embodiments, the phosphate ester is attached to the pentose 3 '-or 5' -carbon. In certain embodiments, the nucleoside is one in which the nucleotide base is a purine, 7-deazapurine, pyrimidine, general nucleotide base, specific nucleotide base, or analog thereof.

Nucleotide analogs refer to those in which one or more of the pentose and/or nucleotide bases and/or nucleoside phosphates may be replaced with their respective analogs. In certain embodiments, exemplary pentose sugar analogs are those described above. In certain embodiments, the nucleotide analogs have a nucleotide base analog described above. In certain embodiments, exemplary phosphate analogs include, but are not limited to, alkylphosphonates, methylphosphonates, phosphoramidates, phosphotriesters, phosphorothioates, phosphorodithioates, phosphoroselenoates, phosphorodiselenoates, phosphorothioanilothioates, phosphoroamidates, boronophosphates, and can include a bound counterion. Other nucleic acid analogs and bases include, for example, embedded nucleic acids (INAs, described in Christensen and Pedersen,2002), and AEGIS bases (Eragen, U.S. Pat. No. 5,432,272). Additional descriptions of various Nucleic acid analogs can also be found, for example, in (Beaucage et al, Tetrahedron 49(10):1925(1993) and references thereto; Letsinger, J.Org.Chem.35:3800 (1970); Sprinzl et al, Eur.J.Biochem.81:579 (1977); Letsinger et al, Nucl.acids Res.14:3487 (1986); Sawai et al, Chem.Lett.805(1984), Letsinger et al, J.Am.Chem.Soc.110:4470 (1988); and Pauwels et al, Chemica script 26: 14191986)), phosphorothioate (Mag et al, Nucleic Acids Res.19:1437 (1991); and U.S. Pat. No. 5,644,048 other nucleic acid analogs include phosphorodithioates (Briu et al, J.am. chem. Soc.111:2321 (1989)), O-methyl phosphoramidate linkages (see Eckstein, Oligonucletides and antibiotics: A Practical Approach, Oxford university Press), those having a cationic backbone (Denpc et al, Proc. Natl. Acad. Sci. USA92:6097 1995 (1995); those having a non-ionic backbone (U.S. Pat. No. 5,386,023, 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863. Kiroedhishhi, Angel et al, model I.Chem.Ed.30: 1991, amino [ 14 ] and No.14,201,201, chemical [ 12 ] chemical et al [ chemical et al. (J.103; chemical et al.;. J.1987: chemical et al., chemical [ 12, J.103; chemical et al., chemical [ 12, 1994, molecular J.11, molecular mineral [ 12, J.11, J.103, chemical et al.) (U.S. 7, chemical et al.),15913, molecular mineral [ 12, J.),190, molecular mineral [ 12, J.),120, molecular mineral [ 12, Nucleotide [ 12, molecular mineral [ 12, J.),35, molecular mineral [ 12, 20, J.),120, 20, molecular mineral [ 12, Nucleotide [ 12, molecular mineral [ 12 ],120, molecular mineral [ 12 ],35, molecular mineral [ 12 ],103 ],35, molecular mineral [ 20, molecular mineral [ 12, molecular mineral [ 20 ],35, molecular mineral [ 20, molecular mineral [ 12 ],120, molecular mineral [ see [ 12, molecular mineral [ see [ 12, molecular mineral [ see [ 20 ]; ] and [ 1, molecular mineral [ see [ 12, molecular mineral [ 12 ],120, molecular mineral [ 12, molecular mineral [ 20 ]; ] and [ 20 ],120, molecular mineral [ 1, molecular mineral [ 12, molecular mineral [ 20, molecular mineral [ 1, molecular mineral [ see [ 20 ],120, molecular mineral [ 1, molecular mineral [ 20, molecular mineral [ 7 ],120, molecular mineral [ 20 ], ] and [ 7 ],120, molecular mineral [ 20 ],32 ],120, molecular mineral [ 20 ],120, molecular mineral [ see [ 20 ],120, molecular mineral [ 1, molecular mineral [ see [ 7 ],120, molecular mineral [ see [ sample [ see [ sample ] and [ see [ sample ] nuclear ] and [ sample [ see [ sample ] C ], ] C ],120, molecular mineral [ sample [ see [ sample [ see [ sample ] C ], ] C ],120, molecular mineral [ sample ] and [ sample ] C ], including those described in U.S. patent nos. 5,235,033 and 5,034,506, and chapters 6 and 7, ASC symposium series 580, "Carbohydrate Modifications in Antisense Research", ed.y.s.sanghuiand.p.dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included in the definition of nucleic acids (see Jenkins et al, chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are also described in Rawls, C & ENews, 1997, 6.2.1997, page 35.

A general nucleotide base or general base refers to an aromatic ring moiety, which may or may not contain a nitrogen atom. In certain embodiments, a general base may be covalently attached to the pentose C-1' carbon to form a general nucleotide. In certain embodiments, a nucleotide base does not, in general, specifically hydrogen bond with another nucleotide base. In certain embodiments, a nucleotide base generally hydrogen bonds with a nucleotide base up to and including all of the nucleotide bases in a particular target polynucleotide. In certain embodiments, a nucleotide base can interact with an adjacent nucleotide base on the same nucleic acid strand by hydrophobic stacking. Typical nucleotides include, but are not limited to, deoxy-7-azaindole triphosphate (d7AITP), deoxy isoquinolone triphosphate (dICSTP), deoxypropioquinolone triphosphate (dPICSTP), deoxymethyl-7-azaindole triphosphate (dM7AITP), deoxyImPy triphosphate (dImPyTP), deoxyPP triphosphate (dPPTP), or deoxypropinyl-7-azaindole triphosphate (dP7 AITP). Other examples of such general bases can be found in, inter alia, published U.S. application No.10/290672 and U.S. patent No.6,433,134.

Polynucleotides and oligonucleotides are used interchangeably and mean single-and double-stranded polymers of nucleotide monomers, including 2' -Deoxynucleotides (DNA) and nucleotides (RNA), linked by internucleotide phosphodiester linkages such as 3' -5' and 2' -5', reverse linkages such as 3' -3' and 5' -5', branched structures, or internucleotide analogs. Polynucleotides have associated counterions such as H +, NH4+, trialkylammonium, Mg2+, Na +, and the like. The polynucleotide may be composed entirely of deoxynucleotides, entirely of nucleotides, or of a chimeric mixture thereof. The polynucleotide may comprise an internucleoside moiety, a nucleobase and/or a carbohydrate analogue. Polynucleotides typically range in size from a few monomeric units, e.g., 3-40 (more frequently referred to in the art as oligonucleotides), up to thousands of monomeric nucleotide units. Unless otherwise indicated, whenever a polynucleotide sequence is present, it is understood that the nucleotides are in 5 'to 3' order from left to right and that "a" represents deoxyadenosine, "C" represents deoxycytosine, "G" represents deoxyguanosine, and "T" represents thymidine, unless otherwise noted.

Nucleobases are intended to mean those heterocyclic moieties, both natural and those non-natural, which are generally known to those employing nucleic acid techniques or employing peptide nucleic acid techniques, to thereby produce polymers capable of sequence-specific binding to nucleic acids. Non-limiting examples of suitable nucleobases include: adenine, cytosine, guanine, thymine, uracil, 5-propynyl-uracil, 2-thio-5-propynyl-uracil, 5-methylcytosine, pseudoisocytosine, 2-thiouracil and 2-thiothymine, 2-aminopurine, N9- (2-amino-6-chloropurine), N9- (2, 6-diaminopurine), hypoxanthine, N9- (7-deaza-guanine), N9- (7-deaza-8-aza-guanine) and N8- (7-deaza-8-aza-adenine). Other non-limiting examples of suitable nucleobases include those nucleobases described by Buchardt et al (WO92/20702 or WO 92/20703).

Nucleobase sequence means any fragment or aggregate of two or more fragments (e.g., an aggregate nucleobase sequence of two or more oligomer blocks) belonging to a polymer comprising nucleobase-containing subunits. Non-limiting examples of suitable polymers or polymer fragments include oligodeoxynucleotides (e.g., DNA), oligonucleotides (e.g., RNA), Peptide Nucleic Acids (PNA), PNA chimeras, PNA combination oligomers, nucleic acid analogs, and/or nucleic acid mimetics.

A polynuclear base chain means an entirely single polymer chain comprising nucleobase subunits. For example, a single nucleic acid strand of a double-stranded nucleic acid is a polynuclear base strand. The nucleic acid is a polymer containing a nucleobase sequence, or a polymer fragment having a backbone formed from nucleotides, or an analog thereof. Preferred nucleic acids are DNA and RNA.

Nucleic acids can also refer to "peptide nucleic acids" or "PNAs," which means any oligomer or polymer fragment (e.g., block oligomer) comprising two or more PNA subunits (residues), but not nucleic acid subunits (or analogs thereof), including, but not limited to, any of the oligomer or polymer fragments mentioned or claimed as peptide nucleic acids in U.S. Pat. nos. 5,539,082, 5,527,675, 5,623,049, 5,714,331, 5,718,262, 5,736,336, 5,773,571, 5,766,855, 5,786,461, 5,837,459, 5,891,625, 5,972,610, 5,986,053, and 6,107,470; which is incorporated herein in its entirety by reference. "peptide nucleic acids" or "PNA" are also applicable to any oligomeric or polymeric fragment comprising two or more subunits of a nucleic acid mimic described in the following disclosure: lagriffoul et al, Bioorganic & Medicinal Chemistry Letters,4: 1081-; petersen et al, Bioorganic & Medicinal Chemistry Letters,6: 793-; diderichsen et al, Tett.Lett.37: 475-; fujii et al, bioorg.Med.chem.Lett.7:637-627 (1997); jordan et al, bioorg.Med.chem.Lett.7: 687-; krotz et al, Tett.Lett.36:6941-6944 (1995); lagriffoul et al, bioorg.Med.chem.Lett.4:1081-1082 (1994); diederichsen, U.S., Bioorganic & Medicinal Chemistry Letters,7:1743-1746 (1997); loweet al, J.chem.Soc.Perkin Trans.1, (1997)1: 539-546; lowe et J.chem.Soc.PerkinTranss.11: 547-554 (1997); lowe et al, J.chem.Soc.Perkin Trans.11: 555-; howarth et al, J.org.chem.62:5441-5450 (1997); altmann, K-H et al, Bioorganic & Medicinal Chemistry Letters,7:1119-1122 (1997); diederichsen, U.S., Bioorganic & Med.chem.Lett.,8:165-168 (1998); diederichsen et al, Angew. chem. int. Ed.,37: 302-; tin et al, Tett.Lett.,38: 4211-; ciapetti et al, Tetrahedron,53: 1167-; lagriffoule et al, chem. Eur. J.,3: 912-; kumar et al, Organic Letters 3(9): 1269-; and peptide-based nucleic acid mimetics (PENAMS), which are described in publication WO96/04000 by Shah et al.

Polymer nanoparticles

Polymeric nanoparticles refer to polymeric nanoparticles containing a therapeutic substance. Polymeric nanoparticles have been developed with a wide range of materials including, but not limited to: synthetic homopolymers such as polyethylene glycol, polylactide, polyglycolide, poly (lactide-co-glycolide), polyacrylic acids, polymethacrylates, polycaprolactone, polyorthoesters, polyanhydrides, polylysine, polyethyleneimine; synthetic copolymers such as poly (lactide-co-glycolide), poly (lactide) -poly (ethylene glycol), poly (lactide-co-glycolide) -poly (ethylene glycol), poly (caprolactone) -poly (ethylene glycol); natural polymers such as cellulose, chitin and alginate, and polymer-therapeutic substance conjugates.

Polymers according to the invention are understood to mean generally high molecular weight compounds which are built up predominantly or completely from a number of similar units bonded together. The polymer includes any of a variety of natural, synthetic and semi-synthetic polymers.

Natural polymers refer to any number of polymer classes derived from nature. Such polymers include, but are not limited to, polysaccharides, cellulose, chitin and alginate.

Synthetic polymers refer to any number of synthetic polymer species not found in nature. The synthetic polymers include, but are not limited to, synthetic homopolymers and synthetic copolymers.

Synthetic homopolymers include, but are not limited to, polyethylene glycol, polylactide, polyglycolide, polyacrylic acid, polymethacrylate, poly (caprolactone), polyorthoester, polyanhydride, polylysine, and polyethyleneimine.

Synthetic copolymers refer to any number of synthetic polymer species that are constructed from two or more synthetic homopolymer subunits. The synthetic copolymers include, but are not limited to, poly (lactide-co-glycolide), poly (lactide) -poly (ethylene glycol), poly (lactide-co-glycolide) -poly (ethylene glycol), and poly (caprolactone) -poly (ethylene glycol).

By semi-synthetic polymer is meant any number of polymers derived by chemical or enzymatic treatment of natural polymers. Such polymers include, but are not limited to, carboxymethylcellulose, acetylated carboxymethylcellulose, cyclodextrins, chitosan, and gelatin.

Polymer conjugates refer to compounds prepared by conjugating, covalently or non-covalently, one or more molecular species to a polymer. The polymer conjugates include, but are not limited to, polymer-therapeutic substance conjugates.

Polymer-therapeutic substance conjugates refer to polymer conjugates in which one or more of the conjugated molecular species is a therapeutic substance. The polymer-therapeutic substance conjugates include, but are not limited to, polymer-drug conjugates.

A polymer-drug conjugate refers to any number of polymer species conjugated to any number of drug species. The polymeric drug conjugates include, but are not limited to, acetyl methylcellulose-polyethylene glycol-docetaxel.

Method of using microfluidic hybrid chip cartridge

In one aspect, a method of forming nanoparticles is provided. In one embodiment, a method includes flowing a first solution and a second solution through a microfluidic mixing cartridge according to any disclosed embodiment and forming a nanoparticle solution in a first mixer.

The method for preparing the nano particles mainly comprises the following steps:

1) preparing a sample 1 and a sample 2, respectively, wherein the sample 1 is a nucleic acid substance, and the sample 2 is a polymer or lipid solution;

2) injecting a sample 1 and a sample 2 from different liquid inlets respectively;

3) collecting the prepared nano particles from a liquid outlet.

Methods of forming nanoparticles with microfluidic hybrid chip cartridges are generally known in the art, and these methods may be used with the disclosed microfluidic hybrid chip cartridges, which essentially provide an improved and simplified way of performing the known methods. Exemplary methods disclosed in the patent literature are incorporated herein by reference. The following examples describe specific methods for producing siRNA lipid nanoparticles using an exemplary microfluidic hybrid chip cartridge.

In one embodiment, the first solution comprises nucleic acids in a first solvent.

In one embodiment, the second solution comprises the lipid particle-forming material in a second solvent.

In one embodiment, a plurality of microfluidic hybrid chip cartridges are used in parallel.

In one embodiment, the microfluidic mixing cartridge comprises a plurality of mixers, and the method comprises flowing the first solution and the second solution through the plurality of mixers to form the nanoparticle solution.

In still other embodiments, a third solution may be introduced to dilute the mixed solution.

Methods using microfluidic hybrid chip cartridges also include methods performed in a sterile environment, such as where certain nanoparticles (e.g., nano-drugs) must be formed sterile. In one embodiment, the method further comprises the step of sterilizing the fluid path prior to the step of flowing the first solution and the second solution through the microfluidic mixing cartridge. In one embodiment, the step of sterilizing the fluid path comprises sterilizing the microfluidic cartridge with radiation. In one embodiment, the step of sterilizing the fluid path comprises sterilizing portions of the microfluidic cartridge prior to assembling the microfluidic cartridge. In one embodiment, the sterile fluid path includes a fluid path from the first inlet connector and the second inlet connector, through the microfluidic structure, and to the outlet opening. In one embodiment, the sterile fluid path further comprises a first syringe containing the first solution coupled to the first inlet. In one embodiment, the sterile fluid path further comprises a second syringe containing a second solution coupled to the second inlet. In one embodiment, the sterile fluid pathway further comprises a sterile container coupled to the outlet opening of the microfluidic mixing cartridge via an outlet opening connector, and wherein the method further comprises the step of delivering the nanoparticle solution from the mixer to the sterile container via the outlet microchannel and the outlet opening. In one embodiment, the method further comprises the step of removing the sterile packaging from the microfluidic mixing cartridge prior to the step of flowing the first solution and the second solution through the microfluidic mixing cartridge.

Drawings

FIG. 1 is a schematic diagram of a microfluidic chip cartridge including microfluidic chips according to an embodiment

FIG. 2 is a schematic diagram of a microfluidic chip including a mixer in one embodiment

FIG. 3 is a schematic perspective view of a mixing unit of a microparticle passage in one embodiment

FIG. 4 is an enlarged schematic view of a mixing unit in one embodiment

FIG. 5 is a schematic structural diagram of a mixer provided in example 1

FIG. 6 is a sectional view taken along line A-A of FIG. 5, and FIG. 3 is a schematic view showing the direction of flow of a sample in the mixer

FIG. 7 is a schematic view showing the direction of flow of a sample in the mixer provided in example 1

Fig. 8 is a schematic structural diagram of a microfluidic hybrid chip provided in embodiment 2

FIG. 9 is a schematic view of a microfluidic hybrid chip cartridge according to embodiment 2

FIG. 10 is a schematic view of a backside structure of a microfluidic hybrid chip cartridge according to embodiment 2

FIG. 11 is a schematic side view of a microfluidic hybrid chip cartridge according to embodiment 2

FIG. 12 is a schematic view of a microfluidic hybrid chip cartridge according to example 2 in an operating state

FIG. 13 is a schematic diagram of a microfluidic hybrid chip cartridge for parallel high-throughput nanoparticle generation consisting of a plurality of microfluidic hybrid chip cartridges in parallel according to example 3

FIG. 14 shows the results of the continuous stability test of lipid nanoparticles prepared by the microfluidic hybrid chip of the present invention and the commercially available fishbone chip of manufacturer PNI in example 5

FIG. 15 shows the comparison of fluorescence intensity of in vitro transfection of eGFP-LPP prepared from microfluidic mixing chip provided by the present invention in example 6 and commercially available fish bone chip from manufacturer PNI

FIG. 16 shows the results of comparing the expression levels of eGFP-LPP transfected in vitro by the microfluidic chip of the invention in example 6 with that of eGFP-LPP prepared by a commercially available fishbone chip from manufacturer PNI

FIG. 17 is a schematic diagram of various other curved structures of a channel of a mixing unit in an embodiment

Detailed Description

In the following, preferred embodiments of the present invention will be described in further detail with reference to the accompanying drawings, it being noted that the following embodiments are intended to facilitate understanding of the present invention without any limitation thereto. The raw materials and equipment used in the examples of the present invention are known products and obtained by purchasing commercially available products.

In the description of the present invention, it is to be understood that the terms "center", "longitudinal", "lateral", "up", "down", "front", "back", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations or positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and for simplicity in description, and do not indicate or imply that the referenced devices or elements must have a particular orientation, be constructed and operated in a particular orientation, and thus, are not to be construed as limiting the present invention. Furthermore, the terms "first", "second", etc. are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of technical features indicated. Thus, a feature defined as "first," "second," etc. may explicitly or implicitly include one or more of that feature. In the description of the present invention, "a plurality" means two or more unless otherwise specified.

In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; they may be connected directly or indirectly through intervening media, or they may be interconnected between two elements. The specific meaning of the above terms in the present invention can be understood by those of ordinary skill in the art through specific situations.

Example 1A mixer according to the invention

Fig. 5, fig. 6 and fig. 7 are schematic diagrams of the mixer provided in this embodiment, where fig. 5 is a schematic structural diagram of the mixer, fig. 6 is a sectional view taken along line a-a of fig. 5, and fig. 7 is a schematic diagram of a sample flow direction in the mixer.

As shown in fig. 5, in the mixer provided in this embodiment, the mixer includes a mixing unit 1, the mixing unit 1 has a first channel 2 and a second channel 3, the first channel 2 is linear, the second channel 3 is curved, and the first channel 2 and the second channel 3 are respectively connected end to end, that is, the head and the head of the first channel 2 and the head of the second channel 3 are connected together, and the tail are connected together.

Preferably, the second channel 3 is semi-circular and has a rectilinear initial segment 4.

Preferably, the length of the initial segment 4 is less than or equal to 1/3 of the length of the second channel.

Preferably, the mixer comprises two or more mixing units 1, each mixing unit being connected end to end; two adjacent mixing units are a mixing unit A1 and a mixing unit B5, the second channel 3 of the mixing unit A is positioned at the right side of the first channel 2, and the second channel 6 of the mixing unit B5 is positioned at the left side of the first channel 7.

Preferably, all channel widths 8 are uniform.

As shown in fig. 6, the mixer of the present invention preferably has a rectangular channel cross-section 9, all channel cross-sections having the same length 10 and all channel widths 8. The channel cross-section 9 of the mixer can be made in various shapes as required, such as circular, semicircular, square, rectangular, triangular, trapezoidal, etc., and for convenience of manufacture, the channel cross-section of this embodiment is preferably rectangular or square.

Preferably, the first channel 2 of each mixing unit is in linear communication with the initial section 4 of the second channel 6 of the next mixing unit.

Preferably, the mixer comprises 6 mixing units 1.

Preferably, further enhancement of the mixing effect can also be achieved by adding more mixing units in series.

Preferably, a plurality of mixers provided in the embodiment can be connected in series to improve the mixing effect according to the requirement of preparing the product.

The flow direction of the sample fluid in the mixer is shown in fig. 7, the sample flows up and down, and is mixed well in the mixer.

Embodiment 2A microfluidic hybrid chip Cartridge according to the present invention

Fig. 8 to 12 show a microfluidic hybrid chip cartridge according to this embodiment, where fig. 8 is a schematic structural diagram of a microfluidic hybrid chip, fig. 9 is a schematic structural diagram of a microfluidic hybrid chip cartridge with an encapsulation box, fig. 10 is a schematic structural diagram of a back surface of a microfluidic hybrid chip cartridge with an encapsulation box, fig. 11 is a schematic structural diagram of a side surface of a microfluidic hybrid chip cartridge with an encapsulation box, and fig. 12 is a schematic structural diagram of a use state of the microfluidic hybrid chip cartridge.

The microfluidic mixing chip cartridge provided by the embodiment comprises the microfluidic mixer provided by the embodiment 1.

As shown in fig. 8-11, the present embodiment provides a microfluidic mixing chip cartridge, which includes a chip 11, the chip 11 is provided with liquid inlets 12 and 312, a liquid outlet 313, liquid inlet pipes 14 and 314, a liquid outlet pipe 15 and a mixer 16, and the liquid inlets 12 and 312 and the liquid outlet 313 are disposed perpendicular to the side wall of the chip; the liquid inlet pipeline 14 is connected with the liquid inlet 12 and the mixer 16, the liquid inlet pipeline 314 is connected with the liquid inlet 312 and the mixer 16, the liquid outlet pipeline 15 is connected with the liquid outlet 313 and the mixer 16, and the packaging box 17 is arranged outside the chip. The liquid inlets 12 and 312 and the liquid outlet 13 are respectively located at two sides of the chip 11.

Preferably, the liquid inlets 12 and 312 and the liquid inlet pipes 14 and 314 are located on the same plane, and the liquid outlet 313 and the liquid outlet pipe 15 are located on the same plane.

Preferably, loading ports 12 and 312, loading ports 14 and 314, loading port 313, unloading port 15 and chip 11 are all substantially in the same plane. The sample is only injected from the side of the chip 11.

As shown in fig. 12, in the microfluidic chip cartridge according to the present invention, the liquid inlets 12, 312 and the liquid outlet 13 are perpendicular to the side wall of the chip 11, when in use, a vertically downward injection manner of the injector is adopted, the chip 11 and the injector are in the same plane, after the injector extracts the liquid sample, the injector is placed vertically downward, the bubbles naturally float to the top end of the injection interior, then the injector is respectively inserted into the liquid inlets 12 and 312 of the chip 11 vertically downward, and then the liquid in the injector is completely injected into the liquid inlets 12 and 312, since the bubbles float to the top of the injector, there is no need to worry about the injection of the bubbles, and the waste of expensive sample liquid caused by the manual operation of driving the bubbles at the head of the injector is also avoided.

Example 3 microfluidic hybrid chip cartridge for parallel high throughput nanoparticle generation provided by the invention

As shown in fig. 13, the present invention provides a microfluidic hybrid chip cartridge for parallel high-throughput nanoparticle generation, which is composed of a plurality of microfluidic hybrid chip cartridges provided in example 2 in parallel. Because the liquid inlets 12 and 312, the liquid outlet 313 and the chip 11 are in the same plane, the microfluidic chip cartridge can be stacked by injecting from the side surface of the chip 11 during sample adding, thereby realizing parallel high-throughput use and being used for generating parallel high-throughput nano particles.

Example 4 comparison of the Performance of different chips

The microfluidic mixed chip provided in example 2 and a fishbone chip of a manufacturer PNI sold in the market are respectively adopted to prepare the lipid nanoparticles, and the influence of different mixing flow rates on the particle size of the lipid nanoparticles is examined, and the method specifically comprises the following steps: an appropriate amount of lipid solution (ionizable lipid MC3, DSPC, cholesterol, mPEG2000-DMG prepared as a 10mg/ml lipid solution at a molar ratio of 50: 10: 38.5: 1.5) was mixed with eGFP-mRNA (dissolved in 1mM ph6.4 citric acid-sodium citrate buffer, mRNA sequence of GFP:

AUGGUGAGCA AGGGCGAGGA GCUGUUCACC GGGGUGGUGC CCAUCCUGGU CGAGCUGGAC GGCGACGUAA ACGGCCACAA GUUCAGCGUG UCCGGCGAGG

101 GCGAGGGCGA UGCCACCUAC GGCAAGCUGA CCCUGAAGUU CAUCUGCACC ACCGGCAAGC UGCCCGUGCC CUGGCCCACC CUCGUGACCA CCCUGACCUA

201 CGGCGUGCAG UGCUUCAGCC GCUACCCCGA CCACAUGAAG CAGCACGACU UCUUCAAGUC CGCCAUGCCC GAAGGCUACG UCCAGGAGCG CACCAUCUUC

301 UUCAAGGACG ACGGCAACUA CAAGACCCGC GCCGAGGUGA AGUUCGAGGG CGACACCCUG GUGAACCGCA UCGAGCUGAA GGGCAUCGAC UUCAAGGAGG

401 ACGGCAACAU CCUGGGGCAC AAGCUGGAGU ACAACUACAA CAGCCACAAC GUCUAUAUCA UGGCCGACAA GCAGAAGAAC GGCAUCAAGG UGAACUUCAA

501 GAUCCGCCAC AACAUCGAGG ACGGCAGCGU GCAGCUCGCC GACCACUACC AGCAGAACAC CCCCAUCGGC GACGGCCCCG UGCUGCUGCC CGACAACCAC

601 UACCUGAGCA CCCAGUCCGC CCUGAGCAAA GACCCCAACG AGAAGCGCGA UCACAUGGUC CUGCUGGAGU UCGUGACCGC CGCCGGGAUC ACUCUCGGCA

701 UGGACGAGCU GUACAAGUAA) at different flow rates of 1,6, 12, 20ml/min, fixed mixing ratio of 3(mRNA solution): 1 (lipid solution), the fixed temperature is 37 ℃, lipid nanoparticles are obtained, the particle size is tested by a dynamic light scattering particle size analyzer, and the test is repeated three times, and the results are shown in table 1.

TABLE 1 comparison of particle size of lipid nanoparticles prepared from different chips

Serial number	Mixed flow rate (ml/min)	PNI fishbone chip (nm)	Microfluidic mixing chip (nm) of example 1
				1	1	164.7±1.1	156.9±7.9
2	6	88.7±8.1	90.6±5.4
				3	12	87.1±4.1	87.2±4.9
4	20	76.7±1.8	83.4±8.5

As can be seen from table 1, the overall difference between the chip structure provided in example 1 of the present invention and the particle size results of the lipid nanoparticles prepared by the fish bone chip of the commercially available manufacturer PNI in each flow rate range (1, 6, 12, 20ml/min) is not large, but the particle size of the lipid nanoparticles prepared by the chip provided in example 1 is more stable, and the particle size difference is smaller at different flow rates. Therefore, the nano particles prepared by the microfluidic mixed chip provided by the invention are more uniform and stable, the flow resistance is smaller, the mixing efficiency is higher, and meanwhile, the parallel high-throughput production can be carried out, and the effect is obviously better than that of the existing microfluidic chip.

Example 5 sustained stability testing of chips

The microfluidic mixed chip provided in example 2 is used for preparing lipid nanoparticles, and the stability of the chip in continuous mixing preparation is examined, and the preparation method specifically comprises the following steps: an appropriate amount of lipid solution was mixed with eGFP-mRNA, respectively, at a fixed mixing ratio of 20ml/min at a fixed mixing flow rate of 3(mRNA solution): 1 (lipid solution), constant temperature of 37 ℃, mixing for 40min, and taking points every 10min to obtain lipid nanoparticles, and testing the particle size with a dynamic light scattering particle size analyzer, and repeating three times, the results are shown in fig. 14 (composition of lipid solution and eGFP-mRNA refer to example 4).

The test results of fig. 14 show that the chip structure provided by the present invention has good continuous stability results, the particle size results obtained after the chip is continuously operated for 40 minutes are equivalent to the initial values, and the polydispersity index PDI is less than 0.05.

Example 6 fluorescent imaging and quantitation of GFP expression of eGFP-LPP prepared on different chips

The microfluidic mixed chip provided in example 2 and a fishbone chip of a manufacturer PNI sold in the market are respectively adopted to prepare lipid nanoparticles, the prepared eGFP-LPP is transfected in vitro, fluorescence imaging and GFP expression quantitative results of eGFP-LPP prepared by different chips are inspected, and the method specifically comprises the following steps: the prepared lipid nanoparticles (containing fluorescent mRNA) containing 100ng eGFP-mRNA prepared by different chips were incubated with 2 × 104 DC2.4 cells for 24 hours, and then the expression of GFP was observed by a fluorescence microscope, and the result is shown in fig. 15, and finally the expression of GFP was quantified by a GFP quantification kit, and the result is shown in fig. 16 (the components of the lipid solution and eGFP-mRNA refer to example 4).

As can be seen in FIG. 15, the results of the tests show that the chip structure provided in example 1 is comparable to the fluorescence intensity of in vitro transfection of eGFP-LPP prepared from a commercially available fishbone chip from manufacturer PNI.

The test results in FIG. 16 show that the chip structure provided in example 1 is comparable to the expression level of eGFP-LPP transfected in vitro by the fish bone chip of the commercial manufacturer PNI, and has no significant difference.

Example 7 comparison of mixing Effect of different mixing element counts

The microfluidic mixing chip provided in embodiment 2 is adopted, wherein the number of mixing units is 2, 4, 6,8, 10, and the lipid nanoparticles are prepared respectively, and the specific steps are as follows: appropriate amounts of lipid solutions were respectively mixed with eGFP-mRNA ((components of lipid solution and eGFP-mRNA refer to example 4): a fixed mixing ratio of 3(mRNA solution): 1 (lipid solution) at a fixed mixing flow rate of 20ml/min, a fixed temperature of 37 ℃, continuously mixed for 40min, and spotting was performed every 10min to obtain lipid nanoparticles, and particle diameters were measured using a dynamic light scattering particle sizer, and a dispersibility index PDI and an encapsulation efficiency were calculated and repeated three times, and the results are shown in table 2.

TABLE 2 comparison of Polymer/mRNA nanoparticle Generation Effect Using different Unit numbers

Number of mixing units	Particle size	PDI (dispersibility index)	Encapsulation efficiency (%)
				2	72.4±2.5	0.145	90.3％
4	79.2±3.7	0.112	92.7％
				6	83.5±8.5	0.040	99.6％
8	84.1±6.3	0.042	99.4％
				10	84.9±5.0	0.043	99.1％

As can be seen from Table 2, the mixing effect when the mixing pipes of 6 mixing units are used for preparing the nanoparticles can completely meet the mixing effect requirement required for preparing the nanoparticles.

Although the present invention is disclosed above, the present invention is not limited thereto. For example, the application range of the micro-fluidic field can be expanded. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

The invention shown and described herein may be practiced in the absence of any element or elements, limitation or limitations, which is specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, and it is recognized that various modifications are possible within the scope of the invention. It should therefore be understood that although the present invention has been specifically disclosed by various embodiments and optional features, modification and variation of the concepts herein described may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The contents of the articles, patents, patent applications, and all other documents and electronically available information described or cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. Applicants reserve the right to incorporate into this application any and all materials and information from any such articles, patents, patent applications, or other documents.