Opposite-flushing mixing device and method for liposome synthesis

Abstract

The invention provides an opposite flow mixing device and method for liposome synthesis, and relates to the field of microflow control, the opposite flow mixing device (100) is provided with a device body (10), the device body (10) is internally provided with a reaction mixing cavity (20), a first fluid channel (30) and a second fluid channel (40), the reaction mixing cavity (20) is provided with a first fluid inlet and a second fluid inlet, and the first fluid channel (30) is communicated with the second fluid channel (40). The first fluid inlet and the second fluid inlet are oppositely arranged, the first fluid channel (30) is communicated with the reaction mixing cavity (20) through the first fluid inlet, and the second fluid channel (30) is communicated with the reaction mixing cavity (20) through the second fluid inlet. The opposed flow mixing device (100) and the method can fully and uniformly mix a nucleic acid drug component and a liposome component in the device, and can be used for stably producing uniform nanoparticles in a standardized manner.

Description

A counter-flow mixing device and method for liposome synthesis

[0001] This application is based on the priority of the prior Chinese patent application (application number 202210340967.9, application date April 2, 2022, invention name “A counter-flow mixing device and method for liposome synthesis”). Technical Field

[0002] The present invention relates to the field of microfluidic control, in particular to a counter-flow mixing device and method for liposome synthesis. Background Art

[0003] Since 2019, the outbreak of the novel coronavirus pneumonia has captured the attention of the international community. As one of the mainstays of COVID-19 prevention, mRNA vaccines have proven their efficacy and market potential. Currently, the mainstream delivery method for mRNA vaccines is LNP delivery, and the primary production equipment for LNP is microfluidic chips. However, current production methods suffer from high costs and low yields. In microfluidics, counterflow is the process of two or more fluid streams flowing in opposite directions and counteracting each other, creating a turbulent region and enhancing heat and mass transfer during fluid mixing. The concept of counterflow has achieved promising results in chemical production processes such as material drying and mixing.

[0004] In multicomponent reaction or assembly systems, efficient mixing of the different components is crucial for controlling the properties and uniformity of the manufactured product. Mixing via flow turbulence is highly effective because turbulence rapidly generates flow structures on a greatly reduced length scale, where mixing between the different components introduced by the different flows can occur on a time scale of tens of milliseconds. For liposome synthesis systems, it is important to match the mixing rate to the reaction rate because if the mixing rate is slow, the reaction will occur in a non-uniform manner in time and space, resulting in heterogeneous products. Therefore, how to provide a mixing device that can synthesize liposomes continuously and stably is a very urgent problem to be solved.

[0005] In view of this, the inventors, based on many years of experience in production design in this field and related fields, have designed a counter-flow mixing device and method for liposome synthesis after repeated experiments, in order to solve the problems existing in the prior art.

[0006] Summary of the Invention

[0007] The purpose of the present invention is to provide a counter-flow mixing device and method for liposome synthesis, which can fully and evenly mix nucleic acid drug components and liposome components in the device and be used to standardize and stably produce uniform nanoparticles.

[0008] To achieve the above-mentioned purpose, the present invention proposes a counter-flow mixing device for liposome synthesis, wherein the counter-flow mixing device has a device body, a reaction mixing chamber, a first fluid channel and a second fluid channel are opened in the device body, the reaction mixing chamber has a first fluid inlet and a second fluid inlet, the first fluid inlet and the second fluid inlet are arranged opposite to each other, the first fluid channel is connected to the reaction mixing chamber through the first fluid inlet, and the second fluid channel is connected to the reaction mixing chamber through the second fluid inlet.

[0009] The present invention also proposes a counter-flow mixing method for liposome synthesis, in which nanoparticles are prepared by the counter-flow mixing device as described above, wherein the liposome mixture enters the reaction mixing chamber from the first fluid channel, and the drug solution enters the reaction mixing chamber from the second fluid channel. When the liposome mixture and the drug solution enter the reaction mixing chamber, counter-flow occurs, and the cationic liposomes in the liposome mixture are rapidly precipitated due to changes in the properties of the solution. The precipitated cationic liposomes wrap the drug components in the drug solution and aggregate and precipitate together to form nanoparticles.

[0010] Compared with the prior art, the present invention has the following characteristics and advantages:

[0011] The present invention proposes a counter-flow mixing device and method for liposome synthesis. The liposome mixture and the drug solution enter the reaction mixing chamber from the first fluid channel and the second fluid channel respectively. Since the first fluid inlet and the second fluid inlet are arranged opposite to each other, the two fluids entering the reaction mixing chamber collide with each other, forming a counter-flow turbulent zone, thereby improving the mixing and reaction efficiency of the two fluids, having more stable performance, and being suitable for high-quality and high-efficiency production. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] The drawings described herein are for illustrative purposes only and are not intended to limit the scope of the present invention in any way. In addition, the shapes and proportional dimensions of the various components in the drawings are merely illustrative and are used to help understand the present invention, and are not intended to specifically limit the shapes and proportional dimensions of the various components of the present invention. Those skilled in the art can select various possible shapes and proportional dimensions to implement the present invention according to specific circumstances under the guidance of the present invention.

[0013] FIG1 is a schematic diagram of the three-dimensional structure of a counter-current mixing device for liposome synthesis proposed by the present invention;

[0014] FIG2 is a side view schematic diagram of a counter-current mixing device for liposome synthesis proposed by the present invention;

[0015] FIG3 is a cross-sectional view taken along line BB in FIG2 ;

[0016] FIG4 is a graph showing the stability results of nanoparticles prepared by the counter-flow mixing device for liposome synthesis proposed by the present invention.

[0017] Description of reference numerals:

[0018] 100. Countercurrent mixing device;

[0019] 10. Device body;

[0020] 20. Reaction mixing chamber;

[0021] 30. a first fluid channel;

[0022] 31. First thin diameter section;

[0023] 32. First thick diameter section;

[0024] 33. First conduit connecting section;

[0025] 40. Second fluid channel;

[0026] 41. Second thin diameter section;

[0027] 42. Second thick diameter section;

[0028] 43. Second conduit connecting section;

[0029] 50. The third conduit connecting section. DETAILED DESCRIPTION

[0030] The following is a detailed description of the technical means used by the present invention to achieve the intended purpose of the invention, with reference to the drawings and preferred embodiments of the present invention.

[0031] As shown in Figures 1, 2 and 3, the present invention proposes a counter-flow mixing device 100 for liposome synthesis, which has a device body 10, in which a reaction mixing chamber 20, a first fluid channel 30 and a second fluid channel 40 are provided. The reaction mixing chamber 20 has a first fluid inlet and a second fluid inlet, which are arranged opposite to each other. The first fluid channel 30 and the second fluid channel 40 are used to introduce fluid into the reaction mixing chamber 20. The first fluid channel 30 is connected to the reaction mixing chamber 20 through the first fluid inlet, and the second fluid channel 40 is connected to the reaction mixing chamber 20 through the second fluid inlet.

[0032] The present invention also proposes a method for liposome synthesis, in which nanoparticles are prepared through a counter-flow mixing device 100. The liposome mixture and the drug solution enter the reaction mixing chamber 20 from the first fluid channel 30 and the second fluid channel 40 respectively and counter-flow. The cationic liposomes in the liposome components are rapidly precipitated due to changes in the properties of the solution. The precipitated cationic liposomes wrap the drug components and aggregate and precipitate together to form nanoparticles.

[0033] The present invention proposes a counter-flow mixing device 100 and method for liposome synthesis. The liposome mixture and the drug solution enter the reaction mixing chamber 20 from the first fluid channel 30 and the second fluid channel 40 respectively. Since the first fluid inlet and the second fluid inlet are arranged opposite to each other, the two fluids entering the reaction mixing chamber 20 are counter-flowed to form a counter-flow turbulent zone, thereby improving the mixing and reaction efficiency of the two fluids, making the performance more stable, and being suitable for high-quality and high-efficiency production.

[0034] The counter-flow mixing device 100 and method for liposome synthesis proposed in the present invention enable cationic liposomes in the liposome components to precipitate rapidly due to changes in the properties of the solution, while simultaneously encapsulating a large amount of nucleic acid drug components and rapidly polymerizing and settling together. This results in more stable performance and is suitable for the production of nanoparticles, thereby achieving high-quality and high-efficiency production of nanoparticles.

[0035] In an optional example of the present invention, the liposome mixture can be replaced by a polymer solution; the drug solution includes a nucleic acid drug solution; and the nanoparticles include nanoparticles for nucleic acid vaccines.

[0036] In a rapid nanocomposite reaction, when the average mixing rate of the liposome mixture (or polymer) and the nucleic acid solution (or drug solution) matches the polyelectrolyte complexation rate, liposome nanoparticles with uniform particle size can be quickly produced. Specifically, the two fluid components provide an initial flow rate through an infusion pump. When the nucleic acid drug component enters the reaction mixing chamber 20 at a constant speed through the second fluid channel 40 of the fluid and is mixed with the liposome component jet that also passes through the first fluid channel 30 at a constant speed, the two fluids collide with each other and mix rapidly. The cationic liposomes in the liposome component are rapidly precipitated due to changes in the properties of the solution, and at the same time, a large amount of nucleic acid drug components are wrapped and quickly aggregated and precipitated together. The performance is more stable, suitable for the production of nanoparticles, and high-quality and high-efficiency production of nanoparticles is achieved.

[0037] The counter-flow mixing device 100 for liposome synthesis proposed in the present invention has a simple structure, is not prone to clogging, is easy to clean, and has a high production speed. The nucleic acid drug components and liposome components can be fully and evenly mixed in the counter-flow mixing device 100, and can be used for standardized large-scale and stable production of uniform nanoparticles.

[0038] In an alternative embodiment of the present invention, the device body 10 is a metal block, with the reaction mixing chamber 20, first fluid channel 30, and second fluid channel 40 formed within the block by punching holes in the center of the block. With this structure, the entire structure of the countercurrent mixing device 100 is integrated within the metal block, effectively preventing solution leakage during the countercurrent mixing process and providing greater safety and reliability.

[0039] In an optional embodiment of the present invention, the axis of the first fluid inlet and the axis of the second fluid inlet are located on the same straight line to ensure that the two fluids will collide after entering the reaction mixing chamber 20 to form a counter-flow turbulent zone.

[0040] In an optional embodiment of the present invention, the first fluid channel 30 includes a first thin-diameter section 31 and a first thick-diameter section 32. The first thin-diameter section 31 is connected to the first fluid inlet, and the inner diameter of the first thin-diameter section 31 is smaller than the inner diameter of the first thick-diameter section 32. With this structure, after the fluid enters the first thin-diameter section 31, the reduced inner diameter of the first thin-diameter section 31 causes jet acceleration, further increasing the flow velocity at the first fluid inlet and enhancing the effect of counteracting turbulence.

[0041] Preferably, the first thick-diameter section 32 has an inner diameter of 1 mm and a length of 2 mm, and the first thin-diameter section 31 has an inner diameter of 0.3 mm and a length of 3.3 mm.

[0042] In an optional example of this embodiment, the first fluid channel 30 further includes a first catheter connecting section 33 for connecting to an infusion catheter. The first catheter connecting section 33 is connected to the first large-diameter section 32, and the inner diameter of the first catheter connecting section 33 is larger than the inner diameter of the first large-diameter section 32. With this structure, the end of the infusion catheter can be inserted into the first catheter connecting section 33, thereby connecting the infusion catheter to the device body 10.

[0043] In an optional example, the infusion catheter is threadedly connected to the first catheter connecting section 33 .

[0044] In an optional embodiment of the present invention, the second fluid channel 40 includes a second thin diameter section 41 and a second thick diameter section 42 . The second thin diameter section 41 is connected to the second fluid inlet and has an inner diameter smaller than that of the second thick diameter section 42 .

[0045] Preferably, the second thick-diameter section 42 has an inner diameter of 1 mm and a length of 2 mm, and the second thin-diameter section 41 has an inner diameter of 0.3 mm and a length of 3.3 mm.

[0046] In an optional example of this embodiment, the second fluid channel 40 also includes a second catheter connecting section 43 for connecting to an infusion catheter, the second catheter connecting section 43 is connected to the second thick diameter section 42, and the inner diameter of the second catheter connecting section 43 is larger than the inner diameter of the second thick diameter section 42.

[0047] In an optional example, the infusion catheter is inserted into the second catheter connecting section 43 and threadedly connected to the second catheter connecting section 43 .

[0048] In an optional embodiment of the present invention, a third conduit connecting section 50 is provided in the device body 10 . The third conduit connecting section 50 is connected to the reaction mixing chamber 20 and discharges the mixed fluid in the reaction mixing chamber 20 .

[0049] In an optional embodiment of the present invention, the reaction mixing chamber 20 is cylindrical, the first fluid inlet and the second fluid inlet are respectively opened on the side wall of the reaction mixing chamber 20, and the third conduit connecting section 50 is arranged along the axis of the reaction mixing chamber 20.

[0050] In an optional example, the inner diameter D of the reaction mixing chamber 20 is 3.3 mm, and the height H of the reaction mixing chamber 20 is 6 mm.

[0051] Preferably, the inner wall surface of the reaction mixing chamber 20 is smooth and easy to clean.

[0052] In an optional example of this embodiment, the axis of the first fluid inlet and the axis of the second fluid inlet are arranged perpendicular to the axis of the reaction mixing chamber 20 .

[0053] Preferably, the first fluid inlet and the second fluid inlet are respectively opened at the center position of the height H of the reaction mixing chamber 20.

[0054] In an optional example, the first fluid channel 30 and the second fluid channel 40 are coaxially arranged.

[0055] Furthermore, the first fluid channel 30 and the second fluid channel 40 are arranged perpendicular to the axis of the reaction mixing chamber 20 .

[0056] The counter-flow mixing device 100 and method for liposome synthesis proposed in the present invention are suitable for preparing lipid nanoparticles at different mixing flow rates and can obtain nanoparticles with uniform particle size. Specifically:

[0057] A counter-flow mixing device 100 for liposome synthesis is provided, wherein the inner diameter D of the reaction mixing chamber 20 is 3.3 mm, and the height H of the reaction mixing chamber 20 is 6 mm. The first fluid inlet and the second fluid inlet are both opened at the center position of the height H of the reaction mixing chamber 20 and are arranged opposite to each other. The first large diameter section 32 has an inner diameter of 1 mm and a length of 2 mm, the first small diameter section 31 has an inner diameter of 0.3 mm and a length of 3.3 mm, the second large diameter section 42 has an inner diameter of 1 mm and a length of 2 mm, and the second small diameter section 41 has an inner diameter of 0.3 mm and a length of 3.3 mm.

[0058] Appropriate amounts of lipid solutions (ionizable lipid MC3, DSPC, cholesterol, and mPEG2000-DMG were prepared at a molar ratio of 50:10:38.5:1.5 to prepare a 10 mg / ml lipid solution) were taken and mixed with mRNA (dissolved in citric acid-sodium citrate buffer at pH = 4). The mixture was stirred at different flow rates of 80, 160, 320, and 480 ml / min, the test temperature was 25°C, the N / P ratio was 6:1, and the flow rate ratio was 3 (mRNA solution): 1 (lipid solution). Lipid nanoparticles were obtained by mixing, and the particle size was measured by dynamic light scattering particle size analyzer. The results are shown in Table 1 and Figure 4:

[0059] Table 1 Comparison of lipid nanoparticle size obtained at different flow rates

[0060] As can be seen from Table 1 and Figure 4, the particle size results of the lipid nanoparticles produced by the counter-flow mixing device 100 for liposome synthesis proposed in the present invention within each flow rate range are not much different overall, and the particle size PDI of the produced lipid nanoparticles is less than 0.1, which can confirm that the nanoparticles produced by the counter-flow mixing device 100 for liposome synthesis proposed in the present invention are uniform and stable, with high mixing efficiency. At the same time, the preparation speed of a single mixing device can reach 480 ml / min, which greatly improves the preparation efficiency, and the effect is significantly better than the existing microfluidic chip.

[0061] The above description is merely a preferred embodiment of the present invention and does not constitute any form of limitation to the present invention. Although the present invention has been disclosed as a preferred embodiment as above, it is not intended to limit the present invention. Any person skilled in the art can make some changes or modifications to the technical contents disclosed above as equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A countercurrent mixing device for liposome synthesis, wherein: The counter-flow mixing device has a device body for counter-flow mixing, and a reaction mixing chamber, a first fluid channel and a second fluid channel are opened in the device body. The reaction mixing chamber has a first fluid inlet and a second fluid inlet. The first fluid inlet and the second fluid inlet are arranged opposite to each other. The first fluid channel is connected to the reaction mixing chamber through the first fluid inlet, and the second fluid channel is connected to the reaction mixing chamber through the second fluid inlet.

2. The countercurrent mixing device for liposome synthesis according to claim 1, wherein: An axis of the first fluid inlet and an axis of the second fluid inlet are located on the same straight line.

3. The countercurrent mixing device for liposome synthesis according to claim 1, wherein: The first fluid channel includes a first thin-diameter section and a first thick-diameter section. The first thin-diameter section is connected to the first fluid inlet, and an inner diameter of the first thin-diameter section is smaller than an inner diameter of the first thick-diameter section.

4. The countercurrent mixing device for liposome synthesis according to claim 3, wherein: The first fluid channel further includes a first catheter connecting section for connecting to an infusion catheter, the first catheter connecting section is connected to the first thick-diameter section, and an inner diameter of the first catheter connecting section is larger than an inner diameter of the first thick-diameter section.

5. The countercurrent mixing device for liposome synthesis according to claim 1, wherein: The second fluid channel includes a second thin-diameter section and a second thick-diameter section. The second thin-diameter section is connected to the second fluid inlet, and an inner diameter of the second thin-diameter section is smaller than an inner diameter of the second thick-diameter section.

6. The countercurrent mixing device for liposome synthesis according to claim 5, wherein: The second fluid channel further includes a second catheter connecting section for connecting to an infusion catheter, the second catheter connecting section is connected to the second thick-diameter section, and the inner diameter of the second catheter connecting section is larger than the inner diameter of the second thick-diameter section.

7. The countercurrent mixing device for liposome synthesis according to claim 1, wherein: A third conduit connecting section is also provided in the device body. The third conduit connecting section is communicated with the reaction mixing chamber and guides out the mixed fluid in the reaction mixing chamber.

8. The countercurrent mixing device for liposome synthesis according to claim 7, wherein: The reaction mixing chamber is cylindrical, the first fluid inlet and the second fluid inlet are respectively opened on the side wall of the reaction mixing chamber, and the third conduit connecting section is arranged along the axis of the reaction mixing chamber.

9. The countercurrent mixing device for liposome synthesis according to claim 8, wherein: The first fluid channel and the second fluid channel are coaxially arranged.

10. The counter-current mixing device for liposome synthesis according to claim 1, wherein: The device body is a metal block, and the reaction mixing chamber, the first fluid channel and the second fluid channel are opened in the metal block.

11. A counter-current mixing method for liposome synthesis, comprising preparing nanoparticles by using the counter-current mixing device for liposome synthesis according to any one of claims 1 to 10, wherein: The liposome mixture enters the reaction mixing chamber from the first fluid channel, and the drug solution enters the reaction mixing chamber from the second fluid channel. When the liposome mixture and the drug solution enter the reaction mixing chamber, they collide with each other. The cationic liposomes in the liposome mixture are rapidly precipitated due to the change in the solution properties. The precipitated cationic liposomes wrap the drug components in the drug solution and aggregate and precipitate together to form nanoparticles.

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