Ethanol injection method device for synthesizing lipidosome
By designing a sealed ethanol injection method device, the problem of bacterial contamination during liposome synthesis is solved, and more stable and pure liposome synthesis is achieved, and product quality is improved.
Patent Information
- Application Number
- CN202421561967.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-03
- Publication Date
- 2025-05-30
- Estimated Expiration
- 2034-07-03
AI Technical Summary
The existing ethanol injection method device is incompletely closed during liposome synthesis, which leads to easy entry of bacteria and other microorganisms, causing liposome contamination and affecting the activity and safety of the product.
An ethanol injection method device for the synthesis of liposomes is designed, which includes a sealed lipid-compatible container and a water-compatible container, which is sealed through a pipe, combined with heating, stirring and driving mechanisms to ensure the sealing reaction of the aqueous phase and the lipid-compatible solution.
It significantly reduces the risk of bacteria and other microorganisms entering the internal environment during the reaction process, ensures the purity of the reaction process, and improves the stability and product quality of the liposome synthesis reaction.
Smart Images
Figure CN222918673U_ABST
Abstract
Description
Technical Field
[0001] The utility model relates to the field of reaction equipment, in particular to an ethanol injection device for synthesizing liposomes. Background Art
[0002] Lipid Nanoparticle (LNP) is a vesicular structure at the nanoscale, and its core is composed of a phospholipid bilayer. This structure is unique, where the hydrophobic tails of phospholipids point to the center of the lipid bilayer, and the hydrophilic heads face the internal and external aqueous phases. Due to its special structure, liposomes have the ability to encapsulate hydrophilic drugs in the aqueous interior or load hydrophobic drugs in the lipid region between the bilayers. Therefore, liposomes show significant advantages in carrying drugs with poor water solubility or high dose toxicity, providing an effective way to improve the biological activity of drugs.
[0003] Although there are various preparation techniques for liposomes, traditionally, it mostly relies on the thin-film evaporation method. This method involves removing harmful organic solvents, and then liposomes are spontaneously formed through the lipid hydration process. However, the liposome vesicles prepared by this method are usually non-uniform and relatively large in size, and further homogenization treatment is required through a liposome extrusion device. This process not only has numerous steps and complex operations, but also consumes a large amount of human resources and time costs, and has high requirements for professional chemical experimental equipment. These factors limit its application in the production of sterile and uniform drug-loaded liposome nanoparticles.
[0004] To overcome the above drawbacks, the ethanol injection process based on the thin-film dispersion method emerged. This method directly produces monolayer or multilayer vesicles with a fixed size by dissolving lipid components in the ethanol phase and injecting them into the aqueous phase containing buffer solution. However, due to the possible incomplete enclosure during the preparation process of the ethanol injection device based on the thin-film dispersion method, bacteria in the environment are more likely to enter the interior through open areas, resulting in liposome contamination. Although ethanol has a certain bactericidal effect, during the preparation process, the concentration and contact time of ethanol may not be sufficient to completely kill all bacteria. Eventually, bacterial contamination may cause liposomes to lose activity or produce adverse reactions during subsequent drug delivery.
[0005] It should be noted that the information disclosed in the above background art section is only used to enhance the understanding of the background of the present disclosure, and thus may include information that does not constitute the prior art known to those of ordinary skill in the art. Summary of the Invention
[0006] (I) Technical Problems to be Solved
[0007] The present utility model provides an ethanol injection method device for synthesizing liposomes, and at least solves the technical problem of how to reduce the risk of bacteria and other microorganisms entering the internal environment during the liposome synthesis reaction.
[0008] (II) Technical solution
[0009] To solve the above technical problems, the present utility model provides the following technical solution:
[0010] An ethanol injection method device for synthesizing liposomes, comprising:
[0011] A lipid phase container, which is a sealed container for storing a lipid phase solution and is provided with an outlet;
[0012] An aqueous phase container, which is a sealed container for storing an aqueous phase solution and is provided with an inlet that is hermetically connected to the outlet through a pipeline;
[0013] A driving mechanism, which is configured to drive the solution in the lipid phase container to enter the aqueous phase container through the pipeline;
[0014] A stirring device, which is configured to stir the solution in the aqueous phase container;
[0015] A heating device, which is configured to heat the solution in the aqueous phase container.
[0016] In some embodiments, the lipid phase container includes a syringe, the needle tube of the syringe is hermetically connected to the pipeline, the driving mechanism is an injection pump, and the injection pump is connected to the piston rod of the syringe for driving the piston rod to move so that the lipid phase solution in the syringe enters the aqueous phase container through the pipeline.
[0017] In some embodiments, the aqueous phase container includes a bottle body and a lid, the lid is hermetically connected to the bottle body and is provided with an outlet hermetically connected to the pipeline.
[0018] In some embodiments, the stirring device is a magnetic stirrer, and a rotor adapted to the magnetic stirrer is provided in the aqueous phase container.
[0019] In some embodiments, the heating device is provided in the magnetic stirrer and heats the bottom of the aqueous phase container through the heating device.
[0020] In some embodiments, the aqueous phase container includes a reaction tank and a tank lid, the tank lid is hermetically connected to the reaction tank, and the tank lid is provided with the inlet.
[0021] In some embodiments, the ethanol injection method device further includes a temperature sensor, which is hermetically connected to the tank cover and is used to detect the temperature of the aqueous solution and feedback the temperature signal to the controller. The controller controls the heating device to operate according to the temperature signal so that the aqueous solution reaches a preset temperature.
[0022] In some embodiments, the driving mechanism is a peristaltic pump. The input end and the output end of the peristaltic pump are respectively connected to the outlet of the lipid phase container and the inlet of the aqueous phase container through pipelines.
[0023] In some embodiments, the stirring device includes a stirring rod, which is rotatably arranged on the tank cover and extends into the reaction tank.
[0024] In some embodiments, a filter tube is provided on the aqueous phase container and / or the lipid phase container, and a filter membrane is provided on the filter tube.
[0025] (III) Beneficial effects
[0026] Compared with the prior art, the ethanol injection method device for synthesizing liposomes provided by the present utility model has the following beneficial effects:
[0027] When the ethanol injection method device for synthesizing liposomes works, the aqueous phase and lipid phase solutions of the ethanol injection method are respectively filled into the aqueous phase container and the lipid phase container. Subsequently, the heating device and the stirring device respectively heat and stir the solution in the aqueous phase container to create an ideal reaction environment for the formation of liposomes. Then, the driving mechanism drives the solution in the lipid phase container to enter the aqueous phase container through the pipeline, so that the aqueous phase and lipid phase solutions react. Among them, the aqueous phase container and the lipid phase container are sealed containers and are hermetically connected through pipelines, significantly reducing the risk of bacteria and other microorganisms entering the internal environment during the reaction process. It not only effectively guarantees the purity of the reaction process, but also greatly improves the stability of the liposome synthesis reaction and the product quality. It can be seen that the ethanol injection method device for synthesizing liposomes of the present utility model has the advantages of significantly reducing bacterial contamination, improving reaction stability and product quality, and provides a new solution for the efficient and safe preparation of liposomes. Description of the drawings
[0028] Figure 1 It is a small-scale test schematic diagram of the ethanol injection method device for synthesizing liposomes in the embodiment.
[0029] Figure 2 It is a pilot-scale test schematic diagram of the ethanol injection method device for synthesizing liposomes in the embodiment.
[0030] Reference numerals:
[0031] Lipid phase container 1, syringe 11, outlet 100, syringe barrel 101, piston rod 102, filter tube 103, filter membrane 104;
[0032] Aqueous phase container 2, bottle body 21, lid 22, reaction tank 23, tank lid 24, inlet 200;
[0033] Driving mechanism 3, injection pump 31, peristaltic pump 32;
[0034] Stirring device 4, magnetic stirrer 41, rotor 42, stirring rod 43;
[0035] Heating device 5, pipeline 6, iron stand 7, temperature sensor 8, controller 9. Specific embodiments
[0036] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention.
[0037] In the existing ethanol injection device based on the thin film dispersion method, there may be an incomplete sealing situation during the preparation process, and bacteria in the environment are more likely to enter the interior, resulting in liposome contamination. Bacterial contamination may cause liposomes to lose activity or produce adverse reactions during subsequent drug delivery.
[0038] To solve the above technical problems, this embodiment provides an ethanol injection method device for synthesizing liposomes. Please refer to Figure 1 and Figure 2 as shown, Figure 1 is a small-scale test schematic diagram of the ethanol injection method device for synthesizing liposomes in the embodiment, Figure 2 is a pilot-scale test schematic diagram of the ethanol injection method device for synthesizing liposomes in the embodiment.
[0039] The ethanol injection method device for synthesizing liposomes in this embodiment includes: a lipid phase container 1, an aqueous phase container 2, a driving mechanism 3, a stirring device 4, and a heating device 5.
[0040] The lipid phase container 1 is a sealed container for storing the lipid phase solution and is provided with an outlet 100 for outputting the lipid phase solution; the aqueous phase container 2 is a sealed container for storing the aqueous phase solution and is provided with an inlet 200 that is hermetically connected to the outlet 100 through a pipeline 6 for inputting the lipid phase solution; the driving mechanism 3 is configured to drive the solution in the lipid phase container 1 to enter the aqueous phase container 2 through the pipeline 6; the stirring device 4 is configured to stir the solution in the aqueous phase container 2; the heating device 5 is configured to heat the solution in the aqueous phase container 2.
[0041] It can be understood that the above-mentioned sealed container refers to a container with outlets and inlets for the necessary solutions and gases in the experiment, and the rest is a sealed structure.
[0042] When the device of the ethanol injection method for synthesizing liposomes works according to the above technical solution, the aqueous phase and lipid phase solutions of the ethanol injection method are respectively filled into the sealed aqueous phase container 2 and lipid phase container 1, effectively avoiding the pollution of the external environment. Subsequently, the heating device 5 and the stirring device 4 heat and stir the solution in the aqueous phase container 2 respectively, creating an ideal reaction environment for the formation of liposomes. Then, the driving mechanism 3 drives the solution in the lipid phase container 1 to enter the aqueous phase container 2 through the pipeline 6, enabling the reaction of the aqueous phase and lipid phase solutions. Among them, the aqueous phase container 2 and the lipid phase container 1 are sealed containers and are hermetically connected through the pipeline 6, significantly reducing the risk of bacteria and other microorganisms entering the internal environment during the reaction. This not only effectively guarantees the purity of the reaction process but also greatly improves the stability of the liposome synthesis reaction and the product quality, providing a new solution for the efficient and safe preparation of liposomes.
[0043] The following content is referred to Figure 1 as shown in
[0044] In one embodiment where the above driving mechanism 3 drives the solution in the lipid phase container 1 to enter the aqueous phase container 2 through the pipeline 6, the lipid phase container 1 includes a syringe 11. The needle tube 101 of the syringe 11 is hermetically connected to the pipeline 6, and the driving mechanism 3 is an injection pump 31. The injection pump 31 is connected to the piston rod 102 of the syringe 11 and is used to drive the piston rod 102 to move, so that the lipid phase solution in the syringe 11 enters the aqueous phase container 2 through the pipeline 6.
[0045] Exemplarily, the injection pump 31 is mainly composed of a stepper motor and its driver, a lead screw, and a bracket, etc. When working, the single-chip microcomputer system issues a control pulse to make the stepper motor rotate. The stepper motor converts the rotational motion into a linear motion through the lead screw, thereby pushing the piston rod of the syringe 11 for injection and infusion, and thus enabling high-precision and smooth pulsation-free liquid transmission.
[0046] In one embodiment of the above aqueous phase container 2, the aqueous phase container 2 includes a bottle body 21 and a lid 22. The lid 22 is hermetically connected to the bottle body 21 and is provided with an outlet 100 hermetically connected to the pipeline 6.
[0047] Exemplarily, the lid 22 and the bottle body 21 are connected by threads, and a rubber ring for sealing is provided at the connection.
[0048] Exemplarily, the bottle body 21 and the lid 22 form a sealed glass bottle.
[0049] In one embodiment of the above stirring device 4, the stirring device 4 is a magnetic stirrer 41. A rotor 42 adapted to the magnetic stirrer 41 is provided in the aqueous phase container 2. When working, the magnetic stirrer 41 drives the rotor 42 to rotate in the aqueous phase container 2, thereby realizing the function of stirring the solution in the aqueous phase container 2.
[0050] In one embodiment of the above heating device 5, a heating device 5 is provided inside the magnetic stirrer 41, and the bottom of the aqueous phase container 2 is heated by the heating device 5. That is, in actual use, a magnetic stirrer 41 with a heating function can be selected.
[0051] Exemplarily, the above pipeline 6 is a flexible hose. To stabilize the flexible hose, an iron stand 7 can also be provided, and the iron stand 7 is used to erect the pipeline 6.
[0052] The ethanol injection method device for synthesizing liposomes of the above technical solutions is applicable to small-scale experiments. It can be understood that the purpose of small-scale experiments is to verify the basic principle and feasibility of the ethanol injection method, and to determine the optimal experimental conditions. The specific working process is exemplified as follows:
[0053] Add mRNA to the sodium citrate solution with pH = 4, mix well with a shaker, add it to the aqueous phase container 2, then place the glass bottle with the rotor 42 on the magnetic stirrer 41, and turn on the heating (temperature 25 °C - 65 °C) and stirring (stirring speed 0 - 1000 rpm) functions of the magnetic stirrer 41; dissolve the four lipid components (such as SM102, DMG-PEG2000, DSPC, Chol) in anhydrous ethanol solution, mix well with a shaker, suck the lipid phase solution with a syringe 11, place the syringe 11 on the syringe pump 31, and set the parameters of the syringe pump 31, such as the injection speed (0.05 ml / min - 120 ml / min) and the injection volume; after preparation, the syringe pump 31 starts to inject until the lipid phase solution is completely injected into the aqueous phase in the glass bottle. Wait for the lipid phase and the aqueous phase to react in the glass bottle for a period of time (1 min - 60 min) to obtain the product. The reaction solution can also be collected into a dialysis cup and placed in 1xPBS or physiological saline for dialysis overnight, and then the sample is collected for subsequent experiments, or the reaction solution is collected into an ultrafiltration tube for ultrafiltration concentration, or tangential flow chromatography technology is used for ultrafiltration concentration and then used for subsequent experiments.
[0054] The working principle of the above small-scale experiment is as follows: The ionizable lipid SM102 in the lipid phase is positively charged in the acidic solution with pH = 4 and can electrostatically bind to the negatively charged mRNA molecules in the aqueous phase. While the reaction is taking place, other lipid components DMG-PEG2000, DSPC, Chol will quickly form a phospholipid bilayer, and the four lipid components will further react to form a stable LNP-mRNA product during dialysis or ultrafiltration.
[0055] The following content refers to Figure 1 As shown, this embodiment provides an ethanol injection method device for synthesizing liposomes applicable to pilot-scale experiments, where:
[0056] In another embodiment of the above aqueous phase container 2, the aqueous phase container 2 includes a reaction tank 23 and a tank cover 24. The tank cover 24 is hermetically connected to the reaction tank 23, and the tank cover 24 is provided with an inlet 200.
[0057] In another embodiment of the above lipid phase container 1, the lipid phase container 1 is an existing sealed glass bottle.
[0058] In order to facilitate the detection of the reaction temperature, the ethanol injection device further includes a temperature sensor 8. The temperature sensor 8 is hermetically connected to the tank cover 24, and is used to detect the temperature of the aqueous phase solution and feed back the temperature signal to the controller 9. The controller controls the heating device 5 to work according to the temperature signal so that the aqueous phase solution reaches the preset temperature.
[0059] Exemplarily, the temperature sensor 8 passes through the tank cover 24, and a sealing member such as a rubber ring is provided at the connection to achieve the effect of hermetic connection.
[0060] In another embodiment of the above driving mechanism 3, the driving mechanism 3 is a peristaltic pump 32. The input end and the output end of the peristaltic pump 32 are respectively connected to the outlet 100 of the lipid phase container 1 and the inlet 200 of the aqueous phase container 2 through pipelines 6.
[0061] In another embodiment of the above stirring device 4, the stirring device 4 includes a stirring rod 43. The stirring rod 43 is rotatably arranged on the tank cover 24 and extends into the reaction tank 23. The stirring rod 43 can be driven by a motor and is provided with blades for improving the stirring efficiency.
[0062] In another embodiment of the above heating device 5, the heating device 5 includes an electric heating rod. The electric heating rod is fixed on the tank cover 24 and extends into the reaction tank 23.
[0063] In order to facilitate the input or output of the corresponding solutions into or out of the aqueous phase container 2 and the lipid phase container 1 and filter the solutions, the aqueous phase container 2 and the lipid phase container 1 are provided with a filter tube 103. A filter membrane 104 is provided on the filter tube 103, and the filter membrane 104 is selected as a thin film with the function of filtering solutions.
[0064] The working process of the above pilot test is as follows: Add mRNA into the sodium citrate solution with a pH of 4, mix well with a shaker, and add it into the reaction tank 23. Then, perform heating (temperature: 25°C - 65°C) and stirring (stirring speed: 0 - 1000 rpm) functions through the heating device 5 and the stirring device 4 respectively. Dissolve the four lipid components (such as SM102, DMG-PEG2000, DSPC, Chol) in an absolute ethanol solution, mix well with a shaker, and add it into the lipid phase container 1. Open the high-precision peristaltic pump 32 and set the parameters of the peristaltic pump 32, such as the pumping speed (0.01 ml / min - 120 ml / min) and the injection volume. After preparation, the peristaltic pump 32 starts to work until the lipid phase solution is completely injected into the aqueous solution in the reaction tank 23. Wait for the lipid phase and the aqueous phase to react in the glass bottle for a period of time (1 min - 60 min) to obtain the product. Alternatively, the reaction solution can be collected into a dialysis cup, placed in 1xPBS or physiological saline for dialysis overnight, and then the sample can be collected for subsequent experiments. Or the reaction solution can be collected into an ultrafiltration tube for ultrafiltration concentration, or ultrafiltration concentration can be performed using tangential flow chromatography technology for subsequent experiments.
[0065] Although the embodiments of the present invention have been shown and described, for those of ordinary skill in the art, it can be understood that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principle and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents.
Claims
1. An ethanol injection method device for synthesizing liposomes, characterized in that: include: The fat phase container is a sealed container used to store the fat phase solution and is provided with an outlet; The water phase container is a sealed container for storing the water phase solution and is provided with an inlet sealedly connected to the outlet through a pipeline; a driving mechanism, the driving mechanism being configured to drive the solution in the lipid phase container into the water phase container through the pipeline; a stirring device, the stirring device being configured to stir the solution in the aqueous phase container; A heating device is configured to heat the solution in the water phase container.
2. The ethanol injection method device for synthesizing liposomes according to claim 1, characterized in that: The fat phase container comprises a syringe, the needle tube of the syringe is in sealed communication with the pipeline; The driving mechanism is a syringe pump, which is connected to the piston rod of the syringe and is used to drive the piston rod to move so that the lipid phase solution in the syringe enters the water phase container through a pipeline.
3. The ethanol injection method device for synthesizing liposomes according to claim 1, characterized in that: The water phase container comprises a bottle body and a cover, wherein the cover is sealedly connected to the bottle body and is provided with the outlet.
4. The ethanol injection method device for synthesizing liposomes according to claim 1, characterized in that: The stirring device is a magnetic stirrer, and a rotor adapted to the magnetic stirrer is arranged in the water phase container.
5. The ethanol injection method device for synthesizing liposomes according to claim 4, characterized in that: The heating device is arranged in the magnetic stirrer, and the bottom of the water phase container is heated by the heating device.
6. The ethanol injection method device for synthesizing liposomes according to claim 1, characterized in that: The water phase container comprises a reaction tank and a tank cover, wherein the tank cover is sealed and connected to the reaction tank, and the tank cover is provided with the inlet.
7. The ethanol injection method device for synthesizing liposomes according to claim 6, characterized in that: The ethanol injection method device also includes a temperature sensor, which is sealed with the tank cover and is used to detect the temperature of the aqueous solution and feed back the temperature signal to the controller. The controller controls the heating device to operate according to the temperature signal so that the aqueous solution reaches a preset temperature.
8. The ethanol injection method device for synthesizing liposomes according to claim 6, characterized in that: The driving mechanism is a peristaltic pump, and the input end and the output end of the peristaltic pump are respectively connected to the outlet of the fat phase container and the inlet of the water phase container through pipelines.
9. The ethanol injection method device for synthesizing liposomes according to claim 6, characterized in that: The stirring device comprises a stirring rod, which is rotatably arranged on the tank cover and extends into the reaction tank.
10. The ethanol injection method device for synthesizing liposomes according to claim 1, characterized in that: The water phase container and / or the fat phase container is provided with a filter tube, and the filter tube is provided with a filter membrane.