Method for automatically preparing phospholipid vesicles

Abstract

The invention provides a method for automatically preparing phospholipid vesicles, which comprises the following steps: spraying a first liquid by using a first automatic pipetting device with a liquid spraying function, the first liquid being a mixture of a first oil phase solution and a first water phase solution, the oil phase solution containing phospholipid, and the first water phase solution containing phospholipid; the first liquid is subjected to liquid spraying treatment to form a water-in-oil emulsion; the water-in-oil emulsion is dropwise added into second liquid through a second automatic pipetting device, the second liquid comprises an upper-layer oil phase and a lower-layer water phase, a phospholipid molecular layer is arranged between the upper-layer oil phase and the lower-layer water phase, water-in-oil emulsion liquid drops penetrate through the phospholipid molecular layer and enter the water phase to be dispersed, and a system containing phospholipid vesicles is formed; and separating to obtain the phospholipid vesicles. The preparation method disclosed by the invention is combined with an integrated automatic platform to complete the whole process of wrapping the biomolecules by the phospholipid vesicles, is simple in process flow and is suitable for wrapping the phospholipid vesicles containing various micromolecular dyes or biomacromolecules.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an automated method for preparing phospholipid vesicles. Background Technology

[0002] Synthetic cells are cells modified or constructed in the laboratory using synthetic biology methods. These cells can be synthesized from the bottom up or obtained by modifying existing cells. Currently, synthetic cell technology is still in its early stages of development, but some important progress has already been made.

[0003] Synthesizing artificial cells by modifying existing cells has become a reality. In 2010, Craig Venter's team used *Mycoplasma capricolum* as a vector and transformed it into a cell possessing the traits and characteristics of *Mycoplasma mycoides* by introducing the genome of a synthetically produced *Mycoplasma mycoides*. This resulted in the first synthesis of an artificial life form completely controlled by artificial genetic instructions, which they named JCVI-syn1.0. In 2016, based on the genome of JCVI-syn1.0, Craig Venter's team designed the smallest genome theoretically capable of guiding the independent replication of a cell—JCVI-syn3.0. However, synthetic cells obtained by modifying intact cells cannot completely unlock the black box of cellular life. Therefore, researchers are attempting to synthesize artificial cells from scratch using a bottom-up approach.

[0004] A bottom-up research strategy requires the polymerization of small biomolecules into macromolecules, followed by assembly into complex cellular structures, ultimately resulting in a complete, living cell. Similar to normal cells, artificial cells also require structures or functions such as cell boundaries and compartmentalization, metabolic systems, and cell communication. Methods for compartmentalizing artificial cells include liposomes, amphoteric polymers, hydrogels, and condensed layers. However, artificial cell synthesis technology still faces many challenges, such as how to design and synthesize sufficiently complex cell structures and functions, how to control cell growth and division, and how to address safety and ethical issues. Therefore, further efforts and exploration are needed in this field.

[0005] Phospholipid vesicle encapsulation technology is a technique that encapsulates small molecules, biomolecules, or other substances within phospholipid bilayer vesicles. Phospholipids consist of a hydrophilic head and a hydrophobic tail. When placed in an aqueous solution, they spontaneously form a bilayer of vesicles, with the hydrophilic head facing outwards and the hydrophobic tail facing inwards. Phospholipids are a major component of cell membranes; therefore, in the field of artificial cell synthesis, phospholipid vesicles represent a more biocompatible artificial cell membrane.

[0006] The most basic method for preparing phospholipid vesicles is the hydration method, which involves dissolving phospholipids in an organic solvent and then drying them to form a thin film. The film is then mixed with an aqueous solution containing the substance to be encapsulated. The mixture is then ultrasonically treated or vortexed to form small monolayer vesicles (SUVs) or large monolayer vesicles (LUVs).

[0007] Building upon this foundation, researchers have developed a more efficient emulsion transfer method. This method boasts higher encapsulation efficiency and shorter incubation time, exhibits high compatibility with charged molecules, and avoids the drawback of hydration methods that may result in multilayered vesicles; however, it requires a high concentration of sugar solution to balance osmotic pressure. Therefore, Tsuji et al. developed a method that does not require a high sugar concentration, providing a physiologically relevant environment for cell simulation. Furthermore, Moga et al. specifically optimized the preparation of high-yield GUVs. The development of microfluidic technology has also provided more possibilities for phospholipid vesicle encapsulation; vesicle encapsulation on chips using PDMS as a raw material allows for more controllable vesicle size and higher size uniformity.

[0008] The development of microfluidic technology has also provided more possibilities for phospholipid vesicle encapsulation. The vesicle encapsulation process on chips made of PDMS can make the vesicle size more controllable and the size uniformity higher.

[0009] In current technologies, the key step in controlling the final size of the vesicles is to manually hold the EP tube and rub it vigorously on the tube rack. This process involves too many uncontrollable factors and lacks good repeatability. Furthermore, manual operation can only perform emulsification one tube at a time, making it impossible to conduct high-throughput experiments. Summary of the Invention

[0010] The purpose of this invention is to provide an automated method for preparing phospholipid vesicles.

[0011] On one hand, the present invention provides a method for preparing phospholipid vesicles, the method comprising:

[0012] A first liquid is sprayed using a first automated pipetting device with a spraying function. The first liquid is a mixture of a first oil phase solution and a first aqueous phase solution, wherein the oil phase solution contains phospholipids. After spraying, the first liquid forms a water-in-oil emulsion.

[0013] Using a second automated pipetting device, an oil-in-water emulsion is dropped into a second liquid, wherein the second liquid comprises an upper oil phase and a lower aqueous phase, with a phospholipid molecular layer between the upper oil phase and the lower aqueous phase. The oil-in-water emulsion droplets pass through the phospholipid molecular layer and enter the aqueous phase to be dispersed, forming a system containing phospholipid vesicles.

[0014] The phospholipid vesicles were isolated.

[0015] According to a specific embodiment of the present invention, preferably, the nozzle diameter for the liquid spraying treatment is 0.2-0.5 mm, the distance between the nozzle and the liquid surface is 2-8 mm, the spraying velocity is 500-900 μL / s, and the spraying acceleration is 8000-12000 μL / s. 2 .

[0016] Preferably, the nozzle diameter for the liquid spraying treatment is 0.25-0.35 mm, the distance between the nozzle and the liquid surface is 4-6 mm, the spray velocity is 800-900 μL / s, and the spray acceleration is 10000-12000 μL / s. 2 .

[0017] According to a specific embodiment of the present invention, preferably, the first automated pipetting device also has a liquid aspiration function. The present invention does not have special requirements regarding the speed of liquid aspiration.

[0018] According to a specific embodiment of the present invention, the first automated pipetting device performs a suction-spray liquid circulation process on the first liquid, and the number of cycles of the suction-spray liquid circulation process is 40-140 times, preferably 70-100 times.

[0019] According to a specific embodiment of the present invention, the first automated pipetting device performs suction-spray liquid circulation treatment on the first liquid to emulsify water and oil to obtain a water-in-oil emulsion.

[0020] According to a specific embodiment of the present invention, preferably, when the water-in-oil emulsion is added dropwise to the second liquid, the distance between the liquid outlet and the liquid surface is 2-8 mm, more preferably 4-6 mm. When adding the water-in-oil emulsion to the second liquid, there are no special requirements for the dripping speed; it is sufficient to control it so that it does not flow down in streams.

[0021] According to a specific embodiment of the present invention, the water-in-oil emulsion droplets penetrate the phospholipid molecular layer and enter the aqueous phase to be dispersed, forming a system containing phospholipid vesicles.

[0022] According to a specific embodiment of the present invention, preferably, the volume ratio of the first oil phase solution to the first aqueous phase solution in the first liquid is 200-250:10-20.

[0023] According to a specific embodiment of the present invention, preferably, the concentration of phospholipid in the first oil phase solution is 350-450 μM; preferably, the first oil phase solution is prepared by dissolving phospholipid in an oil phase solvent, and more preferably, the phospholipid is dissolved in the oil phase solvent by heating and ultrasonication.

[0024] According to a specific embodiment of the present invention, preferably, the phospholipids include POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol). One or more of the following: phosphatidylglycerol, DOPG (1,2-dioleoyl-sn-glycero-3-phospho-glycerol), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine), and cholesterol, preferably POPC.

[0025] According to a specific embodiment of the present invention, preferably, the oil phase solvent is selected from one or more of liquid paraffin, mineral oil, and squalene, and more preferably squalene.

[0026] According to a specific embodiment of the present invention, preferably, the first aqueous phase solution is an aqueous solution containing one or more of small molecule dyes, macromolecule polymers, DNA, and proteins. The concentrations of the small molecule dyes, macromolecule polymers, DNA, and proteins can refer to concentration ranges conventionally used in the art. Typically: the concentration of the small molecule dye can be 0.05-0.5 mM; the concentration of the macromolecule polymer can be 0.01-0.1 mM; the concentration of DNA can be 0.01-0.1 mg / mL; and the concentration of protein can be 0.01-0.1 mg / mL.

[0027] According to a specific embodiment of the present invention, the first liquid is obtained by mixing a first oil phase solution and a first aqueous phase solution. According to a specific embodiment of the present invention, preferably, the second liquid is prepared according to the following method:

[0028] The second oil phase solution and the second aqueous phase solution are mixed, wherein the second oil phase solution contains phospholipids. After the second oil phase solution and the second aqueous phase solution are mixed, they are allowed to stand and separate into layers to obtain the second liquid.

[0029] Preferably, the concentration of phospholipids in the second oil phase solution is 350-450 μM.

[0030] Preferably, the first oil phase solution is prepared by dissolving phospholipids in an oil phase solvent, and more preferably, the phospholipids are dissolved in the oil phase solvent by heating and ultrasonication.

[0031] According to a specific embodiment of the present invention, preferably, the phospholipids include POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphorylcholine), DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine), and POPG (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol). One or more of the following: phosphatidylglycerol, DOPG (1,2-dioleoyl-sn-glycero-3-phospho-glycerol), DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine), DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphorylcholine), and cholesterol, preferably POPC.

[0032] According to a specific embodiment of the present invention, preferably, the oil phase solvent is selected from one or more of liquid paraffin, mineral oil, and squalene, and more preferably squalene.

[0033] According to a specific embodiment of the present invention, preferably, the second aqueous phase solution is an aqueous solution containing glucose. More preferably, the concentration of glucose in the aqueous glucose solution is 200-400 mM.

[0034] According to a specific embodiment of the present invention, preferably, the process of separating the phospholipid vesicles from a system containing phospholipid vesicles includes:

[0035] The system containing phospholipid vesicles is centrifuged to remove the oil phase; preferably, the centrifugation conditions are 200-500×g for 5-10 min.

[0036] According to a specific embodiment of the present invention, preferably, the volume ratio of the second oil phase solution to the second aqueous phase solution in the second liquid is 100-500:100-300.

[0037] According to a specific embodiment of the present invention, the second automated pipetting device and the first automated pipetting device can be the same device. Preferably, the entire process of phospholipid vesicle encapsulation can be completed using a Tecan automated pipetting workstation (Tecan Fluent 1080). Liquid aspiration and dispensing are performed by loading pipettes of different volumes into the FCA (flexible channel arms) channels of the pipetting workstation. FCA50, FCA200, and FCA1000 represent FCAs that can load 50μL, 200μL, and 1000μL pipettes, respectively.

[0038] On the other hand, the present invention provides a phospholipid vesicle prepared by the above method.

[0039] According to a specific embodiment of the present invention, the phospholipid vesicle core is a first aqueous solution, and the first aqueous solution is surrounded by a phospholipid molecule layer.

[0040] According to a specific embodiment of the present invention, preferably, the average diameter of the phospholipid vesicles obtained by the above preparation method is 5-20 μm.

[0041] On the other hand, the present invention provides another method for preparing phospholipid vesicles, the method comprising:

[0042] The first liquid is subjected to peristaltic treatment using a peristaltic pump (from a BioTek MultiFlo FX multi-functional dispenser) or agitation treatment using a Bio-Shaker (BioShake 3000-T, QINSTRUMENTS), wherein the first liquid is a mixture of a first oil phase solution and a first aqueous phase solution, wherein the oil phase solution contains phospholipids, and the first liquid forms a water-in-oil emulsion after peristaltic or agitation treatment;

[0043] Using a second automated pipetting device, an oil-in-water emulsion is dropped into a second liquid, wherein the second liquid comprises an upper oil phase and a lower aqueous phase, with a phospholipid molecular layer between the upper oil phase and the lower aqueous phase. The oil-in-water emulsion droplets pass through the phospholipid molecular layer and enter the aqueous phase to be dispersed, forming a system containing phospholipid vesicles.

[0044] The phospholipid vesicles were isolated.

[0045] This invention develops an integrated automated platform to complete the entire process of encapsulating biomolecules in phospholipid vesicles. The process is simple and applicable to the encapsulation of phospholipid vesicles containing various small or large biomolecules.

[0046] The automated phospholipid vesicle preparation method of the present invention combines emulsion transfer preparation of phospholipid vesicles with an automated pipetting device. The entire process of phospholipid vesicle encapsulation is completed by using an automated pipetting device, and the water-oil emulsification step is completely completed by the suction and spraying of liquid through the FCA channel of the pipetting workstation. On the one hand, it can complete vesicle encapsulation experiments of multiple groups of different substances with high throughput, and can be applied to the encapsulation of various biological dyes and biomolecules. On the other hand, it can stably realize the emulsification process of the inner phase aqueous solution in the oil phase solution of phospholipids. Parallel operation reduces the error of manual experiments and ensures that the properties of vesicles encapsulated in the same batch are similar. Attached Figure Description

[0047] Figure 1 This is a flowchart of the method for preparing phospholipid vesicles according to the present invention;

[0048] Figure 2 The figures show the detection results of phospholipid vesicles in Example 1 and Comparative Example 1, where... Figure 2 In the diagram, A represents the confocal microscopy imaging result, I represents the manual vesicle encapsulation result, and II represents the automated vesicle encapsulation result. Figure 2 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 2 C in the figure represents the statistical bar chart of the coefficient of variation of fluorescence intensity within vesicles; Figure 2 In the figure, E represents the aggregate content statistical bar chart;

[0049] Figure 3 This is a graph showing the detection results of phospholipid vesicles in Example 2, where... Figure 3 In the images, A represents the results of confocal microscopy; I is an image of vesicles encapsulating calcein (green represents calcein); II is an image of vesicles encapsulating λDNA (green represents YOYO-1 stained λDNA); III is an image of vesicles encapsulating dextran (green represents FITC-labeled dextran (40,000 molecular weight)); IV is an image of vesicles encapsulating immunoglobulins (green represents Alexa Fluro 488 labeled IgG protein); and the red areas in all four images represent POPC phospholipids. Figure 3 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 3 In the bar chart, C represents the coefficient of variation of vesicle diameter; Figure 3 In the figure, D represents the statistical bar chart of the coefficient of variation of fluorescence intensity within vesicles; Figure 3 In the figure, E represents the aggregate content statistical bar chart;

[0050] Figure 4 The figures show the detection results of phospholipid vesicles in Examples 3 and 4, where... Figure 4In the diagram, A represents the confocal microscopy imaging result, I represents the result of water-oil emulsification using the FCA 200 automatic aspiration and spraying liquid circulation, II represents the result of water-oil emulsification using the peristaltic pump of the separator, and III represents the result of water-oil emulsification using the high-frequency vibration of the Bioshaker. Red represents phospholipids and green represents calcein. Figure 4 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 4 In the bar chart, C represents the coefficient of variation of vesicle diameter; Figure 4 In the figure, D represents the statistical bar chart of the coefficient of variation of fluorescence intensity within vesicles; Figure 4 In the figure, E represents the aggregate content statistical bar chart;

[0051] Figure 5 This is a graph showing the detection results of phospholipid vesicles in Comparative Example 2. Figure 5 In the diagram, A represents the confocal microscopy imaging result, I represents the result of centrifugation in a 1.5 mL centrifuge tube, and II represents the result of centrifugation in a SensoPlate. Red represents phospholipids, and green represents calcein. Figure 5 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 5 In the bar chart, C represents the coefficient of variation of vesicle diameter; Figure 5 In the figure, D represents the statistical bar chart of the coefficient of variation of fluorescence intensity within vesicles; Figure 5 E in the figure represents the aggregate content statistical bar chart. Detailed Implementation

[0052] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be interpreted as...

[0053] To limit the scope of protection of this invention, conditions not specifically specified in the examples are performed under conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0054] Example 1: Automated preparation of phospholipid vesicles encapsulating calcein

[0055] This embodiment utilizes the Tecan automated pipetting workstation with a suction-spray function to prepare phospholipid vesicles encapsulating calcein.

[0056] For detailed instructions, please refer to [link / reference]. Figure 1 :

[0057] Preparation of oil phase solution containing phospholipids: Add 42 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 3.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0058] Preparation of the first liquid and water-in-oil emulsion: 250 μL of a squalene solution of phospholipids was added to a 1.5 mL centrifuge tube using an FCA 1000 apparatus. 10 μL of a 0.1 mM calcein solution was added to the same solution using an FCA 50 apparatus. The resulting mixture was the first liquid. The first liquid was then subjected to aspiration and spraying treatment using an FCA 200 apparatus with a nozzle diameter of 0.3 mm. The nozzle distance from the liquid surface was 6 mm, the spraying velocity was 900 μL / s, and the spraying acceleration was 12000 μL / s. 2 160 μL of liquid was aspirated and sprayed, and the mixture was circulated 70 times to obtain a water-in-oil emulsion.

[0059] Preparation of the second liquid: Add 200 μL of 300 mM glucose solution to a 1.5 mL centrifuge tube using FCA 1000, and use FCA 200 to suspend 125 μL of squalene solution of phospholipids in the tube. Let stand for 30 minutes to form a phospholipid molecular layer.

[0060] Softening the slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate using FCA 200, let stand for 15 minutes, then add 100 μL of 300 mM glucose solution to each well of the SensoPlate using MCA 200 (MultiChannel Arm) and repeatedly blow and wash the slides. Repeat 3 times.

[0061] Preparation of phospholipid vesicles: Using an FCA 1000 syringe, the water-in-oil emulsion was completely aspirated and added dropwise to a 1.5 mL centrifuge tube where a phospholipid molecular layer had formed. During addition, the droplet outlet was 5 mm above the liquid surface. The centrifuge tube was transferred to a ROTANTA automated centrifuge and centrifuged at 200 × g for 8 minutes. The upper oil phase was discarded in three separate centrifuge cycles using an FCA 200 syringe, with the final cycle involving 24-point rotational aspiration of the oil. A solution containing phospholipid vesicles was obtained. This solution containing phospholipid vesicles can be further centrifuged to remove the aqueous phase, yielding the phospholipid vesicles.

[0062] Microscopic examination: The centrifuge tubes were shaken at 1200 rpm for 3 seconds using a T-shaker, and then phospholipid vesicle solution was aspirated with an FCA 200 and added to the SensoPlate at a rate of 50 μL / well. Multi-field random imaging was performed using the HCA (High Content Analysis) system on a Nikon rotary confocal microscope.

[0063] Comparative Example 1: Manual preparation of phospholipid vesicles encapsulating calcein

[0064] This comparative example is a manually prepared phospholipid vesicle encapsulating calcein.

[0065] Preparation of oil phase solution containing phospholipids: Add 42 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 3.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0066] Preparation of the first liquid and water-in-oil emulsion: Add 250 μL of squalene solution of phospholipids to a 1.5 mL centrifuge tube, then add 10 μL of 0.1 mM calcein solution. The resulting mixture is the first liquid. Hold the tightly capped centrifuge tube and slide it forcefully in one direction on the PCR tube rack, repeating 10 times to obtain the water-in-oil emulsion.

[0067] Preparation of the second liquid: Add 200 μL of external phase aqueous solution to a 1.5 mL centrifuge tube, and suspend 125 μL of squalene solution of phospholipids in the tube, and let stand for 30 minutes.

[0068] Rinse the slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate (96-well glass plate, Greiner), let stand for 15 minutes, discard the liquid, and then add 100 μL of external phase aqueous solution to each well and repeatedly rinse the slides by blowing and rinsing. Repeat 3 times.

[0069] Preparation of phospholipid vesicles: The water-in-oil emulsion obtained after water-oil emulsification was completely aspirated and added dropwise to a 1.5 mL centrifuge tube (3). During addition, the distance between the droplet outlet and the liquid surface was 5 mm. Centrifuge at 1000×g for 8 minutes; carefully discard the upper oil phase. A solution containing phospholipid vesicles was prepared. This solution containing phospholipid vesicles can be further centrifuged to remove the aqueous phase solution, thus obtaining phospholipid vesicles.

[0070] Microscopic examination: The centrifuge tube containing the phospholipid vesicle solution was briefly vortexed once to ensure that all the vesicles attached to the tube wall were shaken off after centrifugation, and the solution changed from transparent to a uniform mist. Then, the phospholipid vesicle solution was aspirated and added to the SensoPlate at a volume of 50 μL / well. Multi-field random imaging was performed using the HCA system on a Nikon rotary confocal microscope.

[0071] The detection results of phospholipid vesicles prepared using the automated platform in Example 1 and manually in Comparative Example 1 are as follows: Figure 2As shown, A represents the confocal microscopy imaging results, I represents the manual vesicle encapsulation results, and II represents the automated vesicle encapsulation results. Red represents phospholipids, and green represents calcein. The scale bar is 20 μm. B is the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution. C is the statistical histogram of the coefficient of variation of fluorescence intensity within the vesicles, P>0.05, n=3. D is the histogram of the coefficient of variation of vesicle diameter, P<0.05, n=3. E is the statistical histogram of aggregate content, P<0.0001, n=90.

[0072] Statistical analysis of the average diameter of phospholipid vesicles prepared by the two methods showed that the average diameter of phospholipid vesicles prepared by the automated platform was 8.14±5.39 μm, while the average diameter of phospholipid vesicles encapsulated by the manual method was 7.80±4.62 μm. The two methods exhibited similar sizes and concentrated distribution, meeting the size requirements for artificially synthesized cells. Figure 2 (B in the middle).

[0073] The uniformity of the phospholipid vesicle encapsulated material prepared by the automated platform was similar to that prepared by the manual method, with coefficients of variation of internal fluorescence intensity of 32.50% and 28.09%, respectively, showing no significant difference. Figure 2 (C in the text). The size uniformity of the automated platform is slightly worse, with a coefficient of variation of 48.40% for diameter, significantly higher than the 36.57% for the manual method, indicating that the size variation of vesicles prepared by the automated platform is relatively greater. Figure 2 (D in the text). However, the automated platform produced significantly fewer phospholipid aggregates encapsulating calcein (1.08%) than the manual method (5.22%), indicating that the automated platform produced cleaner vesicles with fewer impurities. Figure 2 (E in the middle).

[0074] Example 2: Automated preparation of phospholipid vesicles encapsulating biomacromolecules such as calcein, DNA, protein, and dextran.

[0075] This embodiment utilizes the Tecan automated pipetting workstation with a suction-spray function to prepare phospholipid vesicles encapsulating calcein, DNA, protein, and dextran.

[0076] Preparation of oil phase solution containing phospholipids: Add 30 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 2.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0077] Preparation of the first liquid and water-in-oil emulsion: 250 μL of squalene solution containing phospholipids was added to a 1.5 mL centrifuge tube using an FCA 1000. 10 μL of 0.1 mM calcein solution, 0.03 mg / mL λDNA solution, 0.02 mg / mL IgG solution, and 0.1 mM dextran (40000 MW) solution were added to different tubes using an FCA 50. The resulting mixture was the first liquid. This mixture was then aspirated using an FCA 200 with a 0.3 mm nozzle diameter. The nozzle was 6 mm above the liquid surface, the spray velocity was 900 μL / s, and the spray acceleration was 12000 μL / s. 2 160 μL of liquid was aspirated and sprayed, and the mixture was circulated 70 times to obtain a water-in-oil emulsion.

[0078] Preparation of the second liquid: Add 200 μL of 300 mM glucose solution to a 1.5 mL centrifuge tube using FCA 1000, and use FCA 200 to suspend 125 μL of squalene solution of phospholipids in the tube. Let stand for 30 minutes to form a phospholipid molecular layer.

[0079] Softening slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate using FCA200, let stand for 15 minutes, then add 100 μL of 300 mM glucose solution to each well of the SensoPlate and repeatedly blow and wash the slides. Repeat 3 times.

[0080] Preparation of phospholipid vesicles: The water-in-oil emulsion was completely drawn up using FCA 1000 and added dropwise into a 1.5 mL centrifuge tube that had been allowed to stand to form a phospholipid molecular layer. When adding the drop, the distance between the droplet outlet and the liquid surface was 5 mm.

[0081] Transfer the centrifuge tubes to a ROTANTA automated centrifuge and centrifuge at 200×g for 8 minutes. Discard the upper oil phase in three fractions using an FCA200 centrifuge, with the final fraction involving 24-point rotational aspiration. This yields a solution containing phospholipid vesicles. This solution containing phospholipid vesicles can be further centrifuged to remove the aqueous phase, yielding the phospholipid vesicles.

[0082] Microscopic examination: The centrifuge tubes were shaken at 1200 rpm for 3 seconds using a T-shaker, and then phospholipid vesicle solution was aspirated with an FCA 200 and added to the SensoPlate at a rate of 50 μL / well; multi-field random imaging was performed using the HCA system on a Nikon rotary confocal microscope.

[0083] The detection results for the phospholipid vesicles encapsulating small molecule dyes, macromolecules, DNA, and proteins prepared using the automated platform in Example 2 are as follows: Figure 3 As shown, where, Figure 3In the image, A represents the confocal microscopy imaging results. Image I shows an image of vesicles encapsulating calcein, with green representing calcein. Image II shows an image of vesicles encapsulating λDNA, with green representing YOYO-1 stained λDNA. Image III shows an image of vesicles encapsulating dextran, with green representing FITC-labeled dextran (40,000 molecular weight). Image IV shows an image of vesicles encapsulating immunoglobulins, with green representing Alexa Fluro488-labeled IgG protein. In all four images, red represents POPC phospholipids. The scale bar is 20 μm. Figure 3 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 3 C in the figure represents the coefficient of variation of vesicle diameter in a bar chart, where n = 3; Figure 3 D in the figure represents the statistical histogram of the coefficient of variation of fluorescence intensity within the vesicle, n=3; Figure 3 In the bar chart, E represents the aggregate content statistics, n = 90; at the top of the bar, ns indicates P > 0.05, * indicates P < 0.05, ** indicates P < 0.01, and **** indicates P < 0.0001.

[0084] like Figure 3 As shown, the automated phospholipid vesicle encapsulation platform exhibits excellent compatibility in encapsulating various biomolecules. Using DNA, proteins, and dextran as encapsulating materials, it can yield a large number of uniform vesicles with a diameter of approximately 6±2 μm. Figure 3 (B in the text). Except for the vesicle diameter of the IgG protein, which had a coefficient of variation of 50.21%, the vesicle diameters in other groups were all around 30%. Figure 3 The presence of glycerol in the IgG protein buffer (C) may be due to the small amount of glycerol in the buffer, which can affect the self-assembly of phospholipid molecules during encapsulation. The fluorescence intensity inside the vesicles encapsulating λDNA was consistently lower, with a coefficient of variation of 43.61%, significantly higher than the other three groups of vesicles. Figure 3 (D) This is because DNA has the largest molecular weight, and when observed under a confocal microscope, it is not a uniform fluorescence distribution, but rather granular fluorescent spots, which may lead to greater differences in fluorescence intensity between different vesicles during image recognition.

[0085] Example 3

[0086] This embodiment utilizes a peristaltic pump to prepare phospholipid vesicles encapsulating calcein.

[0087] Preparation of oil phase solution containing phospholipids: Add 42 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 3.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0088] Preparation of the first liquid and water-in-oil emulsion: 250 μL of squalene solution of phospholipid was added to a 1.5 mL centrifuge tube using FCA 1000, and 10 μL of 0.1 mM calcein solution was added to different tubes using FCA50. The tubes were then peristalticed for 40 seconds and 60 seconds respectively using a capillary tube connected to a peristaltic pump at high speed to obtain the water-in-oil emulsion.

[0089] Preparation of the second liquid: Add 200 μL of 300 mM glucose solution to a 1.5 mL centrifuge tube using an FCA1000, and use an FCA200 to suspend 125 μL of squalene solution of phospholipids in the tube. Let stand for 30 minutes to form a phospholipid molecular layer.

[0090] Softening slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate using FCA200, let stand for 15 minutes, then add 100 μL of 300 mM glucose solution to each well of the SensoPlate and repeatedly blow and wash the slides. Repeat 3 times.

[0091] Preparation of phospholipid vesicles: Using an FCA 1000 syringe, the water-in-oil emulsion was completely aspirated and added dropwise to a 1.5 mL centrifuge tube where a phospholipid molecular layer had formed. During addition, the droplet outlet was 5 mm above the liquid surface. The centrifuge tube was transferred to a ROTANTA automated centrifuge and centrifuged at 200 × g for 8 minutes. The upper oil phase was discarded in three separate centrifuge cycles using an FCA 200 syringe, with the final cycle involving 24-point rotational aspiration of the oil. A solution containing phospholipid vesicles was obtained. This solution containing phospholipid vesicles can be further centrifuged to remove the aqueous phase, yielding the phospholipid vesicles.

[0092] Microscopic examination: The centrifuge tubes were shaken at 1200 rpm for 3 seconds using a T-shaker, and then phospholipid vesicle solution was aspirated with an FCA 200 and added to the SensoPlate at a rate of 50 μL / well; multi-field random imaging was performed using the HCA system on a Nikon rotary confocal microscope.

[0093] Example 4

[0094] In this embodiment, phospholipid vesicles encapsulating calcein are prepared using Bio-Shaker.

[0095] Preparation of oil phase solution containing phospholipids: Add 42 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 3.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0096] Preparation of the first liquid and water-in-oil emulsion: Water-oil emulsification method: Add 250 μL of squalene solution of phospholipid to a 1.5 mL centrifuge tube using FCA 1000, and add 10 μL of 0.1 mM calcein solution to different tubes using FCA 50. Then shake with Bio-Shaker at 3000 rpm for 6 minutes to obtain water-in-oil emulsion.

[0097] Preparation of the second liquid: Add 200 μL of 300 mM glucose solution to a 1.5 mL centrifuge tube using FCA 1000, and use FCA 200 to suspend 125 μL of squalene solution of phospholipids in the tube. Let stand for 30 minutes to form a phospholipid molecular layer.

[0098] Softening slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate using FCA 200, let stand for 15 minutes, then add 100 μL of 300 mM glucose solution to each well of the SensoPlate and repeatedly blow and wash the slides. Repeat 3 times.

[0099] Preparation of phospholipid vesicles: The water-in-oil emulsion was completely drawn up using an FCA1000 and added dropwise into a 1.5 mL centrifuge tube that had been allowed to stand to form a phospholipid molecular layer. During the addition, the distance between the droplet outlet and the liquid surface was 5 mm.

[0100] Transfer the centrifuge tubes to a ROTANTA automated centrifuge and centrifuge at 200×g for 8 minutes. Discard the upper oil phase in three fractions using an FCA200 centrifuge, with the final fraction involving 24-point rotational aspiration. This yields a solution containing phospholipid vesicles. This solution containing phospholipid vesicles can be further centrifuged to remove the aqueous phase, yielding the phospholipid vesicles.

[0101] Microscopic examination: The centrifuge tubes were shaken at 1200 rpm for 3 seconds using a T-shaker, and then phospholipid vesicle solution was aspirated with an FCA 200 and added to the SensoPlate at a rate of 50 μL / well; multi-field random imaging was performed using the HCA system on a Nikon rotary confocal microscope.

[0102] The detection results for phospholipid vesicles prepared by the three emulsification methods are as follows: Figure 4 As shown, where, Figure 4 In the figure, A represents the confocal microscopy imaging result, I represents the result of water-oil emulsification using the FCA 200 automatic aspiration and spraying liquid circulation, II represents the result of water-oil emulsification using the peristaltic pump of the separator, and III represents the result of water-oil emulsification using the high-frequency vibration of the Bio-Shaker. Red represents phospholipids, green represents calcein, and the scale bar is 20 μm. Figure 4 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 4C in the figure represents the coefficient of variation of vesicle diameter in a bar chart, where n = 3; Figure 4 D in the figure represents the statistical histogram of the coefficient of variation of fluorescence intensity within the vesicle, n=3; Figure 4 In the bar chart, E represents the aggregate content statistics; ns at the top of the bar indicates P>0.05, and **** indicates P<0.0001.

[0103] like Figure 4 As shown, the average diameters of phospholipid vesicles prepared by the three emulsification methods are relatively close, at 6.22 μm, 8.09 μm, and 6.47 μm respectively. However, the vesicle diameter distribution of vesicles prepared by the FCA 200 automatic aspiration and spraying liquid circulation emulsification method is significantly more concentrated, with a standard deviation of 2.52 μm. In contrast, the standard deviation of vesicle diameter prepared by the Bio-Shaker method is 3.37 μm, and the standard deviation of vesicle diameter prepared by the peristaltic pump method is 5.77 μm. This indicates that the vesicle size distribution of vesicles prepared by the automatic aspiration and spraying liquid circulation method is more concentrated, while the vesicle size variation of vesicles prepared by the peristaltic pump method is the greatest. Figure 4 (B in the original text). This result is also reflected in the statistical analysis of the coefficient of variation of vesicle diameter. The coefficient of variation of vesicle diameter prepared by the automatic aspiration and spraying liquid circulation method is only 24.16%, which is significantly lower than the 35.77% of the peristaltic pump and the 38.97% of the Bio-Shaker, indicating that the vesicles prepared by the automatic aspiration and spraying liquid circulation method have more uniform size. Figure 4 (C) The vesicles prepared by the FCA200 automated aspiration and spray liquid circulation emulsification method exhibited the most uniform encapsulation of calcein, with a coefficient of variation of fluorescence intensity within the vesicles of only 22.42%, while the coefficients of variation for vesicles prepared by the other two methods were 47.82% and 43.03%, respectively, significantly higher than the first method. Figure 4 (D) In ​​terms of phospholipid aggregation, the Bio-Shaker method produced significantly more vesicles (5.229%) than the automated aspiration-spray liquid circulation method (2.259%) and the peristaltic pump method (0.672%). Figure 4 (E in the middle).

[0104] Comparative Example 2

[0105] This embodiment utilizes a Tecan automated pipetting workstation (Tecan Fluent 1080) with a suction-and-spray function to prepare phospholipid vesicles encapsulating calcein. SensoPlate was used instead of 1.5 mL centrifuge tubes as the container for spreading the phospholipid molecular layer.

[0106] Preparation of oil phase solution containing phospholipids: Add 42 μL of chloroform solution of POPC (concentration of 25 mg / mL) to a brown glass bottle, blow dry with nitrogen gas, and let stand in vacuum for 30 minutes; add 3.5 mL of squalene to the glass bottle according to the phospholipid concentration of 400 μM, seal the bottle, sonicate at 60°C for 1 hour, and let stand at room temperature overnight.

[0107] Preparation of the first liquid and water-in-oil emulsion: 250 μL of squalene solution containing phospholipids was added to a 1.5 mL centrifuge tube using an FCA 1000 apparatus. 10 μL of 0.1 mM calcein solution was added using an FCA 50 apparatus. The mixture was then aspirated and sprayed using an FCA 200 apparatus with a nozzle diameter of 0.3 mm. The nozzle distance from the liquid surface was 6 mm, the spray velocity was 900 μL / s, and the spray acceleration was 12000 μL / s. 2 160 μL of liquid was aspirated and sprayed, and the mixture was circulated 70 times to obtain a water-in-oil emulsion.

[0108] Preparation of the second liquid: 100 μL of 300 mM glucose solution was added to the SensoPlate using FCA 200. 75 μL of squalene solution of phospholipids was then suspended and dropped into the well using FCA 200 and allowed to stand for 30 minutes to form a phospholipid molecular layer.

[0109] Softening slides: Add 50 μL of 1 mg / mL polylysine polyethylene glycol solution to each well of the SensoPlate using FCA 200, let stand for 15 minutes, then add 100 μL of 300 mM glucose solution to each well of the SensoPlate and repeatedly blow and wash the slides. Repeat 3 times.

[0110] Preparation of phospholipid vesicles: Using an FCA 1000, 40 μL of water-in-oil emulsion was pipetted into each well and added dropwise to the wells where a phospholipid molecular layer had formed. The droplet outlet was 5 mm above the liquid surface during addition. The plate was then transferred to a ROTANTA automated centrifuge and centrifuged at 200 × g for 8 minutes. This yielded a solution containing phospholipid vesicles. This solution containing phospholipid vesicles could be further centrifuged to remove the aqueous phase, thus obtaining the phospholipid vesicles.

[0111] Microscopic examination: Multi-field random imaging was performed using the HCA system on a rotating confocal microscope from Nikon.

[0112] The results of phospholipid vesicle detection in Comparative Example 2 are shown in the figure below. Figure 5 As shown, where, Figure 5 In the figure, A represents the confocal microscopy imaging result, I represents the result of centrifugation in a 1.5 mL centrifuge tube, and II represents the result of centrifugation in a SensoPlate. Red represents phospholipids, green represents calcein, and the scale bar is 20 μm. Figure 5 In this context, B represents the Gaussian distribution fitting curve of the phospholipid vesicle diameter distribution; Figure 5 C in the figure represents the coefficient of variation of vesicle diameter in a bar chart, where n = 3; Figure 5 D in the figure represents the statistical histogram of the coefficient of variation of fluorescence intensity within the vesicle, n=3; Figure 5In the bar chart, E represents the aggregate content statistics, n = 90; ns at the top of the bar indicates P > 0.05, and **** indicates P < 0.0001.

[0113] like Figure 5 As shown, the diameter distribution and internal fluorescence coefficient of phospholipid vesicles obtained by using either 1.5 mL centrifuge tubes or SensoPlate to deposit the phospholipid molecular layer are very similar. However, the phospholipid vesicles encapsulated in the 1.5 mL centrifuge tubes contain more phospholipid aggregates (0.954%), significantly higher than those in the SensoPlate (0.414%). This may be because the phospholipid vesicles prepared in the 1.5 mL centrifuge tubes undergo an additional sample transfer step, which has a certain impact on the state of the phospholipid vesicles. Nevertheless, the proportion of phospholipid aggregates in both methods is at a lower level compared to the vesicles prepared by the manual method described above (5.22%).

[0114] The above description of the embodiments is provided to enable those skilled in the art to understand and use the invention. It will be apparent to those skilled in the art that various modifications can be made to these embodiments, and the general principles described herein can be applied to other embodiments without inventive effort. Therefore, the present invention is not limited to the above embodiments, and any improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the invention should be within the protection scope of the present invention.

Claims

1. A method for preparing phospholipid vesicles, the method comprising: A first liquid is sprayed using a first automated pipetting device with a spraying function. The first liquid is a mixture of a first oil phase solution and a first aqueous phase solution, wherein the oil phase solution contains phospholipids. After spraying, the first liquid forms a water-in-oil emulsion. Using a second automated pipetting device, an oil-in-water emulsion is dropped into a second liquid, wherein the second liquid comprises an upper oil phase and a lower aqueous phase, with a phospholipid molecular layer between the upper oil phase and the lower aqueous phase. The oil-in-water emulsion droplets pass through the phospholipid molecular layer and enter the aqueous phase to be dispersed, forming a system containing phospholipid vesicles. The phospholipid vesicles were isolated.

2. The method according to claim 1, wherein, The nozzle diameter for the liquid spraying treatment is 0.2-0.5 mm, the distance between the nozzle and the liquid surface is 2-8 mm, the spray velocity is 500-900 μL / s, and the spray acceleration is 8000-12000 μL / s. 2 ; Preferably, the nozzle diameter for the liquid spraying treatment is 0.25-0.35 mm, the distance between the nozzle and the liquid surface is 4-6 mm, the spray velocity is 800-900 μL / s, and the spray acceleration is 10000-12000 μL / s. 2 .

3. The method according to claim 1, wherein, The first automated pipetting device also has a liquid suction function. The first automated pipetting device performs a suction-spray liquid circulation process on the first liquid. The number of cycles of the suction-spray liquid circulation process is 40-140 times, preferably 70-100 times.

4. The method according to claim 1, wherein, When the water-in-oil emulsion is added dropwise to the second liquid, the distance between the liquid outlet and the liquid surface is 2-8 mm, preferably 4-6 mm.

5. The method according to claim 1, wherein, The volume ratio of the first oil phase solution to the first aqueous phase solution in the first liquid is 200-250:10-20.

6. The method according to claim 1 or 5, wherein, The concentration of phospholipids in the first oil phase solution is 350-450 mM; preferably, the first oil phase solution is prepared by dissolving phospholipids in an oil phase solvent, and more preferably by dissolving phospholipids in an oil phase solvent by heating and ultrasound. Preferably, the oil phase solvent is selected from one or more of liquid paraffin, mineral oil, and squalene, and more preferably squalene; Preferably, the first aqueous solution is an aqueous solution containing one or more of the following: small molecule dyes, large molecule polymers, DNA, and proteins.

7. The method according to claim 1, wherein, The second liquid was prepared as follows: The second oil phase solution and the second aqueous phase solution are mixed, wherein the second oil phase solution contains phospholipids. After the second oil phase solution and the second aqueous phase solution are mixed, they are allowed to stand and separate into layers to obtain the second liquid. Preferably, the concentration of phospholipids in the second oil phase solution is 350-450 μM; Preferably, the first oil phase solution is prepared by dissolving phospholipids in an oil phase solvent, and more preferably, the phospholipids are dissolved in the oil phase solvent by heating and ultrasonication. Preferably, the oil phase solvent is selected from one or more of liquid paraffin, mineral oil, and squalene, and more preferably squalene; Preferably, the second aqueous phase solution is an aqueous solution containing glucose.

8. The method according to claim 1, wherein, The process of isolating the phospholipid vesicles from a system containing phospholipid vesicles includes: The system containing phospholipid vesicles is centrifuged to remove the oil phase; preferably, the centrifugation conditions are 200-500×g for 5-10 min.

9. Phospholipid vesicles prepared by the method according to any one of claims 1-8.

10. A method for preparing phospholipid vesicles, the method comprising: The first liquid is subjected to peristaltic treatment using a peristaltic pump or agitation treatment using a Bio-Shaker, wherein the first liquid is a mixture of a first oil phase solution and a first aqueous phase solution, wherein the oil phase solution contains phospholipids, and the first liquid forms a water-in-oil emulsion after peristaltic or agitation treatment. Using a second automated pipetting device, an oil-in-water emulsion is dropped into a second liquid, wherein the second liquid comprises an upper oil phase and a lower aqueous phase, with a phospholipid molecular layer between the upper oil phase and the lower aqueous phase. The oil-in-water emulsion droplets pass through the phospholipid molecular layer and enter the aqueous phase to be dispersed, forming a system containing phospholipid vesicles. The phospholipid vesicles were isolated.

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