CN117695961A - Preparation method and device of lipid vesicles - Google Patents
Preparation method and device of lipid vesicles Download PDFInfo
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Abstract
本发明公开了一种脂质囊泡的制备方法及装置，属于生物技术领域。所述制备方法包括，配置磷脂溶液；将磷脂溶液涂覆在反应室的基底材料上；待磷脂溶液干燥后，在基底材料上形成脂质膜；在高离子浓度溶液中对反应室超声处理20～60分钟，使脂质膜从所述基底材料上脱离并自我封闭，制得所述脂质囊泡。所述装置包括超声发射装置、超声换能器、冷却底座和反应室。本发明能够简单高效的制备脂质囊泡，且在制备过程中不受磷脂材料、溶液离子浓度的影响。
The invention discloses a preparation method and device of lipid vesicles, and belongs to the field of biotechnology. The preparation method includes: preparing a phospholipid solution; coating the phospholipid solution on the base material of the reaction chamber; after the phospholipid solution is dried, forming a lipid film on the base material; ultrasonic treatment of the reaction chamber in a high ion concentration solution for 20 ~60 minutes, the lipid membrane is detached from the base material and self-sealed to prepare the lipid vesicle. The device includes an ultrasonic transmitting device, an ultrasonic transducer, a cooling base and a reaction chamber. The present invention can prepare lipid vesicles simply and efficiently, and is not affected by phospholipid materials and solution ion concentration during the preparation process.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a preparation method and a preparation device of lipid vesicles.
Background
Lipid vesicles, also known as lipid vesicles or sometimes simply vesicles, are microscopic spherical structures composed of one or several lipid (usually natural and synthetic phospholipids) bilayers, with a small amount of aqueous phase. Because of good amphiphilicity and biocompatibility, liposomes have been widely used as model biofilms and delivery vehicles for various bioactive materials, such as delivering anticancer and antibacterial agents, genetic material, proteins, DNA, peptides, vaccines, enzymes, and the like, for many years. Another important function of vesicles is to mimic biological membranes. Because the main body of the biological film is a closed bilayer vesicle structure formed by directional arrangement of phospholipid and protein, the biological film plays an important role in living organisms and has the functions of ion migration, immune recognition and the like. Therefore, through the research on vesicles, the understanding of people on biological membranes can be deepened, and a new approach is provided for the bionic research of people. The cell membrane model contains charged lipids and requires physiological conditions. It is therefore important to study the efficient preparation method and apparatus of lipid vesicles.
Currently, methods for preparing lipid vesicles in high ion concentration solutions mainly include modified electroforming and microfluidic methods. Among them, the electroforming method is most commonly used because it can produce lipid vesicles in high yield and quality. However, unless specialized lipid formulations or instruments are used, modified electroforming methods require that the phospholipid bilayer be charged and that the ion concentration of the solution not be too high, typically limited to solutions of low ionic strength (. Ltoreq.50 mM monovalent salt). The best cell membrane model contains charged lipids and requires physiological conditions (154 mM). The use of charged lipids to form large unilamellar vesicles (GUVs) on Indium Tin Oxide (ITO) electrodes in saline solution environments has proven difficult. The microfluidic method needs more complicated microfluidic equipment, a microfluidic chip structure is customized, and organic solvents between phospholipid bilayer are removed after lipid vesicles are prepared, so that the requirements on equipment are high, and the preparation procedure is complicated.
There is therefore a need to develop a lipid vesicle preparation technique whereby lipid vesicles are produced simply and efficiently.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a preparation method and a device for lipid vesicles, which can simply and efficiently prepare the lipid vesicles and improve the efficiency of producing the lipid vesicles.
In order to solve the technical problems, the invention adopts the following technical scheme:
a preparation method of lipid vesicle comprises the following steps,
preparing a phospholipid solution;
coating a phospholipid solution on a substrate material of a reaction chamber;
forming a lipid film on the base material after the phospholipid solution is dried;
and (3) carrying out ultrasonic treatment on the reaction chamber in a high-ion concentration solution for 20-60 minutes to separate and self-seal the lipid membrane from the substrate material, so as to prepare the lipid vesicle.
Preferably, the phospholipid is L-alpha-phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine.
Preferably, the concentration of the phospholipid solution is 1 to 2mg/mL.
Preferably, the reaction chamber is subjected to ultrasonic treatment in a high ion concentration solution, and the formed lipid film is vacuumized for 2 to 3 hours to reach a vacuum degree of 0.08 to 0.1MPa.
Preferably, glass is used as a base material of the reaction chamber.
Preferably, the high ion concentration solution is a sodium chloride solution of 0.65 to 0.9%. Because the ultrasonic wave is adopted, the inhibition effect of high ion concentration on the expansion of the phospholipid membrane can be reduced by utilizing the sound force, so that the lipid vesicle can be prepared in the high ion concentration solution.
Preferably, in the ultrasonic treatment, the driving voltage of the ultrasonic transmitting device is 12-22V.
The invention also discloses a preparation device of the lipid vesicle, which comprises an ultrasonic emission device, an ultrasonic transducer, a cooling base and a reaction chamber;
the reaction chamber comprises two layers of glass and a polydimethylsiloxane layer positioned between the glass; a through hole is vertically formed in the polydimethylsiloxane layer, so that a reaction chamber is formed between the two layers of glass and the through hole; a sample inlet communicated with the reaction chamber is also arranged on the polydimethylsiloxane layer;
the ultrasonic transmitting device is connected with the ultrasonic transducer; the ultrasonic transducer is provided with a piezoelectric ceramic piece and a back lining, the upper side surface of the piezoelectric ceramic piece is connected with glass positioned at the lower side of the reaction chamber through an ultrasonic coupling agent, and the lower side surface of the piezoelectric ceramic piece is abutted with the back lining and then is placed on the cooling base.
The lipid vesicles are prepared by providing appropriate ultrasonic waves for preparing the lipid vesicles by an ultrasonic emission device and an ultrasonic transducer, so that the lipid vesicles can be separated from the substrate material and self-closed in a high-ion concentration solution. The cooling base is used for cooling the piezoelectric ceramic plate which works for a long time, taking away heat, and preventing the temperature of the reaction chamber from rising and affecting the formation of lipid vesicles.
Preferably, the polydimethylsiloxane layer is prepared by using polydimethylsiloxane and polydimethylsiloxane curing agent according to a ratio of 10:1, and after thoroughly mixing and degassing, placing on a petri dish and drying at 65 ℃ for 3 hours to obtain the polydimethylsiloxane layer.
Preferably, the glass is surface treated with a plasma cleaner prior to forming the reaction chamber.
Compared with the prior art, the invention has the following advantages:
1. according to the preparation method provided by the invention, phospholipid is dissolved by chloroform and is prepared into a phospholipid solution, the phospholipid solution is coated on a substrate material of a reaction chamber, and after the phospholipid solution is dried to form a lipid membrane, the lipid membrane can be separated from the substrate material and is self-sealed by ultrasonic treatment of the reaction chamber in a high-ion concentration solution, so that the lipid vesicle is prepared. In the ultrasonic treatment process, the micro-acoustic flow generates acoustic force on the surface of the lipid membrane, promotes the expansion of the lipid membrane in the high-ion concentration solution, and breaks away from the substrate and self-seals under the action of a flow field, thereby forming the lipid vesicle. The method is a non-contact lipid vesicle preparation method and has the advantages of simplicity, high efficiency and capability of being prepared in a high-ion concentration solution.
2. According to the preparation device provided by the invention, the power regulating module transmits electric energy to the ultrasonic transducer, the ultrasonic transducer converts the electric energy into ultrasonic waves, the ultrasonic waves are transmitted to the reaction chamber through the piezoelectric ceramic plate, the damping of the ultrasonic waves in the fluid can form stable flowing sound streams, sound force is generated on the surface of the lipid membrane attached to glass by the sound streams, the expansion of the lipid membrane in the high-ion concentration solution is promoted, and the lipid membrane is separated from the substrate under the action of a flow field and is self-sealed to form lipid vesicles. The device has the advantages of controllable ultrasonic wave size, convenient operation and high efficiency in preparing lipid vesicles.
Drawings
FIG. 1 shows the mechanism of lipid vesicle formation in an example of the present invention.
Fig. 2 is a graph of acoustic volume force and acoustic flow field within a simulated lipid vesicle using a finite element method.
FIG. 3 is a schematic diagram of a device for preparing lipid vesicles.
FIG. 4a is a diagram showing the formation of lipid vesicles prepared in example 1 of the present invention. Fig. 4 b is a diagram showing the formation of lipid vesicles prepared in example 2, and fig. 4 c and d show optical microscopy and electron microscopy characterization of lipid vesicles.
Fig. 5 is a diagram of a finite element simulated acoustic flow field at the cross section of chamber yoz in an embodiment of the invention.
Fig. 6 is a process diagram of the fusion of lipid vesicles in an acoustic flow field in an embodiment of the invention.
In the figure, a piezoelectric ceramic sheet 1, a backing 2, a cooling device 3, a water inlet 31, a water outlet 32, a polydimethylsiloxane layer 4, a sample inlet 41 and glass 5.
Detailed Description
The embodiment of the invention provides a preparation method of lipid vesicles, which comprises the following steps,
preparing a phospholipid solution;
coating a phospholipid solution on a substrate material of a reaction chamber;
forming a lipid film on the base material after the phospholipid solution is dried;
and (3) carrying out ultrasonic treatment on the reaction chamber in a high-ion concentration solution for 20-60 minutes to separate and self-seal the lipid membrane from the substrate material, so as to prepare the lipid vesicle.
The mechanism of lipid vesicle formation is shown in figure 1. Stage a in fig. 1 shows the hydration of the film: the organic solvent in the phospholipid membrane was evaporated by vacuum-pumping, thereby obtaining a stack of lipid membranes on the substrate material (ITO glass) of the reaction chamber. Stages b and c show that in an aqueous environment, lipid molecules diffuse laterally in the bilayer, water flows through the bilayer, and the lipid membrane swells. Stage d in fig. 1 shows that under the action of ultrasonic waves, the micro-acoustic fluid generated by the ultrasonic waves generates acoustic force on the surface of the lipid membrane, promotes the expansion of the lipid membrane in the high-ion concentration solution, and breaks away from the substrate and self-seals under the action of a flow field to form the lipid vesicle lipid membrane. In practice, it is preferable to use a hydrophilic ITO glass as a base material, the surface of which can promote hydration of a solid lipid film and formation of a lipid bilayer, but a high ion concentration solution can inhibit expansion of the lipid film, so that a sound field is introduced at this stage, ultrasonic physical force acts on the lipid film to cause expansion of the lipid film, and finally, the lipid film is detached from the base and self-closed, thereby forming a lipid vesicle lipid film.
The attenuation of ultrasonic waves in a fluid creates a steady flow acoustic stream with a flow velocity proportional to the intensity of sound and the ultrasonic frequency. The acoustic flow may be induced in the fluid by bulk viscous damping of the fluid to induce large-scale acoustic flow, or by thermal viscous damping near the wall to generate acoustic flow near the boundary, as shown in stage c of fig. 1. The ultrasonic force is generated on the surface of the lipid membrane by the micro-sound flow due to the thermal viscous damping near the wall surface, so that the lipid membrane breaks through the inhibition effect of the high-ion concentration solution on the expansion of the phospholipid membrane, and the expansion of the lipid membrane is pushed. Stage d in fig. 1 shows that the lipid vesicles are detached from the surface of the glass substrate and float in solution under the action of stokes drag force Fdrag. Simulation of acoustic volume force f inside lipid vesicles using finite element method aco The distribution and the micro-acoustic flow field distribution reveal the mechanism of forced expansion of the phospholipid membrane in the solution, and the result is shown in figure 2, wherein a in figure 2 is the acoustic force distribution in the vesicle in the process of preparing the lipid vesicle by finite element simulation ultrasound, and b is the acoustic flow field distribution. As can be seen from fig. 2 a and b, under the application of a sound field, the acoustic volume force perpendicular to the phospholipid membrane and the induced i micro-acoustic streaming field push the phospholipid membrane away from the glass substrate, expanding into lipid vesicles.
At Stokes drag force F drag Under the action, the lipid vesicle is separated from the surface of the glass substrate, and the acoustic physical force calculation formula is as follows:
wherein ρ, c 0 W, μ and μb are fluid density, propagation speed of ultrasound in water, ultrasound angular frequency, dynamic viscosity and volumetric viscosity, respectively; p and v are pressure and velocity, respectively. Where Γ is the dissipation factor.
As shown in stage d of FIG. 1, after the lipid vesicles have been inflated to a certain volume, stokes drag force F generated by acoustic flow away from the wall drag So that the lipid vesicles are detached from the ITO glass substrate and self-closed to form lipid vesicles, float in the solution,<v 2 >represents the sound flow velocity and a represents the lipid vesicle radius.
F drag ＝6πμa<v 2 >。
Dissolving phospholipid by chloroform, preparing phospholipid solution, coating the phospholipid solution on a substrate material of a reaction chamber, and carrying out ultrasonic treatment on the reaction chamber in a high-ion concentration solution after the phospholipid solution is dried to form a lipid membrane, so that the lipid membrane is separated from the substrate material and self-closed, thereby preparing the lipid vesicle. In the ultrasonic treatment process, the micro-acoustic flow generates acoustic force on the surface of the lipid membrane, promotes the expansion of the lipid membrane in the high-ion concentration solution, and breaks away from the substrate and self-seals under the action of a flow field, thereby forming the lipid vesicle. The method is a non-contact lipid vesicle preparation method and has the advantages of simplicity, high efficiency and capability of being prepared in a high-ion concentration solution.
The embodiment of the invention also discloses a preparation device of the lipid vesicle, which comprises an ultrasonic emission device, an ultrasonic transducer, a cooling base and a reaction chamber;
the reaction chamber comprises two layers of glass and a polydimethylsiloxane layer positioned between the glass; a through hole is vertically formed in the polydimethylsiloxane layer, so that a reaction chamber is formed between the two layers of glass and the through hole; a sample inlet communicated with the reaction chamber is also arranged on the polydimethylsiloxane layer;
the ultrasonic transmitting device is connected with the ultrasonic transducer; the ultrasonic transducer is provided with a piezoelectric ceramic piece and a back lining, the upper side surface of the piezoelectric ceramic piece is connected with glass positioned at the lower side of the reaction chamber through an ultrasonic coupling agent, and the lower side surface of the piezoelectric ceramic piece is abutted with the back lining and then is placed on the cooling base.
In practice, the preparation device of the lipid vesicle mainly comprises an ultrasonic emission device, an ultrasonic transducer, a cooling base and a reaction chamber, and is shown in fig. 3. The ultrasonic transmitting device shown adopts a piezoelectric ceramic plate 1 with a resonance frequency of 830.0kHz, a diameter of 50.00 mm and a thickness of 2.50 mm as a driving sound source. The precise impedance analyzer 4294 is used for measuring the impedance parameters of the piezoelectric ceramic plate 1, and a matching circuit is designed to perform impedance matching with the piezoelectric ceramic plate 1, so that electric energy is more efficiently converted into acoustic energy. The sound power value is measured by the sound power meter, the sound power output by the device can be regulated by the power regulating module, the back lining 2 of the ultrasonic transducer adopts an air back lining, and a cooling device 3 is additionally arranged behind the back lining 2, so that the piezoelectric ceramic plate 1 can still keep constant temperature under a higher power state, and the piezoelectric ceramic plate 1 is prevented from being influenced by the preparation of lipid vesicles due to the fact that the temperature of the piezoelectric ceramic plate 1 is increased when the piezoelectric ceramic plate 1 works for a long time under a higher driving voltage. In specific implementation, the cooling device 3 adopts a water cooling mode, a water inlet 31 and a water outlet 32 are arranged on the cooling base, heat on the piezoelectric ceramic piece 1 and the backing 2 is taken away through water flow, and the working temperatures of the piezoelectric ceramic piece 1 and the backing 2 are kept stable and proper.
The reaction chamber consists of a polydimethylsiloxane layer 4 (PDMS layer) and glass 5 on both the upper and lower sides of the polydimethylsiloxane layer 4. The glass is preferably ITO glass. The manufacturing process of the reaction chamber is as follows: firstly, mixing PDMS and a PDMS curing agent according to a proportion of 10:1, poured onto a petri dish, then placed in a 65 ℃ dry box for 3 hours, then the resulting PDMS layer was cut and carefully peeled off from the petri dish. A through hole is formed in the middle of the polydimethylsiloxane layer 4, so that a reaction chamber is formed between the polydimethylsiloxane layer 4 and the glass 5 positioned on the upper side and the lower side of the polydimethylsiloxane layer. A sample inlet 41 is also provided on the polydimethylsiloxane layer 4, which runs through the reaction chamber. After the surface treatment of the glass 5 using a plasma cleaner, the bottom glass 5, the PDMS layer 4, and the top glass 5 were bonded together. The PDMS layer 4 between the glasses 5 serves not only as a spacer separating the glasses 5, but also to form the sidewalls of the reaction chamber. The piezoelectric ceramic plate 1 is connected with the glass 5 by using an ultrasonic coupling agent, and the ultrasonic transducer and the reaction chamber are assembled together through a PMMA plate and screws.
The materials and equipment used in the embodiments of the present invention are as follows:
the lipid L- α -phosphatidylcholine was purchased from Avanti Polar lipids (USA).
Molecular probes (3, 3' -octacosyl oxonol perchlorate, ex/em:484/501nm, diO) were purchased from Sigma-Aldrich (USA). The molecular probes are used for staining the phospholipid membrane, for tracking the thickness of the phospholipid membrane, and for better observing the morphology of the lipid vesicles under a microscope, and are added when preparing the phospholipid solution.
Sodium chloride (NaCl) and chloroform (AR) were purchased from Chongqing Chuan Dong chemical (China) and prepared as NaCl solution using Millipore Milli-Q water having a resistivity of 18.0 M.OMEGA.cm at 25 ℃.
Polydimethylsiloxane (PDMS) was purchased from dakangnin (united states).
Polymethyl methacrylate (PMMA) was purchased from yikang (china).
Pipette gun was purchased from eppendorf.
ITO glass was purchased from ZhuhaiKai as a photoelectric technology Co., ltd.
The ultrasonic couplant is purchased from a medical material auxiliary factory of Kai-shi in Tianjin.
Acoustic power meter was purchased from BC GROUP OHMIC, model UPM-DT-1AV, usa).
The piezoelectric ceramic wafer was purchased from Shenzhen Huajida electronics Inc.
Example 1
A preparation method of lipid vesicle comprises the following steps,
l-alpha-phosphatidylcholine powder was taken, dissolved with chloroform and sub-packed into a phospholipid solution having a concentration of 25 mg/ml. Then, 2ul of a phospholipid solution (25 mg/ml) was taken and a phospholipid solution (1 mg/ml) was prepared using chloroform.
And adding 2ul of fluorescent dye (namely molecular probe) into the prepared phospholipid solution, blowing and sucking the solution by using a pipetting gun, uniformly mixing, dripping 5ul of phospholipid solution on the surface of ITO glass subjected to hydrophilic treatment by using a plasma cleaning machine, and uniformly coating the solution by using a syringe needle.
And (3) placing the uniformly coated reaction chamber in a fume hood, taking out the ITO glass sheet formed into a film from the fume hood when the phospholipid film on the surface of the ITO glass is observed to be close to a dry state, placing the ITO glass sheet in a vacuum drying oven, vacuumizing for 2 hours, and completely removing the residual organic solvent. Thus, the phospholipid solution forms a lipid film on the ITO glass plate. In the specific implementation, after the reaction chamber is placed in the fume hood, the illuminating lamp of the fume hood is required to be turned off, on one hand, fluorescence quenching caused by illumination is prevented, on the other hand, chloroform is prevented from being decomposed into extremely toxic substances after being illuminated, and after the phospholipid solution is dried, a lipid film is formed on the substrate material.
Lipid vesicles were prepared using ultrasound in 0.65% NaCl solution. The driving voltage of the ultrasonic emission device is set to be 22V, and the ultrasonic action time of the reaction chamber is 45 minutes. The ultrasonic operation time is divided into three stages, 15 minutes, 3 minutes and 15 minutes, so that the influence of the temperature rise of the reaction chamber caused by the long-time operation heating of the piezoelectric ceramic is reduced. The lipid vesicles are prepared. The formation of the lipid vesicles prepared in this example is shown in FIG. 4 a. As can be seen from fig. 4a, the lipid vesicles prepared in this example had a particle size of approximately 20um.
Compared with the improved electroforming method, the method for preparing the lipid vesicle in high ion concentration based on ultrasonic volume force has the same efficiency as that of the electroforming method, and the ultrasonic method does not need a lipid membrane to have a charged property and does not need to set complex electric parameters. Fluorescence microscopy showed that the fluorescence intensity was higher at the upper right in FIG. 4a, and that the phospholipid vesicles formed in this region had smaller particle size due to thicker phospholipid membrane stacks in this region. In case of an excessive amount of phospholipid membrane, the formed lipid vesicles are too dense, the interaction force between adjacent lipid vesicles and the influence of the viscosity of the phospholipid membrane may hinder the formation of the lipid vesicles, the ultrasonic volume force cannot enable the high concentration of the phospholipid membrane to form larger lipid vesicles at the same time, and the ultrasonic energy is dissipated by a part of the viscous force of the phospholipid membrane and the rest of the energy is absorbed by the dense lipid vesicles. But may be improved by extending the sound field application time or increasing the ultrasonic power or decreasing the phospholipid solution concentration.
Example 2
This example differs from example 1 in that lipid vesicles were prepared using ultrasound in 0.9% NaCl solution.
The formation of the lipid vesicles prepared in this example is shown in fig. 4 b. Compared with the lipid vesicles in fig. 4a, the lipid vesicles prepared in this example had a particle size of much less than 20um, and only a few microns. This is because, at the same phospholipid concentration solution and the same intensity sound field action for the same time, the inhibition of the hydration swelling of the phospholipid membrane is enhanced due to the increase in the ion concentration, and the lipid vesicles prepared in the 0.9% NaCl solution are smaller than those prepared in the 0.65% NaCl solution.
Prepared lipid vesicles are subjected to F induced by acoustic streaming field in a chamber drag Under the action, the glass substrate is separated from the surface of the glass substrate and floats in water. The lipid vesicles prepared in this example were optically characterized as shown in fig. 4 c. In order to better observe the three-dimensional structure morphology of the lipid vesicles, the lipid vesicles are subjected to freeze-drying treatment, and the structure is easily destroyed in the freeze-drying process because the thickness of the phospholipid membrane is only a few nanometers, so that the solution in the chamber is replaced by mannitol solution with the concentration of 2% as a freeze-drying protective agent before the freeze-drying treatment, and the geometry of the lipid vesicles is maintained later, and the freeze-drying process is carried out. The lipid vesicle steric structure was then observed with a Scanning Electron Microscope (SEM). The three-dimensional structure of the lipid vesicles prepared in this example is shown in FIG. 4 d, which shows that the radius is 1.5um, because the lipid vesicles with large particle size are easily damaged during lyophilization, and only the lipid vesicles with small particle size can be retained。
Studies have shown that fusion of adjacent lipid vesicles occurs when subjected to external forces, and fusion of adjacent lipid vesicles is also an important step in the formation of lipid vesicles, which increases the particle size of the lipid vesicles. The shear force of the acoustic flow on the lipid vesicles in the acoustic field promotes the fusion of two adjacent lipid vesicles, and as shown in fig. 5, the finite element simulates the acoustic flow field of the longitudinal section of the reaction chamber yoz, and the lipid vesicles are in the flow field. Lipid bilayers can be viewed as ultrathin liquid layers with surface viscosities, where fluid viscosity at the surface of the lipid vesicles can cause the lipid membrane to flow. As shown in fig. 6 a-f, adjacent target vesicle fusion is indicated in red circles. Under the action of fluid viscosity force and shearing force caused by an acoustic flow field in the acoustic flow field, two adjacent independent lipid vesicles are fused into an elliptical lipid vesicle under the action of ultrasonic power of 3.14W, then the elliptical lipid vesicle is slowly deformed into a spherical lipid vesicle, and the vesicle fusion process is completed within 8 seconds.
Therefore, the preparation method provided by the invention can be used for preparing the lipid vesicle in a non-contact manner through ultrasonic treatment in a high-ion concentration solution, has the advantages of simplicity and high efficiency, and can improve the efficiency of producing the lipid vesicle.
It will be apparent to those skilled in the art that various modifications and variations can be made to the present invention without departing from the spirit or scope of the invention. Thus, it is intended that the present invention also include such modifications and alterations insofar as they come within the scope of the appended claims or the equivalents thereof.
Claims (10)
1. A preparation method of lipid vesicles is characterized by comprising the following steps,
preparing a phospholipid solution;
coating a phospholipid solution on a substrate material of a reaction chamber;
forming a lipid film on the base material after the phospholipid solution is dried;
and (3) carrying out ultrasonic treatment on the reaction chamber in a high-ion concentration solution for 20-60 minutes to separate and self-seal the lipid membrane from the substrate material, so as to prepare the lipid vesicle.
2. The method of claim 1, wherein the phospholipid is L- α -phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, or phosphatidylserine.
3. The method for producing lipid vesicles according to claim 1, wherein the concentration of the phospholipid solution is 1 to 2mg/mL.
4. The method for preparing lipid vesicles according to claim 1 wherein the vacuum of the formed lipid membrane is 0.08MPa to 0.1MPa by evacuating the formed lipid membrane for 2 to 3 hours before the reaction chamber is sonicated in a solution with high ion concentration.
5. The method of claim 1, wherein the reaction chamber is made of glass as a substrate material.
6. The method for producing lipid vesicles according to claim 1, wherein the high ion concentration solution is 0.65 to 0.9% sodium chloride solution.
7. The method for preparing a lipid vesicle according to claim 1, wherein the driving voltage of the ultrasonic emitter is 12-22V during the ultrasonic treatment.
8. The preparation device of the lipid vesicle is characterized by comprising an ultrasonic emission device, an ultrasonic transducer, a cooling base and a reaction chamber;
the reaction chamber comprises two layers of glass and a polydimethylsiloxane layer positioned between the glass; a through hole is vertically formed in the polydimethylsiloxane layer, so that a reaction chamber is formed between the two layers of glass and the through hole; a sample inlet communicated with the reaction chamber is also arranged on the polydimethylsiloxane layer;
the ultrasonic transmitting device is connected with the ultrasonic transducer; the ultrasonic transducer is provided with a piezoelectric ceramic piece and a back lining, the upper side surface of the piezoelectric ceramic piece is connected with glass positioned at the lower side of the reaction chamber through an ultrasonic coupling agent, and the lower side surface of the piezoelectric ceramic piece is abutted with the back lining and then is placed on the cooling base.
9. The apparatus for preparing lipid vesicles according to claim 8, wherein the polydimethylsiloxane layer is prepared from polydimethylsiloxane and a polydimethylsiloxane curing agent according to 10:1, and after thoroughly mixing and degassing, placing on a petri dish and drying at 65 ℃ for 3 hours to obtain the polydimethylsiloxane layer.
10. The apparatus for preparing lipid vesicles according to claim 8, wherein the glass is surface-treated with a plasma cleaner before forming the reaction chamber.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0052322A2 (en) * | 1980-11-10 | 1982-05-26 | Gersonde, Klaus, Prof. Dr. | Method of preparing lipid vesicles by ultrasonic treatment, the use of this method and apparatus for its application |
| US4452747A (en) * | 1982-03-22 | 1984-06-05 | Klaus Gersonde | Method of and arrangement for producing lipid vesicles |
| CN112105346A (en) * | 2018-02-27 | 2020-12-18 | 利宝涂料私人有限公司 | General method for preparing lipid-based coatings |
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2023
- 2023-11-03 CN CN202311456817.5A patent/CN117695961A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0052322A2 (en) * | 1980-11-10 | 1982-05-26 | Gersonde, Klaus, Prof. Dr. | Method of preparing lipid vesicles by ultrasonic treatment, the use of this method and apparatus for its application |
| US4452747A (en) * | 1982-03-22 | 1984-06-05 | Klaus Gersonde | Method of and arrangement for producing lipid vesicles |
| CN112105346A (en) * | 2018-02-27 | 2020-12-18 | 利宝涂料私人有限公司 | General method for preparing lipid-based coatings |
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