Preparation method of aqueous-phase phospholipid membrane vesicles in DNA (Deoxyribose Nucleic Acid)

Abstract

The invention discloses a preparation method of aqueous-phase phospholipid membrane vesicles in DNA (deoxyribonucleic acid). Comprising the following steps: adding an oil phase A into an outer water phase, centrifuging, and forming a phospholipid monomolecular layer at the interface of the oil phase A and the outer water phase; adding the inner water phase into the oil phase B, and emulsifying to obtain emulsified liquid drops wrapped by single-layer phospholipid; and dripping the emulsified liquid to the upper layer of an oil phase of a phospholipid monomolecular layer, centrifuging, absorbing the upper oil phase, and slightly blowing and beating at the bottom of a water phase to obtain the phospholipid. The method comprises the following steps: dissolving DNA and sucrose in pure water to obtain an inner water phase; dissolving glucose and NaCl in pure water to obtain an outer water phase; the preparation method comprises the following steps: dissolving phospholipid in chloroform to obtain phospholipid mother liquor, adding the phospholipid mother liquor into mineral oil, uniformly mixing and oscillating, and supplementing chloroform to obtain an oil phase A and an oil phase B; according to the method, the solubility of phospholipid in an oil phase is improved, the viscosity of the oil phase is properly improved, sucrose and glucose are added into a water phase to provide a density difference, and an emulsification mode is changed, so that stable preparation of the water-phase phospholipid membrane vesicles in the DNA is realized.

Description

Technical Field

[0001] The invention belongs to the technical field of synthetic biology, and in particular relates to a method for preparing a DNA intra-water phase phospholipid membrane vesicle. Background Art

[0002] DNA-encapsulated water vesicles are the basis of artificial cell research experiments. If stable vesicles cannot be obtained in batches, the results of subsequent experiments such as the autonomous growth and division of vesicles will be seriously affected. Therefore, how to stably obtain phospholipid vesicles in the water phase of DNA is a problem that needs to be solved urgently.

[0003] The preparation methods of DNA-in-water vesicles mainly include membrane hydration method, reverse phase evaporation method, microfluidics method and reverse phase emulsion method. (1) Membrane hydration method: By hydrating the phospholipid membrane in the DNA aqueous solution, the phospholipid bilayer spontaneously forms vesicles. The specific operation includes hydrating the dried phospholipid membrane in the DNA solution, and then adjusting the vesicle size through physical treatment (such as vortexing and ultrasound). However, due to the high viscosity of the high-concentration DNA solution, it is difficult to emulsify the phospholipid membrane directly in the aqueous phase, resulting in poor vesicle stability. (2) Reverse phase evaporation method: DNA solution and phospholipid are emulsified in the organic phase, and then the organic phase is removed by evaporation to form vesicles. However, due to the high viscosity of the DNA solution, the viscosity of the organic phase with high phospholipid solubility, such as chloroform and ethanol, is extremely low in the reverse phase evaporation method. When the viscosity difference between the two phases is huge, it is almost impossible to emulsify. (3) Microfluidics method: The flow rate and fluid dynamic conditions of the phospholipid and DNA aqueous phases are precisely controlled by microfluidic chips to achieve high-throughput and uniform preparation of vesicles. However, the microfluidics method is more complex to operate, requires a large learning cost, and has a longer vesicle preparation cycle, so it is suitable for small-scale vesicle preparation. (4) Reverse emulsion method: The reverse emulsion method is a more commonly used method for vesicle preparation. Reverse emulsion refers to an emulsion in which an aqueous solution forms oil-in-water droplets with an organic phase under the action of an oil-soluble surfactant. It is called a reverse emulsion because it is the opposite of the common water-in-oil emulsion. The reverse emulsion method for preparing vesicles refers to the method of dispersing the inner aqueous phase solution into small oil-in-water droplets wrapped in phospholipid monomolecules through a certain emulsification method, placing them on the phospholipid monolayer interface formed by the oil phase and the outer aqueous phase, and applying centrifugal force as a whole. The small droplets wrapped in the monolayer will pass through the interface due to the density difference, thereby wrapping the second layer of phospholipid molecules, thereby generating vesicles with a complete phospholipid bilayer structure. The reverse emulsion method has a simple principle, controllable steps, and is easy to optimize. However, the existing reverse emulsion method does not take into account the effects of phospholipid solubility and oil phase viscosity on vesicle formation. Therefore, when DNA is used as the inner aqueous phase, better results cannot be obtained.

[0004] The existing methods for preparing vesicles are not very applicable. There are cases where vesicles cannot be generated when the internal water is replaced, such as when high-concentration DNA is used as the internal water. In addition, the properties of the vesicles may be unstable, affecting their use in subsequent experiments. Summary of the invention

[0005] In order to solve the deficiencies in the prior art, the present invention aims to provide a method for preparing aqueous phospholipid membrane vesicles in DNA.

[0006] The specific technical solutions of the present invention are as follows:

[0007] The present invention provides a method for preparing a DNA aqueous phase phospholipid membrane vesicle, comprising the following steps:

[0008] (1) Adding oil phase A to the external aqueous phase, after centrifugation, a phospholipid monolayer is formed at the interface between oil phase A and the external aqueous phase;

[0009] (2) adding the internal water phase to the oil phase B and, after emulsification, obtaining emulsion droplets wrapped by a single layer of phospholipids;

[0010] (3) moving the emulsion droplets to the upper layer of the oil phase of the phospholipid monolayer, centrifuging, removing the upper oil phase, and gently blowing at the bottom of the water phase to obtain DNA intra-water phase phospholipid membrane vesicles;

[0011] Wherein, the inner water phase, the outer water phase, the oil phase A and the oil phase B are prepared by the following method:

[0012] Preparation of inner aqueous phase: Dissolve DNA and sucrose in pure water to obtain inner aqueous phase;

[0013] Preparation of external aqueous phase: Dissolve glucose and NaCl in pure water to obtain external aqueous phase;

[0014] Preparation of oil phase A: dissolve phospholipids in chloroform to obtain phospholipid mother liquor, add the phospholipid mother liquor into mineral oil, mix and shake evenly, then add chloroform to obtain oil phase A;

[0015] The preparation method of oil phase B is the same as that of oil phase A.

[0016] Furthermore, the concentration of DNA in the inner aqueous phase is no more than 10 mg / mL.

[0017] Further, the concentration of sucrose in the inner aqueous phase is equal to the concentration of glucose in the outer aqueous phase. The concentration of sucrose only needs to correspond to that of glucose to produce a density difference and an isotonic environment during the preparation process. The presence of a density difference and an isotonic environment is a prerequisite for the preparation of vesicles by the reverse emulsion method. Preferably, the concentration of sucrose in the inner aqueous phase and the concentration of glucose in the outer aqueous phase are 400mM-2M.

[0018] Furthermore, the amount of NaCl added to the outer aqueous phase is calculated to make the osmotic pressure of the outer aqueous phase close to the osmotic pressure of the inner aqueous phase. The purpose of adding NaCl is to supplement part of the osmotic pressure generated by DNA and calcein. After measurement using an ice point osmometer, it was found that the osmotic pressure of an aqueous solution containing only 10 mg / mL calf thymus DNA + 0.1 mM calcein was close to the osmotic pressure of a 20 mM NaCl aqueous solution. If the DNA concentration used is reduced, the NaCl concentration should also be reduced accordingly; if the DNA concentration is so low that it provides almost no osmotic pressure, NaCl may not be added.

[0019] Further, the concentration of phospholipids in the oil phase A and the oil phase B is 1-10 mM;

[0020] Preferably, the phospholipid is selected from palmitoyloleoylphosphatidylcholine.

[0021] Furthermore, the viscosity of the oil phase A and the oil phase B is close to the viscosity of the inner water phase by adjusting the amount of chloroform added, and the density of the oil phase A and the oil phase B is lower than the density of the outer water phase;

[0022] Preferably, the volume fraction of chloroform in the oil phase A and the oil phase B is 14%-16%.

[0023] Furthermore, the inner water phase also includes a fluorescent indicator;

[0024] Preferably, the fluorescent indicator is selected from calcein or propidium iodide;

[0025] Preferably, the concentration of calcein in the inner aqueous phase is 0.1 mM.

[0026] Furthermore, the oil phase A and the oil phase B also include phospholipid dyes;

[0027] Preferably, the phospholipid dye is selected from POPE-Atto 647N.

[0028] Furthermore, in step (2), the volume ratio of the oil phase B to the inner water phase is 50:1-200:1, preferably 100:1.

[0029] Furthermore, the centrifugation speed in step (3) is 8000-9000g, and the time is 30s-1min;

[0030] The emulsification in step (2) is achieved by vortexing;

[0031] Preferably, the rotation speed of the vortex oscillation is 3000 rpm and the time is 60 s.

[0032] The beneficial effects of the present invention are:

[0033] The method for preparing phospholipid membrane vesicles with DNA molecules as the inner water phase provided by the present invention optimizes the vesicle preparation method of the traditional reverse emulsion method, and improves the yield of vesicle preparation and reduces the phenomenon of mutual adhesion between vesicles by increasing the solubility of phospholipids in the oil phase, appropriately increasing the viscosity of the oil phase, adding sucrose and glucose to the water phase to provide a density difference, and changing the emulsification method, so as to obtain DNA inner water phase phospholipid membrane vesicles more stably. The present invention not only provides a more universal vesicle preparation method, solving the problem of unstable results during vesicle preparation; but also can improve the yield of DNA inner water vesicles, and obtain clean vesicles that are convenient for subsequent experiments. The preparation method of the present invention is simple, fast, and repeatable, and the properties of the vesicles are stable and controllable, which is suitable for promotion in the research and subsequent application in the field of artificial cells. Specifically:

[0034] 1. The preparation method of the present invention uses a reverse emulsion method. The various steps of the method are relatively clear and the two layers of phospholipid molecules are formed separately and then wrapped. The influence of the steps on the experimental results is more controllable.

[0035] 2. The optimized reverse emulsion preparation method of the present invention has a simple process, strong adaptability, and is suitable for a variety of artificial cell research applications.

[0036] 3. Adding chloroform to the oil phase not only increases the solubility of phospholipids in the oil phase, thereby making the phospholipid molecules of the vesicles densely arranged, making the properties of the obtained vesicles more stable, and increasing the stability of the vesicles; it also makes the viscosity of the oil phase adjustable. Because the viscosity of chloroform is extremely low, while the viscosity of mineral oil is usually higher, the viscosity of the oil phase can be easily adjusted according to the difference in the internal water phase, making it compatible with the high-concentration DNA solution, making the emulsification process easier, and emulsification can be completed using Vortex shaking.

[0037] 4. Using Vortex oscillation emulsification is more stable, highly controllable and repeatable than the traditional invert emulsion method of hand scraper emulsification. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Principle of the inverse emulsion method; (A) Phospholipid molecules spontaneously aggregate on the surface of water droplets; (B) Phospholipid molecules aggregate and arrange at the oil-water interface; (C) Droplets wrap around the second layer of phospholipids through the interface to form vesicles.

[0039] Figure 2 : DLS estimation of the CAC of POPC in the oil phase; (A) In the absence of chloroform, the CAC of POPC in the oil phase is approximately 10 μM; (B) After adding 10% chloroform, the CAC rises to 20-30 mM, and the solubility increases by about 2000-3000 times.

[0040] Figure 3: Observation results of emulsification of oil phases with different viscosities; (A) 10% chloroform oil phase (85 cP); (B) 15% chloroform oil phase (35 cP).

[0041] Figure 4 : Observation results of the prepared vesicles; (A) vesicles prepared by the conventional inverse emulsion method; (B) vesicles prepared by the method of Example 1. DETAILED DESCRIPTION

[0042] In order to understand the present invention more clearly, the present invention is further described with reference to the following examples and accompanying drawings. The examples are only used for explanation and are not intended to limit the present invention in any way. In the examples, each raw material reagent is commercially available, and the experimental methods without specifying specific conditions are conventional methods and conventional conditions well known in the art, or according to the conditions recommended by the instrument manufacturer.

[0043] Example 1

[0044] This embodiment provides a method for preparing DNA-containing aqueous phospholipid membrane vesicles, and the specific steps are as follows:

[0045] 1. Solution preparation

[0046] (1) Preparation of internal water phase

[0047] Calf thymus DNA was dissolved in pure water at 10 mg / mL, supplemented with 200 mM sucrose and 0.1 mM calcein (488 nm) as a fluorescent indicator to observe the vesicle morphology.

[0048] (2) Oil phase preparation

[0049] POPC (palmitoyloleoylphosphatidylcholine, a phospholipid) and POPE-Atto 647N (phospholipid dye, 640 nm) were dissolved in chloroform respectively, the POPC chloroform mother solution was added to mineral oil (300 cP), mixed and shaken evenly, and a certain volume of chloroform was added to obtain a mineral oil phase with a phospholipid concentration of 1 mM and a chloroform volume fraction of 15%.

[0050] (3) External water phase preparation

[0051] Prepare 200mM glucose and add 20mM NaCl aqueous solution. (The osmotic pressure is basically the same as that of the internal water phase.)

[0052] 2. Vesicle Preparation

[0053] (1) Prepare two centrifuge tubes A and B respectively.

[0054] (2) Take 650 μL of the external aqueous phase and place it at the bottom of a 1 mL centrifuge tube A. Take 150 μL of the oil phase and add it to the external aqueous solution. Centrifuge tube A at 5000 g for 5 min to form a stable phospholipid monolayer at the interface between the oil phase and the external aqueous phase.

[0055] (3) Take 200 μL of the oil phase and add it to a 1.5 mL centrifuge tube B. Add 2 μL of the inner aqueous phase and vortex at 3000 rpm for 60 s to emulsify the mixture to obtain a monolayer of phospholipid-encapsulated emulsion droplets.

[0056] (4) The emulsion droplets were moved to the upper layer of the oil phase in centrifuge tube A and centrifuged at 8000 g for 30 seconds. After centrifugation, the upper oil phase in the tube was aspirated and the bottom of the water phase was gently blown 10 times to obtain the DNA-in-water phospholipid membrane vesicles.

[0057] Example 2

[0058] This embodiment provides a method for preparing DNA-containing aqueous phospholipid membrane vesicles. The difference from Embodiment 1 is that the volume fraction of chloroform in the oil phase in Embodiment 2 is 10%, and the remaining steps are the same as those in Embodiment 1.

[0059] Comparative Example 1

[0060] This comparative example provides a method for preparing DNA-containing aqueous phospholipid membrane vesicles, and the specific steps are as follows:

[0061] 1. Solution preparation

[0062] (1) Preparation of internal water phase

[0063] Calf thymus DNA was dissolved in 10 mM NaCl aqueous solution at 10 mg / mL, and 0.1 mM calcein (488 nm) was added as a fluorescent indicator to observe the vesicle morphology.

[0064] (2) Oil phase preparation

[0065] POPC (palmitoyloleoylphosphatidylcholine, a phospholipid) and POPE-Atto 647N (phospholipid dye, 640 nm) were dissolved in chloroform respectively, and the POPC chloroform mother solution was added into mineral oil (20 cP) and mixed and shaken evenly. After the chloroform was evaporated at high temperature with the lid opened, a mineral oil phase with a phospholipid concentration of 1 mM was obtained.

[0066] (3) External water phase preparation

[0067] Prepare a 20mM NaCl aqueous solution. (The osmotic pressure is basically the same as that of the inner water phase.)

[0068] 2. Vesicle Preparation

[0069] (1) Prepare two centrifuge tubes A and B respectively.

[0070] (2) Take 650 μL of the external aqueous phase and place it at the bottom of a 1 mL centrifuge tube A. Take 150 μL of the oil phase and add it to the external aqueous solution. Centrifuge tube A at 5000 g for 5 min to form a stable phospholipid monolayer at the interface between the oil phase and the external aqueous phase.

[0071] (3) Take 200 μL of the oil phase and add it to a 1.5 mL centrifuge tube B. Add 2 μL of the inner aqueous phase, hold the centrifuge tube vertically on a P96-well CR plate, and rub it quickly for dozens of times to obtain a single layer of phospholipid-wrapped emulsion droplets.

[0072] (4) The emulsion droplets were moved to the upper layer of the oil phase in centrifuge tube A and centrifuged at 8000 g for 30 seconds. After centrifugation, the upper oil phase in the tube was aspirated and the bottom of the water phase was gently blown 10 times to obtain the DNA-in-water phospholipid membrane vesicles.

[0073] Figure 1 This is the principle of the inverted emulsion method (for the principle, see Pautot S, Frisken BJ, Weitz DA. Production of Unilamellar Vesicles Using an Inverted Emulsion [J]. Langmuir, 2003, 19 (7): 2870-2879. DOI: 10.1021 / la026100v.).

[0074] The oil phases in Example 2 and Comparative Example 1 were subjected to DLS (dynamic light scattering detection) to detect the CAC (critical aggregation concentration) of POPC molecules in mineral oil and mineral oil with different concentrations of chloroform added. The results are shown in FIG. Figure 2 As shown, it was found that the solubility of POPC was significantly improved, and the addition of chloroform can effectively increase the solubility of phospholipids in the oil phase.

[0075] Take 200 μL of the oil phase (35 cP) containing 15% chloroform by volume in Example 1 and the oil phase (85 cP) containing 10% chloroform by volume in Example 2, add 2 μL of the internal water phase, and vortex at 3000 rpm for 30 seconds on a Vortex for emulsification. The results of the oil phase emulsification are shown in FIG. Figure 3 It was found that with the increase of chloroform concentration in the oil phase, the emulsion droplets were dispersed more evenly and the emulsification effect was improved. The addition of chloroform can effectively improve the emulsification effect of the oil phase.

[0076] The observation results of the vesicles prepared in Example 1 and Comparative Example 1 are as follows Figure 4 .

[0077] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the invention.

Claims

1. A method for preparing DNA-containing aqueous phospholipid membrane vesicles, characterized in that: The steps include: (1) Adding oil phase A to the external aqueous phase, after centrifugation, a phospholipid monolayer is formed at the interface between oil phase A and the external aqueous phase; (2) adding the internal water phase to the oil phase B and, after emulsification, obtaining emulsion droplets wrapped by a single layer of phospholipids; (3) moving the emulsion droplets to the upper layer of the oil phase of the phospholipid monolayer, centrifuging, removing the upper oil phase, and gently blowing at the bottom of the water phase to obtain DNA intra-water phase phospholipid membrane vesicles; Wherein, the inner water phase, the outer water phase, the oil phase A and the oil phase B are prepared by the following method: Preparation of inner aqueous phase: Dissolve DNA and sucrose in pure water to obtain inner aqueous phase; Preparation of external aqueous phase: Dissolve glucose and NaCl in pure water to obtain external aqueous phase; Preparation of oil phase A: dissolve phospholipids in chloroform to obtain phospholipid mother liquor, add the phospholipid mother liquor into mineral oil, mix and shake evenly, then add chloroform to obtain oil phase A; The preparation method of oil phase B is the same as that of oil phase A.

2. The preparation method according to claim 1, characterized in that: The concentration of DNA in the inner aqueous phase is no more than 10 mg / mL.

3. The preparation method according to claim 1, characterized in that: The concentration of sucrose in the inner aqueous phase is equal to the concentration of glucose in the outer aqueous phase; The amount of NaCl added to the external water phase is calculated to make the osmotic pressure of the external water phase close to the osmotic pressure of the internal water phase.

4. The preparation method according to claim 1, characterized in that: The concentration of phospholipids in the oil phase A and the oil phase B is 1-10 mM; Preferably, the phospholipid is selected from palmitoyloleoylphosphatidylcholine.

5. The preparation method according to claim 1, characterized in that: By adjusting the amount of chloroform added, the viscosity of the oil phase A and the oil phase B is close to the viscosity of the inner water phase, and the density of the oil phase A and the oil phase B is lower than the density of the outer water phase; Preferably, the volume fraction of chloroform in the oil phase A and the oil phase B is 14%-16%.

6. The preparation method according to claim 1, characterized in that: The inner water phase also includes a fluorescent indicator; Preferably, the fluorescent indicator is selected from calcein or propidium iodide; Preferably, the concentration of calcein in the inner aqueous phase is 0.1 mM.

7. The preparation method according to claim 1, characterized in that: The oil phase A and the oil phase B also include phospholipid dyes; Preferably, the phospholipid dye is selected from POPE-Atto 647N.

8. The preparation method according to claim 1, characterized in that: The volume ratio of the oil phase B to the inner water phase in step (2) is 50:1-200:

1.

9. The preparation method according to claim 1, characterized in that: The centrifugal speed in step (3) is 8000-9000g, and the time is 30s-1min; The emulsification in step (2) is achieved by vortexing; Preferably, the rotation speed of the vortex oscillation is 3000 rpm and the time is 60 s.

10. The DNA-in-water phospholipid membrane vesicles prepared by the preparation method according to any one of claims 1 to 9.

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