Liposome manufacturing method

Abstract

To provide a method for producing liposomes which increases the number of liposomes to be formed and can make probability to include a desired inclusions large.SOLUTION: A method for producing liposomes includes the steps (A) to (C) below. (A) A lipid-dispersed oil is layered on a water layer to form a lipid monolayer at an interface, (B) aqueous droplets containing desired inclusions are dispersed in a lipid-dispersed oil to form a lipid monolayer on an aqueous droplet surface, (C) the liquid obtained in step (B) is added to the oil layer of the resultant product in step (A), and by centrifuging, liquid droplets in which the desired inclusions are contained and a lipid monolayer is formed on a surface are passed through the lipid monolayer formed at the interface to form a lipid bilayer on the surface of the droplet to produce liposomes. At least one of the lipid-dispersed oils used in steps (A) and (B) contains a liposome formation promoter, and from the start of steps (A) and (B) to the start of centrifugation in step (C), a standing time of 30 minutes or more is provided, and steps (A), (B), and (C) are performed at a cold temperature.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a method for producing liposomes encapsulating a desired inclusion by an interfacial passage method.

Background Art

[0002] Liposomes are expected to be used in the production of organic compounds that cannot be synthesized organically, cell therapy, sensor and material applications, etc., and extensive basic research is being conducted as a future artificial cell material. Existing liposome production methods include a method of hydrating a lipid bilayer coated on a solid surface with an aqueous solution (hydration method), and a method of covering droplets twice with a lipid monolayer formed at an oil-water interface (interfacial passage method, or centrifugal sedimentation method). For encapsulating biomolecules, the interfacial passage method with excellent encapsulation efficiency is mainly used.

Prior Art Documents

Non-Patent Documents

[0003]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] In order to encapsulate temperature-sensitive biomolecules into liposomes by the interfacial passage method, it is necessary to perform the entire process of liposome formation under cold temperature. However, it has been found that it is difficult to form liposomes using a general interfacial passage method protocol under cold temperature, and the encapsulation efficiency of biomolecules is also low. When the liposome formation efficiency is low, a large amount of rare biomolecules will be lost during the liposome production process, and the production cost will increase significantly. Therefore, it is necessary to improve the interfacial passage method under cold temperature. [Means for solving the problem]

[0005] As a result of diligent research, the inventors of the present invention have discovered that, in a known method for producing liposomes by interfacial passage, by adding a liposome formation promoter to the lipid-dispersed oil layer, and by providing a predetermined standing time in the steps of forming a lipid monolayer at the interface between the aqueous layer and the oil layer, and in the steps of forming a lipid monolayer on the surface of aqueous droplets containing the encapsulant to be encapsulated in the liposome, liposomes encapsulating the desired encapsulant can be efficiently produced, thus completing the present invention.

[0006] In other words, the present invention provides the following: (1) A method for producing liposomes containing a desired internal substance, (A) A step of layering a lipid-dispersed oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, onto a water layer to form a lipid monolayer at the interface, (B) A step of dispersing aqueous droplets containing the desired encapsulant in a lipid-dispersed oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, to form a lipid monolayer on the surface of the aqueous droplets, (C) The liquid obtained in step (B) is added to the oil layer of the result of step (A), and the droplets containing the desired encapsulant and having a lipid monolayer formed on their surface are centrifuged. The droplets are then passed through the lipid monolayer formed at the interface to form a lipid bilayer on the surface of the droplets and generate liposomes. A manufacturing method comprising: at least one of the lipid-dispersed oils used in step (A) and step (B) containing a liposome formation promoter; a standing time of 30 minutes or more is provided from the start of steps (A) and (B) until the start of the centrifugation process in step (C); and steps (A), (B), and (C) are carried out at a cold temperature. (2) The method according to (1), wherein both the lipid dispersion oil used in step (A) and step (B) each contain the liposome formation promoter. (3) The method according to (1) or (2), wherein the liposome formation promoter is at least one selected from the group consisting of a compound in which two or more hydrogen atoms of methane are replaced with chlorine atoms, a saturated or unsaturated chain hydrocarbon having 5 or more carbon atoms, an aromatic hydrocarbon, a monohydric alcohol having 8 or more carbon atoms, and phenols. (4) The method according to (3), wherein the liposome formation promoter is a compound in which two or more hydrogen atoms of methane are replaced with chlorine atoms. (5) The method according to (4), wherein the liposome formation promoter is trichloromethane. (6) The method according to (3), wherein the concentration of the liposome formation promoter in the lipid dispersion oil used in step (A) and / or step (B) is 5 to 20 vol% relative to the total lipid dispersion oil. (7) The method according to (1) or (2), wherein the standing time is 45 minutes or more. (8) The method according to (1) or (2), wherein the cold temperature is 0°C to 8°C. (9) The method according to (1) or (2), wherein the diameter of the liposome is 5 μm or more. [Effects of the Invention]

[0007] The manufacturing method of the present invention provides the effect of increasing the number of liposomes formed and increasing the probability that the desired encapsulating material is contained within, compared to known interfacial passage methods. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows the effect of the waiting time (standing time) τ and the amount of liposome formation promoter added Δ on the total number of liposomes produced from 25 μL of African clawed frog egg extract obtained in the following example (number of experiments N=3) (in the figure, "additive" refers to "liposome formation promoter"). [Figure 2] This figure shows the effect of waiting time (standing time) τ and the amount of liposome formation promoter added Δ on the liposome encapsulation rate of African clawed frog egg extract obtained in the following examples (number of experiments N=3). [Figure 3]This figure shows the changes (number of experiments N=3) in the liposome diameter distribution obtained in the following example, where the amount of liposome formation promoter added and the waiting time (standing time) affect the liposome diameter distribution generated by the interfacial passage method at cold temperatures. [Figure 4] This figure shows the number of liposomes with a diameter of 5 μm or more obtained in the following example (experimental count N=3) (in the figure, "additive" refers to "liposome formation promoter"). [Modes for carrying out the invention]

[0009] The present invention relates to a method for producing liposomes containing a desired encapsulated substance based on a known interface passage method, and is a method that produces liposomes more efficiently than known interface passage methods and increases the probability that the desired encapsulated substance is contained within the liposomes. Preferably, the liposomes are cell-sized liposomes with a diameter of 5 μm or more. There is no particular upper limit to the diameter, but it is usually 30 μm or less, and particularly 15 μm or less.

[0010] As described above, the method of the present invention is (A) A step of layering a lipid-dispersed oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, onto a water layer to form a lipid monolayer at the interface, (B) The process includes dispersing aqueous droplets containing the desired encapsulant in a lipid dispersion oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, thereby forming a lipid monolayer on the surface of the aqueous droplets.

[0011] The "lipid capable of forming a lipid bilayer" used in the above steps (A) and (B) may be the same as the lipids used in known interfacial passage methods. Usually, phospholipids having two long-chain alkyl groups (preferably having 16 to 18 carbon atoms), particularly phosphatidylcholine (also referred to as phosphorylcholine), phosphatidylserine (also referred to as phosphorylserine or phosphatidylserine), phosphatidylethanolamine, and phosphatidylglycerol are used. Specific examples include 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, 1,2-dioleoyl-sn-glycero-3-phosphocholine, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-L-serine, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phosphoethanolamine, 1-hexadecanoyl-2-(9Z-octadecenoyl)-sn-glycero-3-phospho-(1'-rac-glycerol), etc., but are not limited thereto.

[0012] Also, the "oil" used in the above steps (A) and (B) is not particularly limited as long as it can disperse the above-mentioned lipids. Usually, mineral oil (preferably having alkanes with 15 to 50 carbon atoms as the main component), liquid paraffin, hexadecane, squalene, silicone oil, etc. can be mentioned, but are not limited thereto. Dispersing the lipid in the oil can be carried out by well-known methods using, for example, a vortex mixer or ultrasonic waves.

[0013] The concentration of the "lipid" in the "lipid-dispersed oil" used in the above steps (A) and (B) is not particularly limited, but is usually about 0.1 mM to 100 mM, preferably about 1 mM to 10 mM.

[0014] The "aqueous layer" used in the above step (A) may be a layer of pure water, but usually, an aqueous solution such as an aqueous buffer solution is used.

[0015] Examples of the "desired inclusion" used in the step (B) include, but are not limited to, intracellular extracts, cell-free protein synthesis solutions, enzyme-containing solutions, etc. An aqueous droplet containing such an inclusion in an aqueous medium is dispersed in the "lipid-dispersed oil". Dispersing the aqueous droplet in the oil can be performed by adding a liquid containing the inclusion in an aqueous medium to the lipid-dispersed oil and using a well-known method such as a vortex mixer or ultrasonic waves.

[0016] (A) The above description of step (A) and step (B) is the same as the known interface passage method. One important feature of the method of the present invention is that a liposome formation promoter is added to at least one of the "lipid-dispersed oils" used in step (A) and step (B). Here, as the "liposome formation promoter", at least one selected from the group consisting of a compound in which two or more hydrogens of methane are substituted with chlorine, a saturated or unsaturated chain hydrocarbon having 5 or more carbon atoms, an aromatic hydrocarbon, a monohydric alcohol having 8 or more carbon atoms, and phenols is preferable. Compounds in which two or more hydrogens of methane are substituted with chlorine are dichloromethane, trichloromethane, and tetrachloromethane, and among these, trichloromethane (chloroform) is particularly preferable. Examples of the saturated or unsaturated chain hydrocarbon having 5 or more carbon atoms include alkanes and alkenes having 5 to 50 carbon atoms. Examples of the aromatic hydrocarbon include benzene, toluene, xylene, etc. Examples of the monohydric alcohol having 8 or more carbon atoms include alkanols having 8 to 20 carbon atoms. Examples of the phenols include phenol, cresol, etc.

[0017] (A) The concentration of the liposome formation promoter in the lipid-dispersed oil in step (A) and step (B) is preferably about 5% to 20% by volume, more preferably about 7% to 15% by volume.

[0018] Another important feature of the present invention is that a predetermined resting time is provided between the completion of steps (A) and (B) and the start of the subsequent centrifugal processing in step (C). Here, the resting time is 30 minutes or more, preferably 45 minutes or more. There is no particular upper limit to the resting time, but since it is pointless to make it too long, it is usually 2 hours or less.

[0019] In the subsequent (C) step, the liquid obtained in step (B) is added to the oil layer of the result of step (A), and centrifuged to pass droplets containing the desired encapsulant and with a lipid monolayer formed on their surface through the lipid monolayer formed at the interface between the oil layer and the water layer in step (A), thereby forming a lipid bilayer on the surface of the droplets and generating liposomes. Step (C) itself may be the same as known interface passage methods. The centrifugation conditions are usually a centrifugal acceleration of about 1000g to 20000g, preferably about 5000g to 15000g, and can usually be carried out for 3 minutes to 60 minutes, preferably 10 minutes to 45 minutes.

[0020] This centrifugation process causes the aqueous droplets containing the encapsulated material, which were formed in step (B) by being dispersed in the lipid-dispersed oil and having a lipid monolayer formed on their surface, to break through the lipid monolayer formed at the interface between the oil and water layers in step (C) and settle. At this time, the lipid monolayers stack together to form a lipid bilayer, and liposomes are formed. The formed liposomes settle at the bottom of the tube, so they can be collected in a syringe, for example, by inserting a syringe needle through the outside of the tube.

[0021] Steps (A), (B), and (C) are all carried out at low temperatures. Since the contents are often unstable substances of biological origin, it is often necessary to process them at low temperatures. "Low temperatures" usually means 0°C to 8°C, preferably 0°C to 4°C. Particularly preferably, all steps except the centrifugation in step (C) are carried out under ice cooling, and the centrifugation in step (C) is carried out at 4°C or below using a refrigerated centrifuge.

[0022] As specifically described in the following examples, the present invention results in a larger number of liposomes being formed and a higher probability of the desired encapsulation being formed inside, compared to known interfacial passage methods.

[0023] The present invention will be described in detail below based on examples. However, the present invention is not limited to the following examples.

[0024] 1. Reagents and experimental materials used • Purchased mineral oil from Merck (Sigma-Aldrich). • POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) purchased from Avanti Polar Lipids • 18:1 Liss Rhod PE (1,2-Dioleoyl-sn-Glycerol-3-Phosphoethanolamine-N-(Lissamine Rhodamine B Sulfonyl) Ammonium Salt) purchased from Avanti Polar Lipids • Egg Lysis Buffer (ELB) was prepared with the following composition. 250 mM sucrose, 10 mM HEPES, 50 mM KCl, 2.5 mM MgCl2, 1 mM dithiothreitol • African clawed frog egg extract was prepared using a known method (Shintomi, K., Inoue, F., Watanabe, H., Ohsumi, K., Ohsugi, M. and Hirano, T., 2017. Mitotic chromosome assembly despite nucleosome depletion in Xenopus egg extracts. Science, 356(6344), pp.1284-1287.). • African clawed frog sperm nuclei were prepared using a known method (Shintomi et al., cited above). • Energy mix was prepared with the following composition. 190 mM creatine disodium phosphate, 25 mM ATP disodium salt, 25 mM MgCl2 • GFP-NLS was prepared using a known method (Xue, JZ, Woo, EM, Postow, L., Chait, BT and Funabiki, H., 2013. Chromatin-bound Xenopus Dppa2 shapes the nucleus by locally inhibiting microtubule assembly. Developmental cell, 27(1), pp.47-59.).

[0025] 2. Experimental Method • Preparation of lipid-dispersed oils 1) Dissolve POPC (25 mg mL) in chloroform in a glass bottle. -1 ) and 18:1 Liss Rhod PE (RhPE; 1 mg mL -1 Dispense a total volume of 10 μmol (POPC / RhPE = 100 / 0.1 molar ratio) of the substance. 2) Loosen the cap of the glass bottle and evaporate all the chloroform by creating a vacuum in a desiccator connected to a vacuum pump for 60 minutes. 3) Add 4 mL of mineral oil to 10 μmol of dry lipid. At this time, add 0 / 5 / 10% (by volume relative to the mineral oil before addition) of chloroform as a liposome formation accelerator (hereinafter referred to as "accelerator") to the mineral oil, and immediately close the lid. 4) Place the glass bottle in an ultrasonic water bath heated to 60°C and expose it to ultrasound for 1 hour to disperse the lipids into the mineral oil. (Lipid-dispersed oil is now complete.)

[0026] • Formation of liposomes encapsulating African clawed frog egg extract by interfacial passage method 1) From a -80°C freezer, extract African clawed frog eggs (25 μL) and African clawed frog sperm nuclei (1.0 × 10⁶). 8 mL -1 ), GFP-NLS (4.3 μg μL) -1 ), take out the Energy Mix and let it melt on the ice. 2) Add 0.5 μL of Energy Mix and 0.5 μL of GFP-NLS to 25 μL of thawed African clawed frog egg extract and vortex (3220 rpm). Then add 0.5 μL of African clawed frog sperm nuclei, mix the sperm nuclei and egg extract mixture by pipetting, and immediately return to ice (Extract mix). 3) Prepare one 2.0 mL tube, add 1.0 mL of lipid-dispersed oil, and cool on ice for 5 minutes (A1). Simultaneously, prepare two 1.5 mL tubes, add 150 μL of ELB to each, and cool on ice for 5 minutes (B1). 4) Add 25 μL of the Extract mix to tube A1 (A2). At the same time, add 100 μL of lipid-dispersed oil on top of the ELB inside tube B1 (B2). 5) Incubate tubes A2 and B2 on ice (by placing them still) according to the waiting time (standing time) conditions (0 / 60 / 120 minutes). 6) After the waiting period, immediately vortex tube A2 for 1 minute (3220 rpm). After vortexing, layer 500 μL of the contents of tube A2 onto each of the lipid-dispersed oils in tubes B2 (B3). 7) Prepare a new 1.5 mL tube, add 1.0 mL of ELB, and cool it on ice (C1). 8) Centrifuge the two B3 tubes with 9000g at 4°C for 30 minutes (B4). (Interface passage) After centrifugation, confirm that pellets have formed at the bottom of each of the two B4 tubes. 9) Open the cap of the B4 tube and use a syringe needle to make a small through-hole from the outside to the inside of the tube at the pellet formation area at the bottom of the tube. Be careful not to let the contents of the B4 tube leak out of the hole. 10) Press your finger against the open top of tube B4 to compress the inside of the tube, pushing the pellets out of the tube through the through hole at the bottom, dispersing the pellets onto the ELB inside tube C1 which has been pre-cooled on ice, and immediately close the lid of tube C1 (C2). 11) Immediately vortex the C2 tube for 1-3 seconds (3220 rpm) (C3). At this time, take about 5 μL of the solution in C3 and use a microscope to confirm that liposomes have formed within it. 12) Incubate the C3 tube at room temperature for 120 minutes (static). Then, add 5 μM SYTO41 to stain the DNA (C4). 13) Centrifuge the C4 tube at room temperature with 200g for 5 minutes, remove the supernatant using a pipette, and adjust the volume of liquid in the C4 tube to 140μL (C5). Take great care not to aspirate any liposomes that have settled at the bottom of the tube. 14) A frame-sealed slide chamber (base area 17 x 28 mm, chamber volume 140 μL, BIO-RAD) is attached to a glass slide, 140 μL of the liposome solution from the C5 tube is dropped into the chamber, and the liposome solution is sealed inside the chamber using a coverslip. This allows the entire amount of liposomes generated from 25 μL of extract mix to be sealed inside the chamber.

[0027] Microscopic observation For microscopic observation, an inverted microscope (IX-71, Olympus) equipped with a confocal unit (CSU-X1, Yokogawa Electric), a laser and its control unit (ALC5000, Andor Technology), an EM-CCD camera (iXon, Andor Technology), and a 60x oil immersion lens (60x, numerical aperture 1.45, Olympus) was used. A microscope slide containing liposomes was fixed on the stage, the field of view was manually positioned, and automatic imaging of bright-field, red fluorescence (ex 562nm, em 580-660nm), green fluorescence (ex 488nm, em 500-550nm), and blue fluorescence (ex 405nm, em 420-470nm) was performed using a measurement program designed on the control software (iQ2, Andor Technology).

[0028] Image analysis All analysis and figure creation were performed using custom image data processing scripts written in MATLAB (MATHWORKS) software.

[0029] 3. Experimental Results Effects of waiting time and accelerator amount on the number of liposomes formed Figure 1 shows the number of liposomes formed from 25 μL of African clawed frog egg extract. The number of liposomes for each condition of waiting time (standing time) τ and accelerator amount Δ is shown in a bar graph. It can be seen that the number of liposomes increased after the introduction of the accelerator compared to before the waiting time and accelerator introduction.

[0030] Effects of accelerators and waiting time on liposome encapsulation rate in African clawed frog egg extract Figure 2 shows the liposome encapsulation rate of African clawed frog egg extract. A histogram of GFP fluorescence intensity inside liposomes was plotted for liposomes formed from 25 μL of African clawed frog egg extract pre-mixed with GFP-NLS. The GFP intensity outside the liposomes (mean + 3 × standard deviation) is shown by a dashed line. Liposome encapsulation of the egg extract was defined as liposomes with a GFP intensity above the dashed line, and the number of encapsulated liposomes in the egg extract was calculated. The figure shows that the number of encapsulated liposomes in the egg extract increased after the introduction of the accelerator and waiting time compared to before the introduction.

[0031] The effect of accelerators and waiting times on the distribution of liposome diameter and the number of large liposomes formed. Figure 3 shows the diameter distribution of the generated liposomes when the waiting time and amount of additive are varied. A dashed line is drawn on each histogram at the point where the diameter is 5 μm, representing the baseline value. From the histogram in Figure 3, it can be seen that the number of liposomes with a diameter of 5 μm or more increases with increasing waiting time and amount of accelerator. Furthermore, Figure 4 plots the number of liposomes with a diameter of 5 μm or more generated when the accelerator and waiting time are varied. Compared with Figure 1, it can be seen that the number of large liposomes with a diameter of 5 μm or more increases with increasing waiting time and accelerator.

Claims

1. A method for producing liposomes containing a desired internal substance, (A) A step of layering a lipid-dispersed oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, onto a water layer to form a lipid monolayer at the interface, (B) A step of dispersing aqueous droplets containing the desired encapsulant in a lipid-dispersed oil, in which lipids capable of forming a lipid bilayer are dispersed in an oil, to form a lipid monolayer on the surface of the aqueous droplets, (C) The liquid obtained in step (B) is added to the oil layer of the result of step (A), and the droplets containing the desired encapsulant and having a lipid monolayer formed on their surface are centrifuged. The droplets are then passed through the lipid monolayer formed at the interface to form a lipid bilayer on the surface of the droplets and generate liposomes. At least one of the lipid-dispersed oils used in steps (A) and (B) contains a liposome formation promoter, and a standing time of 30 minutes or more is provided from the start of steps (A) and (B) until the start of the centrifugation process in step (C), and steps (A), (B), and (C) are carried out at 0 to 8°C. A method for producing oil, wherein the oil used in step (A) and the oil used in step (B) are mineral oil, liquid paraffin, hexadecane, squalene, or silicone oil, and the liposome formation promoter is a compound in which two or more hydrogen atoms of methane are replaced with chlorine atoms.

2. The method according to claim 1, wherein both the lipid dispersion oil used in step (A) and step (B) each contain the liposome formation promoter.

3. The method according to claim 1 or 2, wherein the liposome formation promoter is trichloromethane.

4. The method according to claim 1 or 2, wherein the concentration of the liposome formation promoter in the lipid dispersion oil used in step (A) and / or step (B) is 5 to 20 vol% relative to the total lipid dispersion oil.

5. The method according to claim 1 or 2, wherein the standing time is 45 minutes or more.

6. The method according to claim 1 or 2, wherein the diameter of the liposome is 5 μm or more.

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