Substrate for manufacturing liposomes, method for manufacturing the same, and method for manufacturing liposome

Abstract

To provide a substrate for manufacturing liposomes that can be repeatedly used by cleaning in a static hydration method or an electroformation method once the substrate is manufactured, and a method for manufacturing liposomes in which giant liposomes of a uniform size can be manufactured simply and at a low cost by using the substrate.SOLUTION: A substrate for manufacturing liposomes of the present invention comprises a substrate and a coating layer having oil repellency which has opening parts on the substrate. The coating layer contains a hydrophilic compound or a fluorine compound. A substrate for manufacturing liposomes of the present invention includes: a first step of coating an organic compound having oil repellency on the surface of the substrate or the surface of photosensitive resin on the substrate; and a second step of forming a region in which an organic compound is attached by a photolithography technique and a region in which the organic compound is removed and a substrate surface is exposed. Lipid solution is coated by using the substrate for manufacturing liposomes of the present invention to manufacture liposomes.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a substrate for manufacturing liposomes having a size of 1 μm or more in diameter and a method for manufacturing liposomes.

Background Art

[0002] Liposomes are spherical closed vesicles having a lipid bilayer with an aqueous phase inside, composed of phospholipids and the like, and have been used as a means for transporting pharmaceuticals and as cosmetics. For example, there are cosmetics in which the permeability to the skin is enhanced by encapsulating an encapsulated component and a hydrogel in liposomes having a diameter of about 100 nm. Among liposomes, liposomes having a size of 1 μm or more in diameter are particularly called giant liposomes. Giant liposomes are widely studied for their potential applications in artificial cells and microreactors due to their structural similarity to living cells.

[0003] As methods for forming giant liposomes, generally, there are a static hydration method, an electroformation method, an oil-water interface passing method, a microchannel method, and the like. Among them, the static hydration method and the electroformation method are known for being able to easily form giant liposomes of uniform size in combination with photolithography technology (for example, Patent Documents 1 and 2).

[0004] In Patent Document 1, a lipid film patterned on a substrate is formed by a photolithography technique, and uniform-shaped liposomes are formed from the lipid film (lipid pattern) by an electroformation method. Further, in Patent Document 2, a substrate having a concave portion formed of a conductive material on the bottom surface is prepared by a photolithography technique, the bottom surface of the concave portion is coated with a lipid solution, and liposomes with a small variation in diameter are manufactured in the concave portion by a static hydration method or an electroformation method.

[0005] However, in Patent Document 1, a parylene resin vapor-deposited on a substrate is patterned to form a perforated sheet. After applying a lipid solution to this and drying it, the perforated sheet made of parylene resin is peeled off to transfer and leave a lipid pattern. In this method, since the perforated sheet for lipid pattern transfer is peeled off from the substrate, there is a problem that the substrate cannot be used again for forming a lipid pattern.

[0006] Also, in Patent Document 2, similar to Patent Document 1, a parylene resin is vapor-deposited on a substrate and the parylene resin is selectively removed by oxygen plasma etching to form recesses made of the parylene resin. However, different from Patent Document 1, there is no step of removing the parylene resin pattern that forms the recesses before manufacturing liposomes. Since the parylene resin pattern remains on the substrate, this substrate with the parylene resin pattern can be repeatedly used in the static hydration method or the electroformation method.

[0007] However, in Patent Document 2, in order to coat the lipid solution only on the bottom surface of the recess, the lipid solution is sprayed from a nozzle facing this substrate while applying a DC high voltage between the bottom surface of the recess and the lipid solution (electrospray method). In the method of Patent Document 2, a special device called electrospray must be used, and there is a problem that it is laborious and costly in the manufacture of liposomes.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0009] The present invention has been made in view of the above circumstances, and provides a substrate for manufacturing liposomes that can be washed and reused repeatedly once manufactured in the static hydration method or the electroformation method. Furthermore, an object of the present invention is to provide a method for manufacturing liposomes that can easily and inexpensively manufacture large liposomes of uniform size using this substrate.

Means for Solving the Problems

[0010] The substrate for manufacturing liposomes according to the first invention is characterized by comprising a substrate and a coating layer having an opening on the substrate and having oil repellency. The substrate for manufacturing liposomes according to the second invention is characterized in that, in the invention according to claim 1, the coating layer contains a hydrophilic compound or a fluorine compound. The substrate for manufacturing liposomes according to the third invention is characterized in that, in the invention according to claim 1, the opening is a circle with a diameter of 1 to 500 μm, an ellipse with a major axis of 1 to 500 μm, or a rectangle with a long side of 1 to 500 μm. The substrate for manufacturing liposomes according to the fourth invention is characterized in that, in the invention according to any one of claims 1 to 3, the substrate has conductivity. The substrate for manufacturing liposomes according to the fifth invention is characterized in that, in the invention according to claim 4, the coating layer is formed on the surface of the insulating layer via the insulating layer on the substrate. The method for manufacturing a substrate for manufacturing liposomes according to the sixth invention includes a first step of applying an oil-repellent organic compound to the surface of the substrate or the surface of a photosensitive resin provided on the substrate, and a second step of forming a region where the organic compound is adhered and a region where the organic compound is removed and the substrate is exposed by photolithography technology. The method for manufacturing liposomes according to the seventh invention is characterized in that liposomes are produced by applying a lipid solution and using the electroformation method using the substrate for manufacturing liposomes according to claim 4.

Effects of the Invention

[0011] According to the first invention, since the coating layer having oil repellency is provided on the substrate, when the lipid is dissolved in the organic solvent, the surface of the coating layer has the property of repelling the lipid solution. On the other hand, since the opening where the substrate surface is exposed does not have the coating layer, a lipid film can be formed on the substrate surface. Thus, by using the substrate according to the present invention, liposomes having uniform diameter sizes can be efficiently produced with allowance in the opening by the static hydration method or the electroformation method. Further, since the surface of the coating layer of the substrate having the opening according to the present invention has the property of repelling the lipid solution, once it is fabricated, it can be washed and reused as a substrate for liposome production repeatedly many times. According to the second invention, by using a hydrophilic compound or a fluorine compound as the coating layer, the oil repellency of the coating layer can be enhanced. According to the third invention, since the size of the hole of the opening (diameter of a circle, major axis of an ellipse, long side of a rectangle) is 1 to 500 μm, giant liposomes having a diameter of 1 μm or more can be easily produced by using the substrate according to the present invention. According to the fourth invention, since the substrate has conductivity, the substrate according to the present invention can be applied to the production of liposomes by the electroformation method. According to the fifth invention, since the coating layer is formed on the surface of the insulating layer via the insulating layer, insulation of the coating layer with respect to the substrate can be ensured. Thereby, liposomes can be stably produced by using the substrate according to the present invention in the electroformation method. Also, scale-up due to large area becomes easy. According to the sixth invention, by the photolithography technique, the region to which the oil-repellent organic compound is attached and the region where the organic compound is removed and the substrate surface is exposed (corresponding to the opening) can be easily and accurately formed. Thus, liposomes having uniform sizes can be produced by using the substrate according to the present invention. According to the seventh invention, since a conductive substrate is used, liposomes can be easily, simply and produced in a short time by the electroformation method.

Brief Description of the Drawings

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Embodiments for Carrying Out the Invention

[0013] Hereinafter, embodiments of the present invention will be described with reference to the drawings. In the drawings, parts common to each figure may be denoted by the same or corresponding reference numerals, and the description thereof may be omitted. Also, the scope of the present invention is not limited to these embodiments.

[0014] <First Embodiment> (Configuration, Structure, Material) First, a first embodiment of the present invention will be described with reference to FIG. 1. FIG. 1 is a (a) top view and (b) cross-sectional view taken along line A-A' of a substrate 10 for manufacturing liposomes according to the first embodiment. The substrate 10 for manufacturing liposomes includes a substrate 1, a coating layer 2, and an insulating layer 4.

[0015] The substrate 1 may have either insulating or conductive properties as long as it is a smooth planar substrate. Also, from the viewpoint of facilitating lipid adhesion, the surface of the substrate 1 preferably has no oil repellency, and more preferably has lipophilicity. Liposomes can be manufactured by the static hydration method without applying an electric field, but the substrate 1 preferably has conductivity in that it can be applied to the electroformation method of applying an alternating electric field. As the conductive substrate, an ITO substrate having a transparent conductive film of ITO (Indium Tin Oxide) can be used.

[0016] Also, the insulating layer 4 is formed by patterning on the substrate 1. The material of the insulating layer 4 is not particularly limited as long as it has insulating properties, but a resin-based material is preferred from the viewpoint of manufacturability, and particularly, it is preferable to use a photosensitive resin (photoresist, hereinafter simply referred to as "resist") in that the opening can be patterned. Also, the film thickness of the insulating layer 4 is preferably 50 μm or less, and more preferably 10 μm or less. This is because when the thickness of the insulating layer 4 exceeds 50 μm, the lipid solution hardly penetrates into the opening 3 and it is difficult for lipids to adhere to the surface of the substrate 1.

[0017] Furthermore, the coating layer 2 is formed on the surface of the insulating layer, and the insulating layer 4 and the coating layer 2 constitute a laminated structure. The coating layer 2 has oil repellency. As the oil-repellent substance, an oil-repellent organic compound, particularly a hydrophilic compound or a fluorine compound is suitable. Examples of the hydrophilic compound include polyvinyl alcohol containing a hydroxyl group, polyvinyl butyral, polysaccharides such as cellulose and its derivatives, and polyethylene glycol-based compounds such as polyethylene glycol, polyethylene glycol diacrylate, polyethylene glycol dimethacrylate, and alkylphenoxy polyethylene glycol acrylate. One or more of these can be used. Examples of the fluorine compound include perfluoropolyether, perfluoroalkyl phosphate esters, perfluoroalkyl phosphates, perfluoroalkyl silanes, perfluoroalkyl silazanes, and perfluoroalkyl-containing cyclic organosiloxanes. One or more of these can be used. Since the coating layer 2 has oil repellency in this way, when the substrate 10 for liposome production is immersed in the lipid solution, it is difficult for lipids to adhere to the surface of the coating layer 2, and the configuration is such that lipids are easily repelled on the surface of the coating layer 2.

[0018] In addition, the laminated film of the insulating layer 4 and the coating layer 2 has an opening 3. The surface of the substrate 1 is exposed at the opening 3. The exposed surface of the substrate 1 does not have oil repellency. Therefore, when the substrate 10 for liposome production is immersed in a lipid solution in which lipids are dissolved in an organic solvent, the lipids can be attached to the substrate surface, and the opening 3 can be used as a lipid attachment region. The shape of the opening 3 can be any of a circular diameter, an ellipse, or a rectangle. In each case, the diameter of the circle, the major axis of the ellipse, and the length of the long side of the rectangle can be 1 to 500 μm. From the viewpoint of easily producing giant liposomes, the diameter of the circle, the major axis of the ellipse, and the length of the long side of the rectangle are preferably 5 to 500 μm, and more preferably 10 to 500 μm. This is because larger giant liposomes can be produced.

[0019] Note that the insulating layer 4 is particularly useful when manufacturing liposomes by the electroformation method using a conductive substrate 1. It can enhance the insulation between the substrate 1 and the coating layer 2. Even if a lipid solution remains on the surface of the coating layer 2 and lipids adhere to the surface of the coating layer 2, the influence of the electric field is weakened by the insulation, making it difficult for liposomes to grow and improving the yield.

[0020] (Manufacturing method) Next, the manufacturing method will be described. FIG. 2 is a diagram for explaining the manufacturing method according to the first embodiment of the present invention. As shown in FIG. 2, (a) First, prepare the substrate 1. As described above, the substrate 1 may have either insulating or conductive properties as long as it is a smooth planar base material when applying the static hydration method. However, when applying the electroformation method, a conductive substrate (for example, an ITO substrate) is preferred. (b) Subsequently, apply a resist to the surface of the substrate 1 using a spin coater or the like and pre-bake (heat) it using a hot plate or the like. The type of resist is not limited to positive or negative types. FIG. 2 shows an example using a negative resist (for example, SU-8, etc.). (c) Then, expose it to ultraviolet light using a photomask, and (d) further heat (post-bake) to cure the resist in the exposed area. (e) Then, apply an oil-repellent organic compound (the hydrophilic compound or fluorine compound exemplified in paragraph

[0017] ) to the surface of the resist using a spin coater or the like, and (f) further heat to cure the oil-repellent organic compound. (g) Thereafter, remove the resist in the unexposed area and the oil-repellent substance thereon with a developer or the like to expose the surface of the substrate 1 under the resist. Thereby, a coating layer 2 made of an oil-repellent organic compound is formed on the surface of the substrate 1 via the insulating layer 4, and an opening 3 with the substrate surface exposed is formed to manufacture a liposome manufacturing substrate according to the first embodiment of the present invention.

[0021] (Second Embodiment) (Configuration, Structure, Material) Next, a second embodiment of the present invention will be described with reference to FIG. 3. FIG. 3 is a (a) top view and (b) cross-sectional view taken along line A-A' of the substrate 10 for manufacturing liposomes according to the second embodiment. The substrate 10 for manufacturing liposomes includes a substrate 1 and a coating layer 2. The structure, configuration, and material of the substrate 1 and the coating layer 2 are basically the same as those of the first embodiment. The difference from the first embodiment is that an insulating layer 4 is not provided under the coating layer 2, that is, the coating layer 2 is a single-layer film and not a laminated film of the insulating layer 4 and the coating layer 2. In the second embodiment, since there is no insulating layer 4, the structure is simplified. In the second embodiment, a coating layer 2 having oil repellency and having an opening 3 is directly formed on the substrate 1. The surface of the substrate 1 has no oil repellency, similar to the first embodiment. Therefore, the opening 3 where the surface of the substrate 1 is exposed becomes a lipid adhesion region.

[0022] (First manufacturing method) Next, the manufacturing method will be described. FIG. 4 is a diagram for explaining the first manufacturing method according to the second embodiment of the present invention. As shown in FIG. 4, (a) First, a substrate 1 is prepared. Similar to the manufacturing method of the first embodiment, the substrate 1 may be any smooth planar substrate material. When applying the static hydration method, it may have either insulating or conductive properties. However, when applying the electroforming method, a conductive substrate (for example, an ITO substrate) is preferred. (b) Next, an oil-repellent organic compound (the hydrophilic compound or fluorine compound exemplified in paragraph

[0017] ) is applied to the surface of the substrate 1. As the coating means, a spin coater can be used to make the film thickness uniform. (c) Subsequently, a metal is formed on the surface of the oil-repellent organic compound. The type of metal is not particularly limited, and for example, aluminum can be used. As the film-forming method, vapor deposition, sputtering, etc. can be used. (d) Further, a resist is applied to the surface of the formed metal using a spin coater or the like and pre-baked (heated) using a hot plate or the like. The type of resist is not limited to positive or negative types, but FIG. 2 shows an example using a negative resist (for example, SU-8, etc.). (e) Then, it is exposed to ultraviolet light through a photomask, (f) and developed. Then, the opened metal is etched to form a metal mask. The etching process may be either chemical etching or physical etching (for example, ion milling using argon gas, etc.). (g) Then, the negative resist is removed by oxygen plasma etching, and the opened oil-repellent organic compound (hydrophilic compound or fluorine compound) is removed to expose the surface of the metal mask and the substrate 1. (h) Then, the metal mask is removed by chemical etching or the like, and further heated to cure the oil-repellent organic compound, thereby forming a coating layer 2 made of the oil-repellent organic compound and an opening 3 where the substrate surface is exposed on the substrate 1, and the liposome manufacturing substrate according to the first embodiment of the present invention can be manufactured.

[0023] In this example (the first manufacturing method), as described above, a negative resist was used in (d), but a positive resist (for example, an AZ-based resist or the like) may also be used. In this case, it is necessary to use a photomask with the black and white reversed for use in (e).

[0024] (The second manufacturing method) Furthermore, other manufacturing methods will be described. FIG. 5 is a diagram for explaining a second manufacturing method according to the second embodiment of the present invention. As shown in FIG. 5, (a) First, a substrate 1 is prepared, and (b) a resist is applied thereon and pre-baked (heated) using a hot plate or the like. The configuration and material of the substrate 1 are the same as those used in the first manufacturing method. The resist may be either positive or negative, but in the example of FIG. 5, a positive resist (for example, an AZ-based resist or the like) is used. (c) Then, exposure is performed using a photomask, and (d) development is performed to pattern the resist, leaving the resist in the unexposed area. (e) Subsequently, an oil-repellent organic compound (the hydrophilic compound or fluorine compound exemplified in paragraph

[0017] ) is applied by spin coating or the like to adhere the oil-repellent organic compound to the surface. (f) Then, the resist in the unexposed area is removed with a dissolving solution or the like. At this time, since the oil-repellent organic compound exists on the resist surface, it is simultaneously removed and lifted off. However, the oil-repellent organic compound attached to the substrate 1 is not removed and remains attached. After the resist and the oil-repellent organic compound in the unexposed area are removed, the surface of the substrate 1 under the resist is exposed. Thereafter, heating is performed to cure the oil-repellent organic compound. Thereby, a coating layer 2 made of an oil-repellent organic compound and an opening 3 with the substrate surface exposed are formed on the substrate 1, and a liposome production substrate according to the first embodiment of the present invention can be manufactured.

[0025] <The third embodiment> Next, the production of liposomes using the liposome production substrate according to the first embodiment and the second embodiment will be described.

[0026] (a) First, dissolve the lipid in an organic solvent to prepare a lipid solution. As the lipid, phospholipids, glycolipids, glycerides, ceramides, fatty acids, steroids, etc. can be used, among which phospholipids are preferred. Regarding phospholipids, although not particularly limited, in the case of anionic phospholipids, POPG (C 40 H 77 O 10 P: 1-palmitoyl-2-oleoyl phosphatidylglycerol), 1-palmitoyl-2-oleoyl phosphatidic acid, 1-palmitoyl-2-oleoyl phosphatidylserine, or, in the case of neutral phospholipids, POPC (C 42 H 82 NO 8 P: 1-palmitoyl-2-oleoyl phosphatidylcholine), 1,2-dioleoyl phosphatidylcholine, 1,2-dipalmitoyl phosphatidylcholine, 1-palmitoyl-2-oleoyl phosphatidylethanolamine, cholesterol, ergosterol, etc. At least one selected from the group consisting of these can be used. Also, in the case of cationic phospholipids, DOTAP (CH 3 O 4 SC 42 H 80 NO 4 : N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulfate), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, or, in the case of cationic steroids, 3β-[N-(N’,N’-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride, etc. At least one selected from the group consisting of these can be used.

[0027] Also, as the solvent of the lipid solution, an organic solvent that can dissolve the lipid, such as methanol, chloroform, hexane, etc., can be used. A hydrophobic substance such as a drug may be mixed with the lipid in the organic solvent. In this case, the drug can be carried on the lipid membrane of the liposome.

[0028] Furthermore, membrane proteins, peptides, hydrophobic drugs, etc. may be added to the lipid solution. In this case, the liposomes can be incorporated into a membrane such as a pharmaceutical and used as a carrier.

[0029] (b) Subsequently, the lipid solution prepared in (a) is dropped onto the liposome production substrate 10 according to the first embodiment or the second embodiment. At this time, on the surface of the coating layer 2, the lipid solution is repelled due to oil repellency, but in the opening 3 (lipid adhesion region), since it does not have oil repellency, the lipid adheres to the surface. Then, in this state, it is dried to remove the organic solvent, and a lipid film 5 is formed in the opening 3.

[0030] (c) Then, the liposome production substrate 10 to which the lipid film 5 is attached is left standing in a container and filled with water. Then, by the static hydration method, liposomes can grow and liposomes can be produced. Note that a water-soluble substance such as a drug may be mixed into the water. In this case, the drug can be carried in the water inside the liposome.

[0031] (d) Also, when the substrate 1 has conductivity, the electroformation method can be applied. For example, the liposome production substrate 10 is used as the lower electrode, and an upper electrode is disposed in the chamber via an insulating spacer (for example, a spacer made of silicon rubber) from the lower electrode, and the chamber is filled with water. Then, the upper electrode and the lower electrode are connected to an AC power supply, and an AC voltage with a frequency of 1 to 10 Hz is applied to the liposome production substrate 10 to apply an AC electric field, and liposomes can be formed in the opening 3 of the liposome production substrate.

[0032] FIG. 6 and FIG. 7 are diagrams for explaining the production of liposomes when using the liposome production substrates according to the first embodiment and the second embodiment, respectively. These are both examples in which the electroformation method is applied using a conductive substrate 1. By applying an AC electric field, the lipid film 5 formed in the opening 3 grows into liposomes in the opening 3 (lipid adhesion region), and liposomes with a substantially uniform diameter reflecting the size of the opening 3 can be produced.

[0033] As described above, by using the liposome manufacturing substrate according to the first or second embodiment of the present invention, liposomes having a uniform diameter corresponding to the size of the lipid adhesion region formed on the substrate surface, particularly giant liposomes, can be produced. The giant liposomes produced using the substrate for producing liposomes according to the first or second embodiment of the present invention are expected to be applied to carriers for immobilizing biomolecules and substrates in the medical and food fields.

Example

[0034] Hereinafter, examples of the present disclosure will be described.

[0035] (Example 1) ITO glass (1001, manufactured by Geomatic Co., Ltd.) cut to 2 cm in length and 3.5 cm in width was used as the conductive planar substrate. The ITO glass was subjected to oxygen plasma treatment at 150 W for 5 minutes for surface cleaning using a small plasma device (PR200, manufactured by Yamato Scientific Co., Ltd.). A mixture of SU-8 10 (manufactured by MicroChem Co., Ltd.) and acetone alcohol (manufactured by Nacalai Tesque Co., Ltd.) with a weight ratio of 1:4 was dropped onto the surface of the ITO glass, and rotated at 400 rpm for 30 seconds and 4000 rpm for 30 seconds using a spin coater (SC-200, manufactured by Okinaga Co., Ltd.), and pre-baked (heated) at 65 °C for 2 minutes and 95 °C for 10 minutes using a hot plate (RSH-1DN, manufactured by AS ONE Co., Ltd.). Subsequently, exposure was performed for 10 seconds through a photomask in which circular light-shielding regions with a diameter of 30 μm were arranged at intervals of 60 μm using a mask aligner (K-307PS95, manufactured by Kyowa Riken Co., Ltd.), and heated at 65 °C for 2 minutes and 95 °C for 5 minutes using a hot plate to cure the exposed portion of SU-8. Next, 0.1 mL of a coating agent NOVEC1720 (manufactured by 3M Co., Ltd.) composed of a fluorinated product was dropped, spin-coated at 100 rpm for 30 seconds and 1000 rpm for 30 seconds, and heated at 150 °C for 30 minutes using a hot plate to cure the coating agent. It was immersed in acetone alcohol to dissolve and remove the unexposed region of SU-8, and a substrate for producing liposomes was obtained.

[0036] FIG. 8 is a diagram for explaining a method of manufacturing liposomes by an electroformation method using the obtained substrate for liposome production. A 1-mm-thick silicon spacer having a circular opening with a diameter of 1 cm was overlaid on the obtained substrate for liposome production, and 30 μL of a methanol solution of 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, manufactured by NOF Corporation) at 20 μg / L and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI, manufactured by Biotium) at 0.5 mol% with respect to POPC was dropped, and dried under reduced pressure to remove methanol, and the lipid was applied to the lipid adhesion region. The opening of the spacer was filled with ultrapure water, and unprocessed ITO glass was overlaid and sealed. An alternating current electric field of 1 Vp-p and 2 Hz was applied for 120 minutes to form liposomes by the electroformation method.

[0037] (Example 2) The same procedure as in Example 1 was carried out except that a photomask in which circular light-shielding regions having a diameter of 50 μm were arranged at intervals of 100 μm was used.

[0038] (Comparative Example 1) The electroformation method was carried out using an unprocessed, i.e., an ITO electrode without a coating layer, as it was.

[0039] (Comparative Example 2) The same procedure as in Example 2 was carried out except that the dropping of NOVEC 1720 and the heating at 150° C. were not performed.

[0040] (Observation of lipid coating, observation of liposomes) The DiI lipids used in Examples 1 and 2 and Comparative Examples 1 and 2 are fluorescent substances that exhibit fluorescence. Utilizing this property, for each substrate of Examples 1 and 2 and Comparative Examples 1 and 2, after applying the lipid solution and drying, the lipids present on the substrate surface were observed using an inverted fluorescence microscope (IX-50, manufactured by Olympus Corporation). Furthermore, after applying an electric field, the liposomes formed on the substrate surface were observed using an inverted fluorescence microscope (IX-50, manufactured by Olympus Corporation). The images obtained by the observation were analyzed to create a distribution of the diameters of the formed liposomes.

[0041] Figure 9 is a microscopic observation image after lipid application. (a) corresponds to Example 1, (b) corresponds to Example 2, (c) corresponds to Comparative Example 1, and (d) corresponds to Comparative Example 2. For Example 1 and Example 2, the lipids are locally applied to lipid adsorption regions in the shape of a 30-μm circle and a 50-μm circle, respectively. On the other hand, for Comparative Example 1, the lipids are uniformly applied over the entire substrate. Also, for Comparative Example 2, the lipids are uniformly applied over the entire substrate. From these results, it can be seen that by using the substrate for liposome production of the present invention, the lipids can be locally applied within the lipid adsorption region reflecting the shape of the lipid adsorption region.

[0042] Figure 10 is a microscopic observation image after electroformation. (a) corresponds to Example 1, (b) corresponds to Example 2, (c) corresponds to Comparative Example 1, and (d) corresponds to Comparative Example 2. For Example 1, giant liposomes were formed directly above the 30-μm circular lipid adsorption region. For Example 2 as well, giant liposomes were formed directly above the 50-μm circular lipid adsorption region. On the other hand, for Comparative Example 1, since the lipids were applied over the entire substrate surface, giant liposomes of various diameters were formed over the entire substrate surface. For Comparative Example 2, although the lipids were applied over the entire substrate surface, since most of the surface is non-conductive, no formation of giant liposomes was observed over the entire substrate surface.

[0043] FIG. 11 is a diagram showing the diameter distribution of liposomes formed by Example 1 and Comparative Example 1. FIG. 12 is a diagram showing the diameter distribution of liposomes formed by Example 2 and Comparative Example 1. Table 1 shows the average value, standard deviation, and coefficient of variation of the diameter of liposomes obtained from these results.

[0044] [Table 1]

[0045] Comparing Example 1, 2 and Comparative Example 1, in Example 1 and 2, the standard deviation and coefficient of variation were small and giant liposomes with a uniform diameter were formed, while in Comparative Example 1, the standard deviation and coefficient of variation were larger than those in Example 1 and 2, and the diameter had a large variation.

[0046] From these results, it can be seen that by using the substrate for producing liposomes of the present invention, giant liposomes with a more uniform diameter can be produced.

Industrial Applicability

[0047] The present invention can be used in the production of artificial cells such as artificial red blood cells, microreactors, biosensors retained in vivo, and the like.

Explanation of Signs

[0048] 1. Substrate 2. Coating layer 3. Opening 4. Insulating layer 5. Lipid film 10. Substrate for liposome production

Claims

1. A substrate for producing liposomes, comprising: a substrate; and a coating layer having openings and oil repellency on the substrate.

2. The substrate for producing liposomes according to claim 1 , wherein the coating layer comprises a hydrophilic compound or a fluorine compound.

3. 2. The substrate for producing liposomes according to claim 1, wherein the opening is a circle having a diameter of 1 to 500 μm, an ellipse having a major axis of 1 to 500 μm, or a rectangle having a major side of 1 to 500 μm.

4. 4. The substrate for producing liposomes according to claim 1, wherein the substrate is conductive.

5. 5. The substrate for producing liposomes according to claim 4, wherein the coating layer is formed on the surface of an insulating layer on the substrate via the insulating layer.

6. A method for producing a substrate for producing liposomes, comprising: a first step of applying an organic compound having oil repellency to a surface of a substrate or to the surface of a photosensitive resin provided on the substrate; and a second step of forming, by photolithography techniques, an area to which the organic compound is attached and an area to which the organic compound is removed and the substrate surface is exposed.

7. 5. A method for producing liposomes, comprising the steps of: applying a lipid solution to the substrate for producing liposomes according to claim 4; and producing liposomes by an electroformation method.

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