Liposomes and methods for producing the same

Abstract

To provide a stable giant liposome with high physical strength, having a diameter of 1 μm or more, and to provide a method for producing the liposome in a simple facility.SOLUTION: A liposome having hydrogel as the substrate has a second lipid coating which comprises second lipid molecules, has anionic or cationic hydrogel particles, and a first lipid coating composed of first lipid molecules having in hydrophilic groups an electric charge of a polarity opposite to a polarity of the hydrogel particles, and aggregated on the surfaces of the hydrogel particles, and forms the first lipid coating and a bimolecular lipid membrane.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present disclosure relates to hydrogel-supported liposomes with diameters of micrometers or larger, and a method for producing the same. [Background technology]

[0002] Liposomes are spherical vesicles with a lipid bilayer, and have been used as a means of transporting pharmaceuticals and in cosmetics. For example, there are cosmetics that have enhanced skin permeability by encapsulating ingredients and hydrogel in liposomes with a diameter of about 100 nm (Patent Document 1). Liposomes with a diameter of 1 μm or more are called giant liposomes. Giant liposomes are being widely studied for their potential use in artificial cells and microreactors due to their structural similarity to living cells.

[0003] Generally, various methods for forming giant liposomes have been investigated, such as the static hydration method (Non-Patent Document 1), the electroformation method (Non-Patent Document 2), the oil-water interface passing method (Non-Patent Document 3), and the microchannel method (Non-Patent Document 4). [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Japanese Patent Application Laid-Open No. 2016-185933 [Non-patent literature]

[0005] [Non-Patent Document 1] MeLeard, P. et al. “Bending elasticities of model membranes: Influences of temtemperature and sterol content.” Biophys. J. 72,2616-2629(1997) [Non-patent document 2] Angelova, MI, Dimitrov, DS “Liposome electroformation”, Faraday Discussions of the Chemical Society, Chem Soc. 81, 303-311(1986) [Non-patent document 3] Pautot, S., Frisken, BJ, Weitz, DA, “Production of Unil amellar Vesicles Using an Inverted Emulsion”, Langmuir 19, 2870-2879(2003) [Non-patent document 4] Carugo, D., Bottaro, E., Owen, J., Stride, E., Nastruzzi, C., Liposome Production by Microfluidics: Potential and Limiting Factors, Sci. Rep. volume 6, Article number: 25876 (2016) Summary of the Invention [Problem to be solved by the invention]

[0006] However, while the static hydration method, electroformation method, and oil-water interface penetration method are simple to operate, they have the problem of difficulty in controlling the particle size of the giant liposomes produced. The microchannel method solves this problem, but has the problem of complicated operation and equipment.

[0007] Furthermore, the giant liposomes produced by any of the above methods are endoplasmic reticulum consisting of only a lipid bilayer membrane, and have problems with stability and physical strength. In other words, since they do not have a cytoskeleton like biological membranes, they have low strength and are likely to collapse due to environmental changes, which has limited the use of giant liposomes as a biological membrane model. [Means for solving the problem]

[0008] A liposome according to one embodiment of the present disclosure is a liposome using a hydrogel as a support, and includes an anionic or cationic hydrogel particle, a first lipid coating formed by assembling first lipid molecules on the surface of the hydrogel particle, the first lipid molecules having a charge in the hydrophilic group of the opposite polarity to that of the hydrogel particle, and a second lipid coating formed by second lipid molecules that form a lipid bilayer membrane with the first lipid coating. When the hydrogel particles are cationic, the first lipid coating contains an anionic phospholipid or a neutral phospholipid exhibiting a neutral charge leaning to a negative charge, and when the hydrogel particles are anionic, the first lipid coating contains a cationic phospholipid. It is something.

[0009] The hydrogel may have a diameter of 1 μm to 1 mm.

[0010] When the hydrogel particles are cationic, the first lipid molecule may be POPG (1-palmitoyl-2-oleoylphosphatidylglycerol), 1-palmitoyl-2-oleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidylserine, and POPC (1-palmitoyl-2-oleoyl phosphatidylcholine) )Yo Alternatively, if the hydrogel particles are anionic, the first lipid molecule may be at least one selected from the group consisting of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride.

[0011] The phrase "the first lipid molecule may be at least one selected from the group consisting of ..." means that the first lipid molecule may be any one selected from the group consisting of the multiple lipid molecules described in "...", or may be a combination of two or more selected from the group.

[0012] The hydrogel may further contain an encapsulated component having a molecular weight of 40,000 or more.

[0013] A method for producing liposomes according to one embodiment of the present disclosure includes the steps of dispersing a hydrogel in a mixed solution containing first lipid molecules to form a lipid-coated gel having a first lipid coating, spreading second lipid molecules at an oil-water interface to form a second lipid coating, and passing the lipid-coated gel through the oil-water interface using a centrifuge to form a lipid bilayer membrane.

[0014] In the first step, the lipid-coated gel may be prepared by adding an oil phase to a dehydrated gel obtained by removing the aqueous phase in which the hydrogel particles are dispersed by suction filtration, and dispersing the gel particles by ultrasonic treatment and vortexing. [Effects of the Invention]

[0015] The above-mentioned method allows for the realization of liposomes with a physically strong and stable hydrogel support, even if the particle size is 1 μm or larger. Furthermore, by using the oil-water interface penetration method with a centrifuge, such giant liposomes can be mass-produced using simple equipment. [Brief explanation of the drawings]

[0016] [Figure 1] FIG. 1 is a conceptual diagram showing an image of a liposome in an embodiment according to one aspect of the present disclosure. [Figure 2] 1 is a fluorescence observation image of a liposome lipid bilayer membrane in Example 1 of the present disclosure. [Figure 3] 10 is a fluorescent observation image showing electrostatic interactions in Example 2 of the present disclosure. [Figure 4] 10 is a fluorescence observation image showing inclusion retention in Example 3 of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0017] An embodiment according to one aspect of the present disclosure will be described in detail below. The liposome according to this embodiment is a liposome using a hydrogel as a support. The hydrogel used as a support may have charged functional groups throughout the gel or at least on the outermost surface of the gel particles. Any hydrogel having charged functional groups, such as sulfonic acid groups, carboxylic acid groups, or phosphonyl groups, as anionic functional groups or quaternary ammonium groups or amine groups, as cationic groups, on the surface of the hydrogel may be used. For example, in the polymer material forming the hydrogel, a compound having the charged functional groups on the outermost surface of an uncharged hydrogel may be used, such as a hydrogel modified or coated with a silane coupling agent or polymer having the charged functional groups. Furthermore, the shape of the hydrogel used as a support is not limited to a spherical shape. Any shape, including non-spherical shapes, may be used as long as the hydrogel has charged functional groups.

[0018] This hydrogel may be anionic or cationic. As an anionic hydrogel, a pre-formed hydrogel, such as SP Sepharose Fast Flow or SP Sepharose XL having sulfonic acid groups, or CM Sepharose Fast Flow having carboxylic acid groups (all manufactured by Cytiva) may be used. As a cationic hydrogel, Q Sepharose Fast Flow or Q Sepharose XL having quaternary ammonium groups, or DEAE Sepharose Fast Flow having diethylaminoethyl groups (all manufactured by Cytiva) may be used. The diameter of the hydrogel may be 1 μm to 1 mm, more preferably 1 μm to 500 μm, and even more preferably 10 μm to 100 μm.

[0019] The inner lipid bilayer membrane covering the hydrogel particles, i.e., the lipid coating on the side directly in contact with the hydrogel (first lipid coating), has a hydrophilic group with an electric charge of the opposite polarity to that of the hydrogel particles. Examples of lipids that can be used include phospholipids, glycolipids, glycerides, ceramides, fatty acids, and steroids.

[0020] In particular, for example, when the hydrogel is a cationic gel, the first lipid coating is preferably, but not limited to, anionic phospholipids such as POPG (C40H77O10P: 1-palmitoyl-2-oleoylphosphatidylglycerol), 1-palmitoyl-2-oleoylphosphatidic acid, or 1-palmitoyl-2-oleoylphosphatidylserine, or neutral phospholipids such as POPC (C42H82NO8P: 1-palmitoyl-2-oleoylphosphatidylcholine), 1,2-dioleoylphosphatidylcholine, 1,2-dipalmitoylphosphatidylcholine, or 1-palmitoyl-2-oleoylphosphatidylethanolamine, or neutral steroids such as cholesterol or ergosterol. Furthermore, a mixed solution containing a neutral lipid as a base and a charged lipid can be used. Specifically, a mixture of anionic phospholipids and neutral phospholipids, or a mixture of anionic phospholipids and neutral steroids, can also be used as the first lipid coating. This is because it makes it easier to retain proteins and other substances within the hydrogel particles in applications such as artificial cells. In this case, when using a mixture of anionic phospholipids and neutral phospholipids or a mixture of anionic phospholipids and neutral steroids, the concentration of the anionic phospholipid in each mixture is preferably in the range of 20 mol% or more and less than 100 mol%.

[0021] Furthermore, when the hydrogel is an anionic-on-gel, the first lipid coating is preferably, but not limited to, a cationic phospholipid such as DOTAP (CHOSCHNO:N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, or a cationic steroid such as 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride. Furthermore, as in the case of a cationic hydrogel, a mixture of a neutral lipid and a charged lipid can be used. That is, a mixture of either a cationic phospholipid or a cationic steroid with either a neutral phospholipid or a neutral steroid can also be used as the first lipid coating. As mentioned above, this is because it facilitates the retention of proteins and the like within the hydrogel particles. In this case as well, the concentrations of the cationic phospholipid and the cationic steroid in the mixed solution are preferably both in the range of 20 mol % or more and less than 100 mol %.

[0022] An image of a liposome in this embodiment using a hydrogel (in the case of a cationic gel) as a support is shown in Figure 1. The outer lipid coating (second lipid coating) of the lipid bilayer is not particularly limited, and may be composed of the same lipid molecules as the first lipid coating.

[0023] The hydrogel may further contain an encapsulated component with a molecular weight of 10,000 or more, more preferably 40,000 or more. The encapsulated component may be a protein such as collagen, hemoglobin, or an enzyme, or a nucleotide. Furthermore, the hydrogel may contain not only organic substances derived from living organisms, but also artificial devices of similar size, such as microchips or micromotors, or small-diameter liposomes.

[0024] As described above, according to this embodiment, it is possible to realize physically stable liposomes (giant liposomes) with a diameter of 1 μm or more, which are supported by a hydrogel.

[0025] The method for producing liposomes according to this embodiment is described below. In this embodiment, liposomes are produced using an improved version of the oil-water interface penetration method. First, a hydrogel is dispersed in a mixed solution containing first lipid molecules (Step 1). As a result, a first lipid coating is formed on the surface of the hydrogel due to electrostatic interaction. Meanwhile, in parallel with Step 1, a second lipid coating is prepared (Step 2). In this step, the second lipid molecules are mixed into a container into which the oil and water phases have been poured in a separated state, and are allowed to spread at the oil-water interface. As a result, a second lipid coating is formed at the oil-water interface.

[0026] In the next step, the hydrogel with the first lipid coating prepared in step 1 is poured into the oil phase of the container, and the lipid-coated hydrogel is passed through the oil-water interface using a centrifuge (step 3). As a result, a lipid bilayer membrane is formed on the hydrogel surface. Using a centrifuge allows a large amount of hydrogel to pass through the oil-water interface in a short period of time, thereby increasing liposome productivity.

[0027] Alternatively, in step 1, the lipid-coated gel may be prepared by adding the oil phase to a dehydrated gel obtained by removing the aqueous phase in which the hydrogel is dispersed by suction filtration, and dispersing the gel particles by ultrasonication and vortexing. Details are provided in the following examples. [Example]

[0028] Hereinafter, examples of the present disclosure will be described.

[0029] Example 1 This example describes an experiment to visualize lipids on the surface of hydrogel particles using a lipophilic carbocyanine dye and the results of the experiment. Positively charged agarose gel (Q Sepharose High Performance) was used for the hydrogel particles. The average particle size was approximately 34 μm. The aqueous phase consisted of TBS (Tris buffer solution; Tris 10 mmol / L, NaCl 150 mmol / L, pH 7.5). The oil phase consisted of a chloroform / hexane mixture (2:8 vol) containing 0.07 mmol / L POPG (C40H77O10P: 1-palmitoyl-2-oleoylphosphatidylglycerol) and 0.5 mol% of the cationic fluorescent lipid DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate, excitation wavelength 549 nm, emission wavelength 565 nm).

[0030] First, 80 μL of the hydrogel suspension was mixed with 80 μL of TBS. The aqueous phase containing the dispersed hydrogel particles was removed by vacuum filtration (Omnipore Membrane Filters JCWP02500, filter holder KGS-25) to prepare a dehydrated gel. Next, 1 mL of the oil phase was added to the dehydrated gel, sonicated for 5 minutes, and then vortexed for 1 hour to disperse the gel particles. This lipid-coated gel was then added to the oil-phase side of a container containing 400 μL of each of the oil and aqueous phases, and passed through the oil-water interface using a centrifuge (5430R, conditions: 120 rcf, 10 minutes) to form a lipid bilayer on the gel surface. A fluorescence image of the resulting liposomes (microscope: FV-1000D confocal laser scanning microscope) is shown in Figure 2.

[0031] In Figure 2, almost no fluorescence was detected inside the hydrogel, but strong fluorescence was observed on the periphery of the hydrogel particles, suggesting that lipid molecules had adhered to the surface of the hydrogel particles, forming liposomes.

[0032] Example 2 In this embodiment, the electrostatic interaction between lipid molecules and the hydrogel support is utilized when attaching lipids to hydrogel particles. Therefore, in this example, we verify the effectiveness of this interaction. For the positively charged hydrogel (Q Sepharose High Performance) and the negatively charged hydrogel (SP Sepharose High Performance), we used DOTAP, which has a positive charge on the hydrophilic side; POPG, which has a negative charge; and POPC, which is neutral and leans toward a negative charge. Lipid coating was attempted using 0.5 mol% fluorescent lipid DiI (lipid concentration 0.07 mmol) for the positively charged hydrogel (Q Sepharose High Performance), and 0.5 mol% anionic fluorescent lipid Fluorescein-DHPE (N-(Fluorescein-5-Thiocarbamoyl)-1,2-Dihexadecanoyl-sn-Glycero-3-Phosphoethanolamine, Triethylammonium Salt) (excitation 495 nm, emission 519 nm, Setareh Biotech) (lipid concentration 0.07 mmol) for the negatively charged hydrogel (SP Sepharose High Performance). A total of six fluorescence observation experiments were performed (inverted optical microscope: IX50, fluorescence observation filter for DiI: U-MWIG, fluorescence observation filter for Fluorescein-DHPE: U-MNIBA). The results are shown in Figure 3.

[0033] The detection of fluorescence indicates the presence of a lipid bilayer membrane. Hydrogel particles exhibited fluorescence when cationic gel was combined with POPC or POPG, which is expected to induce electrostatic interactions (Fig. 3(a) and (b)). Similarly, hydrogel particles exhibited fluorescence when anionic gel was combined with DOTAP (Fig. 3(f)). On the other hand, when cationic gel was combined with DOTAP, which is expected to induce electrostatic repulsion, the fluorescence of DiI was very weak (Fig. 3(c)). Fluorescein-DHPE fluorescence was also very weak when anionic gel was combined with POPC or POPG (Fig. 3(d) and (e)). In both experiments, no change in hydrogel diameter was observed before and after lipid coating. These results suggest that electrostatic interactions between the lipid and hydrogel (support) resulted in the formation of large hydrogel liposomes with an average diameter of 34 μm.

[0034] Example 3 This example describes an experiment investigating the relationship between the molecular weight of the encapsulated component of the liposome of this embodiment and its retention, and the results of the experiment. First, 80 μL of cationic gel suspension was mixed with 20 μL of 0.1 mmol / L FITC-dextran TBS solution and 60 μL of TBS. This 60 μL mixed aqueous solution was subjected to suction filtration to remove the outer aqueous phase of the hydrogel. This hydrogel was dispersed in a lipid (POPC) mixed solution to prepare a lipid-coated gel. In parallel with this, 400 μL of TBS and 400 μL of lipid solution were mixed in a separate microtube and left to stand, allowing a lipid membrane to form at the oil-water interface.

[0035] Next, this lipid-coated gel was poured into the oil phase of a microtube and centrifuged (120 rcf, 10 minutes). After centrifugation, the supernatant oil phase was removed using a Pasteur pipette, and the sample was then placed in a desiccator for 30 minutes to further remove the oil phase. Another 150 μL of the sample was taken, and 250 μL of TBS was added. This washing procedure (1 mL of TBS was added, mixed, and then the hydrogel particles were precipitated in a centrifuge at 120 rcf for 3 minutes, and 1 mL of the supernatant solution was removed) was repeated six times, followed by fluorescence observation (microscope: inverted optical microscope IX50, fluorescence observation device filter: U-MNIBA). The results are shown in Figure 4.

[0036] Figure 4(a) shows the fluorescence of a sample encapsulated with 40,000 molecular weight FITC-dextran during production, while Figure 4(b) shows the fluorescence of a sample encapsulated with 10,000 molecular weight FITC-dextran. The detection of fluorescence indicates that the respective substances are retained within the liposomes. For the hydrogel encapsulating 40,000 molecular weight FITC-dextran, the gel particles retained fluorescence even after washing. Furthermore, in both experiments, no change in hydrogel diameter was observed before and after lipid coating, resulting in giant liposomes that reflected the hydrogel diameter. However, no fluorescence was detected for the hydrogel encapsulating 10,000 molecular weight FITC-dextran. These results suggest that encapsulated components can be retained within liposomes if the molecular weight is 40,000 or greater.

[0037] As described above, according to the present disclosure, it is possible to realize physically stable liposomes with a particle size of 1 μm or more using a hydrogel as a support, and to encapsulate components with a molecular weight of 40,000 or more or a size equivalent to a molecular weight of 40,000 or more in these liposomes. [Industrial Applicability]

[0038] The present invention can be used for applications such as artificial cells such as artificial red blood cells, microreactors, biosensors that remain in the living body, various medical materials, and cosmetic materials.

Claims

1. Using hydrogel as a support, anionic or cationic hydrogel particles; a first lipid coating formed by assembling first lipid molecules on the surface of the hydrogel particles, the first lipid molecules having a charge in the hydrophilic group of opposite polarity to that of the hydrogel particles; The membrane has a second lipid coating made of second lipid molecules that forms a lipid bilayer with the first lipid coating, When the hydrogel particles are cationic, the first lipid coating comprises an anionic phospholipid or a neutral phospholipid exhibiting a neutrality leaning towards a negative charge, and when the hydrogel particles are anionic, the first lipid coating comprises a cationic phospholipid.

2. The liposome according to claim 1, characterized in that the diameter of the hydrogel is 1 μm to 1 mm.

3. 3. The liposome according to claim 1, wherein the first lipid molecule is at least one selected from the group consisting of POPG (1-palmitoyl-2-oleoylphosphatidylglycerol), 1-palmitoyl-2-oleoylphosphatidic acid, 1-palmitoyl-2-oleoylphosphatidylserine, and POPC (1-palmitoyl-2-oleoylphosphatidylcholine) when the hydrogel particles are cationic; or the first lipid molecule is at least one selected from the group consisting of DOTAP (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate), 1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine, and 3β-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol hydrochloride when the hydrogel particles are anionic.

4. The liposome according to any one of claims 1 to 3, wherein the hydrogel further carries an encapsulated component having a molecular weight of 40,000 or more.

5. A method for producing the liposome according to any one of claims 1 to 4, comprising the steps of: A first step of dispersing a hydrogel in a mixed solution containing first lipid molecules to prepare a lipid-coated gel having a first lipid coating; a second step of spreading second lipid molecules onto the oil-water interface to form a second lipid coating; a third step of passing the lipid-coated gel through the oil-water interface using a centrifuge to form a lipid bilayer membrane.

6. 6. The method according to claim 5, wherein in the first step, the aqueous phase in which the hydrogel particles are dispersed is removed by suction filtration, an oil phase is added to the dehydrated gel, and the gel particles are dispersed by ultrasonic treatment and vortexing to prepare the lipid-coated gel.

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