[0032] (2) Preparation method of liposome
[0033] The liposome preparation method described in this embodiment may include a heating step of heating a mixture of one or more lipids and an aqueous solution containing a water-soluble organic solvent, and a cooling step of cooling the mixture after the heating step. Use a well-known preparation method. More specifically, in order to adjust the particle size of liposomes, the method of the present invention can also be combined with an ultrasonic dispersion method, an extrusion method, a French press method, a homogenization method, and the like.
[0034] In the preparation of liposomes of the present invention, soybean lecithin, hydrogenated soybean lecithin, egg yolk lecithin, phosphatidylcholines, phosphatidylserines, phosphatidylethanolamines, phosphatidylinositols, and phosphatidyl sphingomyelin can be used One or two or more lipids from among phospholipids, phosphatidic acids, long-chain alkyl phosphates, gangliosides, glycolipids, phosphatidylglycerols, and sterols. Examples of phosphatidylcholines include dimyristoylphosphatidylcholine, dipalmitoylphosphatidylcholine, distearoylphosphatidylcholine, etc.; examples of phosphatidylserines include dipalmitoylphosphatidylserine , Sodium dipalmitoylphosphatidylserine, sodium phosphatidylserine derived from bovine brain, etc.; as the phosphatidylethanolamines, dimyristoylphosphatidylethanolamine, dipalmitoylphosphatidylethanolamine, distearoylphosphatidylethanolamine Etc.; as phosphatidylinositols, wheat-derived phosphatidylinositol (sodium salt), etc. can be cited; as phosphatidylinositols, sphingomyelin derived from bovine brain, etc.; as phosphatidic acids and long-chain Alkyl phosphates include: dimyristoyl phosphatidic acid, dipalmitoyl phosphatidic acid, distearoyl phosphatidic acid, dihexadecyl phosphate, etc.; as gangliosides, ganglioside GM1 can be cited , Ganglioside GD1a, ganglioside GT1b, etc.; as glycolipids, galactosylceramide, glucose, lactase-based (sphingosine), phospholipids, erythrocyte glycosides, etc.; as phosphatidylglycerols , Including dimyristoylphosphatidylglycerol, dipalmitoylphosphatidylglycerol, distearoylphosphatidylglycerol, etc.; as sterols, cholesterol, dihydrocholesterol, lanosterol, dihydrolanosterol, sitosterol, Campesterol, stigmasterol, brassicasterol, ergosterol, and mixtures of these, namely phytosterols, and hydrogenated phytosterols, as well as 3β-[N-(dimethylethylamino)carbamoyl]cholesterol, N- Positively charged sterols such as (trimethylammonium ethyl) carbamoyl cholesterol. As the lipid constituting the liposome, a combination of phosphorus-containing phospholipids and cholesterol is desirable. In particular, the combination of phosphatidylcholines in phospholipids and cholesterol is better. When using phospholipids and cholesterol to prepare liposomes, it is ideal that the molar ratio of phospholipids to cholesterol is in the range of 1:0 to 1:1.5, and more preferably in the range of 1:0.5 to 1:1.25. .
[0035] Water-soluble organic solvents refer to organic solvents that can be mixed with water, such as alcohols, ethers, esters, ketones, and acetals. As the water-soluble organic solvent, it is desirable to use one or two or more organic solvents selected from tert-butanol, 1-propanol, 2-propanol, and 2-butoxyethanol. The mixing method may be manual shaking, stirring with a stir bar or stirring blade, and an ultrasonic vibrator or the like may also be used for mixing.
[0036] As the concentration of the water-soluble organic solvent in the above-mentioned aqueous solution, the optimal concentration range required for the lipid composition and lipid concentration must be selected. This is because if the concentration of the water-soluble organic solvent increases, the solubility of lipids increases, but liposomes cannot be formed. At the same time, since the water-soluble organic solvent is easy to remain, it is disadvantageous to the organism when the liposome is supplied to the organism. Therefore, it is desirable that the concentration of the water-soluble organic solvent is the lowest concentration that can dissolve the lipid when a mixture of more than one lipid and an aqueous solution is heated. Specifically, it is desirable that the volume percentage of the water-soluble organic solvent to the total capacity of the aqueous solution is 5-30%. When the water-soluble organic solvent is tert-butanol, the volume percentage of the total volume of the aqueous solution is preferably 12-18%; when the water-soluble organic solvent is 1-propanol, the volume percentage of the total volume of the aqueous solution is 5-19%. Better; when the water-soluble organic solvent is 2-propanol, it is better that the volume percentage of the total volume of the aqueous solution is 13~26%; when the water-soluble organic solvent is 2-butoxyethanol, the volume percentage of the total volume of the aqueous solution 6~9% is better.
[0037] The above-mentioned water-soluble organic solvent can be prepared in advance to an aqueous solution of the above-mentioned desired concentration and then added to the lipid; or the water-soluble organic solvent can be added to the lipid first, and after dissolving the lipid component, the water solvent is added to achieve the above concentration.
[0038] The heating method is not particularly limited. For example, in addition to a hot water bath in which the container is placed in a bath containing hot water, a method of directly baking and heating the container with the mixture in the container can also be used, or How to put the container in the electric heater, etc. The heating temperature is not limited to a specific temperature as long as it is the temperature at which the lipid is dissolved in the aqueous solution containing the water-soluble organic solvent, or higher, but the aqueous solution does not produce white turbidity. The heating temperature differs depending on the type of lipid, the concentration of the lipid, the type of water-soluble organic solvent, etc. Generally, the range of 62 to 80°C, and particularly the range of 65 to 72°C is a desirable range. However, when tert-butanol is used as a water-soluble organic solvent and phosphatidylcholine and cholesterol are used as lipids, the ideal heating temperature range is 62 to 72°C.
[0039] The cooling method is not particularly limited. For example, in addition to a method of placing the container in a bath containing cold water, a method of placing the container in a refrigerator with a mixture in the container can also be used. The cooling temperature is not limited as long as it is lower than the heating temperature and can produce liposomes. For example, when phosphatidylcholine and cholesterol are used in lipids, the cooling temperature is preferably 62°C or lower. Especially after heating to above 72°C, it is best to cool to below 62°C. In addition, the cooling rate is preferably 0.5°C/min or more, and more preferably 1°C/min or more.
[0040] When lipids are mixed with an aqueous solution containing a water-soluble organic solvent, sugars such as disaccharides and polysaccharides as osmotic pressure regulators can also be added. The ideal sugar is sucrose among the disaccharides. Sucrose can also be mixed with an aqueous solution containing a water-soluble organic solvent in the state of an aqueous solution. At this time, the suitable concentration of the aqueous sucrose solution is 5 to 70 wt/vol%, and the more suitable concentration is 8 to 50 wt/vol%.
[0041] In the preparation of the liposomes of the present invention, in order to make the particle size of the liposome suspension more uniform, a well-known particle sizing method may be used in combination. For example, air pressure can be used to make a liposome suspension pass through a membrane with a specific pore size to prepare liposomes that can fit the desired pore size. The treatment of the membrane with the specific pore size can be performed once or several times.
[0042] (3) Method for dissolving cholesterol (a method to increase the amount of cholesterol dissolved in an aqueous solution containing a water-soluble organic solvent)
[0043] Cholesterol in lipids is generally hardly soluble in water, and the ratio of dissolving in the above-mentioned water-soluble organic solvent is low. However, when the above-mentioned aqueous solution containing a water-soluble organic solvent and phospholipids coexist, the cholesterol becomes easily dissolved by heating. Specifically, heating a mixture of phospholipids, cholesterol, and the above-mentioned aqueous solution can easily dissolve cholesterol in the aqueous solution.
[0044] The heating temperature may be the temperature at which cholesterol dissolves in an aqueous solution containing phospholipids, water-soluble organic solvents, or the like, or above this temperature, but the aqueous solution does not produce white turbidity. For example, when tert-butanol is used as a water-soluble organic solvent and L-α-dipalmitoylphosphatidylcholine is used as a phospholipid, cholesterol can be fully dissolved in an aqueous solution at a temperature of 62 to 72°C. 1 mole of phospholipid is more soluble in cholesterol in the range of 0.8 to 1.5 moles. In the temperature range of 62~72℃, cholesterol is dissolved in the aqueous solution containing tert-butanol, and all liquids are transparent. When the temperature is further cooled and the temperature of the mixed solution is lower than 62°C, white turbidity occurs, and a liposome suspension containing phospholipids and cholesterol components is formed. Conversely, when the temperature is higher than 72°C, the mixed solution also becomes a white suspension.
[0045] (4) Selection method of organic solvents that can be used
[0046] Through the above preparation method, liposomes with uniform particle size can be obtained. However, some organic solvents are not applicable. Therefore, by using the organic solvent as a candidate to prepare liposomes using the above-mentioned liposome preparation method, and checking whether the prepared liposomes are uniform, it can be judged whether the organic solvent is suitable for this preparation method and selected Practically applicable organic solvents.
