JP2002275052A - Preservation and separation method of vesicles composed of surfactant substance membrane and its products - Google Patents

Abstract

(57) [Summary] [Problem] Since liposomes have poor stability and are difficult to store and transport, it has been difficult to mass-produce them in factories. SOLUTION: By immersing a thin plate-shaped liposome suspension-containing container in a low-temperature liquid having a freezing point or lower, water inside and outside the vesicles is almost simultaneously frozen, and the liposome can be cryopreserved without being destroyed. Incorporation of liposomes into the gel molecules protects the vesicles from physical impact, prevents drying and preserves them.
By these means, liposomes which had to be prepared immediately before use can be manufactured in a factory and used easily.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to the preservation of vesicles composed of a surface active substance film, and is used for cosmetics, pharmaceuticals, feed, compost, food production and the like.

[0002]

2. Description of the Related Art Liposomes, which are used in DDS and the like, are usually produced and administered immediately before administration to patients.

[0003]

An artificial vesicle composed of a membrane of a surfactant such as a liposome has poor stability and is difficult to store and transport for a long period of time. In the manufacturing process, the method for separating vesicles from substances not taken up by vesicles was complicated, costly and time-consuming.

[0004]

According to the present invention, first, a suspension containing vesicles composed of a surface active substance film is placed in a thin container having good heat conductivity, and rapidly frozen in a liquid substance having a low freezing point. By doing so, freezing inside and outside the vesicles occurs almost simultaneously, and cryopreservation becomes possible without destroying the vesicles. Next, a method for preserving vesicles composed of a surface-active substance film, which is characterized by retaining vesicles composed of a surface-active substance film in a gel substance, and a product thereof, A method for separating vesicles composed of a surface-active substance membrane and a product thereof, wherein vesicles composed of a membrane and substances that could not be taken into the vesicles are separated in water or saline by a difference in specific gravity. Is the solution. The vesicles are extremely thin in film thickness and are susceptible to drying and physical impact, so that long-term storage and transportation are made possible by holding the vesicles in a gel as a means of preventing drying and reducing the physical impact. When the membrane is composed of a material having a low specific gravity such as lipids, vesicles and residues can be easily separated due to a difference in specific gravity by releasing an unseparated product into water or saline. Furthermore, since the membrane constituting the vesicle is permeable, a part of the constituents of the gel-like substance is composed of the same substance as a part of the fluid constituent inside the vesicle, so that the inside of the vesicle is unexpectedly formed. Mixing of impurities can be prevented. Further, the surfactant substance membrane may be a monolayer or multilayer phospholipid membrane such as a liposome. By composing the fluid main component inside the vesicles with body fluids of animals and plants, it can be used as compost, feed, cosmetics, and medicine. By adding an antifreezing agent and / or an anticoagulant and / or a preservative and / or a fungicide to blood to the gel-like substance, freezing during refrigerated storage, blood coagulation, decay and mold contamination Can be prevented. By including sodium chloride in the gel component, it is possible to eliminate an extreme difference in osmotic pressure when a body fluid is encapsulated in vesicles. In addition, by adding a digestive enzyme to a gel-like substance containing a carbohydrate or a protein in the component and metabolizing the carbohydrate or the protein to glycogen or an amino acid, it is possible to directly administer the drug to a living body.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The suspension after the preparation of liposomes is sealed in a sealed container, sterilized without heat by irradiation with ultraviolet rays or radiation, rapidly frozen or incorporated into gel molecules, and stored at low temperature to destroy liposomes. Agglomeration can be prevented, and further deterioration of quality can be prevented.

[0006]

[Embodiment 1] In this embodiment, a preservation method by a rapid freezing method will be described. FIG. 1 is a flowchart illustrating the steps of this embodiment. 1 is a step of preparing liposomes, 2 is a step of sealing the suspension in a blood collection container for blood donation and sterilizing by non-heating, 3 is a step of immersing and freezing in liquid nitrogen, 4 is a cryopreservation step, 5 is a thawing step , 6 are administration steps. A liposome is prepared (1), and the suspension is enclosed in a polyethylene blood donation blood collection container. The thickness of the entire container after enclosing should be 10 in consideration of heat conduction.
After sealing, non-heat sterilization is performed by X-ray irradiation (2). Next, the container is immersed in liquid nitrogen at 130 ° C below zero and rapidly frozen (3). Storage is performed in liquid nitrogen (4), and thawing at the time of use is thawing in room temperature water (5). Then, open or use with a syringe (6). If it is necessary to separate the liposome from the suspension, such as when the suspension contains blood, etc., gently release the suspension into water or saline and collect the floating supernatant to separate. I do.

In place of the liquid nitrogen in step 3 described above, a liquid having a freezing point of 30 degrees or less below zero, such as ammonia, alcohol, chloroform, or chlorofluorocarbon, may be infiltrated. However, when using these liquids, it is desirable to use them at a low temperature as close to the freezing point as possible. In addition, it is desirable that a container having a sufficient amount of refrigerant liquid and a thickness as thin as possible is used.

[0008]

Embodiment 2 In this embodiment, a storage method using a gel will be described. FIG.
5 is a flowchart for explaining the steps of this embodiment.
A is a liposome preparation step, B is a suspension of physiological saline,
An addition step of adding starch and its metabolic enzyme, C is a stirring step, D is an encapsulation sterilization step, E is a storage step, F is an incubation step, and G is a stirring and administration step. A liposome is prepared (A), and then physiological saline, starch and its metabolic enzymes, hexokinase, phosphoglucomutase, and phosphorylase, are added to the suspension (B), and the enzyme activity is suppressed by stirring at a low temperature. The suspension is gelled and liposomes are incorporated into the gel molecules (C). Further, it is sealed in a sealed container and sterilized by non-heating (D), and is refrigerated and stored at a low temperature that does not freeze (E). When used, incubate the container with warm water at 37 degrees Celsius while inverting and mixing occasionally for several hours until the gel becomes liquid glycogen (F), and the activity of metabolic enzymes that had been suppressed because of the low temperature was increased. Metabolizes starch. Then, mix well by inversion (G). If the liposome contains no protein and the gel contains a protein, adding a protein-metabolizing enzyme such as pepsin, chymotrypsin, peptidase, papain, or microbial pronase metabolizes the protein to amino acids to prepare the liposome. Suspensions can be administered directly into the body.

[0009]

In the first embodiment, the water inside and outside the vesicles is almost simultaneously frozen by immersing the thin plate-shaped container containing the liposome suspension in a low-temperature liquid below the freezing point, and the liposome can be cryopreserved without being destroyed. . In Example 2, vesicles were protected from physical impact by incorporating liposomes into gel molecules,
Furthermore, drying was prevented and storage was enabled. Furthermore, by metabolizing the gel component into internal substances such as glycogen using metabolic enzymes, it becomes possible to administer the drug-encapsulated liposomes directly into a vein. By these means, liposomes which had to be prepared immediately before use can be manufactured in a factory and used easily.

[Brief description of the drawings]

FIG. 1 is an explanatory diagram showing a flowchart for explaining steps of a first embodiment.

FIG. 2 is an explanatory diagram showing a flowchart illustrating a process of a second embodiment.

[Explanation of symbols]

1 Liposome preparation step 2 Encapsulation, non-heat sterilization step 3 Freezing step 4 Storage step 5 Thawing step 6 Administration step A Liposomal preparation step B Addition step C Stir gelation step D Encapsulation sterilization step E Storage step F Incubation step G Stir administration Process

Claims (9)

[Claims] 1. A surfactant material film, wherein a suspension containing vesicles composed of a surfactant material film is placed in a thin container having good heat conductivity and rapidly frozen in a liquid material having a low freezing point. Preservation method of the composed vesicle and its product. 2. A method for preserving vesicles composed of a surface active substance membrane, characterized by retaining vesicles composed of a surface active substance membrane in a gel substance, and a product thereof. 3. A vesicle composed of a surfactant substance membrane and a substance not taken up inside the vesicles are separated by a difference in specific gravity in water or a saline solution. Vesicle separation method and its products. 4. The surfactant according to claim 2, wherein a part of the component of the gel-like substance is the same substance as a part of a fluid component inside the vesicle. Preservation and separation of vesicles composed of membranes and their products. 5. The surfactant material film according to claim 1, wherein the surfactant material film is an artificial monolayer or multilayer phospholipid film. Preservation and separation method of the composed vesicles and their products. 6. The vesicle comprising a surfactant substance membrane according to claim 1, wherein the main fluid component inside the vesicle is a body fluid of animals and plants. Storage and separation methods and products. 7. The gel-like substance to which an antifreezing agent and / or an anticoagulant for blood and / or a preservative and / or an antifungal agent has been added. A method for storing and separating vesicles comprising the surfactant substance membrane according to any one of the above, and a product thereof. 8. The small-sized surface-active substance film according to claim 2, wherein sodium chloride is contained in said gel-like substance constituent component. Cell preservation and isolation methods and products. 9. The method according to claim 2, wherein a digestive enzyme is added to the gel-like substance containing a saccharide or a protein as a component to metabolize the saccharide or the protein into glycogen or an amino acid. A method for storing and separating vesicles comprising the surfactant substance membrane according to any one of the above, and a product thereof.