JP2011062586A - Method and apparatus for continuously mass-producing liposome - Google Patents

Abstract

An apparatus for producing liposomes encapsulating an antigen or the like homogeneously and in large quantities is provided.
A long endless belt is continuously pulled up through a lipid solution, the lipid is decompressed or warm-air dried and held as a thin film on the surface of the belt, and then the long endless belt is continuously immersed in water or a buffer solution. An apparatus characterized by continuously forming liposomes in water by scraping lipids from the belt with ultrasonic waves or other mechanical vibrations or vibrating brushes while hydrating the belt.
[Selection] Figure 2

Description

The present invention relates to a method for continuous mass production of liposomes by continuously forming a lipid film on an endless belt and an apparatus therefor.

Liposomes, which are closed vesicles of lipids, are biodegradable and non-toxic, and are used as carriers for drug transport because drugs are encapsulated in a relatively impermeable lipid bilayer. In addition, it is often used in vaccine preparations because an adjuvant effect can be expected by using liposome as an antigen carrier. Multilamellar liposomes can be easily obtained by the Bangham method, that is, the rotary evaporator vortexing method. That is, liposome membrane constituents such as lipids are dissolved in an organic solvent, and the organic solvent in the glass container is removed by a rotary evaporator or the like to form a thin film on the container wall. Next, an aqueous solution containing a material to be encapsulated is added to swell the thin film, and the container is shaken (see FIG. 1). However, this method cannot be manufactured in large quantities at a time due to the limitation of the size of the glass container on which the thin film is formed, and the drug entrapment efficiency and the average diameter of the liposome tend to vary depending on the shaking method. (See Non-Patent Document 1).

Regarding a method and apparatus for producing liposomes in large quantities, for example, a method and apparatus for mixing lipids and aqueous solutions with a critical or supercritical fluid (carbon dioxide, nitrous oxide, propane, ethylene, etc.) (see Patent Documents 1 and 2) And a method and an apparatus for continuously supplying the purified liposome in the mixing chamber to the third reservoir from the buffer solution reservoir and the organic lipid container reservoir to the third reservoir (see Patent Document 3). and so on.

JP-T 9-502644 JP 2005-162702 A JP 2005-538967 A

A. D. Bangham et.al. J. Mol. Biol., 13, 238 (1965)

An object of the present invention is to provide a method for producing a liposome having a large amount and a uniform particle diameter by a relatively simple apparatus.

The inventor continuously pulls the long endless belt through the lipid solution, dries the lipid under reduced pressure or warm air and holds it as a thin film on the belt surface, and then continuously continuously the long endless belt in water or a buffer solution. It has been found that the above-mentioned problems can be solved by scraping the lipid from the belt with ultrasonic waves or other mechanical vibrations or vibrating brushes to continuously form liposomes in the water while hydrating through the endless belt.

As the long endless belt, a material having sufficient flexibility and organic solvent resistance is desirable, and for example, synthetic resin, glass fiber, or rolled metal can be used. The length and width of the endless belt can be appropriately selected depending on the production scale, but a range of 1 to 2 m in length and 5 to 20 cm in width is easy to use.

The lipid liquid immersion unit 3 is provided with a sprocket roller for driving the belt, and a thin film is formed on the bald surface by removing excess lipid liquid adhering to the belt with this roller and the next roller. The surface shape of the endless belt is basically a smooth surface, but a belt having a large surface area by providing a large number of shallow depressions on the surface can be used, and the production efficiency of multilamellar liposomes is particularly good.

The lipid membrane drying unit 4 has a structure in which the inlet side and the outlet side of the endless belt are almost sealed with rollers, and the solvent is efficiently removed by reducing the pressure with a vacuum pump while sending warm air of nitrogen or air inside. It is possible to remove, and the removed solvent is cooled and recovered by a solvent recovery trap installed thereafter. If the removal of the solvent is insufficient with one lipid membrane drying unit, it is possible to completely remove the solvent by installing another unit.

Lipids such as egg yolk lecithin, soybean lecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin and their hydrogenated saturated products, sucrose fatty acid One or more sugar fatty acid esters such as esters, trehalose fatty acid esters, and rhamnose fatty acid esters, or a mixture thereof can be used.

Other membrane constituents include sterols such as cholesterol as membrane stabilizers, phosphatidic acid, dicetyl phosphate, ganglioside, stearylamine, etc. as charged substances, and tocopherols as antioxidants, etc. May be added. The composition ratio of these membrane-constituting substances is not particularly limited, but 0 to 0.5 parts by weight of sterols, 0 to 0.1 parts by weight of charged substances, and 0 to 0. It is preferable to add about 1 part by weight. These are used after being dissolved in an organic solvent such as methanol, ethanol, acetone, dimethyl sulfoxide (DMSO), dioxane ethyl ether, or chloroform.

The aqueous solution hydrates and swells and disperses the membrane-constituting lipid dried on the belt surface, and water alone, physiological saline, various buffer solutions, or an aqueous solution in which saccharides are dissolved can be used. Furthermore, substances such as drugs, proteins, viruses and virus-derived antigens, bacteria and bacterial cell-derived substances, and polymers can be dissolved or suspended.

Hereafter, the manufacturing method of the liposome of this invention is demonstrated with reference to FIG.
The apparatus basically removes the drive device (sprocket roller) 2 for rotating the endless belt 1 in the direction of the arrow, the lipid solution immersion unit 3 for immersing the endless belt in the lipid solution, and the lipid solution solvent on the belt. A lipid membrane drying unit 4, a liposome preparation unit 5 for hydrating the dried lipid membrane on the belt with an aqueous solution such as an encapsulated substance suspension, and a belt drying unit 6 for removing water remaining on the belt. The liposome production unit 5 is a substantially sealed cube and sterilizes the belt membrane surface inside, a UV sterilization unit 7, an inclusion substance suspension container 8, an ultrasonic generator 9 that vibrates the suspension, a draining rubber 10, Slits 11 and 12 for entering and exiting the belt are arranged. The endless belt 1 circulates between the units by being guided by the roller guides 13 appropriately installed between the units and in each unit.

The liposome production apparatus of the present invention can produce a large amount of uniform liposomes continuously.

Production method of liposome by bangham vortexing method Liposome production equipment using ultrasonic oscillator for hydration and mixing Liposome production equipment using scraper

Example 1
In the apparatus of FIG. 2, 100 ml of a chloroform solvent containing 400 mg / ml hydrogenated soybean lecithin, 66 mg / ml cholesterol, 30 mg / ml phosphatidylethanolamine membrane-constituting lipid was placed in a lipid-immersed container, A 10 cm wide PVDF endless belt having sprocket holes on the left and right sides of the belt was attached to the liposome production apparatus of the present invention, and the belt was delivered at a speed of 2 cm per second. Subsequently, the PVDF belt with the lipid solution attached in a thin film form is continuously sent to the air-drying unit 4, and the organic solvent is completely removed by sending a low-temperature air at 75 ° C. under suction and vacuum to completely remove the organic solvent. A dry thin film was formed. The organic solvent evaporated and removed here was introduced into a dry ice cooling trap and recovered as a liquid. The belt holding the lipid thin film dried by the drying unit is introduced into the liposome forming unit 5 containing phosphate buffered saline to hydrate the lipid, and then placed at the bottom of the liposome preparation unit. Were peeled off from the belt surface by ultrasonic vibration from the water to form liposomes in water. About 80% of the liposomes prepared by this method were unilamellar liposomes, about 20% multilamellar liposomes, and the diameter was approximately 100 to 150 nm, which was confirmed by electron microscopy and particle analyzer. It is considered that the size of can be adjusted by lipid concentration or ultrasonic output.

Example 2
By filling the liposome-forming unit of Example 1 above with the formalin inactivating antigen (HA value 1: 512 before inactivation) of Newcastle disease virus Ishii strain, and forming liposomes in the same manner as in Example 1, And the vaccine composition which arrange | positioned the antigen on the outer side was obtained. This vaccine composition was instilled into 10 4-week-old SPF chickens, and antibody production in serum and laryngeal wipes was confirmed 3 weeks later. As a result, the serum antibody titer showed a high effective antibody titer of about 1: 256 in average HI titer. It was also confirmed by ELISA that IgA involved in mucosal immunity was also produced in the laryngeal wipe.

Claims (10)

A method for producing a liposome comprising the following steps.
1. 1. Supplying a lipid solution to the surface of the endless belt 2. removing the lipid solution solvent and forming a lipid thin film on the belt surface; 3. Dipping the lipid film on the belt in water or a buffer and applying ultrasonic waves or other mechanical vibrations to peel the lipid film from the belt. 4. Mixing lipid membrane and buffer to form liposomes Cleaning and drying the belt A method for continuously producing liposomes by repeating steps 1 to 5 according to claim 1. The method for producing a liposome according to any one of claims 1 to 2, wherein the material of the endless belt is an organic solvent resistant material, and is selected from a synthetic resin, glass fiber, or metal rolled product. The method of producing a liposome according to any one of claims 1 to 3, wherein the surface shape of the endless belt is a smooth surface or has a structure in which a large number of shallow dents are provided to increase the surface area. The manufacturing method of the liposome in any one of Claims 1-4 which process each process continuously by rotating an endless belt in a fixed direction manually or with an electric motor. Liposomes encapsulating chemicals, proteins, viruses and virus-derived antigens, bacteria and bacterial cell-derived substances, macromolecules and other substances dissolved or suspended in the water or buffer according to claim 1 The method for producing a liposome according to any one of claims 1 to 5, wherein The thin film lipid formed on the belt is immersed in water or a buffer solution, and the lipid membrane is peeled off by ultrasonic waves or other mechanical vibrations. At the same time, the formation of liposomes by hydration and mixing with water or buffer solution is continuously performed. The method for producing liposome according to any one of claims 1 to 6, wherein the method is carried out. Lipids encapsulating drugs, polymers, etc. can be hydrated by adding an aqueous solution or suspension directly to the dry lipid formed on the belt and then scraping it into an aqueous solution containing the encapsulated substance with a scraper. The method for producing liposome according to any one of claims 1 to 6, which is prepared. The apparatus for enforcing the manufacturing method in any one of Claims 1-7. The apparatus for enforcing the manufacturing method of any one of Claims 1-6, or Claim 8.

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