KR800001360B1 - Process for the preparation of liposomes - Google Patents

Abstract

Solns. of amyloglucosidase, penicillamine, imipramine, and disodium betamethasone phosphate were encapsulated in liposomes. The solns. to be encapsulated were mixed with lecithin or other lipids or phospholipids and ethers and the solns. were subjected to ultrasonic vibration to obtain a homogeneous soln. A 2-phase mixt. was then obtained by addn. of NaCl soln. underneath the homogeneous org. phase in a centrifuge tube. The 2-phase mist. was centrifuged at 30000rpm resulting in a colloidal dispersion of liposomes.

Description

Preparation of liposomes

The present invention relates to a method for preparing synthetic liposomes.

Liposomes are fat or oocytes that occur in the plasma. The term “synthetic liposome” refers to at least one compound having a maximum diameter of about 10,000 kPa, preferably between 300 and 2,000 kPa and represented by the general formula XY (where X is a hydrophilic polar group and Y is a hydrophobic nonpolar group). Means a blood cell visible under a microscope bounded by a wall formed by a bilayer of two molecules (having a thickness of about 3,200 μs), the blood cell containing an aqueous solution, for example, an aqueous solution of at least one biologically active substance And are generally present in the form of a colloidal dispersion in an aqueous medium such as saline solution, especially 0.9% by weight sodium chloride solution.

The preparation of liposomes is the most practical and effective for aqueous solutions, particularly useful for the administration of biologically active substances, especially medicaments into living organisms, while prior to reaching the point of action of any substance in vivo, It provides an encapsulation that avoids destruction or inactivation by action.

By selecting the structural XY compound used to form the walls of the liposomes, the wall is attacked only in the presence of certain reagents that are present only in the organs that are resistant to the activity of any part of the body and maintain a biologically active substance. Liposomes having the same can be prepared.

Two methods are known for the preparation of liposomes.

The first of these methods is to contact the fat with the aqueous solution desired for encapsulation, then warm the heterogeneous mixture thus obtained at a temperature slightly above ambient temperature, then stir the mixture vigorously and provide ultrasonic vibration.

Another method of this method is to dissolve a compound of formula XY (where X and Y are as described above) in, for example, a fat, ie a volatile solvent, and evaporate the solvent from the solution so obtained to A film is formed, a liquid to be encapsulated in liposomes is introduced into this container, and finally the liquid contained in the container is provided for the action of ultrasonic vibration.

Thus, these two methods require the total amount of liquid to be encapsulated in an amount much larger than the volume of the liquid finally contained in the liposomes produced by these methods. By these methods, liposomes are formed in the form of colloidal dispersions of blood cells in a liquid phase containing a fraction of the liquid to be encapsulated that is not actually retained inside the liposomes. The ratio of the volume of the liquid encapsulated inside the liposome to the total volume of the surrounding liquid is generally about 1 to 10%.

Therefore, if the liquid to be encapsulated has a high value, then the non-encapsulated liquid is used in further operations to form liposomes, as is often encountered when the liquid is a solution of a biologically active substance. It is necessary to recover the fraction of this liquid before doing so. This recovery is to separate the liposomes from the liquid, to purify the liquid itself, and usually to readjust the concentration of the active substance. In practice, the separation and purification steps use a large amount of solvent, which results in adjusting the concentration of the liquid containing the active ingredient.

The need to take steps to refine the concentration of the tablet and the liquid containing the active ingredient makes these methods difficult to perform on an industrial scale.

It is an object of the present invention to provide only an improved method for preparing liposomes, each producing only a volume of liquid to be encapsulated.

According to the present invention, there is provided a method for preparing a synthetic liposome, wherein the first aqueous solution is in the presence of at least one compound represented by the general formula XY, wherein X represents a hydrophilic polar group and Y represents a hydrophobic non-polar group. Dispersion by ultrasonic vibration in a liquid carrier that is insoluble in water or only slightly soluble in water and less dense than water forms a colloidal dispersion of the first aqueous solution in the liquid carrier, and the dispersed phase is applied to the monomolecular film of the compound of general formula XY. Bounded by; The dispersion is combined with a second liquid to form a heterogeneous two-layer system, the upper liquid layer is formed by the dispersion, the bottom liquid layer is formed as a second aqueous solution, and the two layers are monomolecular films of the compound of compound XY. Separated by; The two-layer system is characterized by centrifuging at a sufficient angular rate against the dispersed blood cells of the first aqueous solution, which will be gravityd through the monomolecular separation film of Compound XY to the bottom layer of the second aqueous solution.

Thus, this method has two steps. In the first step, a dispersion of blood cells of the liquid to be encapsulated is formed under the action of an ultrasonic wave, etc., which are insoluble in water or only slightly soluble in water. Diameter). These blood cells are bounded by a monomolecular film of compound XY, where hydrophilic group X is directed inside the blood cells occupied by the aqueous phase and hydrophobic group Y is directed outside the blood cells in contact with the non-aqueous phase. These cells are not exactly what constitutes blood cells, but they can be referred to as circular liposomes because these cells are not bounded by the bimolecular layer of Compound XY but only by the monomolecular film of the former compound. Each of these blood cells contains the same amount of liquid to be encapsulated, resulting in a liposome. In the following description, these blood cells are referred to as liposome precursors.

By appropriately selecting the relative proportions of the water-soluble liquid agent and liquid carrier to be encapsulated and the compound of the structural formula XY in the first part of this step, encapsulation in the liposome precursor of the whole liquid to be encapsulated can be obtained. Thus, the method according to the present invention avoids the need for recovery, purification, or readjustment of the concentration necessary for the operation of conventional methods as described above.

It can also be seen that the process according to the invention is encapsulated by the formation of liposomes of 0.05-0.1 ml of liquid, which can be used in very small amounts, the amount of electricity being insufficient for use in the methods described above.

Thus, the method according to the present invention can be used in fields such as laboratory research or analytical procedures in which other methods cannot be used.

The second step of the method is to form an appropriate liposome from the liposome precursor. The formation of liposomes passes a monomolecular film of compound XY in the inner phase between the upper liquid and the lower liquid and entrains a portion of the film in which each liposome precursor is associated with the monomolecular film of compound XY surrounding the precursor. This is due to the fact that a bimolecular film of compound XY characteristics of liposomes is formed. The terminal film of compound XY is present in the inner phase between the upper liquid and the lower liquid because the hydrophilic group X is attracted to the solution layer, while the hydrophobic group Y is buried in the non-aqueous solution layer.

Compounds of formula XY include, for example, hydrophilic group X having the following groups (e.g., phosphate groups, carboxyl groups, sulfato groups, amino groups, hydroxyl groups and colin groups) and hydrophobic group Y having the following groups (e.g. And a saturated or unsaturated aliphatic hydrocarbon group (eg, an alkyl or alkylene group) and an aliphatic hydrocarbon group substituted by at least one aromatic or ring aliphatic group.

Preference is given to using phospholipids or substances in close proximity to phospholipids as compounds of formula XY. In particular, the following compounds are useful: lecithin, phosphatidylethanolamine, lysocithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and serabroside.

As the compound of formula XY, it is also possible to use mixtures of at least one phospholipid with at least one other lipid belonging to a different category from that phospholipid. In particular, the following compounds may be used: stearylamine, dicetylphosphate, cholesterol and tocopherol.

Carrier liquids that are slightly soluble or insoluble in water are preferably organic liquids, in particular one of the following: benzene, halo-benzene or alkylbenzenes, aliphatic ethers, aliphatic ketones, aliphatic aldehydes, aliphatic esters, aliphatic hydrocarbons Or cycloaliphatic hydrocarbons or mixtures thereof having a lower density than water are suitable.

The choice of the first aqueous solution, ie the liquid to be encapsulated in liposomes, is limited only to the practicality required for liposomes.

In particular, solutions of at least one or more biologically active substances, for example enzymes or solutions such as antibiotics, can be used.

The second aqueous solution is a pure liquid or other suitable aqueous solution. Suitable as the second aqueous solution are liquids required as carriers for the dispersion of liposomes, for example, aqueous solutions of sodium chloride. In particular, a sodium chloride solution known as a menstrual fluid or a saline solution having a concentration of 0.15 molar sodium chloride (0.9% by weight) per liter can be used to directly obtain a liposome dispersion in a medium which can be injected into the human body in the second step of the method. have. This is thus an additional advantage of the present invention compared to known methods for the preparation of liposomes, in which a suspension of liposomes in the aqueous medium chosen for the end use of liposomes can be obtained directly.

However, it is to be understood that liposomes can be separated from the second aqueous solution if it is desired to remove traces of unencapsulated actives prior to the final use of liposomes. This separation process can be easily carried out by a suitable known method, for example, gel chromatography.

The following examples further illustrate the invention.

Example 1

An aqueous solution of amyloglucosidase containing 10 mg amyloglucosidase per ml of solution was encapsulated in an aqueous sodium chloride solution (0.15 mol per liter) in the following manner.

Lecithin (45 mg) and amyloglucosidase (0.1 ml) were added to dibutyl ether (3 ml), and the heterogeneous mixture thus obtained was subjected to ultrasonic (or nearly ultrasonic) vibration (17 kHZ frequency: power: 70 watts) for 2 minutes. The temperature was kept at a temperature of 30 ° C. or lower by the cooling bath.

A clear liquid with apparently uniform and bluish reflected light was obtained. A layer of this liquid was placed in a centrifuge tube on a bed of 1.5 ml sodium chloride solution (0.15 mole per liter). Thus, containing the top layer formed by the organic phase obtained in the first step of the process by ultrasonic vibration of the bottom layer formed by the aqueous phase (sodium chloride solution) and the mixture of dibutyl ether, lecithin and amyloglucosidase solution. A two-phase “mixture” was obtained.

The two-phase “mixture” was then centrifuged at 30,000 revolutions / minute for 30 minutes. The top layer (organic phase) was then removed and the bottom layer (aqueous phase) was further centrifuged at 30,000 revolutions / minute for 30 minutes. Meaningly a bluish clear liquid was obtained with a very small amount of residue containing non-dispersed lipids (lecithin), the latter being removed. The clear liquid contains a suspension of liposomes (with colloid size) containing an aqueous amyloglucosidase solution in 0.15 M sodium chloride solution and also contains a small amount of non-encapsulated amyloglucosidase.

Depending on how the suspension is used, the suspension may be used when or after the formation of the non-encapsulated amyloglucosidase by gelchromatography (eg, cephalose (dextran) gel).

Example 2

A mixture of lecithin (27 mg), dibutyl ether (2.4 ml) and chloroform (0.6 ml) in which a buffer solution containing 100 mg / ml penicylamine (0.05 m) (phosphate buffer 10 mM, pH 7.2) was involved in ultrasonic vibration. The formation of the organic phase of was encapsulated in 0.5M aqueous sodium chloride solution. This method was carried out in the same manner as in Example 1.

Example 3

In a similar manner to Example 1, an aqueous solution of 0.15 M sodium chloride was prepared using a mixture of lecithin (25 mg) and cholesterol (40 mg) in dibutyl ether (3 ml) containing an aqueous solution containing 300 mg / ml imipramine (0.1 ml). It was encapsulated in the middle.

Example 4

By a method similar to Example 1, an aqueous solution of 150 mg / ml butamethasone disodium phosphate (0.05 ml) was used in a first step using a mixture of dibutyl ether (2.5 ml) and chloroform (0.5 ml) and then Encapsulated in 0.15 M aqueous sodium chloride solution using a mixture of lecithin (15 mg) and phosphatidylethanolamine (12 mg).

Claims (1)

Insoluble or slightly soluble in water in the first aqueous solution in the presence of at least one compound represented by the general formula XY, wherein X represents a hydrophilic polar group and Y represents a hydrophobic nonpolar group, and has a density less than that of water. Dispersion by ultrasonic vibration in the liquid carrier to form a colloidal dispersion of the first water-soluble liquid in the carrier liquid, and the dispersion in which the dispersed phase is bounded by a monomolecular film of a compound of the general formula XY with the second liquid, Forming a heterogeneous two-layer system consisting of an upper liquid layer formed by the first liquid layer and a bottom liquid layer formed by the second aqueous solution, and separating the two-layer system by the monomolecular film of the compound of the general formula XY. Sufficient angular velocity to allow the dispersed globules to enter the second aqueous solution at the bottom through the monomolecular separation film of compound XY by gravity Method for producing a synthetic liposomes by centrifugation.