Background of the Invention

Field of the Invention

This invention relates to improved liposomes. More particularly, the invention is concerned with liposomes which are strengthened in their membrane. Liposomes are closed vesicles consisting of membrane comprising bimolecular lipid layers, and are widely used as model membrane of living cellular membrane in the study of physicochemical properties thereof. Furthermore, liposomes are utilized as carriers for administering substances to target organs of living body since they can keep various substances penned up in an interior aqueous compartment or membrane thereof or fuse with cells or are incorporated thereinto. Researches heretofore directed to utilization of liposomes are those to cover a diversity of scientific fields to which the liposomes are applicable, for example, biology, medical science and pharmacology, wherein the liposomes are utilized as carriers for transporting enzymes or antitumor agents, in the field of immunology, for examination of interaction thereof with cells, and as drug delivery systems.

Description of the Prior Art

As mentioned hereinbefore, liposomes are of very wide application. As often pointed out, however, the problem involved in liposomes is such that the membrane thereof are fragile. That is, the membrane of liposomes is destroyed by chemical or physical change of a lipid which is a membrane-forming substance and thereby to cause leakage of the substance retained by the liposome. Under the circumstances, as is well known, there have heretofore been proposed various processes for strengthening the membrane of liposomes such as a process in which sphingomeylin is incorporated into the membrane so as to impart hydrogen bond region and strengthen said membrane, and a process in which such additives as tocopherol are incorporated into the membrane so as to prevent oxidation of the unsaturated lipid forming said membrane. These processes, however, are not found sufficiently satisfactory, and there has been a growing demand for an advent of a highly efficient process for strengthening the membrane of liposomes.

Summary of the Invention

Extensive studies prosecuted by the present inventors with the view of strengthening the membrane of liposomes, resulted in the finding that the liposome membrane is strengthened by adding an ascorbic acid ester to the liposome constituting lipid, wherein the ascorbic acid ester thus added enters in between the bimolecular lipid layers to strengthen hydrogen bond region as well as hydrophobic bond thereof, and that when the lipid used contains an unsaturated fatty acid, the presence of the ascorbic acid ester thus added prevents oxidation of said lipid and thereby to strengthen the liposome membrane. The present invention has been accomplished on the base of the above finding.

It is an object of the present invention to provide liposomes having strengthened membrane.

Brief Description of the Drawings

In the accompanying drawings, Fig. 1 is a graph showing ethanol resistance of liposomes according to the present invention, and Fig. 2 is a graph showing antioxidation properties of liposomes according to the present invention.

In the graphs of Figs. 1 and 2, respectively, the lines shown below have their respective meanings as indicated.

• X―――――X Composition 1 of the present invention
• ○―――――○ Composition 2 of the present invention
• □―――――□ Composition 3 of the present invention
• control

Detailed Description of the Invention

In the liposomes of the present invention, the membrane constituting the liposome comprises a lipid containing 0.1-20 mol% of an ascorbic acid ester.

Usable as lipids constituting liposome membrane in the present invention are either natural or synthetic lipids, without particular limit, so long as they are capable of forming the liposome. In the case where the lipid contains in the molecule an unsaturated fatty acid, oxidation of said lipid is prevented. Preferably usable as lipids in the present invention are phospholipids, and examples of the pnospholipids which may be used either alone or in combination include lecithin (phosphatidyl choline), phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl serin, phosphatidyl inositol, phosphatidyl glycerol, sphingomyelin, cardiolipin, and those hydrogenated according to the usual method. In particular, phosphatidyl choline or sphingomyelin is preferred among these phospholipids.

Ascorbic acid esters used in the present invention should be fat-soluble, and preferably usable are mono-or diesters of an ascorbic acid with higher fatty acids. Preferably usable higher fatty acids are those having 12 to 22 carbon atoms, particularly 14 to 18 carbon atoms. In particular, palmitic acid and stearic acid are preferred.

The amount of an ascorbic acid ester used is suitably 0.1 to 20 mol%, i.e. the amount of the ester used is 0.1-20 mol per 100 moles of the lipid used. The ascorbic acid esters can be qualitatively analized by thin layer chromatography and determined by liquid chromatography. No strengthening of membrane can be attained if a concentration of the ester used is lower than 0.1 mol%. The use of the ester in a concentration higher than 20 mol% is generally not desirable since physiological activity of ascorbic acid is exhibited simultaneously with strengthening of membrane.

For enhancing the membrane in strength, it is possible to add sterols such as cholesterol to the liposome of the present invention. In order to regulate slow releasing property of medicine kept penned in the present liposome in living body, moreover, it is possible to add charge donating substances, for example, phosphatidic acid, dicetyl phosphate, stearylamine, etc. to the present liposome. Possible destruction of the liposome membrane may be controlled by the presence of these substances mentioned above.

The liposome, per se, of the present invention or those having penned therein various substances are utilized in a wide variety of fields.

For instance, the present liposomes may be used as cell splitting materials, utilizing their affinity for living cellular membrane. Moreover, the present liposomes may be utilized as carriers for medicines which are unstable in vitro or in vivo, or which are desired to be gradually released in living body or to be distributed quickly to specific target organs. As examples of the medicines referred to the above include insulin, heparin, urokinase, ubidecarenone, methotrexate, neomycin, bleomycin, tetracycline, cytochrome C, asparaginase, cytosine arabinoside, etc. In addition thereto, no particular limitation is placed on substances other than medicines which are intended to be carried by the present liposomes so long as they are effectively administered to living body, such as markers, plasmide, DNA, RNA or the like.

The liposomes of the present invention may be prepared by the method known, per se.

For instance, a lipid constituting liposome and an ascorbic acid ester and, if desired, a sterol, a charge donating substance and a fat-soluble medicine, are dissolved in a suitable organic solvent such as chloroform or ethanol. The resulting solution is evaporated to remove the solvent therefrom and thereby to prepare a thin membrane of the lipid. To the thin membrane is added an aqueous solution of a substance which is desired to be incorporated into said thin membrane, followed by vigorous stirring, whereupon there is formed a multilayer liposome. Supersonic treatment of the multilayer liposome gives a o 0 liposome of about 250A - 500A in diameter.

Where the liposome as prepared is intended to use as a medicine retaining liposome, said liposome is washed, if necessary, with physiological saline water or the like, and then formulated into a pellet, suspended preparation, tablet, capsuled preparation, granule preparation, or dust preparation for oral administration purposes, or formulated into an injectable preparation for parenteral administration purposes.

The liposomes of the present invention have membrane comprising a lipid containing 0.1-20 mol% of an ascorbic acid ester. In the liposomes, the membrane is enhanced in its strength because the ascorbic acid ester used enters in between the bimolecular lipid layers of the membrane, whereby hydrogen bond region is strengthened.

Appearance of double concentric circles under a transmission type electron microscope proves that the liposomes comprise bimolecular lipid layers.

Furthermore, in case of the present liposomes having lipids containing unsaturated fatty acids, oxidative degradation of said lipids is prevented by the presence of the ascorbic acid ester used, with the result that the present liposomes are excellent in stability on standing.

The present invention is illustrated more fully below with reference to example and experimental example.

Example

Liposomes having their respective composition as indicated in Table 1 were prepared according to the back phase method (Proc. Natl. Acad. Sci. USA. 75(9). 4194 (1978); Japanese Patent Laid-Open-to-Public Publn. No. 118415/1980).

A solution of predetermined amounts of egg phosphatidyl choline, cholesterol, ascorbic acid esters, sphingomyelin, and α-tocopherol in 20 mℓ of chloroform was placed in a 50 mℓ Erlenmyer's flask. Using a rotary evaporator, the solvent was distilled off to form thin membrane on the inner wall of the flask.

Subsequently, 18 mℓ of diethyl ether was added to the thin membrane in the flask to dissolve the lipid and thereto was added 3 mℓ of a Tyrode's buffer from which Ca , Mg , and glucose had been excluded. Then, after the gaseous phase in the interior of the flask was replaced with nitrogen gas, the flask was irradiated with supersonic wave for about 5 minutes while maintaining the flask at about 20°C. until a uniform suspension was formed. The greater part of the diethyl ether was distilled off at 20-25°C. under reduced pressure (reduced gauge pressure about 400 mmHg) using a rotary evaporator. After mixing the gel-like lipid obtained in the matter now described for 10-15 seconds with Bortex , the diethyl ether was completely removed at 20-25°C. under reduced pressure (reduced gauge pressure about 730 mmHg) therefrom.

The lipid was suspended again in a Tyrode's buffer in the Erlenmyer's flask, from which Ca2+, _Mg²⁺, and glucose had been excluded, and after replacing the gaseous phase in the interior of the flask with nitrogen gas, the flask was irradiated at about 20°C. for 30 minutes with supersonic wave to collect vesicles of the liposome of a single layer or oligolamella structure.

Experimental Example

The liposomes obtained in the above-mentioned Example were tested for ethanol resistance and antioxidation properties.

(1) Ethanol resistance test

The test was carried out in accordance with the method described in S.L.Regen, et. J. Am. Cem. Soc., 102, 6638-6640 (1980).

To 5 mℓ of a 10-time diluted solution of liposomes (diluted with a Tyrode's buffer from which Ca2+, Mg²⁺, and glucose had been excluded) was successively added ethanol in amounts of 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, and 10.0, respectively, and at each time of the addition the diluted solution was measured for turbidity at wavelength of 400 nm. In the graph, showing turbidity on the vertical axis and the amount of ethanol added on the horizontal axis, each measured value was plotted to obtain gradient of the linear regression line between 0-2 mℓ of the amount of ethanol added. The results obtained are shown in Table 2 and Fig. 1. The smaller is the absolute value of gradient, the greater is the ethanol resistance showing a stronger membrane structure.

(2) Antioxidation properties test

The test was carried out according to the method described in "Chemistry of Lipids", compiled by Japan Biochemistry Society, p. 538-539, 1974 (Tokyo Kagaku Dojin).

To 0.5 mℓ of the liposome was added to 2.5 mℓ of 0.2M acetate buffer and was further added 5 mℓ of a 10-4 M 1,1-diphenyl-2-picrylhydrazyl solution (methanol/water = 4:1 mixture), and the resulting mixture was thoroughly stirred. The mixture was incubated at 40-50°C., and absorbance at a wavelength of 517 nm was measured every ten minutes over 60 minutes.

In the graph, showing absorbance on the vertical axis and the warming time on the horizontal axis, each measured value was plotted to obtain gradient of the linear regression line during the warming time of from 10 to 60 minutes. The results are shown in Table 3 and Fig. 2. The smaller is the absolute value of gradient, the higher is the antioxidation properties.

As is clear from Tables 2 and 3, and from Figs. 1 and 2, the ascorbic acid esters show a greater effect of strengthening the membrane of liposomes than sphingomyelin in the ethanol resistance test, and have the effect of improving antioxidation properties of liposomes equal to that of a-tocopherol.