Preparation of liposomes with peg-bound phospholipid on surface

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BACKGROUND OF THE INVENTION

1. Technical Field

The present invention relates to agents for inhibiting adsorption of proteins on the liposome surface.

Further, the invention relates to agents for preventing liposome agglutination.

Furthermore, the invention is concerned with liposomes on which adsorption of proteins is inhibited and which are agglutination-free and a method for preparing the same.

2. Prior Art

Use of liposomes as a carrier for water-soluble or fat-soluble drugs has widely been attempted (Gregoriadis, et al., Ann. N.Y. Acad. Sci., 446, 319 (1985)). Use of liposomes as artificial erythrocytes by incorporating hemoglobin, the oxygen carrier for animals, in the inner aqueous space of liposomes has also been attempted (Japanese Patent Application Laid-Open to Public 178521/1987). Liposome membrane-constituting materials of the liposomes used in these attempts, however, were those composed only of natural or synthetic lipids such as phospholipids and cholesterol.

In order to use liposomes as a carrier for drugs it is necessary to introduce the liposomes into blood vessels in the living body. However, the liposomes composed only of lipids which were conventionally employed were encountered with problems of adsorbing plasma-constituting proteins of the living body (for example, albumin, globulin and fibrinogen) which results in mutual agglutination of the liposomes. The problems were considerable especially of the liposomes which particle size exceeds 0.1 .mu.m. Particle size of the liposomes generally employed is usually 0.1 .mu.m-1 .mu.m. That particle size will not be an obstacle in passing through the blood vessels in the living body because the capillary blood vessels have inner diameter as large as several .mu.m. However, if the liposomes are agglutinated by adsorbing plasma-constituting proteins, the size of the agglutinates becomes tens of micrometers. If the agglutination occurs in the blood vessel, agglutinates of the liposomes will plug the blood vessel to inhibit blood flow possibly causing death of the living body.

Particularly when liposomes are used as artificial erythrocytes, a large dose of liposomes should be administered so that the problem of liposome agglutination in plasma was not negligible. Heretofore, however, there has been developed no technique at all for preventing the agglutination of liposomes in plasma.

In addition, when liposomes are introduced into the living body, antibody protein (immunoglobulin) to the liposome which is an antigen will be adsorbed on the liposomes to produce foreign body recognition in the phagocytes (macrophage) with a result that the liposomes will be included in the macrophage and disappear within a short period of time. Therefore, inhibition of the protein adsorption on liposomes could also delay disappearance of the liposomes in plasma.

It is also noted that the hemoglobin concentration in natural erythrocytes is approximately 30%; as the volume ratio of erythrocytes to the whole blood (hematocrit) is approximately 50%, the hemoglobin concentration in the whole blood is approximately 15%. Accordingly, in the case of artificial erythrocytes which are formed by enclosing hemoglobin in the liposome smaller in particle size than natural erythrocytes, the volume ratio of artificial erythrocytes in an artificial erythrocyte suspension will exceed 50% when the hemoglobin concentration in the artificial erythrocyte suspension is 15%, unless an aqueous solution of hemoglobin with a hemoglobin concentration of 30% or more is subjected to liposome formation. Such suspension, which is poorly fluidized, will produce adverse effects upon circulatory dynamism when administered. In this respect, it is desirable to encapsulate a large amount of hemoglobin in the inner aqueous space of liposomes using lipid in an amount as small as possible. In other words, a method for preparing artificial erythrocytes with a high encapsulation efficiency is desirable. By the dialysis method or the reverse phase method, however, it is difficult to form liposomes of an aqueous solution of hemoglobin with a higher hemoglobin concentration (30% or more) and a higher viscosity. Also by the lamina method in which a liposome-forming lipid is uniformly dissolved in an organic solvent, then the organic solvent is removed and an aqueous solution is added to the lamina of the lipid thus formed to a dispersion, the hydration and dispersion cannot easily be accomplished by the addition of an aqueous solution because the liposome-forming lipid after removal of the organic solvent has been solidified or nearly in loss of fluidity. When the aqueous solution is an aqueous solution of hemoglobin with a high concentration, the proportion of the water combined with the globin protein is high, and the amount of the free water available for hydration of the lipid is small. Thus, liposome formation at a high efficiency was difficult. Therefore, an object of the invention is to provide agents for inhibiting adsorption of proteins on the liposome surface, agents for preventing liposome agglutination, liposomes on which adsorption of proteins in plasma is inhibited and a method for preparing the same. A further object of the invention is to provide a method for preparing artificial erythrocytes comprising forming liposomes of a highly-concentrated hemoglobin at a high efficiency.

SUMMARY OF THE INVENTION

As a result of extensive studies in order to achieve the above-mentioned objects we have found that adsorption of proteins in plasma on the surface of liposomes can be prevented by incorporating a specific agent for inhibiting adsorption of proteins into lipid layer of the liposome, eventually preventing agglutination of the liposomes to each other and further facilitating hydration of the lipid even when artificial erythrocytes are prepared with an aqueous solution of hemoglobin at a high concentration thereby enabling formation of liposomes of a highly-concentrated hemoglobin at a high efficiency. The present invention was completed on the basis of the above findings.

According to the invention, there are provided agents for inhibiting adsorption of proteins on the liposome surface, agents for preventing agglutination of liposomes, liposomes containing these agents and a method for preparing the same as described below.

1) An agent for inhibiting adsorption of proteins on the liposome surface comprising a compound having a hydrophobic moiety at one end and a hydrophilic macromolecular chain moiety at the other end.

2) An agent for inhibiting adsorption of proteins on the liposome surface according to item 1 wherein the hydrophobic moiety and the hydrophilic macromolecular chain moiety are covalently bound.

3) An agent for inhibiting adsorption of proteins on the liposome surface according to item 1 wherein degree of polymerization for the hydrophilic macromolecular chain moiety is 5-1000 moles.

4) An agent for inhibiting adsorption of proteins on the liposome surface according to item 1 wherein the hydrophilic macromolecular chain moiety consists of polyethylene glycol.

5) An agent for inhibiting adsorption of proteins on the liposome surface according to item 1 wherein the hydrophobic moiety and the hydrophilic macromolecular chain moiety are bound via an ether bond.

6) An agent for inhibiting adsorption of proteins on the liposome surface according to item 5 wherein the hydrophilic macromolecular chain moiety is bound with an alcoholic radical of a long chain-aliphatic alcohol, a sterol, a polyoxypropylene alkyl or a glycerin fatty acid ester.

7) An agent for inhibiting adsorption of proteins on the liposome surface according to items 1-4 wherein the hydrophilic macromolecular chain moiety is bound with the hydrophilic group of a phospholipid.

8) An agent for inhibiting adsorption of proteins on the liposome surface according to item 7 wherein the phospholipid is phosphatidylethanolamine.

9) An agent for inhibiting adsorption of proteins on the liposome surface according to item 7 wherein the bond is formed via a triazine ring.

10) An agent for inhibiting adsorption of proteins on the liposome surface according to item 7 wherein the bond is formed via an amide bond.

11) A liposome in which the hydrophobic moiety of the agent for inhibiting adsorption of proteins on the liposome surface according to items 1-10 is fixed to the liposome membrane-constituting lipid layer and the hydrophilic macromolecular chain moiety externally extends from the liposome surface.

12) A liposome according to item 11 wherein hemoglobin is enclosed within the liposome.

13) A method for preparing liposomes on which adsorption of proteins is inhibited which comprises adding an agent for inhibiting adsorption of proteins on the liposome surface according to items 1-10 to a liposome suspension and then collecting the liposomes from said suspension.

14) A method for preparing liposomes on which adsorption of proteins is inhibited which comprises uniformly mixing an agent for inhibiting adsorption of proteins on the liposome surface with a liposome membrane-forming lipid and forming liposomes using the mixture thus obtained.

15) A liposome on which adsorption of proteins is inhibited comprising one end of a hydrophilic macromolecular chain moiety directly bound with a liposome membrane-constituting lipid and the other end externally extending from the liposome surface.

16) A method for preparing liposomes on which adsorption of proteins is inhibited which comprises adding a hydrophilic macromolecular compound activated so as to bind with a liposome membrane-constituting lipid to a liposome suspension and allowing it to react in such a way that one end of the hydrophilic macromolecule is bound with the liposome membrane-constituting lipid and the other end is extended externally from the liposome surface.

DETAILED DESCRIPTION OF THE INVENTION

The agents for inhibiting adsorption of proteins on the liposome surface or the agents for preventing agglutination of liposomes in the present invention are compounds which have a hydrophobic moiety at one end and a hydrophilic macromolecular chain moiety at the other end.

As preferred examples of the hydrophobic moiety are mentioned alcoholic radicals of a long chain aliphatic alcohol, a sterol, a polyoxypropylene alkyl or a glycerin fatty acid ester and phospholipids. As preferred examples of the hydrophilic macromolecular chain moiety are mentioned polyethylene glycols.

Especially preferable in the invention are non-ionic surface-active agents of PEG addition type in which a polyethylene glycol (called PEG hereinbelow) and an alcoholic radical of the hydrophobic moiety are bound by ether bond or PEG-bound phospholipids in which PEG and a phospholipid are covalently bound.

The polyethylene glycol-bound phospholipid in the invention is a molecule of such a structure that polyethylene glycol (PEG) is covalently bound with the hydrophilic moiety (polar head) of a phospholipid which contains one or more PEG chains per molecule. The end of the PEG chain that has not been bound with the phospholipid may also be a hydroxyl group or an ether with a short chain such as with methyl or ethyl or an ester with a short chain such as with acetic acid or lactic acid.

In order to achieve the objects of the invention, PEG chain length in the PEG-bound phospholipid molecule is desirably in the range of 5-1000 moles, more preferably 40-200 moles in terms of the average degree of polymerization. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will hardly be produced. Beyond the above-defined range, water-solubility of the PEG-bound phospholipid will be too high to be readily fixed inside the liposome membrane.

In order to produce a covalent bond between PEG and a phospholipid a reaction-active functional group is necessary at the polar moiety of the phospholipid. The functional group includes amino group of phosphatidylethanolamine, hydroxyl group of phosphatidylglycerol, carboxyl group of phosphatidylserine and the like; the amino group of phosphatidylethanolamine is preferably used.

For the formation of a covalent bond between the reaction-active functional group of a phospholipid and PEG are mentioned a method employing cyanuric chloride, a method employing a carbodiimide, a method employing an acid anhydride, a method employing glutaraldehyde and the like. The method employing cyanuric chloride (2,4,6-trichloro-striazine) is preferably used for binding the amino group of phosphatidylethanolamine with PEG. For example, treatment of monomethoxypolyethylene glycol and cyanuric chloride by known reaction procedures affords 2-O-methoxypolyethylene glycol-4,6-dichloro-s-triazine (activated PEG1 ) or 2,4-bis-(O-methoxypolyethylene glycol)-6-chloro-s-triazine (activated PEG2) {Y. Inada, et al., Chem. Lett., 7, 773-776 (1980)}. Binding of these with the amino group by a dehydrochloric acid condensation reaction yields a phospholipid with PEG covalently bound with the polar head of phosphatidylethanolamine. In the above reaction there is contained one PEG chain in one phospholipid molecule when employing activated PEG1 and two PEG chains with activated PEG2. Phospholipids bound with PEG via an amide bond is also produced by reacting monomethoxy PEG with succinic anhydride to introduce a carboxyl group into the end of the PEG and reacting the product with phosphatidylethanolamine in the presence of a carbodiimide.

In order to prepare a liposome with the PEG-bound phospholipid of the invention contained in the lipid layer, a PEG-bound phospholipid may uniformly be mixed with a liposome-forming lipid in advance, and the lipid mixture may be treated by a conventional method to form liposomes. The liposome-forming lipids as herein referred to contain as the main component phospholipids obtained from natural materials such as egg yolk and soybean or those which are produced by organic chemical synthesis used alone or in combination. Representative are phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. In addition, sterols such as cholesterol and cholestanol as a membrane-stabilizing agent, phosphatidic acid, dicetyl phosphate and higher fatty acids as a charged substance and other additives may be added. Mixing ratio of the PEG-bound phospholipid with the liposome-forming lipid is 0.1-50 mol %, preferably 0.5-20 mol % and more preferably 1-5 mol % in terms of the molar ratio to the phospholipid of the main component. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will not be sufficiently high. Beyond the above-defined range, solubilizing capacity of the PEG-bound phospholipid will cause instability of the liposome.

In effecting in advance uniform mixing of the liposome-forming lipid with the PEG-bound phospholipid, for example, the two may be dissolved in a volatile organic solvent and then the organic solvent can be removed by evaporation. If a fat-soluble drug is to be contained in the liposome, it may be mixed with the liposome-forming lipid during the above procedures. Formation of liposomes from the mixed lipids thus obtained may be carried out according to liposome formation methods usually employed. For example, any of such methods as shaking, sonication and French pressure cell may be employed. Liposomes of particle sizes between 0.1 .mu.m and 1 .mu.m are produced allowing for carrying a sufficient amount of a water-soluble drug or physiologically active substance in the inner aqueous space, provided that the above-mentioned PEG-bound phospholipid is used within the above-defined ranges. The PEG-bound phospholipid is contained in the lipid layer of liposomes thus obtained, but the content is not necessarily the same as that based upon the proportion originally mixed with the lipid. If the water solubility of the PEG-bound phospholipid is high, part of it will possibly be eluted into the aqueous phase outside the membrane. Although the form of the PEG-bound phospholipid present in the lipid membrane of the liposome is not clear, it is believed that the hydrophobic moiety of the PEG-bound phospholipid is present in the hydrophobic region of the liposome membrane, and the hydrophilic PEG chain is present from the hydrophilic region in the membrane over to the aqueous medium outside the membrane. It follows therefore that the PEG chain of the PEG-bound phospholipid in the liposome obtained by the method of the invention is present in both of the outer aqueous phase and the inner aqueous space of the liposome.

The PEG-bound phospholipid of the invention need not necessarily give a clear solution when dissolved in water. However, if the PEG-bound phospholipid of the invention is uniformly dissolved in water, the liposome of the invention may also be prepared by an alternative method. As a matter of fact, liposomes containing the PEG-bound phospholipid in the lipid layer may also be prepared as follows: To a suspension of liposomes carrying a water-soluble or fat-soluble drug or the like (which have been prepared by a conventionally employed liposome formation method) is added the PEG-bound phospholipid of the invention either as it is or in aqueous solution. In this case, the PEG-bound phospholipid appears to be in dispersion in the form of micelle-like molecular aggregates in the aqueous solution. When liposomes are co-existent in the dispersion, the hydrophobic moiety in the PEG-bound phospholipid molecule is fixed in the hydrophobic region in the liposome membrane by hydrophobic interaction thereby taking a structure in which the hydrophilic PEG chain is exposed on the surface of liposomes on the side of the outer aqueous phase only.

Addition of the PEG-bound phospholipid in aqueous solution may be made at the critical micelle concentration or higher. At a lower concentration, however the, amount of the phospholipid adsorbed on the liposome will not be sufficient to maintain the effect of preventing agglutination of liposomes in plasma. At a concentration which is too high, the liposome will be so unstable as eventually to cause leakage of the water-soluble drug or the like present in the inner aqueous space. Therefore, the concentration is preferably 0.01-20%, more preferably 0.05-20% in terms of the concentration in the liposome suspension.

Liposomes containing the PEG-bound phospholipid in the lipid layer can also be prepared by an alternative method. As a matter of fact, liposomes containing a phospholipid with a reaction-active functional group are prepared by a conventional method, and subsequently a PEG activated at one end is added to the outer solution of the liposomes to allow for binding with the phospholipid. For example, liposomes containing 1-50 mol % of phosphatidylethanolamine in the whole phospholipid are prepared, activated PEG2 in a basic buffer solution (pH 9 or higher) is added at a concentration of 1-20% and the mixture is allowed to react at room temperature for 1-24 hours. There is formed a structure in which the hydrophilic PEG chain is exposed on the surface on the side of the outer aqueous phase of the liposomes.

The non-ionic surface-active agent of polyoxyethylene ether addition type as referred to in the invention is a non-ionic surface active agent having a molecular structure that contains a polyoxyethylene chain as the hydrophilic moiety and in which the polyoxyethylene chain is bound with an alcoholic radical of the lipophilic (hydrophobic) moiety by an ether bond. It includes, for example, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene alkylphenyl ethers, polyoxyethylene polyoxypropylene block polymers, polyoxyethylene polyoxypropylene alkyl ethers, polyoxyethylene glycerin fatty acid esters, polyoxyethylene sorbitan fatty acid esters and the like.

Among non-ionic surface active agents of polyoxyethylene addition type, a non-ionic surface active agent of polyoxyethylene ester addition type that has a molecular structure in which the polyoxyethylene chain is bound with the lipophilic moiety by ester bond is contained in the lipid layer of liposomes will produce a low effect in inhibiting adsorption of proteins in plasma and preventing agglutination of liposomes.

In order to achieve the objects of the invention the polyoxyethylene chain length in the non-ionic surface active agent of polyoxyethylene ether addition type is desirably in the range of 5-1000 moles, more preferably 10-40 moles in terms of the average degree of polymerization of ethylene oxide. Below the above-defined range, the effect of preventing agglutination of liposomes in plasma will hardly be developed. Beyond the above-defined range, water solubility of the non-ionic surface active agent will become too high to be readily fixed in the liposome membrane.

Among a variety of non-ionic surface active agents of polyoxyethylene ether addition type, polyoxyethylene alkyl ethers, polyoxyethylene sterol ethers, polyoxyethylene polyoxypropylene alkyl ethers and polyoxyethylene glycerin fatty acid esters are particularly effective in producing liposomes of high protein adsorption-inhibitory and agglutination-preventive effects when contained in the lipid layer of liposomes.

Polyoxyethylene alkyl ethers have a structure in which a polyoxyethylene and a saturated or unsaturated aliphatic alcohol are bound by an ether bond. Aliphatic alcohols having 8-22 carbon atoms are preferably employed.

The polyoxyethylene sterol ethers are compounds having a molecular structure in which a polyoxyethylene and a sterol are bound by an ether bond. The sterol includes animal sterols (zoosterols) such as cholesterol and cholestanol, plant sterols (phytosterols) such as sitosterol and stigmasterol and fungal sterols (mycosterols) such as ergosterol and zymosterol. Although it is not necessary to specify the nature of the sterol in the polyoxyethylene sterol esters, those which have the same structure in the side chain as that of cholesterol are preferably used.

The polyoxyethylene polyoxypropylene alkyl ethers have a molecular structure in which a polyoxypropylene is added to a saturated or unsaturated aliphatic alcohol by an ether bond, and to the end hydroxyl group of the polyoxypropylene is further added a polyoxyethylene by ether bond. Average degree of polymerization for the polyoxypropylene is preferably 2-8, and aliphatic alcohols having 8-22 carbon atoms are preferably employed.

The polyoxyethylene glycerin fatty acid esters have a molecular structure in which a polyoxyethylene is added to the free hydroxyl group of a glycerin fatty acid ester (monoglyceride or diglyceride). Either saturated or unsaturated fatty acids having 8-22 carbon atoms are preferably employed.

In order to prepare a liposome containing the non-ionic surface active agent of polyoxyethylene ether addition type in the lipid layer according to the invention, a non-ionic surface active agent of polyoxyethylene ether addition type may uniformly be mixed with a liposome-forming lipid in advance, and the lipid mixture may be treated by a conventional method to form liposomes. The liposome-forming lipid as herein referred to contains as the main component phospholipids obtained from natural materials such as egg yolk and soybean or those which are produced by organic chemical synthesis used alone or in combination. Representative are phosphatidylcholine, sphingomyelin, phosphatidylethanolamine and phosphatidylserine. In addition, sterols such as cholesterol or cholestanol as a membrane-stabilizing agent, phosphatidic acid, dicetyl phosphate and higher fatty acids as a charge-providing substance and other additives may be added. The mixing ratio of the non-ionic surface active agent of polyoxyethylene ether addition type with the liposome-forming lipid is 0.5-20 moles, preferably 1-5 moles of ethylene oxide unit per mole of the phospholipid of the main component. For example, when dipalmitoylphosphatidylcholine (molecular weight 752) as the phospholipid and polyoxyethylene phytostanol ether with an average degree of polymerization of 25 for ethylene oxide (molecular weight ca. 1500) as the non-ionic surface active agent of polyoxyethylene ether addition type are used, the molar ratio of the non-ionic surface active agent of polyoxyethylene ether addition type is 0.02-0.8 moles, preferably 0.04-0.2 moles per mole of the phospholipid, and the weight ratio is 0.04-1.6 parts by weight, preferably 0.08-0.4 parts by weight of the non-ionic surface active agent of polyoxyethylene ether addition type per part by weight of the phospholipid. Below the above-defined range, the effect of preventing