CA1062556A - Liposomes preparation - Google Patents
Liposomes preparationInfo
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- CA1062556A CA1062556A CA243,293A CA243293A CA1062556A CA 1062556 A CA1062556 A CA 1062556A CA 243293 A CA243293 A CA 243293A CA 1062556 A CA1062556 A CA 1062556A
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Abstract
ABSTRACT OF THE DISCLOSURE
Synthetic liposomes, for example containing a biologically active substance, are prepared by dispersing a first aqueous liquid with the aid of ultrasonic vibration in a water-immiscible carrier liquid less dense than water in the presence of a compound of the formula XY where X is a hydrophilic polar group and Y is a hydrophobic non-polar group to form dispersed globlules of the first aqueous liquid bounded by a monomolecular layer of the compound XY; adding this dispersion to a second aqueous liquid to form a two layer system bounded by a monomolicular layer of the compound XY and centrifuging the system to force the dispersed globules through the boundary layer of the compound XY and into the second aqueous liquid.
Synthetic liposomes, for example containing a biologically active substance, are prepared by dispersing a first aqueous liquid with the aid of ultrasonic vibration in a water-immiscible carrier liquid less dense than water in the presence of a compound of the formula XY where X is a hydrophilic polar group and Y is a hydrophobic non-polar group to form dispersed globlules of the first aqueous liquid bounded by a monomolecular layer of the compound XY; adding this dispersion to a second aqueous liquid to form a two layer system bounded by a monomolicular layer of the compound XY and centrifuging the system to force the dispersed globules through the boundary layer of the compound XY and into the second aqueous liquid.
Description
lO~Z5S6 The present invention is concerned with the preparation of synthetic liposomes.
~iposomes are fatty or oily globules occurring in cytoplasm. ~he term "synthetic liposomes" refers to microscopic globules, having a maximum diameter of the order of 10,000 A and preferably having a diameter between 300 and 2,000 A, bounded by a wall formed by at least one bimolecular layer (having a thickness of the order ~,200 A) of a compound of the general formula XY, where X is a hydrophilic polar group and Y is a hydrophobic non-polar group, the globules containing an aqueous liquid, for example an aqueous solution of at least one biologically active substance, and existing generally in the form of a collodial dispersion in an aqueous medium such as an aqueous saline solution, in particular a 0.9% by weight sodium chloride solution.
~he preparation of liposomes provides a method of encapsulation which is most practical and effective for aqueous liquids a~d which is particularly useful for administration of biologically active substances, particularly medicaments, into living organisms, while a~oiding the destruction or inactivation of the substance in the organism, for example by the action of gastric or intestinal juices, before the substances reach the site where they are required to act.
1(~6Z556 By selection of the compound of formula XY
used to-form the wall of the liposomes, it is possible to produce liposomes having walls which resist the activity of certain zones in the organism and are only --attacked in the presence of partic-ular agent~ which only exist in the organs where the biologically active substance is to be liberated.
~wo processes for the preparation of liposomes are known.
One of these processes consists of placing a lipid in contact with an a~ueous liquid which it is wished encapsulate and then warming the heterogeneous mixture ~ -thus obtained at a temperature slightly above ambient temperature and then submitting the mixture to vigorous agitation following ultrasonic vibration.
~he other process consists of dissolving a compound of formula XY (where ~ and Y are as defined above), for example a lipid, in a volatile solvent, forming a film of the compound on the walls of a receptacle by evaporating the solvent from the solution thus obtained, introducing in the same receptacle the liquid which is wished to encapsulate in the liposomes, and finally submitting the li~uid in the receptacle to the action of ultrasonic vibrations.
1~62556 ~ he two processes thus require the use of a total volume of the llquid which it is desired to encapsulate very much larger than the volume of that liquid which is finally contained in the liposomes produced by the process.
Aeeording to these processes, the liposomes are formed in effeet in the state of a eollodial dispersion of globules in a li~uid phase which eomprises the fraction of the liquid to be encapsulated which has not been retained in the interior of the liposomes. ~he ratio of the volume of encapsulated liquid in the interior of the liposomes to the total volume of the surrounding liquid is in general of the order of 1 to 10%.
.
In eonsequence, if the liquid to be encapsulated - has a high value, as is the case most generally met when the liquid is a solution of a biologically active substance, it is necessary to reeover ~he fraction of that liquid whieh has not been eneapsulated before using it in further operations to form liposomes. This recovery requires the separation of the liposomes from the liquid then purification of the liquid itself and, usually, the readjustment of the eoneentration of the aetive substance. In practice the separation and purification steps require the use of large volumes of solvents and in eonsequenee the eoneentration of the liquid containing the active substanee has to be
~iposomes are fatty or oily globules occurring in cytoplasm. ~he term "synthetic liposomes" refers to microscopic globules, having a maximum diameter of the order of 10,000 A and preferably having a diameter between 300 and 2,000 A, bounded by a wall formed by at least one bimolecular layer (having a thickness of the order ~,200 A) of a compound of the general formula XY, where X is a hydrophilic polar group and Y is a hydrophobic non-polar group, the globules containing an aqueous liquid, for example an aqueous solution of at least one biologically active substance, and existing generally in the form of a collodial dispersion in an aqueous medium such as an aqueous saline solution, in particular a 0.9% by weight sodium chloride solution.
~he preparation of liposomes provides a method of encapsulation which is most practical and effective for aqueous liquids a~d which is particularly useful for administration of biologically active substances, particularly medicaments, into living organisms, while a~oiding the destruction or inactivation of the substance in the organism, for example by the action of gastric or intestinal juices, before the substances reach the site where they are required to act.
1(~6Z556 By selection of the compound of formula XY
used to-form the wall of the liposomes, it is possible to produce liposomes having walls which resist the activity of certain zones in the organism and are only --attacked in the presence of partic-ular agent~ which only exist in the organs where the biologically active substance is to be liberated.
~wo processes for the preparation of liposomes are known.
One of these processes consists of placing a lipid in contact with an a~ueous liquid which it is wished encapsulate and then warming the heterogeneous mixture ~ -thus obtained at a temperature slightly above ambient temperature and then submitting the mixture to vigorous agitation following ultrasonic vibration.
~he other process consists of dissolving a compound of formula XY (where ~ and Y are as defined above), for example a lipid, in a volatile solvent, forming a film of the compound on the walls of a receptacle by evaporating the solvent from the solution thus obtained, introducing in the same receptacle the liquid which is wished to encapsulate in the liposomes, and finally submitting the li~uid in the receptacle to the action of ultrasonic vibrations.
1~62556 ~ he two processes thus require the use of a total volume of the llquid which it is desired to encapsulate very much larger than the volume of that liquid which is finally contained in the liposomes produced by the process.
Aeeording to these processes, the liposomes are formed in effeet in the state of a eollodial dispersion of globules in a li~uid phase which eomprises the fraction of the liquid to be encapsulated which has not been retained in the interior of the liposomes. ~he ratio of the volume of encapsulated liquid in the interior of the liposomes to the total volume of the surrounding liquid is in general of the order of 1 to 10%.
.
In eonsequence, if the liquid to be encapsulated - has a high value, as is the case most generally met when the liquid is a solution of a biologically active substance, it is necessary to reeover ~he fraction of that liquid whieh has not been eneapsulated before using it in further operations to form liposomes. This recovery requires the separation of the liposomes from the liquid then purification of the liquid itself and, usually, the readjustment of the eoneentration of the aetive substance. In practice the separation and purification steps require the use of large volumes of solvents and in eonsequenee the eoneentration of the liquid containing the active substanee has to be
2~ adjusted.
lO~Z556 ~he necessity of carrying out the steps of purification and the readjustment of the concentra-tion of the liquid containing the ac-tive substance render these two processes difficult to put in-to practice on an industrial scale.
he present invention has as an object an improved p^rocess for the preparation of liposomes, using for each preparation only that volume of liquid which is to be encapsula-ted.
According to the present invention there is provided a process for the preparation of synthetic liposomes, characterised in that a first aqueous liquid is dispersed with the aid of ultrasonic ~-ibration in a carrier liquid which is insoluble in water or only slightly soluble in water and has a density less than that of water, in the presence of at least one compound of the general formula XY, where X
represents a hydrophilic polar group and Y represents a hydrophobic non-polar group, so as to form a colloidal dispersion of the first aqueous liquid in the carrier liquid, the dispersed phase being bounded by a monomolecular film of the compound of the general formula XY; this dispersion is combined with the second liquid to form a heterogeneous two layer system comprising an upper liquid layer formed by the dispersion and a lower liquid layer formed by the second aqueous liquid, the two layers being separated by the monomolecular film of the compound of formula XY; and then the two layer system is centrifuged at an angular velocity sufficient for the 1(~6Z556 dispersed globules of the first a~ueous li~uid to be forced by gravity through the monomolecular separa-ting film of the compound XY and into the lower la~er of the second aqueous liquid.
Thus the process comprises two steps In the first step a dispersion of globules of the liquid to be encapsulated is formed under the action of ultrasonic vibrations, these globules having colloidal dimensions (i.e. a diameter of the order of 200 to 1,000 A) in a liquid which is either insolu~le in water or only slightly soluble in water. These globules are bounded by a mono-molecular film of the compound XY in which the hydrophilic groups X are turned towards the interior of the globules which is occupied by the aqueous liquid and the hydrophobic groups Y are turned towards the outside of the globules which is in contact with a non-aqueous phase These globules, although they do not properly speaking constitute liposomes, since they are not bounded by a bimolecular layer of the compound XY but only by a monomolecular film of that compound, can nevertheless be regarded as prototype liposomes, each of them containing the same volume of the liquid to be encapsulated as the liposomes finally obtained. In the following description, these globules are referred to liposome precursors.
By choosing suitable relative proportions of the aqueous liquid to be encapsulated and the carrier liquid and the compound of formula ~Y in this first part of the process it is possible to obtain encapsulation in the liposome precursors of the whole of the liquid to be encapsulated. ~he process according to the present - invention thus avoids the need for recovery, purification or readjustment of the concentration, which, as indicated above, are necessary in the operation of the known processes.
It will also be understood that the process according to the invention allows the encapsulation by the formation of liposomes, of liquias of which only a very small quantity is available, for example quanti-ties of the order of 0.05 to 0.1 millilitre, which would be insufficient for use in the processes described above.
~ hus the process according to the invention has applications in fields, such as certain laboratory research or analytical procedures, in which the other processes are inapplicable.
~he second step of the process consists of forming the liposomes proper from the liposome precursors. It may be supposed that this formation results from the fact that, in passing through the monomolecular film of the compound XY at the interphase between the upper liquid !
and the lower liquid, each li.posome precursor e~*rains a par.t of the film which becomes associated with the monomolecular film of compound XY which surrounds the . precursor and thus forms a bimolecular film of the compound XY characteristic of liposomes. ~he monomolecular film of the compound XY exists at the interface be-tween the upper and lower liquids because of the fact -that the hydrophilic groups X are attracted to the aqueous liquid layer whlle the hydrophobic groups Y remain embedded in the non-aqueous layer.
~he compound of formula XY may, for example, comprise a compound i.n which the hydrophilic group X
is one of the following groups: phospha-to, carboxylic, sulphato, amino, hydroxyl and choline and in which the hydrophobic group Y is one of the following groups:
a saturated or unsaturated alipkatic hydrocarbon group (for example an alkyl or alkYlene group), and an aliphatic hydrocarbon group substituted by at least one aromatic or cycloaliphatic group Preferably, a phospholipid or a substance closely related to phospholipids is used as a compound of formula XY
In particular the following compounds are useful:
lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, ~06Z556 phosphatidic acid and the cerebrosides.
It is possible also to use as the compound of formula XY a mixture of at least one phospholipid and at least one other lipid belonging to a category different -from the phospholipids In particular the following eompounds may be used: stearylamine, dicetyl phosphate, eholesterol and tocopherol.
~he carrier liquid having only a small solubility in water or being insoluble in water is preferably an organic liquid, partieularly one of the following eompounds: benzene, a halo-benzene or an alkyl benzene, an aliphatie ether, a aliphatic ketone, an aliphatic aldehyde, an aliphatic ester, an aliphatic hydrocarbon or a eyeloaliphatie hydrocarbon or a mixture thereof having a density less than that of water ~he selection of the first aqueous li~uid, i.e.
the liquid to be eneapsulated in the liposomes is limited B only ~ the required utility for the liposomes.
In particular, it is possible to use a solution of at least one biologically active substance, such as an enzyme, or a soIution of a medicament such as an antibiotie.
~he second aqueous liquid may be pure water or any other l~ZSS6 aqueous liquid apprcpriate For -preference the second a~ueous liquid is the liquid which is required to be the carrier for the dispersion of the liposomes when they are used, for example an a~ueous solution of sodium chloride. In particular an a~ueous sodiu~ chloride solution known as physiological serum or physiological saline,having a concentration of 0 15 mole of sodium chloride per litre (0 9% by weight) can be used in order to obtain directly in the second step of the process a liposome dispersion in a medium which is injectible in the human body. It is thus a further advantage of the process of the invention as compared with the known processes for the preparation of liposomes, tha-t it is possible to obtain directly a suspension of liposomes in an aqueous medium chosen with regard to the final use of the liposomes.
It is to be understood, however, that it is equally possible to sep æ ate the liposomes from the second a~ueous li~uid, for example if it is wished to remove every trace of the active substance which has not been encapsulated, before the final utilisation of the liposomes ~his separation may be easily effected by any appropriate known method, for example by gel chroma-tography ~he following ~xamples illustra-te the invention lO~Z556 Example 1 .
An a~ueous solution of amyloglucosidase containing 10 mg of amyloglucosidase per millilitre of solution is encapsulated in an aqueous sodium chloride solu-tion (0.15 mole per litre) in the following manner:
~ecithin (54 mg) and the aqueous solution of amyloglucosidase (0.1 ml) are added to dibutyl ether (3 ml~ and *he heterogeneous mixture thus obtained is subjected to ultrasonic (or near-ultrasonic) vibration (17 kHz freque~cy: output: 70 Watts) for 2 minutes while maintaining a temperature less than 30C by means of a cooling bath.
A transparent liquid, apparently homogeneous and having a bluish reflection, is obtained. A layer f this liquid is placed in a centrifuge tube on a layer o 1.5 ml of an aqueous solution of sodium chloride (0.15 mole per litre). ~here is thus obtained a 2-phase "mixture"
- comprising a lower layer formed by an aqueous phase (the sodium chloride solution) and an upper layer formed by the organic phase obtained in the first step of the process by ultrasonic vibration of the mixture of dibutyl ether, lecithin and the amylglucosidase solution.
~he 2-phase mixture is then centrifuged at 30,000 rev/min for 30 minutes. ~he upper layer (organic phase) is then .
lO~Z556 removed and the lower layer (aqueous phase) is submitted to a further centrifugation a-t ~0,000 rev/min for 30 minutes A faintly bluish clear liquid is obtained together with a very small amount of residue comprising non-dispersed lipid (lecithin), which is rejected. ~he clear liquid comprises a suspension of liposomes (having collodial dimensions) containing the aqueous amyloglucosidase solution, in a 0.15 M sodium chloride solution, and also contains a small quantity of non-encapsulated amyloglucosidase.
L0 ~ Depending on how the suspension is to be used, it is possible to use the suspension as formed or after elimination of non-encapsulated amyloglucosidase by gel chromatography (for example on a sepharose (dextran) gel).
L5 ~ æ~
A buffered solution (phosphate buffer 10 mM, pH
7.2) containing 100 mg/ml penicillamine (0.05 ml) is encapsulated in a 0.15 M aqueous solution of sodium chloride, using for the formation of the organic phase subjected to ultrasonic vibration lecithin (27 mg) and a mixture of dibutyl ether (2.4 ml) and chloroform (0.6 ml). ~he process is carried out in the same way as in ~xample 1.
106255~i Example 3 In a method similar to that of Example 1, an aqueous solution containing 300 mg/ml imipramine (0.1 ml) is encapsulated in a 0,15 M a~ueous solution of sodium chloride, using a mixture of lecithin (25 mg) and cholesterol (40 mg) - in dibutyl ether (3 ml).
Example 4 By a method similar to that of E~ample 1, an aqueous solution of 150 mg/ml betamethasone disodium phosphate (0.05 ml) is encapsulated in a 0.15 M aqueous solution of _sodium chloride using a mi~ture of lecithin (15 mg) and phosphatidylethanolamine (12 mg) is a mixture of dibutyl ether (2.5 ml) and chloroform (0.5 ml) in the first step.
lO~Z556 ~he necessity of carrying out the steps of purification and the readjustment of the concentra-tion of the liquid containing the ac-tive substance render these two processes difficult to put in-to practice on an industrial scale.
he present invention has as an object an improved p^rocess for the preparation of liposomes, using for each preparation only that volume of liquid which is to be encapsula-ted.
According to the present invention there is provided a process for the preparation of synthetic liposomes, characterised in that a first aqueous liquid is dispersed with the aid of ultrasonic ~-ibration in a carrier liquid which is insoluble in water or only slightly soluble in water and has a density less than that of water, in the presence of at least one compound of the general formula XY, where X
represents a hydrophilic polar group and Y represents a hydrophobic non-polar group, so as to form a colloidal dispersion of the first aqueous liquid in the carrier liquid, the dispersed phase being bounded by a monomolecular film of the compound of the general formula XY; this dispersion is combined with the second liquid to form a heterogeneous two layer system comprising an upper liquid layer formed by the dispersion and a lower liquid layer formed by the second aqueous liquid, the two layers being separated by the monomolecular film of the compound of formula XY; and then the two layer system is centrifuged at an angular velocity sufficient for the 1(~6Z556 dispersed globules of the first a~ueous li~uid to be forced by gravity through the monomolecular separa-ting film of the compound XY and into the lower la~er of the second aqueous liquid.
Thus the process comprises two steps In the first step a dispersion of globules of the liquid to be encapsulated is formed under the action of ultrasonic vibrations, these globules having colloidal dimensions (i.e. a diameter of the order of 200 to 1,000 A) in a liquid which is either insolu~le in water or only slightly soluble in water. These globules are bounded by a mono-molecular film of the compound XY in which the hydrophilic groups X are turned towards the interior of the globules which is occupied by the aqueous liquid and the hydrophobic groups Y are turned towards the outside of the globules which is in contact with a non-aqueous phase These globules, although they do not properly speaking constitute liposomes, since they are not bounded by a bimolecular layer of the compound XY but only by a monomolecular film of that compound, can nevertheless be regarded as prototype liposomes, each of them containing the same volume of the liquid to be encapsulated as the liposomes finally obtained. In the following description, these globules are referred to liposome precursors.
By choosing suitable relative proportions of the aqueous liquid to be encapsulated and the carrier liquid and the compound of formula ~Y in this first part of the process it is possible to obtain encapsulation in the liposome precursors of the whole of the liquid to be encapsulated. ~he process according to the present - invention thus avoids the need for recovery, purification or readjustment of the concentration, which, as indicated above, are necessary in the operation of the known processes.
It will also be understood that the process according to the invention allows the encapsulation by the formation of liposomes, of liquias of which only a very small quantity is available, for example quanti-ties of the order of 0.05 to 0.1 millilitre, which would be insufficient for use in the processes described above.
~ hus the process according to the invention has applications in fields, such as certain laboratory research or analytical procedures, in which the other processes are inapplicable.
~he second step of the process consists of forming the liposomes proper from the liposome precursors. It may be supposed that this formation results from the fact that, in passing through the monomolecular film of the compound XY at the interphase between the upper liquid !
and the lower liquid, each li.posome precursor e~*rains a par.t of the film which becomes associated with the monomolecular film of compound XY which surrounds the . precursor and thus forms a bimolecular film of the compound XY characteristic of liposomes. ~he monomolecular film of the compound XY exists at the interface be-tween the upper and lower liquids because of the fact -that the hydrophilic groups X are attracted to the aqueous liquid layer whlle the hydrophobic groups Y remain embedded in the non-aqueous layer.
~he compound of formula XY may, for example, comprise a compound i.n which the hydrophilic group X
is one of the following groups: phospha-to, carboxylic, sulphato, amino, hydroxyl and choline and in which the hydrophobic group Y is one of the following groups:
a saturated or unsaturated alipkatic hydrocarbon group (for example an alkyl or alkYlene group), and an aliphatic hydrocarbon group substituted by at least one aromatic or cycloaliphatic group Preferably, a phospholipid or a substance closely related to phospholipids is used as a compound of formula XY
In particular the following compounds are useful:
lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, ~06Z556 phosphatidic acid and the cerebrosides.
It is possible also to use as the compound of formula XY a mixture of at least one phospholipid and at least one other lipid belonging to a category different -from the phospholipids In particular the following eompounds may be used: stearylamine, dicetyl phosphate, eholesterol and tocopherol.
~he carrier liquid having only a small solubility in water or being insoluble in water is preferably an organic liquid, partieularly one of the following eompounds: benzene, a halo-benzene or an alkyl benzene, an aliphatie ether, a aliphatic ketone, an aliphatic aldehyde, an aliphatic ester, an aliphatic hydrocarbon or a eyeloaliphatie hydrocarbon or a mixture thereof having a density less than that of water ~he selection of the first aqueous li~uid, i.e.
the liquid to be eneapsulated in the liposomes is limited B only ~ the required utility for the liposomes.
In particular, it is possible to use a solution of at least one biologically active substance, such as an enzyme, or a soIution of a medicament such as an antibiotie.
~he second aqueous liquid may be pure water or any other l~ZSS6 aqueous liquid apprcpriate For -preference the second a~ueous liquid is the liquid which is required to be the carrier for the dispersion of the liposomes when they are used, for example an a~ueous solution of sodium chloride. In particular an a~ueous sodiu~ chloride solution known as physiological serum or physiological saline,having a concentration of 0 15 mole of sodium chloride per litre (0 9% by weight) can be used in order to obtain directly in the second step of the process a liposome dispersion in a medium which is injectible in the human body. It is thus a further advantage of the process of the invention as compared with the known processes for the preparation of liposomes, tha-t it is possible to obtain directly a suspension of liposomes in an aqueous medium chosen with regard to the final use of the liposomes.
It is to be understood, however, that it is equally possible to sep æ ate the liposomes from the second a~ueous li~uid, for example if it is wished to remove every trace of the active substance which has not been encapsulated, before the final utilisation of the liposomes ~his separation may be easily effected by any appropriate known method, for example by gel chroma-tography ~he following ~xamples illustra-te the invention lO~Z556 Example 1 .
An a~ueous solution of amyloglucosidase containing 10 mg of amyloglucosidase per millilitre of solution is encapsulated in an aqueous sodium chloride solu-tion (0.15 mole per litre) in the following manner:
~ecithin (54 mg) and the aqueous solution of amyloglucosidase (0.1 ml) are added to dibutyl ether (3 ml~ and *he heterogeneous mixture thus obtained is subjected to ultrasonic (or near-ultrasonic) vibration (17 kHz freque~cy: output: 70 Watts) for 2 minutes while maintaining a temperature less than 30C by means of a cooling bath.
A transparent liquid, apparently homogeneous and having a bluish reflection, is obtained. A layer f this liquid is placed in a centrifuge tube on a layer o 1.5 ml of an aqueous solution of sodium chloride (0.15 mole per litre). ~here is thus obtained a 2-phase "mixture"
- comprising a lower layer formed by an aqueous phase (the sodium chloride solution) and an upper layer formed by the organic phase obtained in the first step of the process by ultrasonic vibration of the mixture of dibutyl ether, lecithin and the amylglucosidase solution.
~he 2-phase mixture is then centrifuged at 30,000 rev/min for 30 minutes. ~he upper layer (organic phase) is then .
lO~Z556 removed and the lower layer (aqueous phase) is submitted to a further centrifugation a-t ~0,000 rev/min for 30 minutes A faintly bluish clear liquid is obtained together with a very small amount of residue comprising non-dispersed lipid (lecithin), which is rejected. ~he clear liquid comprises a suspension of liposomes (having collodial dimensions) containing the aqueous amyloglucosidase solution, in a 0.15 M sodium chloride solution, and also contains a small quantity of non-encapsulated amyloglucosidase.
L0 ~ Depending on how the suspension is to be used, it is possible to use the suspension as formed or after elimination of non-encapsulated amyloglucosidase by gel chromatography (for example on a sepharose (dextran) gel).
L5 ~ æ~
A buffered solution (phosphate buffer 10 mM, pH
7.2) containing 100 mg/ml penicillamine (0.05 ml) is encapsulated in a 0.15 M aqueous solution of sodium chloride, using for the formation of the organic phase subjected to ultrasonic vibration lecithin (27 mg) and a mixture of dibutyl ether (2.4 ml) and chloroform (0.6 ml). ~he process is carried out in the same way as in ~xample 1.
106255~i Example 3 In a method similar to that of Example 1, an aqueous solution containing 300 mg/ml imipramine (0.1 ml) is encapsulated in a 0,15 M a~ueous solution of sodium chloride, using a mixture of lecithin (25 mg) and cholesterol (40 mg) - in dibutyl ether (3 ml).
Example 4 By a method similar to that of E~ample 1, an aqueous solution of 150 mg/ml betamethasone disodium phosphate (0.05 ml) is encapsulated in a 0.15 M aqueous solution of _sodium chloride using a mi~ture of lecithin (15 mg) and phosphatidylethanolamine (12 mg) is a mixture of dibutyl ether (2.5 ml) and chloroform (0.5 ml) in the first step.
Claims (13)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A process for the preparation of synthetic lipo-somes, comprising the steps of dispersing a first aqueous liquid with the aid of ultrasonic vibration in a carrier liquid which is insoluble in water or only slightly soluble in water and has a density less than that of water, in the presence of at least one compound of the general formula XY, where X represents a hydrophilic polar groups and Y represents a hydrophobic non-polar group, selected from the group consisting of phospholipids and lipid-like substances in which the hydrophilic polar group X is selected from phosphato, carboxylic, sulphato, amino, hydroxyl and choline; and in which the non-polar hydrophobic group Y is selected from saturated or unsaturated hydrophobic aiiphatic hydrocarbon groups and such aliphatic hydrocarbon groups which are substituted by at least one aromatic or cycloaliphatic group and mixtures of such phospholipids and lipid-like substances, so as to form a colloidal dispersion of the first aqueous liquid in the carrier liquid, the dispersed phase globules, each being bounded by a monomolecular film of the compound of the general formula XY; combining this dispersion with a second aqueous liquid to form a heterogeneous two-layer system comprising an upper liquid layer formed by the dispersion and a lower liquid layer formed by the second aqueous liquid, the two layers being separated by the monomolecular film of the compound of formula XY; and then centrifuging the two-layer system at an angular velocity sufficient for the dispersed globules of the first aqueous liquid to be forced by gravity through the monomolecular separating film of the compound XY and into the lower layer of the second aqueous liquid.
2. The process of claim 1 wherein the compound of the general formula XY is a phospholipid.
3. The process of claim 2 wherein the phospholipid is selected from the group consisting of lecithin, phosphatidyl-ethanolamine, lysolecithin, lysophosphatidylethanolamine, phos-phatidylserine, phosphatidylinositol, sphingomyelin, cardio-lipin, phosphatidic acid and the cerebrosides.
4. The process of claim 1 wherein the compound of the general formula XY comprises at least one other lipid belonging to a category of lipids other than the phospholipids.
5. The process of claim 4 wherein the said other lipid is selected from the group consisting of stearylamine, dicetyl phosphate, cholesterol and tocopherol.
6. The process of claim 1 wherein the said carrier liquid is an organic liquid.
7. The process of claim 6 wherein the said organic liquid is selected from the group consisting of benzene, halo-benzenes or alkyl benzenes, aliphatic ethers, aliphatic ketones, aliphatic aldehydes, aliphatic esters, aliphatic hydrocarbons and cycloaliphatic hydrocarbons and mixtures thereof.
8 The process of claim 1 wherein said first aqueous liquid is an aqueous solution of at least one biologically active substance.
9. The process of claim 8 wherein the said active substance is an enzyme.
10. The process of claim 8 wherein the said active substance is an antibiotic.
11. The process of claim 1 wherein the second aqueous liquid is an aqueous solution of sodium chloride.
12. The process of claim 11 wherein the said solution of sodium chloride contains 0.15 moles of sodium chloride per litre.
13. A process for the preparation of synthetic liposomes, comprising the steps of (a) dispersing a first aqueous liquid consisting of a solution of a biologically active substance, with the aid of ultrasonic vibration in a carrier liquid which is insoluble in water or only slightly soluble in water and has a density less than that of water, in the presence of at least one interface compound selected from the group consisting of lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cardiolipin, phosphatidic acid and the cerebrosides or a mixture thereof with at least one lipid selected from the group consisting of stearylamine, dicetyl phosphate, cholesterol and tocopherol, so as to form a colloidal dispersion of the first aqueous liquid in the carrier liquid, the dispersed phase being bounded by a monomolecular film of the said interface compound; (b) combining this dispersion with a second aqueous liquid to form a heterogeneous two layer system comprising an upper liquid layer formed by the dispersion and a lower liquid layer formed by the second aqueous liquid, the two layers being separated by the monomolecular film of the said interface compound; and (c) centrifuging the two layer system at an angular velocity of about 30,000 rev/min to force the dispersed globules of the first aqueous liquid by gravity through the mono-molecular separating film of the interface compound and into the lower layer of the second aqueous liquid.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA243,293A CA1062556A (en) | 1976-01-12 | 1976-01-12 | Liposomes preparation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA243,293A CA1062556A (en) | 1976-01-12 | 1976-01-12 | Liposomes preparation |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1062556A true CA1062556A (en) | 1979-09-18 |
Family
ID=4104949
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA243,293A Expired CA1062556A (en) | 1976-01-12 | 1976-01-12 | Liposomes preparation |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1062556A (en) |
-
1976
- 1976-01-12 CA CA243,293A patent/CA1062556A/en not_active Expired
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