Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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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.
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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.
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~ 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.
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~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
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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
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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
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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,
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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
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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
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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
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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.
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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.
