 |
Description  |
|
|
The present invention concerns a process for the preparation of solutions
or suspension of liposomes in aqueous media.
Liposomes are microscopic vesicles, generally spherically shaped, formed
from one or several concentric layers (lamellae) of lipid molecules, i.e.
compounds having a lipophobic hydrophilic moiety and a lipophilic
hydrophobic moiety. The lamellae of a water-soluble liposome are formed of
at least one bimolecular lipid layer (which lipid can be represented
hereinafter by the formula XY, wherein X is the hydrophilic moiety and Y
is the hydrophobic moiety), the molecules of this layer being so oriented
that the hydrophilic functions thereof stay in contact with the aqueous
phase. Since the liposomes lamellae are being separated from each other by
a water film, they have a wall-like structure which can be schematically
represented, in section, by a series of molecular composites XY--YX
stacked together in the plane of the paper. The size of the liposomes
vesicles is extremely variable and depends, as will be described
hereinafter, on the methods used for the manufacture thereof; in general,
they have a 25 to 30,000 nm diameter and the lipid film around is about 3
to 10 nm. The smaller liposomes of this range in general have a
mono-lamellar envelope, that is a monolayer of the following molecular
association:
##STR1##
The aqueous phase in which the liposomes are in solution or suspension is
generally different from the aqueous phase contained in the inside
thereof. Hence, the preparation of liposomes constitutes a very practical
encapsulation method for trapping an aqueous liquid and such vesicles are
particularly useful for introducing biologically active substances into
living organisms, namely drugs or medicines, while preventing an early
degradation thereof (for instance by stomach or intestine fluids) before
these substances have a chance to reach the specific organ to be cured. By
a proper selection of the compound(s) alone or together with other
surfactants ZW wherein Z has the same definition as X and W has the same
definition as Y, XY to form the liposomes walls, it is effectively
possible to form liposomes with walls resisting the action of specific
categories of organic fluids and being dissolved only by the media which
exist within the organs wherein the biologically active substances must be
liberated. Consequently, in general, the liposomes contain within their
inside an aqueous solution of the encapsulated product and, in order to
constitute the liposome solution itself, they will be dissolved or
dispersed in water or in any other aqueous phase, for instance in aqueous
NaCl of isotonic concentration.
Acknowledgment is made to the fact that the content of the liposomes of the
present invention is not limited to medicines, drugs and other
biologically active compounds but covers other water-soluble materials. As
such other materials, the followings can be recited: dyestuffs, perfumes,
flavours or, in general, ingredients to be used within specific industrial
processes or compositions of matter but which should be released with some
delay relative to their initial addition, or used up progressively in the
course of time. It would be therefore wrong to consider that liposomes are
only pharmaceutical products per se, although they may, as mentioned
above, be the vehicles for drugs or alike.
It will also be noted that the term "lipid" as used herein is taken in its
widest sense, i.e. it comprises most compounds belonging to the definition
XY or ZW given before, either natural or synthetic, such as, for instance,
many kinds of surfactants used in the industries of pharmacy, cosmetics,
textiles, detergents, foodstuffs, etc . . .
There exists already several methods for the preparation of liposomes
solutions in the above defined sense, which methods are described in the
following reference: "New Aspects of liposomes" by D. A. Tyrrell, T. D.
Heath, C. M. Colley & B. E. Ryman, Biochimica & Biophysica Acta, 457
(1976), 259-302. One of these processes comprises heating a heterogeneous
mixture of a lipid and the aqueous liquid to be encapsulated to a
temperature above room temperature, then subjecting the mixture to violent
agitation followed by sonication, that is, to vibrations of sonic or
ultra-sonic frequencies.
Another method comprises dissolving a compound of formula XY (X and Y
having the above defined meaning), e.g. a lipid, in a volatile solvent,
evaporating said solution contained in a vessel thus forming a film of the
lipid of the walls of the vessel, introducing the liquid to be
encapsulated in the flask and, finally, subjecting the latter to sonic or
ultra-sonic frequencies whereby part of said liquid will be divided into
droplets surrounded by lipid envelopes. More prolonged is the treatment,
more of the liposomes tend to acquire a mono-lamellar shell.
It is realized that both of the above methods lead to a solution of
liposomes suspended in the liquid to be encapsulated which, in general, is
not the ultimate objective. It is therefore necessary, afterwards, to
separate the liposome vesicles from the carrier liquid and, thereafter, to
redisperse them in a different aqueous phase. Such a separation of the
liposomes from their initial liquid carrier can be effected for instance
by chromatography on molecular sieves, on silica-gels or sephadexes
(granulated polymers), or by repeated centrifugation, all methods which
are tedious and not economical.
It is also possible, according to another process for the preparation of
liposomes solutions, to inject an ethanol-lipid solution into the solution
to be encapsulated which leads to the formation of about 25 nm liposome
vesicles. However, such method is only applicable when the product to be
encapsulated does not denature in the presence of alcohol and, on the
other hand, the separation of the obtained vesicles from the excess of
non-encapsulated liquid and their subsequent redispersion into another
aqueous carrier is plagued with the same drawbacks as those mentioned
above.
One has also used a method which consists in mixing a lipid and a detergent
with the liquid to encapsulate and emulsifying the mixture; then,
afterwards, the detergent is eliminated by dialysis. Thus, here again one
obtains the liposomes dispersed in the excess of the liquid to be
encapsulated from which they must be separated and purified.
It will be further remarked that the prior processes described hitherto all
require to have available a volume of the starting aqueous liquid much
larger than the quantity thereof which is ultimately encapsulated within
the liposomes. Effectively, as explained above, in these processes the
liposomes are formed as hollow beads in solution or colloidal suspension
in a liquid carrier which is constituted by the portion of the original
liquid to be encapsulated which has not been retained within said beads.
The ratio of the liposomes encapsulated liquid to the total volume of
liquid is, in general, in the region of 1 to 10% only. Consequently, if
the liquid to be encapsulated is expensive--and this is generally the case
with biologically active solutions--it becomes necessary to recover that
portion of non-encapsulated liquid for further recycling. This recovery
parallels the separation of the liposomes from that liquid. Then, after
separation, the liquid must be freed from undesirable impurities and its
concentration of active substances must be restored since separation and
purification operations may require large volumes of solvents leading to
unacceptable dilution of the active principles. Therefore, it is difficult
and expensive to adapt the above processes of manufacture of the liposomes
solutions to an industrial scale because of the very large volumes of
liquids to be handled as compared with the relatively poor yield
efficiency experienced.
There has been recently described a new method for the preparation of
liposomes in aqueous solution (published German patent application DOS No.
2,532,317 and U.S. Pat. No. 4,089,901) which largely remedies the
above-mentioned drawbacks. Following this method, the solution to be
encapsulated is simply added to a solution of the surfactant in an organic
solvent insoluble in water and of density below 1 and the mixture is
sonicated as in the prior-art. After this step, there is obtained in the
organic liquid a suspension of microscopic aqueous vesicles called
hereinafter "liposomes precursors" which result form the dispersion of the
solution to be encapsulated in the organic solvent, which vesicles are
surrounded by a monolayer of lipid molecules each of which would be
oriented as follows: the X function contacts the aqueous phase and,
therefore, it is directed toward the inside of each droplet whereas the Y
function is turned toward the outside thereof, i.e. it protrudes out of
the shell and dips in the organic medium which still contains, in the
dissolved state, an excess of the lipid.
Thereafter, the suspension of "precursors" is centrifugated in the presence
of the aqueous medium in which it is desired to produce the liposomes
solution. Since this aqueous medium is denser than the said organic
solvent, it forms the bottom phase in the centrifugating tubes. During
centrifugation, the "liposome precursors" leave the upper organic phase
and, when subjected to the centrifugal force, they will penetrate into the
aqueous phase. When doing so, they will get across the organic--water
interphase which, of course, comprises a lipid barrier resulting from the
presence of the excess of such lipid dissolved in the organic solvent. The
molecules of the lipid in this interphase will have naturally oriented
themselves according to the relative positions of the two phases, i.e. the
X functions being wetted by the water and the Y function being wetted by
the solvent. Hence, when crossing the barrier, the "precursors" will
acquire a second layer of the surfactant, the molecules of which will be
upturned relative to that of the first layer, the two layers thus
constituting a normal liposome lamella of structure XY--YX. Therefore, the
method directly affords the liposome solution in the desired aqueous
medium.
Thus, in other words, this recent process comprises two phases: in the
first phase, one forms under the effect of sonication a dispersion of
vesicles or globules of the liquid to be encapsulated in another liquid
insoluble or nearly insoluble in water (such globules having colloidal
dimensions, i.e. 20 to 100 nm). These globules are delineated by a
monomolecular pellicle of the compound XY the hydrophilic moiety X of
which is turned towards the inside of the globules which contain the
encapsulated liquid and the hydrophobic moiety Y of which is,
contrariwise, turned toward the outside of the globules, which outside
comprises the non-aqueous phase. These globules, although they are not
true liposomes since they are not limited by a double molecular layer of
the XY compound, can still be considered as the skeleton of the liposomes
because each of them contains the same volume of the aqueous liquid that
will be contained in the ultimately formed liposomes. It is therefore
justified to call such globules under the name of "liposomes precursors".
By properly adjusting the respective proportions of the aqueous liquid to
be encapsulated, the organic solvent and the compound of formula XY, it is
possible to encapsulate within said liposome precursors the near totality
of the initial aqueous liquid. The yield of the complete method is
therefore often close to theory, e.g. around 80%, which constitutes a
considerable improvement over the classical methods in which the yields
are in the range of 1 to 20%.
The second phase of the reference process comprises forming the liposomes
themselves. It can be theorized that this formation is connected with the
crossing by the precursors of the monomolecular layer of the compound XY
at the interphase between the non-aqueous upper-layer and the aqueous
under-layer. It should be noted that the existence of such monomolecular
pellicle is known per se since it intrinsically results from the
properties of the surfactant XY of which the hydrophilic groups X are
attracted by water whereas the lipophilic Y groups remain in contact with
the organic phase. Therefore, when moving across the boundary layer, each
precursor will entrain a portion of this pellicle which will tie up with
the first monomolecular film surrounding the precursor and thus form the
characteristic head-to-head bimolecular lamellae of the liposomes.
This method is therefore convenient as it avoids the normal operations of
recovery and replenishment of the concentration in active ingredients of
the initial aqueous liquid which are, as mentioned heretofore, necessary
when carrying out the classical methods for the preparation of liposomes
solutions. It will be also readily understandable that the reference
method enables the encapsulation in liposome form of very small quantities
of liquids, e.g. 0,05 to 0,1 ml, which volumes would be insufficient in
case of applying the older processes. Thus, this recent reference method,
which enables the direct preparation of liposomes solutions with no need
to first separate the liposomes from the remainder of the initial aqueous
liquid and, thereafter, to redisperse these liposomes into another desired
aqueous medium, can be used in many applications in which the classical
methods would not be suitable, such as biological and medical work and
analyses.
It should however be remarked that, despite its strong advantages, this
last method still has two drawbacks: first, the organic water-insoluble
solvent must necessarily be lighter than water (to ensure the ready
formation of an aqueous under-layer in the centrifugation tubes), which
imposes specific solvent choice limitations. Second, the centrifugation
operation is, in itself, undesirable because common high-speed centrifuges
do not permit treating large quantities of liquids in one operation.
Therefore, centrifugation is time consuming and expensive. Further, some
sensitive biological products poorly stand the enormous accelerations (of
about 10.sup.3 to 10.sup.5 g) involved in ultra-centrifugation.
The present invention fully remedies these drawbacks. Thus, the process of
the invention involves, as in the above-described prior art, forming
"liposome precursors", and contacting said "precursors" with an aqueous
medium in a manner such that a liposome solution or suspension in said
aqueous medium is produced, said "liposome precursors" consisting of small
vesicles of a first aqueous phase surrounded by an envelope of a lipid or
surfactant of formula XY wherein X is a hydrophilic lipophobic group and Y
is lipophilic hydrophobic group, said "liposome precursors" being produced
by dispersing said first aqueous phase in a water-insoluble or hardly
insoluble solvent in the presence of an amount of compound XY. The process
of the invention comprises emulsifying the "liposome precursors" in said
aqueous medium in the presence of an excess of compound XY or another
surfactant ZW wherein Z is a hydrophilic group and W is a hydrophobic
group, said water insoluble or hardly soluble solvent being removed prior
to or after said emulsifying.
For dispersing the first aqueous phase in the water-insoluble solvent,
classical means such as sonication, violent mixing, homogenization, gas
blowing, spraying, etc. can be used. Sonication is preferred for
convenience. When separating the water-insoluble solvent prior to
emulsifying, classical methods such as distillation, evaporation and
centrifugation can be used. However, such removal of the solvent prior to
emulsification can lead to undesirable partial agglomeration of the
"precursors"; therefore, it is preferred to proceed with said removal of
the solvent after emulsification of the "precursors" containing solvent in
said aqueous medium. Said removal can be effected for instance by
selective distillation or evaporation. A preferred method comprises
emulsifying the two phases together and subjecting said emulsion to
evaporation conditions which cause the non aqueous solvent to evaporate
thus providing a solvent-free solution or suspension of liposomes in said
aqueous medium.
To practically implement the operational factors of this embodiment of the
invention, one can introduce a non water-soluble organic solvent into a
reactor, add a lipid or a mixture of products of formula XY constituting a
lipidic fraction, then add the aqueous solution to be encapsulated,
homogenize the mixture and, by sonication with sonic or ultrasonic
vibrations, form therein the vesicles or beads of trapped aqueous liquid
called "liposome precursors". Then, one adds the desired portion of
aqueous medium e.g. a diluted solution of one or several salts dissolved
in water, or more simply pure water, and if necessary a further portion of
compound XY or another ZW as defined above, and one subjects the mixture
to the action of a stirrer-emulsifier until a homogeneous emulsion is
formed. This emulsion comprises micro-drops of solvent dispersed in the
aqueous phase and each micro-drop of solvent contains, dissolved, an
excess of the lipid fraction and, in suspension, a variable number of
liposome precursors. Then, while maintaining the emulsion in a stable
condition by stirring (or even after having stopped the stirrer if the
emulsion is intrinsically stable), one subjects the content of the reactor
to evaporative conditions. For instance, at room pressure, one can blow
air (or another gas) at the surface of the liquid (or tangentially
thereto), one collects the air loaded with the solvent vapors and one
circulates this air into a condenser cooled to a low temperature which
effects condensation of the vapors. During evaporation and because of the
consecutive size reduction undergone by the solvent drops, the liposome
precursors are progressively driven off said drops and dispersed through
the solvent-water boundary film therebetween and, consequently, through
the lipid membrane separating the phases, thus acquiring the complementary
lipid layer which converts them from the "precursor" state to the true
liposome state. The resulting aqueous liposome solution therefore contains
the vesicles in the dissolved or dispersed form in the said added aqueous
medium. It may be advantageous to use in the last phase of the method a
lipid or surfactant ZW different from XY, particularly in the case where
the mutual affinity of the hydrophobic-lipophilic moieties Y and Z toward
each other is greater than the affinity between two Y groups together or
two W groups together. In such case (for instance when having XY being
rather a good lipophilic dispersing agent and ZW being a rather good
hydrophilic dispersing agent) liposomes having a shell of
##STR2##
structure are obtained which are particularly stable.
This method, the yield of which is very high (about 50 to 80%), is
therefore extremely simple to carry out compared to the prior-art methods.
Large quantities of material can be worked up at one time and, if wanted,
solvents heavier than water can be used. Solvent selection is therefore
wider than in the case of the method of DOS No. 2,532,317 thus making it
possible to use a great variety of surfactants including, for example,
some lipids which are solids at room temperature and which, normally, had
to be heated and melted, when used in connection with the older methods,
at temperatures often detrimental to the products to be encapsulated.
Further, this greatest choice of solvents facilitates a proper selection
thereof when solvents must be found which are inert toward the active
ingredients to be encapsulated.
It should be remarked that means other than those described above can be
used for preparing the above emulsion and for subsequently evaporating the
solvent. For instance, one can emulsify by a shaking treatment and
evaporate under reduced pressure. However whatever method, the partial
vapor pressure of the solvent should be significantly larger than that of
water; otherwise, to prevent water exhaustion repeated water replenishment
during evaporation will be required.
As solvents, one can use hydrocarbons such as benzene, toluene,
cyclohexane, petroleum ether, octane, etc . . . , ethers such as diethyl-,
di-isopropyl- and dibutyl-ether, etc . . . , esters such as ethyl, propyl
or butyl-acetate, ethyl carbonate, etc . . . , halogenated solvents, e.g.
CCl.sub.4, CHCl.sub.2, chloroform, benzyl chloride, etc . . .
As surfactants of formula XY or ZW, one can use, for instance, ternary or
complex lipids, glycerides, cerides, etholides and sterids an, namely, one
or several compounds in which the hydrophilic X respectively Z group is
selected from the following phosphato, carboxyl, sulfato, amino, hydroxyl
and choline and the hydrophobic Y respectively W group is one of the
following: aliphatic saturated or unsaturated groups (e.g. alkyl or
alkylene), polyoxyalkylene and aliphatic hydrocarbon groups substituted by
at least one aromatic or cycloaliphatic rest.
It will be noted that when using XY or ZW compounds with acidic hydrophilic
groups (phosphato, sulfato, etc . . . ) the obtained liposomes will be
anionic (called (-) liposomes); with basic groups such as amino, cationic
(+) liposomes will be obtained and with polyethyleneoxy or glycol groups,
neutral liposomes will be obtained. One can find many compounds suitable
for the invention in the following references: Mc Cutcheon's Detergents &
Emulsifiers and Mc Cutcheon's Functional Materials, Allured Publ. Company,
Ridgewood, N.J. USA. Preferably, one uses, as compounds XY or ZW,
substances related to phospholipids, namely the following compounds:
lecithin, phosphatidyl-ethanolamine, lysolecithin,
lysophosphatidyl-ethanolamine, phosphatidylserine, phosphatidyl-inositor,
sphingomyeline, cephaline, cadiolipine, phosphatidic acid, cerebrosides,
dicetyl phosphate, phosphatidyl-choline and
dipalmitoyl-phosphatidylcholine. As lipids with no phosphorus, one can
use, for instance, stearylamine, dodecylamine, hexadecylamine (Kodak
Ltd.), cetyl palmitate, glyceryl ricinoleate, hexadecyl stearate,
isopropyl myristate, amphoteric acrylic polymers, triethanolamine--lauryl
sulfate, alcoyl-aryl sulfonates, polyethoxylated fatty acid amides, etc .
. . The lipid fraction can further contain, dissolved therein, other
substances for controlling the stability and the permeability of the
liposome membrane. As such, the followings can be mentioned: Sterols, e.g.
cholesterol, tocopherol, phytosterols, and lanolin extracts, etc . . .
In the present process, one can encapsulate practically all hydrosoluble
substances which do not have a too strong affinity for the compounds of
the liposome shells or which would permeate such shells. In this respect,
one can recite, besides the products already mentioned hereinbefore,
aqueous solutions of biologically active substances, e.g. heavy metal
chelating agents, enzymes, drugs, antibiotics, etc . . . Examples of
suitable substances are listed in the following reference: "Targetting of
Drugs", G. Gregoriadis, Nature 265, [2] (1967), 407.
As aqueous dispersing medium in which the liposomes are ultimately
dissolved, one can use pure water or any other convenient aqueous liquid.
Preferably, one uses a liquid which is intended to be the final dispersing
medium for the liposomes when used, e.g. a diluted NaCl solution. In
particular, one can use an aqueous NaCl solution having 0.15 mole of
NaCl/liter (0.9% by weight) called physiological serum in order to
directly obtain, in the course of the second step of the method, a
liposome solution in a medium which can be directly injected into the
body. Thus, another advantage of the present process becomes apparent: the
possibility of directly obtaining a liposome suspension in a medium
selected according to the end-uses of such liposomes.
Naturally, it is always possible to separate the liposomes prepared
according to the present method from their aqueous dispersion medium, for
instance, if it is desired to avoid all traces of non encapsulated active
compound dissolved in said medium. Such separation can be carried out by
any usual means, e.g. by sephadex chromatography.
The following Examples illustrate the invention in a more detailed manner.
EXAMPLE 1
In a 10 ml pyrex flask were introduced 2 ml of dibutyl ether and 1 ml of
cyclohexane, then there was added 0.2 ml of a 10 mg/l insulin solution in
aqueous 9.permill. NaCl at pH 3 (HCl N/10). Then, there was added 150 mg
of dipalmitoyl-phosphatidyl-choline (hydrophilic-lipophilic compound) and,
at room temperature, the heterogeneous mixture was subjected to
ultra-sonics for 1 min. by means of a "BRANSON" generator (Model B-12, 20
KHz, 150 W). There was obtained a clear solution containing the liposome
precursors in the form of microvesicles of the insulin solution the walls
of which were made of a layer of surfactant and having molecules oriented
in such direction that the hydrophilic function thereof
(phosphatidylcholine) was pointing toward the inside of the vesicles and
that the hydrophobic groups (hydrocarbon chains) were turned outwards
towards the organic solvent. Such microvesicles were suspended in
colloidal form within the organic solvent containing, dissolved, an excess
of the surfactant.
Then, the organic solution was introduced into another flask containing 30
ml of neutral aqueous NaCl at 0.9% and the mixture was emulsified at
30.degree. C. by means of an emulsifying stirrer rotating at high speed.
After this step, the mixture would consist, consequently, of a dispersion
of fine droplets of the organic solvent in the water phase. Each droplets
would contain, in suspension, the liposome percursors described above and
an excess of lipids.
By means of an appropriate tube, a current of air was introduced into the
flask and made to sweep on the emulsion very close to its surface while
stirring at 30.degree. C.; the mixture of air and solvent vapors was
cooled in a condenser at -10.degree. C. whereby the vapors condensed into
liquid. This operation was continued until condensation had ceased (about
20-30 min).
After this step, there remained in the flask about 20 ml of clear solution
(9.permill. NaCl) in which the liposomes capsules were suspended in the
form of vesicles the shell of which consisted of a double molecular layer
of surfactant with molecules oriented head-to-head and the tails of which
were directed toward the inside as well as toward the outside thereof; the
external layer of this shell was acquired from the excess of lipid
dissolved in the organic solvent and made available by evaporation.
Two ml of the liposome solution were chromatographed on a column containing
2.5 g of Sephadex G-50 (eluant: 0.15 M aqueous NaCl+0.05 M phosphate
buffer, pH 7.5) and, by analysis of the eluate (spectrometric), it was
measured that only 20-25% of the original insuline solution did escape
encapsulation. Therefore, in most cases, the liposome solution obtained
according to this Example can be therepeutically used as prepared without
further purification.
EXAMPLE 2
An aqueous 10 g/l amyloglucosidase solution was encapsulated and dispersed
into 0.15 M NaCl solution as follows: a mixture of lecithin (74 mg), 0.1
ml of the amyloglucosidase solution and diisopropyl ether (3 ml) were
sonicated for 2 min (20 kHz, 150 W) while cooling below 30.degree. C. by
means of a cooling bath. There was obtained a clear homogeneous bluish
liquid to which there was added 15 ml of 0.15 M aqueous NaCl. The mixture
was emulsified as described in Example 1, after which the fine emulsion
was swept with a current of nitrogen which evaporated the organic solvent.
This blowing was continued until completion of the evaporation (20-30
min). There was obtained a clear suspension of colloidal liposomes--in
which the amyloglucosidase solution was trapped--in aqueous 0.15 M NaCl
and containing a very small amount (about 5%) of untrapped enzyme.
Depending on the desired use, this solution can be used as such or after
the non encapsulated amyloglucosidase has been separated (e.g. by gel
chromatography on "sepharose").
EXAMPLE 3
The process of Example 1 was repeated but using, as the solution to be
encapsulated, 0.05 ml of an aqueous buffered solution (phosphate buffer 10
mM, pH 7.2) of 0,5 mg/ml of arabinose citosine. The dispersing phase was
0.15 M NaCl (10 ml), the lipid was cardiolipin (47 mg) and the organic
solvent dibutyl ether (2.4 ml) and CHCl.sub.3 (0.6 ml).
EXAMPLE 4
The process of Example 1 was repeated but using the following products:
penicillamine to be encapsulated at 100 mg/ml (0.1 ml); dispersing medium
0.15 M aqueous NaCl; surfactants lecithin (105 mg) and cholesterol (35
mg); solvent butyl acetate (3 ml).
EXAMPLE 5
The process of Example 1 was repeated but using the following ingredients:
to be encapsulated, betamethasone at 150 mg/l in aqueous disodium
phosphate (0.05 ml); dispersing phase 0.15 M aqueous NaCl; lipid lecithin
(85 mg) and phosphatidyl-ethanolamine (45 mg); solvent 3-heptanone (3 ml).
EXAMPLE 6
The method of Example 1 was repeated using as the ingredients 0.1 ml of
aqueous solution of the acid complex polyinosinic-polycytidilic acids
(poly(I)-poly(C)) at 1 mg/ml to be encapsulated; dispersing phase 0.15 M
NaCl; surfactant lecithin (60 mg), stearylamine (20 mg) and cholesterol
(15 mg); solvent a 1:1 by volume mixture of diisopropyl ether and butyl
acetate (3 ml).
EXAMPLE 7
To 20 ml of diisopropyl ether were added 1.5 g of lecithin, 0.4 g of
phosphatidylserine and 0.5 g of cholesterol, then a solution of 1.8 mg of
actinomycin D in 3 ml of phosphate buffer (0.1 M, pH 7). The mixture was
sonicated for 5 min as described in Example 1. Then there was added 100 ml
of aqueous phosphate buffer (0.1 M, pH 7) and emulsification was carried
out as in Example 1. Without stopping the emulsifying stirrer, the flask
was connected to a vacuum tap and the pressure was progressively reduced
to 10 Torr while controlling the temperature to 20.degree.-22.degree. C.
After about 45 min, the diisopropyl ether was completely removed and the
residual mixture was in the form of a clear liposome solution. By
chromatography of a sample on Sephadex, it was measured that 88% of the
original actinomycin D had been entrapped.
EXAMPLE 8
1 g of trypsin in 30 ml of a 0.1 M (pH 7) phosphate buffer was sonicated in
the presence of a solution of 30 g of phosphatidylinositol in 100 ml of
dipropyl ether. Then 460 ml of aqueous 0.5% NaCl were added and
emulsification was carried out. The fine emulsion was evaporated for 3 hrs
at 15.degree. C. under 10 Torr which gave a clear liposome solution. By
analysis of a sample (chromatography on Sephadex G-50) it was ascertained
that the encapsulation yield was about 85%.
EXAMPLE 9
In this Example, there is described the formation of Liposomes having an
asymmetrical envelope. This envelope has an inside phospholipid layer
consisting of dipalmitoyl-phosphatidyl choline and an outside layer
consisting of a mixture of egg-lecithin and phosphatidylserine. The
following method was used:
To 3 ml of dibutyl-ether, there were added 0.1 ml of an aqueous solution at
0.001 M of 3,9-bisdimethylaminophenazothionium chloride and at 0.015 M of
NaCl and 55 mg of dipalmitoylphosphatidylcholine; then the mixture was
sonicated as described in Example 1 and there was obtained a transparent
organic solution containing microvesicles (liposomes precursors) of the
above mentioned colored aqueous solution surrounded by a layer of
dipalmitoyl-phosphatidylcholine. This organic solution was thereafter
centrifugated for 20 min. at 8000 g. After the end of this operation,
there was obtained a clear supernatant phase and a translucent blue bottom
phase containing the microvesicles agglomerated together under the effect
of the centrifugal force. This phase which was in the form of a rather
consistent gel was transformed into a 10 ml container, there it was mixed,
by means of a spatula, with 0.3 ml of a 200 mg/ml solution of a 20:1 (mole
ratio) mixture of egg-lecithin and phosphatidylserine in dibutylether.
Then there was added to the gummy mass 5 ml of a 0.15 M NaCl water
solution and the mixture was subjected to vigourous agitation with a
magnetic stirrer. The mass progressively dispersed itself and, after 10-15
min, there was obtained a well homogeneous, transparent solution
containing, in suspension, the liposomes with a mixed envelope. The
outside layer of this envelope consisted of the mixture of egg-lecithin
and phosphatidylserine and the inside layer thereof consisted of
dipalmitoyl-phosphatidylcholine.
EXAMPLE 10
Liposomes precursors containing insulin were prepared as described in
Example 1. Then, instead of continuing as further described in Example 1,
i.e. directly emulsifying the organic solution containing the
microvesicles in an aqueous 0.9% NaCl solution, the organic solution was
first concentrated by subjecting to reduced pressure (20 Torr) at room
temperature. After evaporation of the organic solvents, there was obtained
at the bottom of the container an oily translucent layer consisting of
agglomerated microvesicles. There were then added in the flask 7 ml of an
aqueous 0.9% NaCl solution and, by means of a magnetic stirrer, this oily
phase was dispersed into the aqueous medium. This oily phase progressively
disappeared and, after 10-15 min, there was obtained a transparent
homogeneous solution containing, in suspension, the desired liposomes.
After chromatographing this solution on "Sephadex 4B" (Pharmacia, Sweden),
analysis of the non encapsulated phase showed that 52% of the starting
insulin had been effectively encapsulated in the liposomes.
EXAMPLE 11
A mixture of lecithin (40 g), dibutylether (600 ml) and an aqueous 0.9%
NaCl solution containing 10 g/l of insulin (400 ml) were homogenized by
means of a MINISONIC homogenizer (ULTRASONICS Ltd., Great-Britain). There
was obtained a stable milky-looking solution which was introduced into a 4
l flask containing 2 l of an aqueous 0.9% NaCl solution; then, by means of
an emulsification stirrer, the organic suspension and the aqueous medium
were emusified together (15 min). Thereafter the organic solvent was
removed by air-stripping, i.e. the mixture was circulated downwards a
column traversed by an ascending air current, the latter being collected,
when loaded with the solvent vapors, at the top of the column; there was
thus obtained a homogeneous translucent solution of liposomes containing
the insulin.
EXAMPLE 12
Dibutylether (3 ml), aqueous 9.permill. NaCl containing 10 mg/ml of insulin
(1 ml) and lecithin (125 mg) were introduced in a strong 10 ml Pyrex tube,
together with 2 g of 1 mm diameter glass beads. The tube was stoppered,
was placed on a shaker and shaked for 30 min at 100 cycles per min. There
was thus obtained a rather homogeneous milky-looking solution which was
poured into a 100 ml flask containing aqueous 9.permill. NaCl (30 ml).
Then, the organic solvent was evaporated as described in Example 2 by
using a current of nitrogen. After sweeping for 20-30 min, there was
obtained a limpid solution of liposomes containing the insulin under
encapsulated form.
* * * * *
|
|
 |
|
 |
Description |
|
