Note: Descriptions are shown in the official language in which they were submitted.
~;~'75;~
~ETHOD FOR PRE6ERVING LIPOSOMES
Field of the Invent on
The present invention relates generally to
liposomes, and more particularly relates to a method of
preserviny liposomes containing biologically acti~e mol-
ecules. This process is useful in applications such as in
vivo drug delivery and preservation of diagnostic agents.
This invention was made with U.S. Government
support under Grant No.~PCM 82-17538 with the National
Science Foundation and the University of CaIifornia. The
U~S.~Government has certain rights in this invention.
Backqround of th`e Invention
Lipo~somes are unilamel:Lar or multilamellar
~lipid vesicles which~enclose~a flu~id space. The walls o~
the vesicles are formed by a bimolecular layer of one or
more lipid components having polar heads and non-polar
tails. In~an aqueous (or polar) solution, the polar heads
of one layer orient outwardly ~o extend into the
surrounding medium, and the non-polar tail portions of
the~lipids associate with each other, thus providing a
polar~surface and a non-polar core in the wall of the
vesic~e. Unilamellar liposomes have one such bimolecu-
lar layer, whereas multilamellar liposomes generally have
a plurality oE substantially concentric bimolecular
layers.
Liposomes are well recognized as use~ul for
encapsulation Oe drugs and other therapeutic agents and
or carrying these agents to in vivo sites. For example,
~ :~. .
:: , . : :
- .. . . .
- , ~ : ,. .
'. , ' .
~L~'7S~
U.S. Patent 3,g93,754, inventors Rahman et al., issued
November 23, 1976, discloses an improved chemotherapy
method in which an anti-tumor drug is encapsulated within
liposomes and then injected. U.S. Patent 4,263,42~,
inventors Apple et al., issued April 21, 1981, discloses
an antitumor drug which may be more effectively delivered
to selective cell sites in a mammalian organism by
incorporating the drug within uniformly sized liposomes.
Drug administration via liposomes can have reduced
toxicity, altered tissue distribution, increased drug
effectiveness, and an improved therapeutic indexO
Liposomes have also been used successfully for
introducing various chemicals~ biochemicals, genetic
material and the like into viable cells ln vitro, and as
carriers for diagnostic agents.
A variety of methods for preparing liposomes
are known, many of which have been described by Szoka and
Papahadjopoulos, Ann. Rev. BiophYsics Bioeng. 9: 467-508
(1~80). Also, several liposome encapsulation methods
are disclosed in the patent literature, notably in U.S.
Z- patent 4,235,871, to Papahadjopoulos et al., issued
November 25, 19~0, and in U~S. patent 4,016,100 to Suzuki
et al., issued April 5, 1977.
Although encapsulation of therapeutic agents
and biologically active compounds in liposomes bas sig-
nificant commercial potential, a major dif~iculty that
has been encountered in the commercial use of liposome
encapsulates is with their long term stability. Although
liposome structures may be maintained intact under
certain storage conditions, such conditions are often
3a. inconvenient or unavailable. It is as a solution to this
problem that the method of this invention is presented.
~, , : -. .:
~.~75~4~
Summary of the In ention
Accordingly, it is an object o~ the present
invention t~ provide a commercially feasible method of
preserving liposomes.
It is another object of the present invention
to provide a commercially feasible method o~ preserving
liposomes by means of freeze-drying, wherein upon rehy~
dration, resultant liposomes can retain as much as lOU~
of their original encapsulated material.
It is still another object of the present
invention to provide a method oE preserving liposomes by
means of a carbohydrate compound capable of preserving
structure and function in biological membranes.
It is a further object o~ the present invention
to provide a method of preserving liposomes by means o~ a
preserving agent such as trehalose, present either as
encapsulated material inside the liposome or externally
in solution during freeze-drying, or both.
It is yet another object of the present
invention to provide a lyophilized composition which upon
rehydration retains up to 100% of original encapsulated
material.
Further objects an~ advantayes oE this inven-
tion will become apparent from the study of the following
portion of the specification, the claims and the attached
drawings~
In one aspect the present invention is directed
to a method for preserving liposomes, comprising:
providing i-nitial liposomes, said liposomes having an
initial quantity of water-soluble material encapsulated
therein, said material including a first preserving agent;
contacting said initial liposomes with a second preserving
agent in an aqueous solution; and, lyophilizing said
c~
.~ :
. , . -. . :
,
~ ' . . - ' ' , ':
. ., -
,' : ' ~ . ... .
~S;~4~
3a
initial liposomes in the presence of said second preserving
agent to form lyophilizates.
In a further aspect of the present invention, a
method for preserving liposomes inc1udes freeze-drying
liposomes in the presence of a preserving agent capable of
preserving structure and function in biological membranes.
Preferred preserving agents include carbohydrates having
at least two monosaccharide units, and especially
preferred compounds include the disaccharides
. . - -
~: : . . . .
sucrose, maltose, and trehalose.
In another aspect of the present invention, the
method comprises freeze-drying liposomes which in addi-
~ion to containing biologically active molecules or
therapeutic agents contain a preserving agent such as
trehalose internally. In a preferred embodiment of the
inventive method, an appropriate compound such as trehal-
ose is present both inside and outside the lipid membrane;
preferred weight ratios of total preserving agent to
lipid range from about 0.1:1 to 3.0:1~ An especially
preferred weight ratio is about 1~:1Ø
The invention also embodies a lyophiliæed
composition such that when reconstituted by rehydration,
resultant liposomes retain substantially all of their
originally encapsulated material. Such a lyophilized
composition may be prepared by the method as outlined
above.
Detailed Description of the Invention
The invention comprises a method for preserving
liposomes containing biologically active molecules using
a preserving agent. The method involves either freeze-
drying liposomes in the presence of a preserving agent, or
- freeze-drying liposomes which contain a preserving agent
internally in addition to encapsulated medicaments, or
both. Preferred preserving agents are carbohydrates
having at least two monosaccharide units joined in
glycosidic linkage, and particularly preferred pre-
serving agents include sucrose, maltose and trehalose.
Of these, trehalose has been found to be the most
effective preserving agent for use with the inventive
method.
Trehalose is a naturally occurring sugar found
.: ~, . . . . - . ..................... . - .
.. : . ~ .. . .
.: , :. .
7S~8
at high concentrations in organisms capable of surviving
dehydrationO Trehalose is especially effective in pre-
serving structure and function in dry biological mem-
branes. Liposomes which are freeze-dried in the presence
of trehalose and which additionally contain encapsulated
trehalose, exhibit particularly good retention of encap~
sulates. That is, when liposomes are exposed to tre-
halose both internally and externally during freeze-
drying, they can retain as much as 100% of their original
encapsulated contents upon rehydrakion. This is in sharp
contrast to liposomes which are freeze-dried without any
preserving agent, which show extensive fusion between
liposomes and loss of contents to the surrounding medium.
Representative phospholipids used in forming
liposomes which may be used in this process include phos
phatidylcholine, phosphatidylserine, phosphatidic acid
and mixtures thereof. Both natural and synthetic phos-
pholipids may be successfully usedO
The biologically active or therapeutic encap-
sulated material is preferably water soluble. Examples
of suitable therapeutic agents with which this preserva-
tion method can successfully be carried out include
sympa~homimetic drugs such as amphetamine sulfate, epi-
nephrine hydrochloride, or ephedrine hydrochloride; an-
tispasmodics such as atropine or scopalamine; broncho-
dilators such as isoproternol; vasodilators such as
dilthiazen; hormones such as insulin: and antineoplastic
drugs such as adriamycin. Suitable biologically active
molecules include, for example, RNA, DNA, enzymes and
immunoglobulins.
Small unilamellar vesicles (SUV's) are pre-
pared as starting materials prior to encapsulation of
trehalose, and may be prepared by any of the available
... . , : :
5~4~3
techniques. Suitable techniques include injection of
the lipid in an organic solvent into water, extrusion from
a French pressure cell, and sonication. The material to
be trapped may be added at any s~age during preparation of
the small unilamellar vesicles, but in practice it is most
convenient to mix the small unilamellar vesicles with an
aqueous solution of the material to be trapped immedi-
ately be~ore preparation of large unilamellar vesicles.
Preferred weight ratios of encapsulate to lipid are about
l.li:l.O.
Large unilamellar vesicles (LUV's) with in-
creased trapping e~ficiency may tben be prepared by
either freeze-thawing or rotary evaporation. An exemplary
rotary evaporation method and one which is especially
effective in conjunction with the method disclosed herein
is illustrated in Deamer, D.W., "A Novel Method for
Encapsulation of Macromolecules in Liposomes" in Gregori-
adis, G. (ed.) Liposome Technolo~Y (19~4). The method
comprises providing a polar solution having initial
liposomes and a quantity of material to be encapsulated.
Substantially~all of the solution is removed, and the
resultant liposomes are then recove~ed by hydration o~
the concentrated admixture. ThiS method is also the
subject of U.S. Patent No. 4,515,736, inventor Deamer, et al.,
issued May 7, 1985. The resulting vesicles may then be made
more uniform by filtration, centrifugation or gel permeation
chromatography.
; Trehalose may be added at any stage during
preparation of the large unilamellar vesicles, but greatly
improved preservation is attained with trehalose present on
both sides of the phospholipid bilayer. ~herefore, trehalose
is preferably added be~ore the large
~' ' ' . : ..
. ~ ~
75~
unilamellar vesicles are prepared, so that trehalose is
trapped inside. The preferred weight ratio of total
trehalose to lipid ranges from about 0.1:1 to about 3.0:1;
a particularly preferred weight ratio is approximately
1.0:1Ø The large unilamellar vesicles are then frozen
in liquid nitrogen and lyophilized Under some circum-
stances, as when lipids are used which are susceptible to
damage due to the presence of oxygen, it may be desirable
to seal the dry preparations under vacuum. Rehydration
is accomplished simply by adding water to the dry mixture.
Although in a preferred embodiment of the
invention, the liposomes are exposed to trehalose, it
should be understood that a variety of preseeving agents
may be substituted for trehalose, including carbohydrate
compounds which are composed of at least two monosac-
charide units. In particular, sucrose and maltose are
suitable alternatives.
The follo~ing examples illustrate certain as-
pects and embodiments oE the present invention, and are
not intended to limit the scope of the invention as
defined in the appended claims.
Exam~le_l
! A phospholipid mixture consisting of approximately 40
mg. dipalmitoyl phosphatidylcholine and phosphatidic
acid in a molar ratio of 95:5 was sonicated to optical
clarity in a bath sonicator. Large unilamellar vesicles
were prepared by freeze-thawing in a 50 mM solution of
isocitric acid in water as the compound to be encap-
sulated. Excess isocitric acid was removed by dialysis.
Trehalose (2.0:1.U trehalose:phospholipid weight ratio)
was added either after freeze-thawing or beforehand, thus
providin~ so~e large unilamellar vesicles with external
~, .
- .
.~: . . .- ' . . ~:
'' ~ '' .' : . ., :
~ - . ,.~ ,
- .
trehalose only and some vesicles with trehalose both
externally and internally. Isocitric acid was assayed by
adding isocitrate dehydrogenase and NADP to the outside
of the vesicles according to the method of Plaut, et al.
(Eds.), Methods in Enz~molo~y, Volume 5 (New York: Aca-
demic Press). Isocitrate external to the vesicles was
oxidized by the isocitrate dehydrogenase, resulting in
reduction of NADP to NADPH, the rate and amount of which
may be recorded fluorometrically. Total isocitric acid
in the vesicles was assayed following addition of Triton
X-lO0 (octylphenoxy polyethoxyethanol, a detergent and
emulsifier manufactured by Rohm ~ Haas Co., Philadelphia,
PA; "TRITON" is a registered trademark of Rohm & Haas
Co~), which releases the trapped isocitric acid into the
surrounding medium. Isocitric acid trapped in the vesi-
cles was assayed before and after both lyophilization and
rehydration, thus providing an estimate of the efficiency
with which the trapped isocitrate was retained. As may
be seen in Table 1, the results show that over sixty
percent (60%) of the trapped isocitrate was retained when
the vesicles were lyophilized with trehalose both inside
and outside the vesicles. When trehalose was present
externally only, there was still a significant increase
in the efficiency of retention, but to a lesser degree
than in the case where trehalose was present on both sides
of the lipid membrane. Examination of lipid concen-
tration at time of freezing showed that such had no
significant effect on retention of trapped material
folIowing lyophilization.
. :: .:,
.: ~ .
~.~'75~4~3
Table 1
Method of .
Preparing Concentration g Trehalose Trehalose %Reten-
LUV's of Lip_d /g Lipid External Internal tion
(mg/ml)
FT* 10.8 0 - - 0
FT 11.1 0.08 - + 0
FT 10.8 1.78 + - 42
FT 11.1 1.78 + ~ 61 :~.
:
*FT = freeze-thaw
:`:
,
,:
'
:
:
: , . . . ;, . . . . . .
: . : , : ..
- . , . ~ . -
'7S~
1~
~e~
Small unilamellar vesicles of were made by
sonication of 43 mg egg phosphatidylcholine in 4 ml of
water. Large unilamellar vesicles were prepared by
rotary drying the phospholipid in the presence of 32 mg of
trehalose and 13 mg of isocitric acid. The weight ratios
of phospholipid:trehalose:isocitric acid were approxi-
mately 4:3:1. Excess isocitric acid and trehalose were
removed by dialysis a~ainst distilled water, and the
amount oE isocitric acid trapped in the vesicles was
determined by the enzyme assay described in Example 1.
Trehalose was added to the dialyzed liposomes to give a
final weight ratio of phospholipid:trehalose of 1.0:1.4,
and the sample was lyophilized. The sample was then
rehydrated with distilled water, and the amount of
isocitric acid remaining in the liposomes was determined
by enzyme assay. The lyophilized vesicles retained 75%
of their original contents.
Example 3
A phospholipid mixture of palmitoyloleoyl
phosphatidylcholine (9U~) and phosphatidylserine (10~)
was hydrated to 10 mg./ml., and small unilamellar
vesicles were then prepared by sonication. Large uni-
lamellar vesicles were prepared by rotary drying in the
presence of isocitric acid, which served as the encap-
sulated molecule. Essentially the same techniques as
previously described in Examples 1 and 2 were used.
Efficiency of retention o isocitric acid following
lyophilization and rehydration was recorded as before,
with large unilamellar vesicles lyophili~ed first in the
presence and then in the absence of trehalose. As may be
seen in Table 2, the results show that 100~ of the trapped
..
.
~ ~ 7~j~ 4~
isocitric acid is retained when the large unilamellar
vesicles are lyophilized and rehydrated under the stated
conditions. As the previous examples demonstrated, tre-
halose is preferably present both externally and inter-
nally to optimize retention of the encapsulate.
~ 4
One of the damaging events presumed to be
occurring during lyophilization is close approach of the
large unilamellar vesicles to each other, leading to -
fusion and leakage of the vesicular contents. Fusion has
been assayed by resonance energy transfer, a fluorescence
method which depends upon energy trans~er from an excited
probe (the "donor probe") to a second probe (the "acceptor
probe"). The acceptor probe fluoresces when the energy
transfer occurs. In order for the transfer to occur the
two probes must be in close proximity. Thus probe
intermixing can be used as an assay for fusion between
vesicles during lyophilization. Large unilamellar vesi-
cles were prépared with donor probe in one preparation and
acceptor probe in anotherj and the two preparations were
mixed be~ore lyophilization. Following lyophilization
and rehydration, probe intermixing was measured, with the
results listed in Table 3~ The results show that with
increasing trehalose concentration there is a decrease in
probe intermixing. Furthermore, the presence of tre-
halose inside the liposomes alone significantly reduce
probe mixing. Thus, use of trehalose tends to reduce
fusion of the vesicles.
, . ~ . . , ~ ,
- : . : ,, , : `: ~, : ~ ' ' ` :
:. : . : ' ' ' , '
.
1 ~ 7~3~ 4
12
Table 2
Method of
Preparing g Trehalose Trehalose ~Reten-
LUV's ~ External Internal tion
_
RD* 0.06 - + 0
RD 3.2 + + 1~0
RD O -- -- O
RD 3.9 + - 26
RD 0.11 + + 22
RD 0.19 + + 49
RD 0.33 + + 69
RD ~.63 + + 76
RD 0.91 + + 86
RD 1.76 + + 99
; 15
....
*RD = rotary drying
. "
.:
- ' , . ' ` ` ' ',
. . .
~-~'75~ 8
Table 3
Method of
Preparing g ~rrehalose Trehalose ~ Probe
LUV's /~ L~pid External Internal
RD* 0.05 - + 72
RD 0.15 + + 39
RD 0.25 + + 29
RD 0.50 + + 12
~D 0.95 + + 8
FT** 0. - _ 93 ~
FT 0.4 + + 79 0
FT 0.8 + + 59
FT 1.2 + + 54,0
FT 1.6 ~ + 38.0
FT ~2.0 + + 15.0
.
*RD = rotary drying
~ ~*FT = fre~eze-thaw
: -
FU9ION BETWEEN PALMITOYLOLEOYL PHOSPHATIDYLCHOLINE: -
PHOSPHATIDYLSERINE (90:10) LARGE UNILAMELLAR VESICLES,
AS ASSAYED BY RESONANCE ENERGY TRANSFER BETWEEN FL~ORES-
CENT PROBES
' ' ' '. ': ' ". ;':
75~
Example ~
A further experiment was carried out identical
to that set forth in Example 3, with first maltose and
then sucrose as the preserving agent. Results are set
forth in Tables 4 and 5. As may be concluded from those
tables, both maltose and sucrose provide good retention
of encapsulated material following lyophilization.
Table 4
.
Method of
Preparing g Trehalose Trehalo e ~Reten-
LUV's /g Li~id Externel Internal tion
RD 0.05 - + ~ 3
RD U.lS + + 41
RD Q.25 + + 88
: RD 0 49
RD : ~.64 ~ + + lO0
,
Table 5
':
. . .
~: : Method of
~ Preparing g Trehalose Trehalose %Reten- -~
: 2:5 LUV'~ ~LipidExternal Internal tion :
RD 0.07 - + 20
RD 0.3S + + 57
RD 0.49 + + ~9
RD 0,83 + + 86
RD l.lS + + 91
.
