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Description  |
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FIELD OF THE INVENTION
The present invention relates generally to liposomes, and more particularly
to a method for encapsulating materials, such as drugs, nucleic acids,
proteins, reporter molecules, enzymes and the like, into liposomes.
Liposomes formed in accordance with the present invention are useful in
applications such as in vivo drug delivery and as diagnostic agents.
BACKGROUND OF THE INVENTION
Liposomes are unilamellar or multilamellar lipid vesicles which enclose a
three-dimensional space. The membranes of liposomes 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 to extend into the aqueous, or polar, solution and
to form a continuous, outer surface. Unilamellar liposomes have one such
bimolecular layer, whereas multilamellar vesicles generally have a
plurality of substantially concentric bimolecular layers arranged rather
like an onion.
Liposomes are well recognized as useful for encapsulating therapeutic
agents, such as cytotoxic drugs or other macromolecules capable of
modifying cell behavior, and carrying these agents to in vivo sites. For
example, U.S. Pat. No. 3,993,754, inventors Rahman et al., issued Nov. 23,
1976, discloses an improved method for chemotherapy of malignant tumors in
which an antitumor drug is encapsulated within liposomes and the liposomes
are injected into an animal or man. U.S. Pat. No. 4,263,428, inventors
Apple, et al., issued Apr. 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
index.
Liposomes have also been used in vitro as valuable tools to introduce
various chemicals, biochemicals, genetic material and the like into viable
cells, and as 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 (1980). Also, several liposome encapsulation methods are
disclosed in the patent literature.
For example, U.S. Pat. No. 4,235,871, inventors Papahadjopoulos and Szoka,
issued Nov. 25, 1980, describes a method whereby large unilamellar
vesicles can be formed which encapsulate large macromolecules. A principle
disadvantage of this method is the exposure of the material to be
encapsulated to organic solvent, such as diethyl ether, which may result
in denaturation of sensitive proteins.
U.S. Pat. No. 4,016,100, inventors Suzuki et al., issued Apr. 5, 1977,
describes the entrapment of certain pharmaceuticals in lipid vesicles by
freezing the aqueous phospholipid dispersion of pharmaceutical and lipid.
It is not clear as to the bio-availability of the total material
encapsulated, and the technique may not be efficient for pharmaceuticals
of a relatively polar nature. Also, the necessity for freezing, thawing
and then separating large volumes is expensive for large-scale, commercial
preparation.
Although encapsulation of therapeutic agents and biologically active
compounds in liposomes has significant potential for delivering such
materials to targeted sites in the human body and for diagnostic
applications, producing encapsulated materials on a commercially feasible
scale has been a problem. The current methods involve organic solvents or
detergents which are expensive, difficult to remove, or present health
hazards, and which may interact unfavorably with the therapeutic agents or
biologically active molecules to be encapsulated.
It is an object of the present invention that a method be provided which is
suitable for the encapsulation of a wide variety of materials, including
biologically active macromolecules such as nucleic acids, polypeptides,
and enzymes, and which has trapping efficiencies up to about fifty percent
of the original material utilized for encapsulation.
It is a further object of the present invention to provide a method which
is simple, avoids the use of organic solvents or detergents, and which is
feasible and inexpensive for large-scale, commercial production of
liposomes having materials encapsulated therein.
SUMMARY OF THE INVENTION
A method for encapsulating materials into liposomes comprises providing a
first polar solution. The first polar solution has initial liposomes and a
quantity of material to be encapsulated dispersed therein. Substantially
all of the first polar solution is removed and a concentrated admixture of
the initial liposomes and the quantity of material to be encapsulated is
formed. The resultant liposomes are then readily recovered by hydrating
the concentrated admixture. The resultant liposomes encapsulate from about
1 weight percent to about 50 weight percent (or greater) of the material.
This value can be controlled by the original weight ratio of lipid to
solute, and often reaches a maximum at approximately 10:1 lipid:solute
ratios.
A preferred embodiment is wherein the first polar solution is aqueous. Use
of water as a single, aqueous phase in which both the initial liposomes
and the material to be encapsulated are dispersed avoids possible
unfavorable interactions with the selected material for encapsulation and
obviates the removal of organic solvents or detergents of prior methods.
Instead, the water of the preferred embodiment can be readily removed with
conventional equipment while forming the concentrated admixture of initial
liposomes and material to be encapsulated. The inventive method can be
conveniently practiced in commercial quantities, such as for
pharmaceutical or diagnostic preparations.
A mechanism for the inventive encapsulation process is believed to be that
the initial liposomes are unable to maintain a stable bilayer structure
during removal of the first polar, or aqueous, solution. Thus, as the
initial liposomes are concentrated during removal of the solution, they
flatten and fuse around the flattened edges. During fusion, the material
to be encapsulated is believed to be sandwiched between the resulting
lamellae of the fused liposomes. When the concentrated admixture is
subsequently rehydrated, the lamallae swell and disperse into larger,
vesicle structure, that is the resultant liposomes, with a significant
fraction of the original material present being encapsulated within the
three-dimensional space.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Generally, and as well known, the lipid membranes of liposomes are formed
by a bimolecular layer of one or more naturally occurring and/or synthetic
lipid compounds having polar heads and nonpolar tails.
Representative, suitable phospholipids or lipid compounds for forming
initial liposomes useful in the present invention are phosphatidylcholine
("PC"), both naturally occurring and synthetically prepared, phosphatidic
acid ("PA"), phosphatidylserine ("PS"), phosphatidylethanolamine ("PE"),
sphingolipids, phosphatidyglycerol ("PG"), spingomyelin, cardiolipin,
glycolipids, gangliosides, cerebrosides and the like used either
singularly or intermixed such as in soybean phospholipids.
More particularly useful phospholipids include egg phosphatidylcholine
("EPC"), dilauryloylphosphatidylcholine ("DLPC"),
dimyristoylphosphatidylcholine ("DOPC"), dipalmitoylphosphatidylcholine
("DPPC"), distearoylphosphatidylcholine ("DSPC"),
1-myristoyl-2-palmitoylphosphatidylcholine ("MPPC"),
1-palmitoyl-2-myristoyl phosphatidylcholine ("PMPC"),
1-palmitoyl-2-stearoyl phosphatidylcholine ("PSPC"),
1-stearoyl-2-palmitoyl phosphatidylcholine ("SPPC"),
dioleoylphosphatidylycholine ("DOPC"), dilauryloylphosphatidylglycerol
("DLPG"), dimyristoylphosphatidylglycerol ("DMPG"),
dipalmitoylphosphatidylglycerol ("DPPG"), distearoylphosphatidylglycerol
("DSPG"), dioleoylphosphatidylglycerol ("DOPG"), dimyristoyl phosphatidic
acid ("DMPA"), dipalmitoyl phosphatidic acid ("DPPA"), dimyristoyl
phosphatidylethanolamine ("DMPE"), dipalmitoyl phosphatidylethanolamine
("DPPE"), dimyristoyl phosphatidylserine ("DMPS"), dipalmitoyl
phosphatidylserine ("DPPS"), brain phosphatidylserine ("PS"), brain
sphingomyelin ("BSP"), dipalmitoyl sphingomyelin ("DPSP"), and distearoyl
sphingomyelin ("DSSP").
The lipid composition of both the initial liposomes and the resultant
liposomes, formed in accordance with the inventive encapsulation method,
is normally the same. Where the resultant liposomes are intended for in
vivo applications (such as drug delivery), then it is normally desirable
that the lipid composition have a transition temperature below body
temperature. Liposomes which are composed of phospholipids and which are
at temperatures below the characteristic gel-liquid crystalline phase
transition temperature are considered "solid," and when above this
transition temperature are considered "fluid. " Another consideration in
selecting the composition of lipid or lipids for liposome applications is
that alkyl-ether linked lipids (rather than ester linked) are more stable
to hydrolysis, and hence alkyl-ether linked lipids for the resultant
liposomes may be particularly desirable for diagnostic applications.
In addition, other lipids such as steroids, cholesterol, aliphatic amines
such as long chain aliphatic amines and carboxylic acids, long chain
sulfates and phosphates, dicetyl phosphate, butylated hydroxytoluene,
tocophenol, retinol, and isoprenoid compounds may be intermixed with the
phospholipid components to confer certain desired and known properties on
the initial liposomes and hence the resultant liposomes. Further,
synthetic phospholipids containing either altered aliphatic portions, such
as hydroxyl groups, branched carbon chains, cycloderivatives, aromatic
derivatives, ethers, amides, polyunsaturated derivatives, halogenated
derivatives, or altered hydrophllic portions containing carbohydrate,
glycol, phosphate, phosphonate, quaternary amine, sulfate, sulfonate,
carboxy, amine, sulfhydryl, imidazole groups and combinations of such
groups, can be either substituted or intermixed with the phospholipids.
The material to be encapsulated is preferably soluble in the first polar
solution, most preferably is substantially water-soluble where the
solution is aqueous, but may be substantially insoluble in the selected
polar solution so long as the material is of a suitably small size to be
dispersed in the polar, or aqueous, solution and subsequently encapsulated
within the resultant liposomes. That is, since the diameter of the
resultant liposomes (before filtration) will typically range up to about
100 microns, substantially insoluble materials, such as particulate
materials, should be sufficiently minute as to be encapsulated within the
three-dimensional, confined space of the resultant liposomes. Also,
suitable materials for encapsulation will not contain exposed hydrophobic
portions which would prevent entrapment, and are less volatile than the
polar solution in which the materials are dispersed.
Suitable therapeutic agents for encapsulation include, for example,
symphathomimetic agents such as amphetamine sulfate, epinephrine
hydrochloride or ephedrine hydrochloride; antispasmodics such as
hyosthiamine, atropine, scopolamine hydrobromide, timepidium bromide;
bronchodilators such as tretoquinol hydrochloride or isoproterenol
hydrochloride; vasodilators such as dilthiazem hydrochloride or
dipyridamole, hemostatics such as carbazo-chrome sodium sulfate; vitamins
such as bisbutylthiamine; hormones such as insulin; antibiotics such as
amino benzylpenicillin, alpha-phenoxypropylpenicillin or
alpha-carboxybenzylpenicillin; and antineoplastic agents such as
daunorubicin and adriamycin.
Suitable biologically active compounds and diagnostic agents for
encapsulation include, for example, RNA, DNA, enzymes, and
immunoglobulins, such as IgG and Fab' fragments. Also, various natural and
synthetic enzyme substrates for enzyme analyses may be encapsulated.
Suitable reporter molecules for encapsulation include, for example,
radioactive ions, chemiluminescent molecules and fluorescent molecules.
The initial liposomes may be formed and dispersed in the polar solution by
a variety of known techniques, such as sonication, injection of an alcohol
solution of lipid into the aqueous phase, extrusion with a French press
under very high pressure, and homogenization, where the majority of
initial liposomes therefrom are unilamellar. A particularly preferred
technique is by sonication of the lipid composition in distilled water.
Preferred lipid concentrations are from about 1 mg/ml to about 50 mg/ml,
more preferably from about 5 mg/ml to about 20 mg/ml. The initial
liposomes may also be treated so as to be relatively homogeneous in size
by means such as sequential extrusion through defined pore size
polycarbonate membranes, as described by Olson, et al., Biochem. Biophys.
Acta. 557: 9-23 (1979).
The material to be encapsulated may be dispersed in the polar solution
before, after, or during the formation and dispersal of initial liposomes.
It is normally preferable to simply combine the material to be
encapsulated with already formed initial liposomes in the selected polar
solution at a desired mass ratio. Typical mass ratios of initial liposomes
and material are from about 1:1 to about 100:1, more preferably from about
2:1 to about 50:1.
The critical solution removal step to form a concentrated, intimate
admixture of the initial liposomes and the material for encapsulation in
accordance with the present invention is preferably effected by
evaporation of the single phase, polar solvent, either under a reduced
pressure (such as about 10-50 mm Hg) or by passing a dry gas over the
solution.
For most efficient removal of the aqueous phase, the vessel containing the
dispersion can be rotated to spread the dispersion over a larger surface
area. Alternatively, the dispersion can be sprayed into an evacuated
vessel (flash drying). If the material for encapsulation, or solute, is
not sensitive to heat, the solution can be warmed to speed the evaporation
process. As the lipid approaches dryness, the originally dispersed
vesicles touch and fuse, forming a multilamellar structure that
"sandwiches" the solute. Where solution removal is effected by evaporation
under a reduced pressure or by means of a dry gas, the concentrated
admixture of initial liposomes and material to be encapsulated typically
forms a highly viscous, gel-like residue.
Water, or any desired aqueous solution, may then be simply added to
redisperse the resultant liposomes, which take the form of a heterogeneous
population of unilamellar and multilamellar vesicles containing up to
about half of the originally present material. These resultant liposomes
may be made more uniform by filtration, centrifugation or gel permeation
chromatography. If it is desired to remove solute external to the
resultant liposomes, the latter procedure can be used simultaneously.
The following experimental methods, materials and results are described for
purposes of illustrating the present invention. However, other aspects,
advantages and modifications within the scope of the invention will be
apparent to those skilled in the art to which the invention pertains.
EXAMPLE I
A lipid mixture (100 mg lipid) containing 50 mole % phosphatidylserine, 25
mole % phosphatidyl ethanolamine and 25 mole % cholesterol is probe
sonicated for 10 min in 10 ml water to form small unilamellar vesicles.
Calf thymus DNA (10 mg) in 1 ml water is then added, and the mixture is
placed in a rotary evaporator and warmed to 50.degree. C. with nitrogen
gas being blown over it during rotation to remove solvent. After removing
substantially all the solvent (about one hour), the concentrated, intimate
admixture of lipids and DNA is easily hydrated by adding 10 ml of water
while rotation is continued, which causes redispersion of the sample. The
dispersed sample is withdrawn and passed once through a 1.2 .mu.m
polycarbonate filter, followed by gel filtration to remove soluble DNA
external to the vesicles. Upon analysis, the vesicles are found to have
encapsulated 47% of the original DNA present, and range in size up to 2
.mu.m in diameter. The biologically active material so encapsulated is
suitable, for example, for delivery of DNA to viable cells.
EXAMPLE II
Egg phosphatidylcholine (100 mg) is dispersed by agitation in 10 ml water
containing 1.0 mM 6-carboxylfluorescein ("6-CF"), followed by passage
through a French press at 400 kg/cm.sup.2 pressure. The solution is taken
to substantial dryness as described in Example I, then redispersed by
addition of 10 ml water while the flask is rotating. The dispersed
liposomes are sized by passage through 1.2 .mu.m polycarbonate filter,
followed by appropriate gel permeation chromatography to remove the
external 6-CF. Upon analysis, 22% of the 6-CF is found to encapsulated.
EXAMPLE III
Mixed soybean phospholipid (100 mg) is dissolved in 1.0 ml ethanol and
dispersed as small unilamellar vesicles by injection into 10 ml water.
Hemoglobin is then added to a final concentration of 1.0 mg/ml, and the
solution is dried by rotary evaporation as described in Example I,
followed by polycarbonate filtration and gel permeation chromatography to
remove external hemoglobin. The resulting vesicles range up to 2 .mu.m in
diameter and were found to encapsulate 36% of the original hemoglobin
present.
EXAMPLE IV
Encapsulation of salmon sperm DNA was carried out generally as described by
Example II, but with varying amounts of lipid with respect to DNA. Thus,
100 .mu.g DNA was combined with 0.2 mg/ml, 0.5 mg/ml, 1.0 mg/ml, 2.0
mg/ml, 5.0 mg/ml and 10 mg/ml lipid, respectively. Following rehydration,
1 ml of buffer was added (50 mM Tris-HCl, 5 mM MgCl.sub.2, 0.2 mM
beta-mercaptoethanol), followed by 20 units pancreatic DNAse to hydrolyze
and external DNA that might be binding to vesicle surfaces. After one
hour, the liposomes were pelleted (10 kg, 60 min), washed once in buffer,
and the DNA content was precipitated by addition of 4 ml ethanol, followed
by freezing in liquid nitrogen and centrifugation (30 min, 10,000 X g,
-10.degree. C.). The DNA pellet was resuspended in 2 ml potassium
phosphate buffer (0.1M, pH 8.0) and scanned spectrophotometrically. The
absorbance at 260 nm was then compared with that of the original DNA
present and expressed as percent encapsulated. Table I, below, illustrates
the encapsulation efficiency as the mass ratio of lipid/DNA is varied
between 2:1 to 100:1.
TABLE I
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Phosphatidylcholine (mg)
0.2 0.5 1.0 2.0 5.0 10
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Deoxyribo- 1.1 5.0 9.8 24 45 39
nucleic acid
(% encapsulated)
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While the invention has been described in connection with specific
embodiments thereof, it will be understood that it is capable of further
modification, and this application is intended to cover any variations,
uses or adaptations of the invention following, in general, the principles
of the invention and including such departures from the present disclosure
as come within known or customary practice in the art to which the
invention pertains and as may be applied to the essential features
hereinbefore set forth, and as fall within the scope of the invention and
the limits of the appended claims.
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