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Description  |
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FIELD OF THE INVENTION
The present invention relates generally to liposomes, and more particularly
to liposomes which may encapsulate materials, such as drugs, nucleic
acids, proteins, reporter molecules and the like, and which have a
plurality of thiol reactive groups connected to and extending from the
lipid bilayer. These thiol reactive liposomes may be readily and
efficiently covalently bound to a variety of ligands having thiol groups
for uses such as the specific targeting of chemotherapeutic agents, as
immunodiagnostic agents, and the like.
The invention described herein was made in the course of work under a grant
or award from the Department of Health and Human Services.
BACKGROUND OF THE INVENTION
Liposomes are unilamellar or multilamellar lipid vesicles which enclose a
three-dimensional space. The lipid 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 solution, the polar heads of one layer
orient outwardly to extend into the aqueous 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. Thus,
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.
However, a deficiency of liposomal drug delivery has been the inability to
quantitatively or selectively direct the liposomes' contents to specific
sites of action over a therapeutically meaning time frame.
It has been suggested that target, or site, specificity might be conferred
on liposomes by their association with specific antibodies or lectins.
Methods of associating antibodies with liposomes have been described and
may be generally divided into two groups--nonspecific association and
covalent attachment.
Non-specific association appears to rely upon the affinity of the Fc
portion of the antibody for the hydrophobic region of the lipid bilayer.
This has little practical value because the liposomes are rendered more
permeable to their encapsulated contents and may themselves be aggregated.
Further, it is not believed that this complex would be sufficiently stable
in plasma for the considerable periods of time believed necessary in many
potential clinical applications.
Considerable effort has ensued in attempts to covalently attach protein to
liposomes, with several promising results. For example, Heath et al., have
reported efficiently covalently binding liposomes to biologically active
proteins by periodate oxidation of glycosphingolipids. Science, Vol. 210,
pp. 539-541 (1980). This method of liposome-antibody conjugation has bound
up to about 200 .mu.g of protein per .mu.mole of total lipid.
SUMMARY OF THE INVENTION
It is an object of the present invention that liposomes be provided which
may be readily and efficiently covalently bound to a variety of ligands
bearing thiol groups to achieve reproducible, high coupling ratios without
vesicle aggregation.
It is a further object of the present invention that the liposomes,
following coupling with ligands, result in a highly stable ligand-vesicle
linkage, and particularly result in a linkage which is stable in serum or
in the presence of reducing agents.
It is a further object of the present invention that ligands, particularly
antibodies, retain a substantial amount of antigen binding capacity after
having been coupled to the inventive liposomes.
These and other objects of the present invention are provided by liposomes
having a lipid bilayer defining an outer surface. A plurality of thiol
reactive groups are integrally connected to the lipid bilayer and extend
outward with respect to the outer surface. Particularly preferred
embodiments of the present invention are maleimide moieties as the thiol
reactive groups. A representative one of such a thiol reactive liposome is
illustrated by the following structure (wherein a portion which includes a
maleimide moiety is enlarged relative a diagrammatic liposome
representation):
##STR1##
The thiol reactive liposomes may be separated from impurities by
conventional techniques after formation and then stored.
Thiol reactive liposomes in accordance with the present invention form
quite stable covalent bonds with ligands having thiol groups, such as Fab'
fragments. For example, liposomes as above illustrated, when coupled with
Fab', resulted in no coupled Fab' being lost during incubation for 24
hours in 50% human serum.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Both naturally occurring and synthetic lipids are known and useful in
forming liposomes. For example, naturally occurring lipids such as
phosphoglycerides, sphingolipids, and glycolipids are all characterized by
having polar head regions and non-polar tail regions which form
bimolecular layers readily in aqueous systems. A variety of synthetic
lipids (often differing from the naturally occurring lipids simply by
having different hydrocarbon chain lengths in the non-polar tail regions)
are also known and have been used to form liposomes. In addition,
components such as vitamin E (normally considered to be a lipid since it
is insoluble in water but extractable with organic solvents) and the like
may be included in liposomal membranes.
The fluid encapsulated by liposomes normally includes a polar liquid, or
aqueous, phase into which the polar heads of the membranes' inner layer
extend. The fluid may carry, either dissolved or undissolved, a wide
variety of other components. For example, the fluid may include
biologically active molecules, pharmaceuticals, nutrients, and reporter
molecules such as radioactive ions, chemiluminescent molecules and
fluorescent molecules.
Liposomes may be prepared by any of various of conventional methods known
to the art. These various known methods may be generally characterized as
yielding either unilamellar vesicles or multilamellar vesicles. Either
liposomal structure is suitable for the present invention; however, due to
the generally larger internal space available in unilamellar liposomes,
the inventive liposomes are preferably prepared by the reverse-phase
evaporation vesicle (REV) method, as is described in U.S. Pat. No.
4,235,871, issued Nov. 25, 1980, inventors Szoka, Jr., and
Papahadjopoulos, which description is incorporated herein by reference.
Liposomes in accordance with the present invention include a plurality of
thiol reactive groups. These thiol reactive groups are adapted to form
either thioether bonds or disulfide bonds with ligands bearing thiol
groups. The thiol reactive groups are at polar head regions of nitrogen
containing lipids which are components of at least the lipid bilayer which
defines an outer surface for the liposomes. (e.g. in the instance of
unilamellar vesicles this lipid bilayer is the solid lipid bilayer,
whereas in the instance of multilamellar vesicles, the nitrogen containing
lipids are components of at least the most outward of the lipid bilayers).
A suitable nitrogen containing lipid is normally one component of two or
more lipid components, or lipid mixture, constituting the liposomal
membrane. When liposomes in accordance with the present invention are
formed from a mixture of two or more lipid components, then the nitrogen
containing lipids having thiol reactive groups bound thereto may
constitute up to about 80 mole % with respect to the total lipid content.
Primary considerations in selecting the amount of nitrogen containing
lipids having thiol reactive groups are that too large a concentration may
lead to aggregation of the vesicles or of insufficient integrity (such as
permeability) of the liposomal membrane; alternatively, too little of the
nitrogen containing lipids bearing thiol reactive groups may result in
inadequate coupling ratios of ligands per vesicle lipid content. For most
applications, the mole % of nitrogen containg lipids having thiol reactive
groups will be in an amount of about 0.01 mole % to about 80 mole %. For
example, coupling ratios in excess of 250 microgram Fab' per micromole of
total lipid have been reproducably obtained with about 2.5 mole %.
Any of the amphiphilic substances known to produce liposomes may be
utilized. Nevertheless, some mixtures of lipids may tend to be permeable
to small molecules, and cholesterol is frequently a desirable addition to
some of these lipid mixtures for reducing the permeability thereof. Other
components may also be utilized to reduce liposome permeability. For
example, a phosphatidyl choline having the fatty acid saturated aliphatic
chain, or non-polar tails, of a length of 18 (rather than the usual
unsaturated 16 to 18 carbon chain obtainable from egg yolks) may be
utilized.
A variety of nitrogen containing lipid precursors may be derivatized in
order to bear the suitable thiol reactive groups. Thus, for example,
nitrogen containing lipid precursors having primary or secondary amino
groups within the polar head region may be reacted with a suitable
activating group, or reagent (further discussed hereinafter), to form an
amide or an amidine linkage. Suitable nitrogen containing lipid precursors
include, for example, phosphatidylethanolamine, phosphatidylserine,
stearylamine, glycolipids with amino substituted sugars, and the like.
The ligand is anchored to the liposome surface via a covalent bond with the
nitrogen containing lipids, which are structural parts of the lipid
bilayer. This may be accomplished either by derivatizing the primary or
secondary amino groups of lipids in preformed liposomes, or by first
derivatizing the nitrogen containing lipid precursors and then forming the
inventive liposomes. The latter is preferred because of convenience in
preparing the liposomes, since the derivatized lipid precursors can be
prepared in advance, used to form liposomes when desired, and the
resultant liposomes will automatically bear the thiol reactive groups.
As has previously been noted, thiol reactive groups of the liposomes are
adapted to form disulfide or thio ether bonds with ligands bearing thiol
groups. Preferred thiol reactive groups adapted to form disulfide bonds,
for example with IgG fragments bearing sulfhydryl groups, are
2-Pyridyldithiol, 4-Pyridyldithiol, and thiosulphate. A particularly
preferred thiol reactive group of the type adapted to form thio ether
bonds, for example with IgG fragments bearing sulfhydryl groups, includes
maleimide moieties.
In both instances, it is preferred that the thiol reactive groups be spaced
from the amide or amidine linkages of the nitrogen containing lipids by
organic spacer arms. These organic spacer arms may be composed of a wide
variety of organic moieties, such as carbon chains (branched or unbranched
and saturated or unsaturated) as well as rings, particularly aromatic
rings such as substituted or unsubstituted phenyl moieties. Suitable
organic spacer arms will not interfere with coupling reactions between the
thiol reactive groups and ligands, and function to position the thiol
reactive groups to extend outward of the liposomal outer surface. This
positioning favors coupling reactions with ligands.
Derivatives of nitrogen containing lipids may be formed by reaction with
suitable reagents. A suitable reagent may be viewed as having an amino
reactive moiety at one end of the molecule, the thiol reactive group at
the other end of the molecule, and the organic spacer arm therebetween.
Where the liposomes are preformed and include nitrogen containing lipid
precursors, the thiol reactive groups may be incorporated via amide or
amidine linkages as follows. Where the amino reactive moiety is an
aldehyde, the primary or secondary amino group of a nitrogen containing
lipid precursor in an aqueous solution may be reductively aminated in the
presence of a reducing agent such as sodium cyanoborohydride or sodium
borohydride. Where the amino reactive moiety is, for example, methyl
imidate, an amidine linkage will form spontaneously in aqueous solution
with a primary amino group of a nitrogen containing lipid precursor. Where
the amino reactive moiety is N-succinimide, an amide linkage forms
spontaneously with a primary amino group of the lipid precursor.
Where the liposomes are to be formed from a lipid mixture, the nitrogen
containing lipid precursor may be derivatized as follows. A lipid solution
may be formed and a suitable reagent admixed. Where the amino reactive
moiety of the reagent added is an aldehyde, then the lipid may be
solubilized in, for example, chloroform:methanol (1:1). In the presence of
a reducing agent, such as sodium cyanoborohydride, sodium borohydride or
lithium cyanoborohydride, primary or secondary amino groups of the lipid
precursor will be reductively aminated. Where the amino reactive moiety of
the added reagent is methyl imidate, a primary amino group of the lipid
precursor will react, in the presence of triethylamine, to form an amidine
linkage. Similarly, use of a reagent having N-succinimide as the amino
reactive moiety results in an amide linkage.
Once formed, the inventive liposomes having thiol reactive groups may be
separated from impurities by one or a combination of techniques, such as
gel chromatography, flotation in polymer gradients, and the like. The
liposomes may be stored at low temperature (for example about 4.degree.
C.) as an aqueous suspension under an inert atmosphere. The liposomes may
also be extruded to control their size, and may be subjected to
manipulations which remove non-encapsulated materials.
Preparation of several embodiments of the present invention will now be
more particularly described. Various abbreviations will sometimes be used,
many of which are listed along with their definitions below.
PE (transesterified egg phosphatidylethanolamine)
PC (phosphatidylcholine)
DPPC (dipalmitoylphosphatidylcholine)
DTNB (5,5-dithiobis 2-nitrobenzoic acid)
CDI (carbonyldeimidazole)
DDT (dithiothreitol)
SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate)
2-TP (2-Thiopyridinone)
PDP-PE (N-[3-(2-Pyridyldithiopropionyl] phosphatidylethanolamine)
SMPB (succinimidyl 4-(p-maleiminidophenyl) butyrate)
MPB-PE (N-[4-(p-maleimidophenyl) butyryl] phosphatidyethanolamine
SUV (small unilamellar vesicles)
LUV (large unilamellar vesicles)
REV (reverse phase evaporation)
Buffer I (100 mM NaCl, 100 mM borate, 50 mM citrate, and 2 mM EDTA)
Buffer II (35 mM NaCl, 100 mM borate, 50 mM citrate, and 2 mM EDTA)
Buffer IA (35 phosphate, 20 mM citrate, 108 mM NaCl and 1 mM EDTA)
EXAMPLE I
PDP-PE LIPOSOMES
Synthesis of PDP-PE.
PE (50 .mu.mol) was dissolved in 3 mL of anhydrous methanol containing 50
.mu.mol of triethylamine and 25 mg of SPDP. The reaction was carried out
at 25.degree. C. under an argon atmosphere. Following 5 h, TLC of the
reaction mixture revealed quantitative conversion of the PE to a faster
running product. Methanol was removed under reduced pressure, and the
products were redissolved in chloroform and applied to a 10-mL silica gel
column which had been activated (150.degree. C. overnight) and prewashed
with 100 mL of chloroform. The column was washed with an additional 20 mL
of chloroform followed by 20 mL of each of the following
chloroform-methanol mixtures 40:1, 30:1, 25:1, 20:1, and 15:1 and,
finally, with 60 mL of 10:1 chloroform-methanol. The phosphate-containing
fractions eluting in 15:1 and 10:1 chloroform-methanol were pooled and
concentrated under reduced pressure.
Analysis by TLC (silica gel H; solvent chloroform-methanol-acetic acid,
60:20:3) indicated a single phosphate-positive, ninhydrin- and
sulfhydryl-negative spot. Identification of the product as the
(pyridyldithio)propionyl derivative of PE was confirmed by our observation
that a stoichiometric amount of 2-thiopyridinone (2-TP) is released upon
the addition of excess DTT. No detectable decomposition of PDP-PE was
observed for periods of up to 6 months when stored in glass ampules under
argon at -50.degree. C.
Figure I, below, generally illustrates the above described reaction scheme.
##STR2##
(where R.sub.1 and R.sub.2 are traditionally carbon chains of various
lengths)
Preparation of Vesicles.
Vesicles were prepared by the reverse-phase evaporation method of Szoka &
Papahadjopoulos (1978) Proc. Natl. Acad. Sci. U.S.A., Volume 75, No. 9,
pp. 4194-4198, also disclosed by U.S. Pat. No. 4,235,871. Briefly, 10
.mu.mol of cholesterol, 9 .mu.mol of PC, 1 .mu.mol of PDP-PE and a trace
amount of [.sup.3 H]DPPC were dissolved in 1 mL of freshly distilled
diethyl ether. Buffer I (pH 6.0) (0.3 mL) was added, and the two phases
were emulsified by sonication for 2 min at 25.degree. C. in a bath-type
sonicator. Ether was removed under reduced pressure at 30.degree. C. The
resulting vehicle dispersion was extruded through 0.4- and 0.2- .mu.m pore
Bio-Rad Laboratories Uni-Pore polycarbonate membranes, as is described in
U.S. Pat. No. 4,263,428, issued Apr. 21, 1981, to produce uniformly sized
vehicles. For determination of internal volumes, vesicles were prepared in
the presence of 0.3 M sucrose and a trace amount of [.sup.14 C] sucrose.
The internal volume was calculated from the amount of sucrose (specific
activity of [.sup.14 C] sucrose) remaining after removal of unentrapped
solute by gel filtration on Sephadex G-25. Sucrose efflux, expressed as
the proportion of sucrose remaining entrapped for periods up to 24 h, was
determined by dialysis.
Characterization of Vesicles.
Electron microscopic observations reveal that vesicles composed of PC,
cholesterol, and PDP-PE (45:50:5), prepared by the reverse-phase
evaporation method and extruded through 0.2- .mu.m pore membranes, are
spherical in shape and range in diameter from about 500 .ANG. to 0.5
.mu.m. The vast majority of vesicles, however, fall in the size range of
1000-3000 .ANG., the mean diameter being about 1900 .ANG.. Occasional
multilamellar vesicles are visible in such EM preparations.
The encapsulated volume of such vesicles, calculated from the specific
activity of [.sup.14 C] sucrose remaining associated with vesicles
following removal of the unentrapped solute by gel filtration, is
4.5.+-.0.3 .sup..mu. L/.sup..mu. mol of vesicle phospholipid, slightly
less than the predicted value of 6.4 .mu.L/mol, assuming that all vesicles
are single layered and 0.2 .mu.m in diameter. The permeability of these
vesicles to sucrose was found to be quite low. The rate of sucrose efflux
is less than 1%/h at 25.degree. C.
The low value for sucrose encapsulation together with the EM results
suggests that a small proportion of the PC-cholesterol-PDP-PE vesicles
used in this study are multilamellar. In order to determine more precisely
the average number of lamellae per vesicle, we have synthesized a reducing
agent, DHLA-dextran T-20, which cannot permeate vesicle bilayers but is
capable of reducing the pyridyl disulfide moiety of PDP-PE molecules that
are exposed in the outer monolayer of vesicles. We have measured the
appearance of 2-TP, which is released as a product of PDP-PE reduction, to
determine the proportion of PDP-PE molecules present in preformed vesicles
that are accessible to this impermeable reducing agent. 54.5 nmol of 2-TP
is released within 5 min following the addition of excess DTT (which
freely permeates vesicle bilayers) to a suspension of PDP-PE-containing
vesicles (0.5 .mu.mol of total phospholipid). This corresponds closely to
the expected value of 50 nmol (0.1 mol fraction of the total phospholipid
in these vesicles is PDP-PE).
Figure II, below, illustrates a thiol reactive liposome of the PDP-PE
species, with the PDP-PE lipid component being enlarged relative the
diagrammatic representation of the lipid bilayer which forms the liposomal
outer surface.
##STR3##
As may be understood, the particular --(CH.sub.2).sub.2 -- organic spacer
arm of the Figure II structure, above, may vary (for
example--(CH.sub.2).sub.n -- where n is the integer 1 or greater).
EXAMPLE II
MPB-PE LIPOSOMES
Synthesis of MPB-PE:
Transesterified egg PE (100 .mu.mol) was dissolved in 5 ml anhydrous
methanol containing 100 .mu.mol freshly distilled triethylamine and 50 mg
succinimidyl 4-(p-maleimindo phenyl) butyrate (SMPB). The reaction was
carried out under an argon atmosphere at room temperature. Thin layer
chromatography of the mixture following two hours revealed quantitative
conversion of the PE to a faster running product (R.sub.f 0.52, silica gel
H, solvent: chloroform-methanol water, 65:25:4). Methanol was removed
under reduced pressure and the products redissolved in chloroform. The
chloroform phase was extracted twice with 1% NaCl to remove unreacted SMPB
and water soluble byproducts. The MPB-PE was further purified by silicic
acid chromatography as described for PDP-PE. Following purification, TLC
indicated a single phosphate positive, ninhydrin-negative spot. MPB-PE is
stable for at least 4 months when stored at -50.degree. C. as a chloroform
solution sealed in glass ampules under argon. Figure III, below, generally
illustrates the above described reaction scheme.
##STR4##
Preparation of Vesicles:
LUV were prepared by the reverse phase evaporation method of Szoka and
Papahadjopoulos, supra, with minor modifications. Briefly, 10 .mu.mol
cholesterol, 0.5 .mu.mol PC, 0.5 .mu.mol MPB-PE and a trace amount of
(.sup.3 H)DPPC were dissolved in 1 ml diethyl ether. Buffer (20 mM citric
acid, 35 mM disodium phosphate, 108 mM NaCl, 1 mM EDTA, pH 4.5) was added
(300 .mu.l) and the two phases emulsified by sonication for 1 min at
25.degree. C. in a bath-type apparatus. Ether was removed under reduced
pressure at room temperature and the resulting vesicle dispersion extruded
through 0.4.mu. and 0.2.mu. Uni-Pore polycarbonate membranes (Bio-Rad
Laboratories).
The size, encapsulated volume and substantially unilamellar characteristics
of the MPB-PE vesicles were substantially as described for PDP-PE
vesicles, above.
Figure IV, below, illustrates a thiol reactive liposome of the MPB-PE
species, with the MPB-PE lipid component being enlarged relative the
diagrammatic representation of the lipid bilayer forming the liposomal
outer surface.
##STR5##
As may be understood, the particular --(CH.sub.2).sub.3 --.phi.-- organic
spacer arm of the Figure IV structure, above, may vary. For example, where
--(CH.sub.2).sub.n -- and n is 1 to about 6, and where the maleimide
moiety is substituted at a meta or ortho position on the phenyl group.
EXAMPLE III
S-SULFONATE LIPOSOMES
S-sulfonate liposomes are prepared in a manner analogous to Examples I and
II, with the reaction scheme for sulfonation of PE being in accordance
with the method of Oeltmann and Heath, J. Biol. Chem. 254: 1022-1027
(1979) and generally represented by Figure V, below, and the particular
S-sulfonate species of the thiol reactive liposomes being illustrated by
Figure VI in a similar manner to that of Figures II and IV.
##STR6##
As may be understood, the --(CH.sub.2).sub.n -- organic spacer arm of
Figure VI, above, may vary (and originates from n of Figure V), normally
wherein n is an integer from 1 to about 6.
EXAMPLE IV
HALOACETYL LIPOSOMES
Haloacetyl liposomes are prepared in a manner analogous to Examples I and
II, with the reaction scheme for sulfonation of PE being in accordance
with the method of Rector, et al., J. Immuno. Methods 24: 321-336 (1978)
and generally represented by Figure VII, below, and the particular
haloacetyl species of the thiol reactive liposomes being illustrated by
Figure VIII in a similar manner to that of Figures II and IV.
##STR7##
wherein n is normally 1 to about 6.
Coupling of Ligands to the Thiol Reactive Liposomes
The thiol reactive liposomes are adapted to react with ligands having
reactive thiol groups. As used herein, ligand means a compound which can
interact specifically but non-covalently with a ligand-binding molecule,
or receptor. One type of such interaction is antigen-antibody, another is
hormone-receptor, and yet another is carbohydrate-lectin.
Particularly preferred ligands for coupling to the thiol reactive liposomes
are Fab' antibody fragments, each of which contains a single reactive
thiol group at a defined position on the molecule. As is well known, the
IgG immunoglobulin molecule has a molecular weight of about 150,000 d and
possesses four peptide chains linked together by disulfide bonds. Upon
enzymatic digestion with pepsin, the Fc portions of the heavy chains are
cleaved. Treatment of the F(ab').sub.2 antibody fragments with DTT under
suitable conditions results in the selective reduction of the
interheavy-chain disulfide bond of this molecule and thereby produces two
monomeric Fab' fragments. Each monomer produced by this method contains
about one sulfhydryl group which is at one end of the monomer, while the
antigen binding site is distal therefrom. Use of Fab' fragments for
coupling to the inventive liposomes is also preferred for many
applications, as the absence of a Fc region eliminates the possibilities
of Fc-mediated binding and complement activation, and reduces the
likelihood of anti-idiotypic antibody production in vivo.
Where the ligands to be coupled to the thiol reactive liposomes of the
present invention do not contain a reactive thiol group, then such ligands
will be thiolated.
The thiol reactive liposomes form covalent bonds with suitable ligands.
These covalent bonds may be generally characterized as disulfide bonds or
thio ether bonds. Thus, for example, the PDP-PE and S-sulfonate liposomes
couple with Fab' fragments by means of disulfide linkages, whereas the
MPB-PE and haloacetyl liposomes couple by means of a thioether linkage.
As will be more fully discussed hereinafter, the MPB-PE liposomes are most
preferred for coupling with thiol reactive ligands, particularly Fab'
fragments, due to the substantially irreversible coupling of
immunoglobulin fragments to the MPB-PE vesicles. Thus, extremely stable
couplings result. For example, serum does not cause elution of conjugated
Fab' from the MPB-PE vesicles nor does it interfere with binding of
liposomes to cells. This is believed to be particularly important, as many
clinical applications of coupled liposome-ligand conjugates will require
exposure to serum for considerable periods of time.
Examples V and VI, below, illustrate preparation of Fab' antibody fragments
and coupling thereof to PDP-PE vesicles and MPB-PE vesicles respectively.
EXAMPLE V
Preparation of Fab' Antibody Fragments.
The coupling method for the covalent attachment of antibody fragments to
the surfaces of lipid vesicles depends on the availability of thiol groups
on the antibody fragments capable of participating in a disulfide
interchange reaction with the (pyridyldithio)propionyl moiety of PDP-PE
molecules present in the outer monolayer of preformed vesicles. For
minimization of vesicle aggregation due to cross-bridging, a single thiol
group per antibody fragment is desirable. Conditions for the preparation
of 50,000-dalton Fab' antibody fragments, each of which contains a single
reactive thiol group at a defined position on the molecule, were as
follows.
Treatment of rabbit F(ab').sub.2 antibody fragments with DTT (20 mM) at low
pH (5.5) for 90 min at 25.degree. C. results in the selective reduction of
the inter-heavy-chain disulfide bond of this molecule and thereby produces
two monomeric Fab' fragments. Titration of Fab' fragments with Ellman's
reagent reveals that each monomer produced by this method contains, on the
average, 0.95 sulfyhdryl group. Gel filtration on Sephadex G-75 indicates
that greater than 95% of the F(ab').sub.2 fragments is converted to the
50K Fab' during such a reduction. Moreover, when antihuman erythrocyte
F(ab').sub.2 fragments are subjected to similar DTT treatment, the
capacity of the fragments to agglutinate human erythrocytes is reduced
64-fold (the HA titer of a 10 gm/mL solution falls from 8192 to 128).
Upon the removal of DTT, Fab' monomers are unstable and tend to re-form
F(ab').sub.2 dimers as the result of an oxidative reaction between the
sulfyhydryl groups exposed on each Fab' fragment. The rate of F(ab').sub.2
formation (measured as the reduction in the number of titratable thiol
groups) is dependent on the pH and the availability of molecular oxygen.
Two hours after the removal of DTT, in the absence of O.sub.2, the number
of thiol groups per Fab' monomer is reduced to 0.75 at pH 6.0 and to 0.50
at pH 8.0. In the presence of molecular oxygen, the rate of F(ab').sub.2
formtion is accelerated, and essentially complete reannealing is observed
within 2 h at pH 8.0.
Despite the tendency of Fab' molecules to recombine into the dimer form at
alkaline pH, this competing reaction does not appear to be rate limiting
with respect to vesicle coupling. The addition of freshly reduced Fab'
fragments at 30-min intervals during the course of a coupling reaction
does not significantly improve coupling ratios. The preparation as above
described is illustrated by Figure IXA, below.
Coupling of Fab' Fragments to PDP-PE-Containing Vesicles.
The protocol we have followed in order to obtain covalent coupling of Fab'
antibody fragments to PDP-PE-containing vesicles is illustrated by Figure
IXB, below. PDP-PE vesicles are mixed with Fab' fragments (about 3 .mu.mol
of phospholipid and 1-12.5 mg of Fab') immediately following the removal
of DTT (see preceding section). The pH is adjusted to 8.0 and the coupling
reaction allowed to proceed for 2 h under argon. Unreacted antibody
fragments are then removed by gel filtration.
A mixture of control vesicles (PC-cholesterol, 50:50) and nonspecific
rabbit Fab' fragments was chromatographed on Sephadex G-150. From the
elution profile, the vesicles appear in the void volume of such a column
while the antibody fragments elute with the included volume. No binding of
Fab' fragments to control vesicles is evident. However, when 5 mol % of
PDP-PE is included in the vesicle membrane, a significant proportion
(approximately 30%) of the added Fab' coelutes with the vesicles. When
fractions from this coeluant are pooled, concentrated, and
rechromatographed on Sephadex G-150, all of the Fab' coelutes with the
vesicle peak, indicating a stable association between Fab' molecules and
vesicles. This Fab'-vesicle binding is completely reversible, however, in
the presence of 50 mM DTT at pH 8.0. These results suggest that Fab'
binding results from the formation of reversible disulfide cross-linkages
between Fab' fragments and vesicles.
##STR8##
EXAMPLE VI
Preparation of Antibody Fragments:
Fab' fragments of nonspecific rabbit IgG and anti hRBC-F(ab').sub.2
fragments were prepared and purified as described in Example V, above,
except Buffer IA(pH 5.0) containing 20 mM dithiothreitol was used for the
reduction step. F(ab').sub.2 fragments were radiolabeled with .sup.125 I
to a specific activity of .about.2.times.10.sup.6 cpm/mg prior to
reduction.
Coupling of Fab' Fragments to MPB-PE Vesicles:
The protocol of covalently coupling Fab' antibody fragments to MPB-PE
vesicles is illustrated by Figure X, below. Vesicles
(PC-Cholesterol-MPB-PE; 9.5:10:0.5) prepared by the reverse-phase
evaporation method and extruded through 0.2 .mu.Uni-Pore membranes,
entrapped about 15% of the original aqueous volume (4.73 .mu.l/.mu.mol
phospholipid). Sucrose efflux was less than 0.5% per hour in Buffer I at
25.degree. C. and less than 3% per hour in 50% serum. The half-life of the
maleimide was greater than 4 hours in Buffer I at pH 4.5-6.5.
Fab' fragments prepared as described above contained an average of 0.85-SH
groups per molecule. The half-life of the --SH was 4-5 hours in Buffer IA
(pH 6.5).
MPB-PE containing vesicles (1.4 .mu.mol/ml) were reacted with freshly
reduced Fab' fragments (0.5-0.4 mg/ml) for 8 hours at 25.degree. C. When
such mixtures were chromatographed on Sephadex G-200, 20-30% of the Fab'
coeluted with vesicles in the void volume. The Fab' remained with vesicles
during rechromatography, indicating a stable association. When exposed to
a 1:32 dilution of goat anti-rabbit IgG serum, greater than 95% of both
the (.sup.125 I)Fab' and (.sup.3 H)DPPC labels coprecipitated suggesting a
rather homogeneous lipid to protein ratio. Nonspecific binding of Fab' to
control vesicles (PC-cholesterol, 1:1) was less than 4 .mu.g/.mu.mol
phospholipid at Fab' concentrations below 5 mg/ml.
##STR9##
We found a linear relationship between the amount of Fab' bound to vesicles
(in 8 hours) and the initial Fab' concentration. For antibody
concentrations of 0.5, 2.0 and 4.0 mg/ml, we obtained coupling ratios of
70.+-.15, 330.+-.20 and 584.+-.40 .mu.g Fab' per .mu.mol vesicle
phospholipid, respectively. Some aggregation of vesicles occurred at Fab'
concentrations above 4 mg/ml.
In a typical coupling reaction, approximately 340 .mu.g of the Fab' was
coupled to vesicles in 8 hours. This value corresponds to greater than
3000 Fab' molecules per each vesicle (0.2.mu. diameter). The time course
of Fab' coupling to PDP-PE vesicles at pH 8.0 and equivalent protein and
lipid concentrations, by comparison, was less efficient than the reaction
of Fab' with maleimide-PE at pH 6.5.
Thiol reactive liposomes in accordance with the present invention form
quite stable covalent bonds with Fab' fragments. For example, about 92% of
the original Fab' remains associated with PDP-PE vesicles during an 8 hour
incubation at pH 8.0 in DTT, and about 62% of the original Fab' remains
associated in 50% human serum. The most preferred embodiment of MPB-PE
vesicles, when coupled with Fab', results in no coupled Fab' being lost
from the MPB-PE vesicles during incubation for 24 hours in DTT (50 mM, pH
7.5) or human serum (50%, pH 7.4). Table I, below, illustrates stability
data for Fab' coupled with PDP-PE vesicles and MPB-PE vesicles,
respectively.
TABLE I
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(a) Fab' fragments covalently coupled to PDP-PE vesicles:
.mu.g of Fab'/.mu.mol
of phospholipid
Fab' remaining
8 h, coupled after 8 h
start 25.degree. C.
(%)
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pH 6.0 286 277 97
pH 7.0 286 272 95
pH 8.0 286 263 92
25% human serum
286 212 74
50% human serum
286 177 62
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(b) Fab' fragments covalently coupled to MPB-PE vesicles:
.mu.g of Fab'/.mu.mol
of phospholipid
Fab' remaining
24 h, coupled after 24 h
start 25.degree. C.
(%)
______________________________________
pH 7.5* 340 326 96
50% human serum
340 319 94
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*50 mM DTT
As has previously been noted, where the ligand to be coupled does not have
reactive thiol groups, then it will be thiolated prior to coupling with
the thiol reactive liposomes. Proteins, and particularly antibodies, are
desirably thiolated (assuming fragments such as Fab', which already bear
sulfhydryl groups, are not being used) for various immunodiagnostic
applications. This is illustrated by Example VII, below.
EXAMPLE VII
MPB-PE was synthesized as has already been described. Liposomes were then
prepared by the method of Szoka and Papahadjopoulos, supra, from 10:10:1
phosphatidylcholine:cholesterol:MPB-PE in a buffer at pH 6.0-6.7. A
suitable buffer is 0.05 M morpholino-ethanesulfonic acid, 0.096 M NaCl, pH
6.4. It is preferred to prepare the vesicle below pH 7.0 to ensure the
maximal stability of the maleimide function.
Six antibody preparations were pyridylthiolated and reduced by the method
of Carlsson et al. Biochem. J., 173, pp. 723-737 (1978). Reaction of
protein with 10 mole of SPDP per mole of protein results in the
substitution of 3-5 mole of pyridyldithiol groups per mole protein. After
reduction with dithiothreitol, the protein was separated from the reducing
agent on a polyacrylamide column (50 to 100 mesh) equilibrated in
argon-purged (de-oxygenated) buffer, pH 6.0-6.5. The protein fractions
were pooled and concentrated to a suitable volume under argon in an amicon
type concentrator. Commonly, the protein is concentrated to around 3
mg/.mu.l. MPB-PE liposomes were then added to the protein solution with
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