Note: Descriptions are shown in the official language in which they were submitted.
NOVEL COMPOSITION FOR TARGETING, STORING A~D
LO~DING OF LIPOSO~ES
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BACKGROUND OF THE INVENTION
The present invention is directed to liposomes. More particularly,
the present invention is directed to the covalent and non-covalent
coupling of liposomes to proteins for purposes of ln vivo targeting, or
for use in diagnostics.
Liposomes are completely closed structures composed of lipid bilayer
membranes containing an encapsulated aqueous volume. Liposomes may
contain many concentric lipid bilayers separated by aqueous phase
(multilamellar vesicles or MLVfi), or may be composed ~f a single membrane
bilayer (unilamellar vesicles).
Liposome preparation has typically been achieved by the process of
Bangham et.al., (1965 J. Mol. biol., 13: 238-252) whereby lipid6
suspended in organic solvent are evaporated under reduced pressure to a
dry film in a reaction ve6sel. An appropriate amount of aqueous phase is
then added to the vessel and the mixture agitated, then allowed to stand,
essentially undisturbed for a time sufficient for the multilamellas
vesicles to form. The aqueous phase entrapped within the liposomes may
comprise bioactive agents including, but not limited to, drugs, hormones,
proteins, dyes, vitamins, or imaging agents.
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The currcnt state of the art is such that liposomes may be
reproducibly prepared using a number of techniques. Liposomes
resulting from some of these techniques are small unilamellar vesicles
(SUVs) Papahadjopoulos and Mil]er, Biochem. Biophys. Acta, 135, p. 624-
638 (1967), reverse-phase evaporation vesicles (REV) U.S. Patent No.
4,235,871 issued November 25, 1980, stable plurilamellar vesicles
(SPLV) U.S. Patent No. 4,522,803 issued June 11, 1985, and large
unilamellar vesicles produced by an extrusion technique as described in
PCT Publication No. WO 86/00238, published January 16, 1986 Cullis et
al., entitled "Extrusion Technique for Producing Unilamellar Vesicles".
One of the primary uses for liposomes is as carriers for a variety
of materials, such as drugs, cosmetics, diagnostic reagents, bioactive
compounds, and the like. Such systems may be designed for both
diagnostics and ln vivo uses. In this regard, the ability to produce
an antibody-directed vesicle would be a distinct advantage over similar
undirected systems (Gregoriadis, G., Trends Pharmacol Sci, 4, p. 304-
307, 1983). Useful applications would be in the selective targeting of
cytotoxic compounds entrapped in vesicles to circulating tumor cells
(Wolff et al., Biochim Biophys. Acta, 802, p. 259-273, 1984), or ap-
plications of these immunoglobulin-associated vesicles in the devel-
opment of diagnostic assays. As is well known in the art, liposomes
may be covalently coupled to proteins, antibodies and immunoglobins.
Heath et al. (Biochim. Biophys. Acta., 640, p. 66-81, 1981), describe
the covalent attachment of immunoglobulins to liposomes containing
glycosphingolipid. Leserman et al. (Liposome Technology, III, 1984,
CRC Press, Inc., CA., p. 29-40; Nature, 288, p. 602-604, 1980) and
Martin et al., (J Biol. Chem., 257, p. 286-288, 1982) have described
procedures whereby thiolated IgG or protein A is covalently attached to
lipid vesicles, and thiolated antibodies and Fab' fragments are at-
tached to liposomes, respectively. These protocols and various modifi-
cations (Martin et al., Biochemistry, 20, p. 4229-4238, 1981; and
Goundalkar
A
lX9~
et.al., J. Pharm. Pharmacol. 36, p. 465-466, 1984) represent the most
versatile approaches to coupling. Avidin-coupled and avidin and
biotinyl-coupled phospholid liposomes containing actinomycin D have
successfully targeted tumor cells expressing ganglio-N-triosylceramide
(Urdal et al., J. Biol. Chem., 255, p. 10509-10516, 1980). Huang et al.
(Biochim. Biophys. Acea., 716, p. 140-150, 1982) demonstrate the binding
of mouse monoclonal antibody to the major histocompatibility antigen H-2
(K), or goat antibody to the major glycoprotein of Molony Leukemia Virus,
to palmitic acid. These fatty acid modified IgGs were incorporated into
liposomes, and the binding of these liposomes to cells expressing the
proper antigens characterized. Other in vitro efforts to specific
binding of liposomes coated with specific immunoglobins have been
performed (Sharkey et al., Fed.Proc., 38, p. 1089, 1979). In still other
coupling studies, Rahman et. al. found that tissue uptake of liposomes
t 15 could be altered by attachment of glycolipids to the liposomes (J. Cell
Biol., 83, p. 268a, 1979).
One aspect of the present invention is to couple biotinylated
proteins such as immunoglobulins and antibodies to liposomes with
covalently-attached streptavidin. Methods for this coupling are herein
provided. The nature of this covalent attachment between streptavidin
and the liposomes is a chemical bonding between the streptavidin, and
derivatized phosphstidylethanolamine incorporated in the liposome
bilayer. In a second aspect of the invention, Applicants provide a
two-step method for the non-covalent coupling of these biotinylated
proteins to biotinylated-phosphatidylethanolamine (PE)-containing
liposomes through the same streptavidin linker. This non-covalent
attachment of streptavidin and liposomes occurs through a specific
association between four specific biotin binding sites on streptavidin,
and the biotin. These antibody-liposome complexes bind specifically to
target cells as directed by the coupled antibody. Such liposomes may be
made to contain bioactive agents such as drugs.
In accordance with a primary use for liposomes, the entrapment of
antineoplastic agents inside liposomal bilayers has resulted in more
~297~8~
efficacious therapy as compared to direct administration of the drug.
(Forssen et al., Cancer Res., 43, p. 546, 1983; and Gabizon et a].,
Cancer Res., 4 , p. 4734, 1982). A prob]em with the encapsu]ation of
antineop]astic drugs is in the fact that many of these drugs have been
found to be rapidly released from liposomes after encapsulation. This
is an especial]y undesirable effect, in view of the fact that toxicity
of these agents can be significantly reduced through liposome encapsu-
lation as compared to direct administration. See, for example, Forssen
et al. Cancer Res., 43, 546 (1983) and Rahman et al. Cancer Res., 42,
1817 (1982). Clearly, a method whereby drug could be loaded into pre-
formed liposomes would be advantageous. To achieve this object, the
invention, in accordance with one of its aspects, provides a method for
loading liposomes with ionizable antineoplastic agents wherein a trans-
membrane potential is created across the walls of the liposomes and the
antineoplastic agent is loaded into the liposomes by means of the
transmembrane potential. See also PCT Publication No. WO 86/01102,
published February 27, 1986, ~alley et al. entitled "Encapsulation of
Antineoplastic Agents in Liposomes".
In accordance with these needs, a liposome composition is pre-
sented which describes the use of protein-coupled liposomes which may
be stored stably for an indefinite period, in a dehydrated state, with
loading of the liposomes on an "as needed" basis.
SU~ARY OF THE INVENTION
We have prepared a liposome composition whereby the glycoprotein
streptavidin is coupled to liposomes for purposes of liposome target-
ing. The streptavidin may in turn couple biotinated proteins such as
Immunoglobulin G or monoclonal antibodies and be loaded with a variety
of bioactive agents, depending on use. Such agents may be the anti-
neoplastic agents such as daunorubicin, doxorubicin, and vinblastine.
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The liposomes are preferably prepared in such a way as to create a
transmembrane potential across their lamellae in response to a
concentration gradient. This concentration gradient may be created by
either Na /K potential or pH (H ). The difference in internal
versus external potential is the mechanism which drives the loading of
the liposomes with ionizable bioactive agents; delayed loading of
preformed liposomes will occur in response to the transmembrane
potential. These liposomes may be dehydrated in the presence of one or
more protecting sugars such as the disaccharides trehalose and sucrose,
stored in their dehydrated condition, and subsequently rehydrated with
retention of the ion gradient and associated ability to accumulate the
bioactive agent. Such bioactive agents may be those used as in vivo
pharmaceutical preparations, such as antineoplastic agents including
doxorubicin. These preparations may be administered to a subject for
treatment of disease. Alternatively, the coupled liposome preparations
may be used in diagnostic assays. Methods are provided for the
preparation of liposomes either covalently or non-covalently coupled to
streptavidin, which in turn are complexed with biotinylated proteins such
as IgG or monoclonal antibodies. In the case of non-covalent binding of
liposomes to streptavidin, the liposomes comprise biotinylated
phosphatidylethanolamine. Such liposomes are incubated with, for
example, about 10-fold molar excess streptavidin to biotinylated
phosphatidylethanolamine (PE), to complete the coupling reaction. The
liposomes may be large unilamellar vesicles, and may also comprise egg
phosphatidylcholine (EPC).
In preparations containing EPC and biotinylated PE, the latter i8 in
an about O.l to 0.5% mole ratio with the EPC, preferably an about O.l~
mole ratio.
Compositions of protein-streptavidin-biotinylated PE liposomes
wherein the protein is a monoclonal antibody or Immunoglobulin G are
claimed. These liposomes may also comprise a bioactive agent. They may
be used in vivo as a pharmaceutical preparation in a subject or
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alternatively in in vitro diagnostic a3says by contacting a sample with
the composition.
BRIEF DESCRIPTION OF THE DRAWI~G
Figure l is a graph showing coupling of anti-rat erythrocyte IgG to
EPC LUVETs (labeled with H-DPPC) containing PDP-PE (closed triangle)
and MPB-PE (closed circle) as a function of cholesterol content.
Figure 2 is a graph characterizing the covalent coupling reaction of
IgG to vesicles with regards to time course, of (A), MPB-PE concentration
(B) and IgG concentration (C).
Figure 3 is a graph showing the influence of reaction pH on covalent
coupling of anti-human erythrocyte IgG (closed circle) and streptavidin
tclosed triangle) to EPC/Chol (50:45) vesicles containing 5 mol~ MPB-PE.
Figure 4 is a graph showing the efficiency of covalent coupling of
anti-human erythrocyte IgG to vesicles of variable size.
Figure 5 is a graph showing the elution profile for biotinated
anti-human erythrocyte IgG to vesicles which have covalently coupled
streptavidin.
Figure 6 is a graph showing the accumulation of adriamycin into
covalently-coupled streptavidin-vesicles ("avisomes") which were prepared
with a transmembrane pH gradient. Vesicles composed of EPC (closed
circle) or EPC/Chol (closed triangle).
Figure 7 is a graph showing the non covalent coupling of
fitreptavidin to LWs containing biotinylated phosphatidylethanolamine.
DETAILED DESCRIPTION OF THF. INVENTION
As described above, the present invention describes a liposome
composition that results from the coupling of the liposomes to
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strcptavidin. In addition, the composition can be dehydrated and re-
hydrated. The liposomc portions can bc loaded with a chosen bioactive
agent by potential difference of ions across the bilayer membranes dur-
ing thc rehydration step or subsequently thereto. Alternatively, the
bioactive agent may be added to the liposomes prior to dehydration.
The streptavidin-coupled liposomes can be coupled to proteins such as
Immunoglobulin G or monoclonal antibodies which have been biotinated by
coupling to biotin. Quite surprising is the observed stability of the
streptavidin-liposome-biotinated protein complex which makes streptavi-
din an attractive coupler between the liposomes and the targeting pro-
teins. The proteins bound to the liposomes aid in targeting the lipo-
somes and their contents to a specific site in the body.
In one embodiment of the present invention, liposomes are formed
using the LUVET apparatus described in PCT Publication No. W0 86/00238,
published January 16, 1986, entit]ed "Extrusion Technique for Producing
~nilamellar Vesicles", and covalently coupled to streptavidin using a
modified technique of Leserman et al., (Liposome Technology, III, 1984,
CRC Press, Inc., N.Y., p. 29-40). Liposomes are formed with a trans-
membrane potential i.e. Na+/K+ gradient or H+ potential difference
across the bilayers, see PCT Publication No. W0 86/01102, published
February 27, 1986, Bally et al., entitled "Encapsulation of Antineo-
plastic Agents in Liposomes"; this potential difference effected by the
ionic concentrations of the internal versus the external media of the
liposome. Liposomes are then dehydrated either in the presence or
absence of sugars such as trehalose, and may be stored in this state
for indefinite periods of time; see PCT Publicàtion No. W0 86/01103,
published February 27, 1986, JanoEf et al., entitled "Dehydrated
Liposomes".
In another embodiment of the present invention, biotinylated
proteins are non-covalently coupled to biotinylated PE-containing
~L297~7~37
liposomes via streptavidin. The non-cova]ent binding of the streptavi-
din to the liposomes, the first step, involves incorporation of biotin-
PE in the liposomcs, followed by a second step of binding the strept-
avidin to the biotinylated protein. The proteins are prepared for this
binding by the use of fluorescent derivatizing reagents such as the
fluorescent amine reagent fluorescein-isothiocyanate (FITC).
There are four biotin binding sites on the streptavidin, which
makes liposomes containing biotin aggregate with streptavidin in an
excess of biotinylated PE. Thus, the amount of biotinylated PE to
incorporate into the liposomes was titrated in order to prevent this
aggregation, while maximizing the streptavidin coupling. Values for
biotinylated PE may range from about 0.05 to 0.5 mole% (with respect to
total lipid in the liposome preparation); if the amount of biotin is
increased further than about 0.5%, complete aggregation and precipi-
tation of liposomes is observed on addition of streptavidin. This
aggregation phenomenon may bc exploited in the use of these systems in
an aggregation-type diagnostic assay.
The biotinylated antibody is then attached to the streptavidin
coated liposome. These liposomes effectively targeted specifically to
their target cells with little non-spccific binding.
The liposomes used in the present invention can have a variety of
compositions and internal contents, and can be in the form of multila-
mellar, unilamellar, or other types of liposomes, or more generally,
lipid-containing particles, now known or later developed. For example,
the lipid-containing particles can be in the form of steroidal lipo-
somes, U.S. Patent No. 4,721,612, alpha-tocopherol containing lipo-
somes, PCT Publication No. W0 87/02219, stable plurilamel]ar liposomes
(SPLVs), U.S. Patent No. 4,522,803, issued June 11, 1985, monophasic
vesicles (MPVs), U.S. Patent No. 4,588,578, issued May 13, 1986, or
lipid matrix carriers (LMC), U.S. Patent No. 4,610,868, issued
September 9, 1986. Within the class of liposomes that may be used in
the present invention is a preferred
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subclass of liposomes characterized in having solute distribution
substantially equal to the solute distribution environment in which
prepared. This subclass may be defined as stable plurilamellar vesicles
(SPLV), monophasic vesicles (MPVs), and frozen and thawed multilamellar
S vesicles (FATMLVs) as described in "Solute Distributions and Trapping
Efficiencies Observet in Freeze-Thawed Multilamellar Vesicles" Mayer et
al. Biochimica et Biophysica Acta 817:1983-196 (1985). It is believed
that the particular stability of the SPLV type liposomes arises from the
low energy state attendant to solute equilibrium.
Alternatively, techniques used for producing large unilamellar
liposomes (LUVs), such as, reverse-phase evaporation, infusion
procedures, and detergent dilution, can be used to produce the
liposomes. A review of these and other methods for producing liposomes
can be found in the text Liposomes, Marc ~. Ostro, ed., Marcel Dekker,
Inc., New York, 1983, Chapter 1.
Compounts which are bioactive agents can be entrapped with;n the
liposomes of the present invention. Such compounds include but are not
limited to antibacterial compounds such as gentamycin, antiviral agents
such as rifampacin, antifungal compounds such as amphotericin B,
anti-parasitic compounds such as antimony derivatives, tumoricidal
compounds such as adriamycin, anti-metabolites, peptides, proteins such
85 albumin, toxins such as diptheriatoxin, enzymes such as catalase,
polypeptides such as cyclosporin A, hormones such 85 estrogen, hormone
antagonists, neurotransmitters such as acetylcholine, neurotransmitter
antagonists, glycoproteins such as hyaluronic acid, lipoproteins such as
alpha-lipoprotein, immunoglobulins such as IgG, immunomotulators such as
interferon or interleuken, vasodilators, dyes such as Arsenazo III,
radiolabels such as 14C, radio-opaque compounds such as 90Te,
fluorescent compounds such as carboxy fluorscein, receptor binding
molecules such as estrogen receptor protein, anti-inflammatories such as
indomethacin, antigalucoma agents such as pilocarpine, mydriatic
compounds, local anesthetics such as lidocaine, narcotics such as
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i297'7E~
codeine, vitamins such as alpha-tocopherol, nucleic acids such as
thymine, polynucleotides such as RNA polymers, psychoactive or anxiolytic
agents such as diazepam, mono- di- and polysaccharides, etc. A few of
the many specific compounds that can be entrapped are pilocarpine, a
polypeptide growth hormone such as human growth hormone, bovin growth
hormone and porcine growth hormone, indomethacin, diazepam3
alpha-tocopherol itself and tylosin. Antifungal compounds include
miconazole, terconazole, econazole, isoconazole, tioconazole, bifonazole,
clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole,
fenticonazole, nystatin, naftifine, amphotericin B, zinoconazole and
ciclopirox olamine, preferably miconazole or terconazole, The entrapment
of two or more compounds simultaneously may be especially desirable where
such compounds produce complementary or synergistic effects. The amounts
of drugs administered in liposomes will generally be the same as with the
free drug; however, the frequency of dosing may be reduced.
The liposomes of the present invention are preferably dehydrated
using standard freeze-drying equipment or equivalent apparatus, and, if
desired, the liposomes and their surrounding medium can be frozen in
liquid nitrogen before being dehydrated. Alternatively, the liposomes
can also be dehydrated without prior freezing, by simply being placed
under reduced pressure. Dehydration with prior freezing requires the
presence of one or more protective sugars in the preparation, A variety
_ of sugars can be used, including such sugars as trehalose, maltose,
sucroæe, glucose, lactose, and dextran. In general, disaccharide sugars
have been found to work better than monsaccharide ~ugars, with the
disaccharide sugars trehalose and sucrose being most effective.
The one or more sugars are included as part of either the internal
or external media of the liposomes. Most preferably, the su~ars are
included in both the internal and external media so that they can
interact with both the inside and outside surfaces of the liposomes'
membranes. Inclusion in the internal medium is accomplished by adding
the sugar or sugars to the solute which the liposomes are to
encapsulate. Since in most cases this solute also forms the bathing
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medium for the finished liposomes, inclusion of the sugars in the solute
also makes them part of the external medium. Of course, if an external
medium other than the original solute is used, e.g., to create a
transmembrane potential (see below), the new external medium should also
include one or more of the protective sugars.
In the case of dehydration without prior freezing, if the liposomes
being dehydrated have multiple lipid layers and if the dehydration is
carried out to an end point where there is sufficient water left in the
preparation so that a substantial portion of the membranes retain their
integrity upon rehydration, the use of one or more protective sugars may
be omitted. Tt has been found preferable if the preparation contains at
the end of the dehydration process at least about 2%, and most preferAbly
between about 2% and about 5~, of the original water present in the
preparation prior to dehydration.
Once the liposomes have been dehydrated, they can be stored for
extended periods of time until they are to be used. When the dehydrated
liposomes are to be used, rehydration is accomplished by simply adding an
aqueous solution, e.g., distilled water9 to the liposomes and allowing
them to rehydrate.
As discussed above, in accordance with another of its aspects, the
present invention provides a method for loading liposomes with ioniæable
antineoplastic agents wherein a transmembrane potential is created across
the bilayers of the liposomes and the antineoplastic agent is loaded into
the liposomes by means of the transmembrane potential. ~he transmembrane
potential is generated by creating a concentration gradient for one or
more charged species (e.g., Na , K and/or H ) across the liposome
membranes. The concentration gradient is created by producing liposomes
having different internal and external media, i.e., internal and external
media having different concentrations of one or more charged species.
Specifically, liposomes are prepared which encapsulate a first
medium having a first concentration of the one or more charged species.
~,.,
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For a typical liposome preparation technique (see discussion above), this
first medium will surround the liposomes as they are formed, and thus the
liposomes' original external medium will have the same composition as the
first medium. To create the concentration gradient, the original
external medium is replaced by a new external medium having a different
concentration of the one or more charged species. The replacement of the
external medium can be accomplished by various techniques, such as, by
passing the liposome preparation through a gel filtration column, e.g., a
Sephadex column, which has been equilibrated with the new medium, or by
centrifugation, dialysis, or related techniques.
In accordance with the invention, it has been found that this
transmembrane potential can be used to load ionizable antineoplastic
agents into the liposomes. Specifically, once liposomes having a
concentration gradient and thus a transmembrane potential of the
appropriate orientation have been prepared, the process of loading
antineoplastic agents into the liposomes reduces to the step of adding
the agent to the external medium. Once added, the transmembrane
potential will automatically load the agent into the liposomes.
The transmembrane potential loading method can be used with
essentially any antineoplastic agent which can exist in a charged state
when dissolved in an appropriate aqueous medium (e.g., organic compounds
which include ~n amino group which can be protonated). Preferably, the
agent should be relatively lipophilic so that it will partition into the
liposome membranes. Examples of some of the antineoplastic agents which
can be loaded into liposomes by this method include doxorubicin,
. mitomycin, daunorubicin, streptozocin, vinblastine, vincristine,
mechlorethamine hydrochloride, melphalan, cyclophosphamide,
triethylenethiophosphoramide, carmustine, lomustine, semustine,
hydroxyurea, thioguanine, decarbazine, cisplatin, procarbazine, and
pharmaceutically acceptable salts and derivatives thereof.
In addition to loading a single antineoplastic agent, the method can
be used to load multiple antineoplastic agents, either simultaneously or
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sequentially. Also, the liposomes into which the ionizable
antineoplastic agents are loaded can themselves be pre-loaded with other
antineoplastic agents or other drugs using conventional encapsulation
techniques ~e.g., by incorporating the drug in the buffer from which the
liposomes are made).
Turning now to the aspects of the invention relating to reducing the
rate of release of an ionizable antineoplastic agent or other ionizable
biologically-active agent (drug) from liposomes, it has been surprisingly
found that the rate of release can be markedly reduced by creating a
transmembrane potential across the liposome membranes which is oriented
to retain the agent in the liposomes. That is, for an agent which is
positively charged when ionized, a transmembrane potential is created
across the liposome membranes which has an inside potential which is
negative relative to the outside potential, while for an agent which is
negatively charged, the opposite orientation is used.
As with the transmembrane loading aspects of the invention, the
transmembrane potentials used to reduce the rate of drug release are
created by adjusting the concentrations on the inside and outside of the
liposomes of a charged species such as Na , K and/or H . Indeed,
if the liposomes have been loaded by means of a transmembrane potential
produced by such a concentration gradient, simply keeping the liposomes
in an external medium which will maintain the original concentration
gradient will produce the desired reduction in the rate of release.
Alternatively, if a transmembrane potential has not already been created
across the liposome membranes, e.g., if the lipoæomes have been loaded
using a conventional technique, the desired transmembrane potential can
be readily created by changing the composition of the external medium
using the exchange techniques described above.
Turning next to the aspects of the invention relating to the
dehydration protocols, two basic approaches are provided: l) the
liposomes can be loaded with antineoplastic agents (e.g., using
conventional techniques or the transmembrane potential loading technique
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described above), dehydrated for purposes of storage, shipping, and the
like, and then rehydrated at the time of use; or 2) pre-formed liposomes
can be dehydrated for storage, etc., and then at or near the time of use,
they can be rehydrated and loaded with an ionizable antineoplastic agent
using the transmembrane potential loading technique described above.
When the dehydrated liposomes are to be used, rehydration is
accomplished by simply adding an aqueous solution, e.g., distilled water
or an appropriate buffer, to the liposomes and allowing them to
rehydrate. The liposomes can be resuspended into the aqueous solution by
gentle swirling of the solution. The rehydration can be performed at
room temperature or at other temperatures appropriate to the composition
of the liposomes and their internal contents.
If the antineoplastic agent which is to be administered was
incorporated into the liposomes prior to dehydration, and no further
composition changes are desired, the rehydrated liposomes can be used
directly in the cancer therapy following known procedures for
administering liposome encapsulated drugs.
Alternatively, using the transmembrane potential procedures
described above, ionizable antineoplastic agents can be incorporated into
the rehydrated liposomes just prior to administration~ In connection
with this approach, the concentration gradient used to generate the
transmembrane potential can be created either before dehydration or after
rehydration using the external medium exchange techniques described above.
For example,, liposomes having the same internal and external media,
i.e., no transmembrane potentials, can be prepared, dehydrated, stored,
rehydrated, and then the external medium can be replaced with a new
medium having a composition which will generate transmembrane potentials,
and the transmembrane potentials used to load ionizable antineoplastic
agents into the liposomes. Alternatively~ liposomes having internal and
external media which will produce transmembrane potentials can be
prepared, dehydrated, stored, rehydrated, and then loaded using the
transmembrane potentials.
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Liposomes of the present invention may be administered to a subject
such as a mammal including humans. For administration to humans in the
treatment of afflictions, the prescribing physician will ultimately
determine the appropriate dose for a given human subject, and this can be
expected to vary according to the age, weight, and response of the
individual as well as the nature and severity of the patient's symptoms.
The mode of administration may determine the sites and cells in the
organism to which the compound will be delivered. For instance, delivery
to a specifric site of infection may be most easily accomplished by
topical application (if the infection is external e.g., on areas such as
eyes, skin, in ears, or on afflictions such as wounds or burns) or by
absorption through epithelial or mucocutaneous linings (e.g., nasal,
oral, vaginal, rectal, gastrointestinal, mucosa, etc.). Such topical
t 15 application may be in the form of creams or ointments. The
liposome-entrapped materials can be administered alone but will generally
be administered in admixture with a pharmaceutical carrier selected with
regard to the intended route of administration and standard
pharmaceutical practice. They may be injected parenterally, for example,
intravenously, intramuscularly, or subcutaneously. For parenteral
administration, they are best used in the form of a sterile aqueous
solution which may contain other solutes, for example, enough salts or
glucose to make the solution isotonic.
For the oral mode of administration, liposome composition of this
invention can be used in the form of tablets, capsultes, lozenges,
troches, powders, syrups, elixirs, aqueous solutions and suspensionsJ and
the like. In the case of tablets, carriers which can be used include
lactose, sodium citrate, and salts of phosphoric acid. Various
disintegrants such as starch, and lubricating agents such as magnesium
stearate, sodium lauryl sulfate and talc, are commonly used in tablets.
For oral administration in capsule form, useful diluents are lactose and
high molecular weight polyethylene glycols. When aqueous suspensions are
required for oral use, certain sweetening and/or flavoring agents can be
addet.
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~2~3778~
The liposomes of this invention may also be used in diagnostic
assays; in this case the amount of the composition used will depend on
the sensitivity of the liposome-coupled antibody to the target components
in the sample.
~5ATERIALS AND MET~ODS
Egg phospatidylcholine (EPC) was isolated from hen egg yolk
employing established procedures. Egg phosphatidylethanolamine (EPE~ was
obtained from EPC utilizing the headgroup exchange capacity of
phospholipase D (Kates et al., Methods in Enzymology, 14, Lavenstein, J.
ed., 1969, Academic Press, Inc., p. 197-211).
Dioleoylphosphatidylcholine (DOPC) and dipalmitoylphosphatidylcholine
(DPPC) were obtained from Avanti Polar Lipids. Strepavidin, cholesterol,
trehalose, dithiothreitol (DTT), and fluorescein isothiocyanate Celite
(Celite FITC) were purchased from Sigma. N-Succinimidyl
3-(2-pyridyldithio) propionate (SPDP), N-succinimidyl
4-(p-Maleimidophenyl)butyrate (SMPB), biotinyl-N-hydroxysuccinimide (BHS)
biotin-phosphatidylethanolamine, and N-biotinoyldipalmitoyl-
phosphatidylcholine were obtained from Molecular Probes. Rabbit
anti-human red blood cell IgG and Rabbit anti-rat red blood cell IgG were
supplied by Cooper Biomedical. Sephadex G-50 fine, Sepharose 4B-CL, and
Sepharose C14B were purchased from Phsrmacia. [3H]-DPPC and ¦3H~-BHS
were obtained from NEN, carrier free Na 125I (100 mCi/ml) was supplied
by Amersham and iodogen was obtained from Pierce. Adriamycin was
obtained from B.C. Cancer Control Agency. Biotinylated anti-rat
erythrocyte IgG was obtained from Cappel. All other chemicals were of
analytical grade.
Lipid was estimated by the standard lipid phosphate assay or by
incorporation of trace quantities of [ H-DPPC] introduced in the
original lipid film, and later monitored using a Packard Tri-Carb 4000
series scintillation cou~ter. FITC was assayed by monitoring the
fluorescence at 520nm using a SLM-Aminco SPF-500C spectrofluotometer with
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125
an excitation wavelength of 495 nm. I was measured using a Packard
Auto-Gamma 5650 gamma counter. Vesicle size distributions were
determined by quasi-elastic light scattering (QELS) analysis utilizing a
Nicomp Model 270 submisron particle sizer operating at 632.8nm and 5mW.
Example 1
Covalent Coupling
I. Synthesis of N-[4-(p-Maleimidophenyl)butyryl] phosphatidyl-
~ethanolamine (MPB-PE)
MPB-PE was prepared according to the procedure of Martin et al.,
J.Biol.Chem., 257, 286-288, (1982) EPE (200 mg) was dissolved in 5 ml
of freshly distilled anhydrous methanol containing 200 umol of freshly
distilled triethylamine and 100 mg SMBP. The reaction was carried out at
room temperature under nitrogen and its progress followed using thin
layer chromatography (TLC, running solvent:chloroform/methanol/water,
65:25:4). Following an 18 hour incubation, 95% of the EPE ~as converted
to MPB-PE. Methanol was removed under reduced pressure and redissolved
in chloroform and this mixture washed extenæively with 1% NaCl to remove
unreacted SMPB and residual triethylamine. The product of this reaction
was characterized by two dimensional TLC and proton NMR~ This analysis
indicated the presence of two components, one being MPB-PE and comprising
approximately 60% of the product. The product of the reaction mixture
described above was incorporated into vesicles without additional
purification. The product was stored at -20C and was shown to be
stable for at least 6 month6.
II. Synthesis of N-[3(2-Pyridyldithio)proprionyl]
phosphatidylethanolamine (PDP-PE)
PDP-PE was prepared according to the procedure of Leserman et al.,
Liposome Technology, III, Gregoriadis, ed., 1984, CRC Press, Inc., CA.,
p. 29-40. Briefly, 50 umol EPE was dissolved in 3.5 ml
chloroform/methanol (9:1) and added to 1.5 ml methanol containing 60 umol
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SPDP and 100 umol tr;ethylamine. After a four hour incubation at room
temperature, TLC (solvent: chloroform/methanol/water, 65:25:4) analysis
indicated 99% conversion of EPE to a faster running product. This
reaction mixture was washed with 10 ml of phosphate buffered saline (O.lM
NaCl, O.lM potassium phosphate, pH 7.4). This was repeated three
additional times prior to removal of the organic phase under reduced
pressure. Further analysis by two dimensional TLC and proton NMR
indicated a single product which appeared greater than 98~ pure. PDP-PE
was stored under N2 in chloroform at -20 C and was stable for at
least six months.
III. Vesicle Preparation
Large unilamellar vesicles (LUVs) were prepared employing the LUVET
(LUV's by extrusion techniques) procedure described by Hope et al.,
Biochim, Biophys. Acta, 812, p. 55-65 tl985). Appropriate mixtures of
lipid were deposited as a dry lipid film by evaporation from chloroform
under a stream of nitrogen gas, placed under vacuum for at least two
hours and subsequently hydrated with appropriate buffer by vortex mixing
to produce multilamellar vesicles (MLVs). Frozen and thawed MLV (FATMLV)
systems as described in Mayer et.al, Biochim. et Biophys. Acta, 817,
193-196 (1985), were obtained by freezing the MLV's in liquid nitrogen
and thawing at 40C, a cycle which was repeated five times. These
FATMLV's were then extruded under nitrogen pressure ten times through two
stacked Nucleopore polycarbonate filters of defined pore size. Generally
a pore size of lOOnm was employed resulting in a preparation of
unilamellar vesicles (VETloo) with an average diameter of llOnm as
determined by QELS messurements. Vesicles of variable sizes were
produced by similar extrusion techniques through filters with pore sizes
ranging from 30nm to 400nm. Unless specified differently vesicles were
prepared in a NaCl/EPPS buffer (150mM NaCl, 20mM EPPS, (N-2-Hydroxy-
ethylpiperazinepropanesulphonic acid) pH 8.0) at a final lipid
concentration of 10 umol/ml. In vesicles which contained PDP-PE, the
presence of this lipid was verified by measuring the release of
2-thiopyridinone upon addition of DTT (25mM final concentration) as
indicated previously.
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IV. Preparation of Proteins Used for Coupling
IgGs and streptavidin were modified with the amine reactive
heterobifunctional reagent SPDP according to Leserman et al. (supra.).
Biotin-conjugated antibodies were prepared according to Bayer et al.,
Biochim et Bio~hys Acta, S , p. 464-473, (1979).
V. Covalent Coupling of Proteins to Vesicles
The steps utilized to crosslink proteins to liposomes are as
follows: thio-reactive lipids were generated, PDP-PE and MPB-PE,
according to the procedures of Leserman, et al~, supra., and Martin et
al., supra., respectively. These derivatized PE's were incorporated into
vesicles, at levels of 5 mol~ based on total modified lipid content.
Since the MPB-PE was shown to be less than 60% pure by two dimensional
TLC and 1H-NMR, the vesicles prepared with "MPB-PE" actually contained
only 3% of this modified lipid. Thus, for vesicles prepared as described
above, 100 ul of vesicles were added to 100 to 500 ul of the protein
solution depending on protein concentration. This reaction mixture
(pH=8.0) was incubated in the dark under nitrogen for 12 to 18 hours
unless specified differently. Subsequently the liposomes were separated
from unassociatet protein employing a Sepharose 4B-CL column (column
volume equivalent to a least 20 times the sample volume) equilibrated
with NaCl/Hepes buffer. Protein and total lipid concentrations in the
fractions were determined directly or calculated from the specific
activities of 5I-labeled protein and [ H]-DPPC, respectively.
Where FITC-labeled proteins were used fractions were dissolved in ethanol
to a total volume of 2ml and the fluorescence was determined and compared
to the fluorescence associated with a known quantity of fluoresceinated
protein. The presence of lipid was shown not to influence fluorescence
in this assay system.
Binding of biotinated-IgGs to avidin-coupled vesicles was assessed
as described above after a 30 minute incubation at room temperature. IgG
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~2~7'7~37
was gPnerally added at equimolar concentrations with respect to avidin
present in the incubation mixture.
Protein modification with the heterobifunctional reagent SPDP was
shown to be extremely reproducible provided the DPDP was freshly prepared
and stored anhydrously. This reaction resulted in the substitution for
IgG in the range of 4 to 5 moles 2-dithiopryidinone (2-DT) per mole IgG
~regardless of the IgG used) and for protein A and streptavidin 6 to 7
moles 2-DT per mole protein based on an incubation ratio of lO moles SPDP
per mole protein. It was demonstrated that this extent of modification
did not influence the binding activity of a monoclonal IgG
(anti-transferrin receptor IgG) or the ability of streptavidin to bind
biotin.
The reaction mixture used for coupling consisted of vesicles
containing one of the thio-reactive lipids plus the modified protein
bearing several thiol group6, 8S judged by the release of 2-DT on
addition of DTT. Following an incubation period of 18 hours, the
vesicles and associated protein were fractionated from free protein on
Sepharose 4B-CL columns and the lipid and protein were quantified as
indicated in the methods.
Martin et al. (Ann. N.Y. Acad.Sci., 446, p. 443-456, 1985) had
suggested that the thio-reactive PE derivatives function better in the
presence of cholesterol. The amount of IgG coupling obtained with
vesicles prepared with PDP-PE or MPB-PE was compared as a function of
cholesterol content. The results, shown in Figure 1, indicate that IgG
crosslinking to vesicles containing PDP-PE does not occur unless the
composition includes greater than 20 mol% cholesterol. Conversely,
levels of 12 ug IgG per umole lipid were obtained for vesicles containing
the SMPB derivative of PE even in the absence of cholesterol. This
amount of association increased linearly with respect to the amount of
cholesterol included in the vesicles to levels approaching 30 ug IgG per
umole lipid for vesicles containing 45 mol~ cholesterol.
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The reaction of SPDP modified proteins with MPB-PE was extensively
characterized. The time course for coupling IgG (Fig.2A) suggests that
the reaction proceeds in two phases. Approximately 25 ug of IgG was
coupled to vesicles within one hour. Subsequently, the rate of
association decreased several fold, but continued linearly for at least
18 hours. As indicated in Fig. 2B, maximum coupling was obtained when
the vesicles were prepared with 5 mol% MPB-PE. Similar levels of IgG
coupling were obtained with 10 mol~ MPB-PE, however there was a
significant amount of vesicle crosslinking in this preparation as judged
by an increases in the optical density of the reaction mixture and
greater than 50% loss of both lipid and protein on the Sepharose 4B-CL
gel filtration column. The amount of IgG coupling to vesicles increased
linearly with increased amounts of modified IgG present in the incubation
mixture (Fig. 2C). Assuming a vesicle diameter of 100 nm, levels of 170
ug IgG per umole lipid, also resulted in vesicle crosslinking.
The extent of protein coupling was shown to be dependent on the pH
of the reaction mixture (Fig. 3~. Contrary to previous work which
performed similar coupling reactions at pH 6.7, (Heath et.al, Proc. Natl.
Acad.Sci USA, 80, p. 1377-1381, (1983), Bragman et al., Biochim. Biophys.
Acta, 730, p. 187-195, (1983), and Bragman et al., JNCI, 73, p. 127-131,
(1984), modified IgG and streptavidin coupling to vesicles occurred only
at pHs greater than 8Ø At pH values below 7.0, only background
protein-lipid association was observed.
As shown in Fig. 4, the amount of IgG crosslinking was dependent on
the size of the vesicle employed, The maximum efficiency of the coupling
reaction never exceeded 45% based on the amount of protein present in the
reaction mixture. However, for vesicles which had been sized through the
200 nm pore size filters, showing an average diameter of 162 ~/- 41 nm by
QELS measurements, the ratio of available MPB-PE to IgG approached 1
indicating that the maximum level of crosslinking was obtained. Based on
the QELS approximated average diameters, 3950, 75, and 20 IgG molecules
were bound to the 162 nm, 115 nm and 82 nm vesicles, re~pectively.
Similar results have been obtained for sonicated vesicles when compared
~377~3'7
to vesicles produced by the reverse phase-evaporation technique and sized
through 0.2 and 0.4 um pore size filters.
Finally, the influence of lipid composition on the coupling of
proteins to vesicles containing MPB-PE was determined. The results shown
in Table 1 emphasize previous results (Fig. 1), and demonstrate that IgG
coupling to vesicles is much more efficient when cholesterol is
incorporated. Crosslinking of the smsller streptavidin (68,000 daltons
vs. 150,000 daltons for IgG) was not influenced by addition of this
sterol. These data also indicate that incorporation of either a negative
charge (phosphatidylserine or "PS") or a positive charge (stearylamine or
"SA") into the liposome does not influence the extent of coupling for
either protein.
VI. Binding of biotinated-IgG to stepavidin coupled vesicles-
("Avisomes")
An advantage can be obtained if a single vesicle preparation is
employed to couple a variety o~ different immunoglobulins. It has been
shown that not all IgGs can be modified with the reagent SPDP such that
they retain binding activity (Heath et.al, supra), and that certain IgGs
which have been extensively modified with SPDP could not be crosslinked
efficiently by the procedures described above. Leserman et. al, supra.,
recognized these limitations and circumvented them somewhat by coupling
protein A to liposomes. Since protein A binds the Fc portion of IgGs of
certain subclass (IgG2a), protein A coupled vesicles specifically bound
to cells preincubated with a variety of antibodies. This allowed for a
comparison of a number of different parameters using a single vesicle
preparation. A similar yet more general approach can be achieved by
taking advantage of the strong affinity (Kd=10 15M) of biotin for
streptavidin. Since IgGs can be easily biotinated, Bayer et al., supra;
and Heitzmann et al. Proc. Natl. Acad. Sci. USA, 715 p. 3537-3541,
(1974), a streptavidin coupled vesicle can be used to bind any number of
IgGs w~th differing specificities. This application of "Avisomes" is
illustrated in Fig. 5, which shows the elution profile for "avisomes")
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~29~'7i~7
following a thirty minute incubation with biotinated-IgG (4 biotins per
IgG). More than 40 ug IgG were bound per umole lipid which corresponded
to approximately 100 IgGs per vesicle. Similar levels of association
we~e obtained for three different IgG and could be obtained at biotin/IgG
ratios of less than 2. In addition, the efficiency of this association
was better than that observed for the chemical crosslinking procedures
employed previously.
VII. Dehydration of protein-coupled vesicles and entrapment of adriamycin
Strepavidin-coupled vesicles ("avisomes") were dehydrated according
to the procedure of Madden et 81., Biochim. Biophys Acta, 817, p. 67-74,
(1985). Vesicles were prepared as described above in a citrate buffer
(lOOmM citric acid, 150 mM KOH, pH 4.5~ which contained 250mM trehalose.
Subsequently the external buffer was exchanged for NaCl/EPPS buffer,
thereby establishing A transmembrane electrical potential which can be
utilized for accumulating adriamycin. Following the previously described
coupling reaction, unassociated protein was separated from vesicles as
described above employing a Sepharose 4B-CL column equilibrated with
NaCl/Hepes buffer containing 250mM trehalose. Vesicles with bound
protein were divided into lml aliquots and dried in 10ml Kimex tubes at
room temperature under vacuum for 24 hours.
Following dehydration, samples were rehydrated by addition of 900ul
distilled water. The resulting preparation was characterized wit~
respect to binding of several biotinated-IgG and the ability to
accumulate adriamycin. Adriamycin was quantitated by determining the
absorbance at 480nm of a triton X-100 solubilized sample which has been
fractionated on Sephadex G-50 to remove unassociated adriamycin.
As shown in Fig. 6 "avisomes" can accumulate adriamycin in response
to a preexisting pH ~radient, where levels of 180 and 120 nmoles
adriamycin per umole lipit are obtained for streptavidin coupled vesicles
composed of EPC and EPC/Chol (i:l), respectively.
~ * Trade Mark
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12~37787
The ab;lity to store "avisomes" in a dehydrated form was then
demonstrated (Table 2). In these experiments vesicles were prepared in
the presence of trehalose with removal of untrapped trehalose performed
prior to coupling, by gel filtration. Following the coupling reaction
trehalose was added (final concentration = 250mM) to the streptavidin
coupled vesicles and then dehydrated as described previously. There was
little change in the amount of streptavidin bound to rehydrated
"avisomes". Moreover, the biotin binding activity of streptavidin
associated with vesicles (units per umole lipid) was not influenced by
this dehydration step. This is also reflected in the ability of these
preparations to bind biotinated-IgG to the same extent observed prior to
dehydration.
Example 2
Non-Covalent Coupling
Biotinylated PE was incorporated into egg phosphatidylcholine (EPC)
at a molar ratio of 0.1% (with respect to PC) and LUVs produced by an
extrusion procedure through 100 nm filters (Hope et al., supra.), and
using a freeze and thaw technique(Bally et al., 1985, Biochim. et
Biophys. Acta., 812, 66-76), resulting in vesicles of approximately lOOnm
diameter. The vesicles were incubated at 25C for 30 minutes at pH 8.0
in 10 fold molar excess of streptavidin (with respect to PE) in 20mM EPPS
buffered saline. At 5, 10, 15 and 20 minute intervals, aliquots were
fractioned on Sepharose C14B columns (5 ml) to separate liposomally-bound
streptavidin from free streptavidin (Fig. 7). The ratio of streptavidin
bound per umole total lipid was determined to be at
~- streptavidin:biotinylated PE ratios of 1:12 (mol/mol). This resulted in
a maximum of 5.8 ug of streptavidin bound per umol lipid.
Biotinylated anti-rat erythrocyte IgG was prepared with 1-5 biotins
covalently bound per mole of antibody by the method of Bayer et al.,
supra. FITC-labelling of biotinated antibodies was performed by
incubation of antibody (5 mg/ml in PBC) with celite-FITC (2.5 mg/ml in
0.1 mM NaCl, 0.2M Na bicarbonate, pH 8.8) for 20 minutes at 25C, followed
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~97787
by gel filtration on Sepharose G50 (50 ml column).
Antibody-streptavidin-liposomes were prepared by incubation of
FITC-labelled biotin antibody (1 mg/ml) with streptavidin liposomes
(1-2.5 umoles/ml) for 30 minutes at 25 C, at a 4-fold mole ratio of
antibody to streptavidin. The final product was separated from free
antibody by gel filtration on a Sepharose C14B column (15 ml).
Phospholipid was assayed using the standard phosphate assay of Bartlett
et al.)
Example 3
Non-Covalent Couplin~
The methods of Example 2 were followed using 0.05, 0.15, 0.25, 0.35,
and 0.5 mole% biotinylated PE with EPC in the liposomes. This linearly
increased the amount of streptavidin bound per liposome, by increasing
the number of sites available for biotinylated antibody to couple to
liposomes. A constant ratio of streptavidin was maintained with respect
to total lipid.
Example 4
Targeting of Non-Covalently Coupled Systems
Biotinylated anti-rat IgG or Biotinylated F(ab)2 fragments were
coupled to LW s as in Example 2. Thèse LUVs contained 125I-labelled
tyramine inulin (25 umoles EPC, 25 nmoles biotin PE, 0.027 uCi 125I
inulin/umol lipid), according to the procedures of Sommerman et al.,
1984, Biochim. et Biophys. Res Comm., 122, 319-324. The high specific
activity of this entrapped marker allowed the in vitro distribution of
the vesicle antibody complexes to be determined. For the preparation of
biotinated F(ab)2 fragments, biotinated anti-rat erythrocyte IgG (4
biotins/IgG) was digested with pepsin in 0.1 N Na acetate, pH 4.5 at
37C overnight (Nisoff et al., 1960, Arch. Biochem. Biophys.~ 89,
230-244). The products were fractionated on a Sephadex G150 column and
fractions containing F(ab)2 fragments, as determined by 10~ SDS
polyacrylamide gel (Laemmli, 1970, Nature, 227, 680-685) were pooled.
~ -25-
12~ 7
F(ab)2 or antibody streptavidin liposomes were prepared as in ~xample
2. For erythrocyte cell binding studies, rat or human erythrocytes were
washed with 20 mM EPPS buffered saline, pH 8, three times. Lipid (0.62
umol/ml) was incubated with 109 erythrocytes in each experiment for 1
hour at 4C, with the exception of (a) (see Table 1), where the lipid
concentration was 1.76 umoltml. Cells were washed three times with 20 mM
EPPS buffered saline, pH 8, and were counted to determine levels of
erythrocyte-associated liposomes.
As shown in Table 3, little non-specific binding of biotinylated
liposomes to rat or human erythrocytes was observed. Anti-rat
erythrocyte IgG or F(ab)2 liposome complexes bind specifically to rat
erythrocytes but not to human erythrocytes.
-26-
~2~7~87
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Table 3 - Binding of targeted liposomes to rat erythrocytes
. . .
No. of liposomes bound per
rat human
Sample erythrocyte erythrocyte
Liposomes 18 ND
Streptavidin-liposomes 20 ND
(a)Pre-inc~bation with IgG + streptavidin-liposomes 542 ND
IgG streptavidin-liposomes 416 11
IgG streptavidin-liposomes ~ biotin73 ND
F(ab)2 streptavidin liposomes 302 11
F(ab)2 streptavidin liposomes ~ biotin 50 ND
ND = Not Done
-29-
, .
