U.S. patent application number 11/359964 was filed with the patent office on 2006-08-03 for modular targeted liposomal delivery system.
This patent application is currently assigned to Transave, Inc.. Invention is credited to Patrick Ahl, Donna Cabral-Lilly, Ravi Erukulla, Andrew Janoff, Paul R. Meers, Tong Shangguan.
Application Number | 20060172003 11/359964 |
Document ID | / |
Family ID | 36576404 |
Filed Date | 2006-08-03 |
United States Patent
Application |
20060172003 |
Kind Code |
A1 |
Meers; Paul R. ; et
al. |
August 3, 2006 |
Modular targeted liposomal delivery system
Abstract
A liposome including a fusogenic liposome, a linking moiety and
a targeting moiety. The fusogenic liposome is a lipid bilayer
encapsulating contents. The linking moiety is electrostatically
bound to the lipid bilayer, and the targeting moiety is covalently
bound to the linking moiety. The liposome may also include a
stabilizing moiety interposed between the linking and targeting
moieties, and covalently bound to both. Alternatively, the
stabilizing and targeting moieties may be covalently bound to
separate linking moieties, the linking moieties being
electrostatically bound to the lipid bilayer.
Inventors: |
Meers; Paul R.; (Princeton
Junction, NJ) ; Shangguan; Tong; (Princeton, NJ)
; Cabral-Lilly; Donna; (Princeton, NJ) ; Ahl;
Patrick; (Princeton, NJ) ; Erukulla; Ravi;
(Plainsboro, NJ) ; Janoff; Andrew; (Princeton
Junction, NJ) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Assignee: |
Transave, Inc.
Monmouth Junction
NJ
|
Family ID: |
36576404 |
Appl. No.: |
11/359964 |
Filed: |
February 22, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10130951 |
May 24, 2002 |
7060291 |
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PCT/US00/31712 |
Nov 17, 2000 |
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11359964 |
Feb 22, 2006 |
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60167297 |
Nov 24, 1999 |
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60209088 |
Jun 2, 2000 |
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Current U.S.
Class: |
424/450 |
Current CPC
Class: |
A61K 9/1271
20130101 |
Class at
Publication: |
424/450 |
International
Class: |
A61K 9/127 20060101
A61K009/127 |
Claims
1-9. (canceled)
10. A method of introducing a bioactive agent into the cytoplasm of
a cell, said method comprising: a) preparing a fusogenic liposome,
said fusogenic liposome comprising a lipid bilayer encapsulating a
bioactive agent; b) electrostatically linking a targeting moiety to
said fusogenic liposome through a linking moiety to form a targeted
liposome; c) contacting said targeted liposome with a cell; d)
dissociating said targeting moiety from at least a portion of the
lipid bilayer of said targeted liposome to form an exposed lipid
bilayer portion; e) interacting said exposed lipid bilayer portion
of said targeted liposome with the membrane of said endosome such
that said bioactive agent is released into the cytoplasm of said
cell.
11. The method of claim 10, further comprising the steps of
electrostatically linking a stabilizing moiety to said fusogenic
liposome, and dissociating said stabilizing moiety from said at
least a portion of the lipid bilayer contemporaneously with said
dissociating of said targeting moiety.
12. (canceled)
13. The method of claim 1 1, wherein said stabilizing moiety is at
least one moiety selected from the group consisting of polyethylene
glycol, polyvinylpyrolidone, a dextran, a polyamino acid,
methyl-polyoxazoline, polyglycerol, poly(acryloyl morpholine), and
polyacrylamide.
14. The method of claim 10, wherein the lipid bilayer comprises an
N-acylphosphatidylethanolamine (NAPE).
15. The method of claim 10, wherein said targeting moiety is at
least one moiety selected from the group consisting of a vitamin,
transferrin, an antibody, sialyl Lewis X antigen, hyaluronic acid,
mannose derivatives, glucose derivatives, cell specific lectins,
galaptin, galectin, lactosylceramide, a steroid derivative, an RGD
sequence, EGF, EGF-binding peptide, urokiase receptor binding
peptide, a thrombospondin-derived peptide, an albumin derivative
and a combinatorial molecule.
16. The method of claims 10, wherein said linking moiety is at
least one moiety selected from the group consisting of polylysine,
protamine, polyethyleneimine, polyarginine, polyacrylate, a
spermine derivative, cytochrome c, an annexin, heparin sulfate, an
aminodextran, polyaspartate, polyglutamate, a polysialic acid, and
poly(2-ethylacrylic acid).
17. The method of claim 10, wherein the bioactive agent is selected
from the group consisting of a nucleic acid, an antiviral agent, an
antibacterial agent, an antifungal agent, an antimetabolic agent,
an antineoplastic agent, a sterol, a carbohydrate, an amino acid, a
peptides, a protein, a dye, a radiolabel, a radiopaque compound, a
fluorescent compound, a mydriatic compound, a bronchodilator and a
local anesthetic.
18. The method of claim 16, wherein the bioactive agent is a
condensed nucleic acid.
Description
FIELD OF THE INVENTION
[0001] The invention relates to a targeted liposome capable of
efficient intracellular delivery of one or more bioactive agents, a
pharmaceutical composition including the targeted liposome, and a
method of delivering the contents of a liposome to an in vivo or in
vitro target site such as a tumor cell, site of inflammation or
infection.
BACKGROUND OF THE INVENTION
[0002] Pharmaceutical usefulness of bioactive agents depends upon
the ability to position therapeutically-effective quantities of
intact agent at the target site in the patient. Delivering intact
bioactive agents to target sites can be difficult: In vivo
degradation of bioactive agents can occur, as well as
absorption/retention of the agent by non-targeted systems. Even if
pharmaceutically effective amounts of intact agent can be delivered
to the vicinity of the target site, accessing the functional
location of the site for the bioactive agent can be challenging,
particularly if that location is intracellular: For example,
certain polar compounds and many large molecules can not enter
cells at all because of their inability to cross target cell
membranes. In addition, dilution of the bioactive agent by
non-specific binding to non-target sites reduces the amount of
bioactive agent available to the target site.
[0003] Yet another challenge in the therapeutic delivery of drugs
or bioactive agents is limiting the toxicities often associated
with therapeutically effective concentrations of drugs or bioactive
agents. Delivery to a specific target site can also reduce some
toxicity normally associated with the administration of a drug or
agent. Even when a drug or bioactive agent has no toxicity
associated with it, the "loss" of agent through degradation,
removal by non-target organs and other delivery failures can
significantly and prohibitively increase the cost of the therapy or
decrease the efficacy.
[0004] One method used to deliver bioactive agents or drugs to
tissues and cells is to encapsulate the bioactive agents or drugs
in liposomes. Often, an added advantage to this type of formulation
is the reduction of the toxicity associated with certain drugs or
bioactive agents. The intracellular targeting possibilities that
liposomes provide are especially intriguing. While certain cells
are known to engulf liposomes, delivery of most liposomes to a
target site is not sufficient to deliver the encapsulated contents
to the interior of the cell. Fusogenic liposomes are known that
allow the liposome's bilayer to fuse with the cell membrane and
thus, deliver the encapsulated bioactive agents or drugs to the
cell. However, often these fusogenic liposomes lack stability when
incubated in serum. In addition, most fusogenic liposomes have not
heretofore been able to be targeted to the specific site where the
bioactive agent or drug is required. Efficient liposomal delivery
to cells in vivo requires specific targeting and substantial
protection from the extracellular environment, particularly serum
proteins. Unfortunately, most known targeting and protecting
strategies also generate large steric barriers on the surface of
the liposomes that limit or prohibit fusogenic delivery of the
liposomal contents into the interior of the cell. In addition,
known targeting and protecting strategies attach
targeting/protecting molecules to the liposome through hydrophobic
bonding to the liposome's lipid bilayer. These hydrophobic bonds
usually inhibit the function of fusogenic membranes. When the
hydrophobic moiety is designed to allow dissociation of the
targeting/protecting moiety, its dissociation does not occur
specifically at the target site.
SUMMARY OF THE INVENTION
[0005] Briefly, the invention is a modular liposomal targeting and
delivery system comprising a fusogenic liposome, a linking moiety
and a targeting moiety. In an alternate embodiment, the system may
comprise a stabilizing moiety, either instead of or in addition to
the targeting moiety. The fusogenic liposome comprises a lipid
bilayer encapsulating contents to be delivered to a target site
such as a tumor, a site of inflammation or infection and/or to the
cytoplasm of a cell. The liposomes may contain one or more
bioactive agents or drugs or may contain a combination of bioactive
agents and drugs. In one embodiment, the bioactive agent is a
nucleic acid, preferably DNA. The targeting and/or the stabilizing
moiety is covalently bound to one or more linking moieties, and the
linking moiety electrostatically binds at neutral pH to the lipid
bilayer of the fusogenic liposome. The linking moiety is selected
from the group consisting of polylysine, protamine,
polyethyleneimine, polyarginine, polyacrylate, a spermine
derivative, cytochrome c, an annexin, heparin sulfate, an
aminodextran, polyaspartate, polyglutamate, a polysialic acid,
and/or poly(2-ethylacrylic acid). The targeting moiety is a
molecule that will bind selectively to the surface of targeted
cells. For instance, the targeting molecule may be a ligand that
binds to the cell surface receptor found on a particular cell type
or expressed at a higher frequency on target cells than on other
cells. The targeting moiety is selected from the group consisting
of a vitamin, transferrin, an antibody or fragment thereof, sialyl
Lewis X antigen, hyaluronic acid, mannose derivatives, glucose
derivatives, cell specific lectins, galaptin, galectin,
lactosylceramide, a steroid derivative, an RGD sequence, EGF,
EGF-binding peptide, urokinase receptor binding peptide, a
thrombospondin-derived peptide, an albumin derivative and/or a
molecule derived from combinatorial chemistry. The targeting moiety
enhances the ability of the liposome to bind to a targeted cell and
to deliver the liposomal contents to the target site. While not
being limited to the following explanation of the mechanism by
which the present invention delivers the liposomal contents to the
cellular interior, it has been theorized that after the liposome
binds to a cell, an endocytosis pathway may be provoked, wherein
the liposome is engulfed and sequestered in an endosomal
compartment within the cell. The low pH (relative to the
extra-cellular plasma) within the endosomal compartment weakens the
electrostatic bond between the linking moiety and the liposome,
wherein at least a portion of the linking moiety (and targeting
moiety covalently bound thereto) dissociates at least temporarily
from the liposome to expose the fusogenic lipid bilayer and enable
fusion of the liposomal membrane and endosomal membrane. Release of
the liposomal contents into the cytoplasm of the cell results from
the fusion. Even if the endocytosis pathway is not initiated when
the liposome binds to the cell, a low pH immediately surrounding
the cell (relative to the plasma pH beyond the immediate vicinity
of the cell) could cause at least temporary dissociation of the
linking moiety from the liposome as described above, thereby
enabling release of the liposomal contents into the cell
cytoplasm.
[0006] Preferably, the invention also comprises a stabilizing
moiety that is indirectly attached to the lipid bilayer of the
fusogenic liposome through a linking moiety. The stabilizing moiety
is covalently bound to the linking moiety, which linking moiety is
electrostatically bound or can be electrostatically bound to the
lipid bilayer at or near neutral pH, i.e., the pH of normal serum.
A targeting moiety may also be covalently bound to the stabilizing
moiety. Preferably, the stabilizing moiety is selected from the
group consisting of polyethylene glycol, polyvinylpyrolidone, a
dextran, a polyamino acid, methyl-polyoxazoline, polyglycerol,
poly(acryloyl morpholine), and/or polyacrylamide.
[0007] In one embodiment, the invention is a composition comprising
a fusogenic liposome comprising a lipid bilayer encapsulating
contents; and a linking moiety electrostatically bound to said
lipid bilayer; wherein a targeting moiety is covalently bound to
said linking moiety.
[0008] The invention also encompasses a method of introducing a
bioactive agent into a cell, comprising preparing a fusogenic
liposome having a lipid bilayer which encapsulates a bioactive
agent, electrostatically linking a targeting moiety to said
fusogenic liposome to form a targeted liposome, contacting the
targeted liposome with a cell such that the targeting moiety is
released from at least a portion of the lipid bilayer of the
liposome to expose a portion of the lipid bilayer, and fusing the
exposed lipid bilayer portion with a cell membrane such that the
bioactive agent is released into the cell. Preferably, the method
also includes electrostatically binding a stabilizing agent to the
lipid bilayer of the fusogenic liposome.
BRIEF DESCRIPTION OF THE DRAWINGS
[0009] FIG. 1 depicts transfection of ovarian cancer cells by
charge reversal liposomes and its enhancement by the use of a
targeting/stabilizing module.
[0010] FIG. 2 depicts the quantitation of enhanced transfection
efficiency for charge reversal liposomes when targeted by modules
with transferrin or folate.
[0011] FIG. 3 demonstrates that enhanced transfection efficiency of
fusogenic ANacylPE liposomes by a folate targeting module is
dependent on folate.
[0012] FIG. 4 demonstrates that folate targeting modules
electrostatically linked to the liposomes enhance transfection more
than folate conjugated to lipids.
[0013] FIG. 5 depicts the maintenance of liposomal integrity when
an antibody conjugate is electrostatically bound to the liposome
surface.
[0014] FIG. 6 demonstrates enhanced liposomal uptake mediated by an
antibody conjugate electrostatically bound to the liposome
surface.
[0015] FIG. 7 shows enhanced transfection as a result of increasing
amounts of an antibody conjugate electrostatically bound to the
surface of liposomes containing plasmid DNA.
[0016] FIG. 8 depicts enhanced green fluorescent protein expression
as a result of antibody targeting.
[0017] FIG. 9 depicts the binding of transferrin-targeted liposomes
of the present invention to OVCAR-3 cells in the presence and
absence of serum.
[0018] FIG. 10 depicts the effect of polylysine-transferrin
congugates on liposome binding and transfection.
[0019] FIG. 11 is a fluorescent photomicrograph depicting the
effect of pKT on liposome binding to cells and transfection.
[0020] FIG. 12 demonstrates the inhibition of transfection mediated
by pKT-targeted liposomes by anti-transferrin antibodies.
[0021] FIG. 13 depicts the transfection efficiency of conjugates
compared to free polylysine.
[0022] FIG. 14 demonstrates the binding of fluorescent liposomes to
OVCAR-3 cells derived from the ascites of a mouse xenograft with
and without a targeting agent (pKT).
[0023] FIG. 15 demonstrates the beta-galactosidase assay for in
vivo transfection.
[0024] FIG. 16 demonstrates the beta-galactosidase assay for in
vivo transfection.
DETAILED DESCRIPTION OF THE INVENTION
[0025] Delivery of liposomally encapsulated drugs or nucleic acids
to specific cells via membrane fusion is a highly desirable goal
because of protection of the cargo from degradation, potential for
targeting and decrease of drug toxicity. Furthermore, it is
advantageous to protect and/or stabilize the liposome itself until
it reaches its desired site of delivery, because the liposome can
be degraded in vivo. However, many targeting molecules such as
antibodies and most protecting or stabilizing moieties such as
polyethylene glycol (PEG) sterically inhibit the interaction
between the liposomal membrane and the cell membrane, even though
the liposome has bound to the cell surface. Because of this steric
hindrance, it is generally not possible for fusogenic liposomes to
efficiently deliver contents to the cytoplasm of the cell. This
invention provides a targeted liposome, or targeted and stabilized
liposome, where at least a portion of the targeting/stabilizing
moieties are at least temporarily removable at the target site. The
invention involves non-covalent adherence of a targeting moiety,
and preferably a stabilizing moiety, to the surface of a liposome
by electrostatic interactions, preferably multivalent electrostatic
interactions. Such interactions can be quite strong if the valency
of interaction is high enough. However, the reduced pH (relative to
the extra-cellular plasma beyond the immediate vicinity of the
cell) at certain in vivo sites such as the site of infection,
inflammation or within certain cellular organelles or compartments
such as endosomes, loosens or, in certain cases, reverses the
interaction between the adherent moiety and the liposome. Once a
fusogenic liposome, so modified, binds to the targeted cell type
and encounters the reduced pH environment, the
targeting/stabilizing moiety may be completely or partially removed
if at least one of the electrostatically interacting species, i.e.,
membrane lipids or targeting/stabilizing agent is at least
partially neutralized by the low pH environment in combination with
other ions. Furthermore, a portion of the electrostatically bound
targeting/stabilizing agent may exchange onto the cellular membrane
following binding of the liposome and internalization into an
endosome, also eliminating steric interference to fusion. For
example, a fusogenic, anionic liposome can be protected by a
polylysine conjugate with PEG and further targeted by a linked
molecule, such as folate, transferrin or an antibody. If the
anionic lipid is at least partially neutralized at lower pH, the
targeting/stabilizing agent may completely or partially dissociate
allowing exposure of the bare fusogenic liposomal membrane and
subsequent fusion or partial fusion of the liposome with the
endosomal membrane. This releases the liposomal contents into the
cytoplasm. The contents may include any therapeutically relevant
molecule(s).
[0026] A second advantage of this method of targeting and
stabilizing the liposome is that a single type of liposome can be
produced to which any number of targeting/stabilizing agents can be
subsequently bound electrostatically. This embodiment merely
requires mixing a fusogenic liposome, encapsulating a relavant drug
or bioactive agent, with a linking moiety covalently bound to a
targeting moiety that will bind to the specific targeted site.
Thus, the system is modular. Selected targeting agents covalently
linked to linking moieties that will bind electrostatically to the
lipid bilayer can be prepared. Fusogenic liposomes encapsulating
different drug or bioactive agents can be independently prepared.
Then, depending on the target site and the therapeutic agent(s) one
desires to administer, the appropriate combination of targeting
agent and liposome encapsulating bioactive agent or drug can be
mixed to provide a specific therapeutic agent that is targeted to
the specific cellular site. This will allow patient-specific
targeting with a single type of liposomal preparation that can be
modified by an array of targeting agents.
[0027] The basic elements of the invention are a fusogenic
liposome, a linking moiety and a targeting moiety. The fusogenic
liposome comprises a lipid bilayer encapsulating contents to be
delivered to the cytoplasm of a cell, the contents preferably being
a bioactive agent, and more preferably condensed DNA. The targeting
moiety is positioned exteriorly of the liposome, and is covalently
bound to the linking moiety. The linking moiety is
electrostatically bound to the lipid bilayer of the fusogenic
liposome, thus indirectly linking the targeting moiety
electrostatically to the lipid bilayer. Preferably, the invention
also comprises a stabilizing moiety which, like the targeting
moiety, is indirectly electrostatically bound to the lipid bilayer
through a linking moiety. Specifically, the stabilizing moiety is
covalently bound to the linking moiety, and the linking moiety is
electrostatically bound to the lipid bilayer of the fusogenic
lipid. A targeting moiety may be covalently bound to the
stabilizing moiety. These individual elements of the invention will
now be described in detail.
[0028] "Liposomes" are self-assembling structures comprising one or
more lipid bilayers, each of which comprises two monolayers
containing amphipathic lipid molecules oppositely oriented.
Amphipathic lipids comprise a polar (hydrophilic) headgroup region
covalently linked to one or two non-polar (hydrophobic) acyl
chains. Energetically unfavorable contacts between the hydrophobic
acyl chains and the surrounding aqueous medium induce the
amphipathic lipid molecules to arrange themselves such that their
polar headgroups are oriented towards the bilayer's surface, while
the acyl chains reorient towards the interior of the bilayer. An
energetically stable structure is thus formed in which the acyl
chains are effectively shielded from coming into contact with the
aqueous environment. A "fusogenic" liposome as defined herein is a
liposome that is capable of interacting with a cell membrane in a
way that permits the contents of the liposome to enter the
cytoplasm of the cell. Without limitation, this interaction can
take the form of complete or partial fusion of the membranes, or
adjacent, contemporaneous and localized disruptions of the cellular
and liposomal membranes that allows passage of the liposome
contents into the cytoplasm of the cell.
[0029] Liposomes (see, e.g., Cullis et al., 1987; New, 1995) can
have a single lipid bilayer (unilamellar liposomes, "ULVs"), or
multiple lipid bilayers (multilamellar liposomes, "MLVs" or
"SPLVs"). Each bilayer surrounds, or encapsulates, an aqueous
compartment. Given this encapsulation of aqueous volume within a
protective barrier of lipid molecules, liposomes are able to
sequester encapsulated molecules, e.g., nucleic acids, away from
the degrading effects of factors, e.g., nuclease enzymes, present
in the external environment.
[0030] Liposomes can have a variety of sizes, e.g., an average
diameter as low as 25 nm or as high as 10,000 nm or more. Size is
affected by a number of factors, e.g., lipid composition and method
of preparation, well within the purview of ordinarily skilled
artisans to determine and account for, and is determined by a
number of techniques, such as quasi-elastic light scattering, also
within the artisans' purview.
[0031] Various methodologies, also well within the purview of
ordinarily skilled artisans, such as sonication, homogenization,
French Press application and milling can be used to prepare
liposomes of a smaller size from larger liposomes. Extrusion (see,
e.g., U.S. Pat. No. 5,008,050) can be used to size reduce
liposomes, that is to produce liposomes having a predetermined mean
size by forcing the liposomes, under pressure, through filter pores
of a defined, selected size. Tangential flow filtration
(WO89/008846), can also be used to regularize the size of
liposomes, that is, to produce a population of liposomes having
less size heterogeneity, and a more homogeneous, defined size
distribution. The contents of these documents are incorporated
herein by reference.
[0032] Liposomes of this invention can be unilamellar, or
oligolamellar, and can have a size equal to that of liposomes
produced by any of the methods set forth hereinabove. However, in
preferred embodiments of this invention, the liposomes are
unilamellar liposomes having number average sizes of about 50-500
nm.
[0033] Liposomes are composed of a variety of lipids, both
amphipathic and nonamphipathic, obtained from a variety of sources,
both natural and synthetic. Suitable liposomal lipids include,
without limitation, phospholipids such as phosphatidylcholines
("PC's"), phosphatidylethanolamines ("PE's"), phosphatidylserines
("PS's"), phosphatidylglycerols ("PG's"), phosphatidylinositols
("PI's") and phosphatidic acids ("PA's"). Such phospholipids
generally have two acyl chains, these being either both saturated,
both unsaturated or one saturated and one unsaturated; said chains
include, without limitation: myristate, palmitate, stearate,
oleate, linoleate, linolenate, arachidate, arachidonate, behenate
and lignocerate chains.
[0034] Phospholipids can also be derivatized, by the attachment
thereto of a suitable reactive group. Such a group is generally an
amino group, and hence, derivatized phospholipids are typically
phosphatidylethanolamines. The different moieties suited to
attachment to PE's include, without limitation: acyl chains
(WO98/16199), useful for enhancing the fusability of liposomes to
biological membranes; peptides (WO 98116240), useful for
destabilizing liposomes in the vicinity of target cells; biotin and
maleimido moieties (U.S. Pat. Nos. 5,059,421 and 5,399,331,
respectively), useful for linking targeting moieties such as
antibodies to liposomes; and, various molecules such as
gangliosides, polyalkylethers, polyethylene glycols and organic
dicarboxylic acids (see, e.g., U.S. Pat. Nos. 5,013,556, 4,920,016
and 4,837,028). The contents of the above-cited documents are
incorporated herein by reference.
[0035] Accordingly, in the most preferred embodiments of this
invention, the liposomes comprise a derivatized phospholipid,
adapted so as to enhance delivery of their contents. The liposomes
may also, but are not required to, comprise additional lipids as
well, said additional lipids being incorporated into the liposomes
for a number of reasons apparent to artisans of ordinary skill in
the field of liposomology. Such reasons include, without
limitation, stabilizing or targeting the liposomes, as well as
further altering the liposomes' pharmacokinetic behavior. Suitable
additional lipids include any of those lipids commonly recognized
as suitable for incorporation in liposomes, including, without
limitation, phospholipids, glycolipids and sterols.
[0036] Preferably, liposomes of this invention have a lipid
component which comprises a derivatized phospholipid and an
additional lipid. Suitable liposomes and the methods of preparing
them are described in U.S. patent application Ser. No. 08/951,056,
incorporated herein in its entirety by reference.
[0037] Preferably, the derivatized phospholipid is an N-acylated
PE. Such NAPEs are useful in preparing fusogenic liposomes and are
preferred for preparing liposomes comprising the drug or bioactive
agent complexes of the present invention.
[0038] NAPE-induced bilayer destabilization induces the bilayers to
fuse to biological membranes in the vicinity and hence, enhances
the bilayers' fusogenicity (Shangguan et al., 1998). Enhanced
fusogenicity, in turn, can be used to deliver encapsulated
bioactive agents, such as nucleic acids or other agents that can
not cross the cell membrane, to cells, by combining the cells with
the liposomes under conditions, e.g., the presence of appropriate
concentrations such as Ca.sup.t+ and Mg.sup.2+. Liposome-cell
contact results in release of the liposome-encapsulated bioactive
agents local to the cells, and/or directly into the cells'
cytoplasm as a result of fusion between liposome and cell
membranes. Such delivery is either in vivo or in vitro.
[0039] An alternative preferable formulation of liposomes for
modular targeting are referred to below (example 1) as "charge
reversal" liposomes. The composition of such liposomes also allows
electrostatically bound targeting/stabilizing groups. Such
liposomes reverse charge at low pH, dissociating the
targeting/stabilizing conjugates and attaining a positive charge to
enhance interaction with the cellular membrane.
[0040] The liposomal lipid can also comprise a "headgroup-modified
lipid," i.e., a lipid having a polar group derivatized by the
attachment thereto of a moiety capable of inhibiting the binding of
serum proteins to a liposome incorporating the lipid. Incorporation
of headgroup-modified lipids into liposomes thus alters their
pharmacokinetic behavior, such that the liposomes remain in the
circulation of an animal for a longer period of time then would
otherwise be the case (see, e.g., Blume et al., 1993; Gabizon et
al., 1993; Park et al., 1992; Woodle et al., U.S. Pat. No.
5,013,556; Allen et al., U.S. Pat. Nos. 4,837,028 and 4,920,016;
the contents of these documents being incorporated herein by
reference).
[0041] Nucleic acids that may be encapsulated in the liposome are
DNA, including genomic DNA, plasmid DNA and cDNA, or RNA;
preferably, the encapsulated nucleic acid is DNA, more preferably,
closed (circular) plasmid DNA. "Encapsulated" or "containing" as
used herein with regard to the contents of the liposome describes
materials that are within the interior aqueous volume of the
liposome, intercalated in the lipid bilayer of the liposome, or
partly intercalated in the lipid bilayer of the liposome.
[0042] Liposomes of the invention can contain one or more bioactive
agents. Bioactive agents which may be associated with liposomes
include, but are not limited to: antiviral agents such as
acyclovir, zidovudine and the interferons; antibacterial agents
such as aminoglycosides, cephalosporins and tetracyclines;
antifungal agents such as polyene antibiotics, imidazoles and
triazoles; antimetabolic agents such as folic acid, and purine and
pyrimidine analogs; antineoplastic agents such as the anthracycline
antibiotics and plant alkaloids; sterols such as cholesterol;
carbohydrates, e.g., sugars and starches; amino acids, peptides,
proteins such as cell receptor proteins, immunoglobulins, enzymes,
hormones, neurotransmitters and glycoproteins; dyes; radiolabels
such as radioisotopes and radioisotope-labeled compounds;
radiopaque compounds; fluorescent compounds; mydriatic compounds;
bronchodilators; local anesthetics; and the like.
[0043] Liposomal bioactive agent formulations can enhance the
therapeutic index of the bioactive agent, for example by buffering
the agent's toxicity. Liposomes can also reduce the rate at which a
bioactive agent is cleared from the circulation of animals.
Accordingly, liposomal formulation of bioactive agents can mean
that less of the agent need be administered to achieve the desired
effect.
[0044] The liposome of this invention may be dehydrated, stored and
then reconstituted such that a substantial portion of their
internal contents are retained. Liposomal dehydration generally
requires use of a hydrophilic drying protectant such as a
disaccharide sugar at both the inside and outside surfaces of the
liposomes' bilayers (see U.S. Pat. No. 4,880,635, the contents of
which are incorporated herein by reference). This hydrophilic
compound is generally believed to prevent the rearrangement of the
lipids in liposomes, so that their size and contents are maintained
during the drying procedure, and through subsequent rehydration.
Appropriate qualities for such drying protectants are that they be
strong hydrogen bond acceptors, and possess stereochemical features
that preserve the intermolecular spacing of the liposome bilayer
components. Alternatively, the drying protectant can be omitted if
the liposome preparation is not frozen prior to dehydration, and
sufficient water remains in the preparation subsequent to
dehydration.
[0045] Also provided herein is a pharmaceutical composition
comprising a pharmaceutically acceptable carrier and the liposome
of this invention. Said composition is useful, for example, in the
delivery of nucleic acids to the cells of an animal.
"Pharmaceutically acceptable carriers" as used herein are those
media generally acceptable for use in connection with the
administration of lipids and liposomes, including liposomal
bioactive agent formulations, to animals, including humans.
Pharmaceutically acceptable carriers are generally formulated
according to a number of factors well within the purview of the
ordinarily skilled artisan to determine and account for, including
without limitation: the particular liposomal bioactive agent used,
its concentration, stability and intended bioavailability; the
disease, disorder or condition being treated with the liposomal
composition; the subject, its age, size and general condition; and
the composition's intended route of administration, e.g., nasal,
oral, ophthalmic, topical, transdermal, vaginal, subcutaneous,
intramammary, intraperitoneal, intravenous, or intramuscular (see,
for example, Naim (1985), the contents of which are incorporated
herein by reference). Typical pharmaceutically acceptable carriers
used in parenteral bioactive agent administration include, for
example, D5W, an aqueous solution containing 5% weight by volume of
dextrose, and physiological saline. Pharmaceutically acceptable
carriers can contain additional ingredients, for example those
which enhance the stability of the active ingredients included,
such as preservatives and anti-oxidants.
[0046] Lipids useful in the practice of this invention are, as
described hereinabove, those lipids recognized as suitable for
incorporation in liposomes, either on their own or in connection
with additional lipids; these include, phospholipids, glycolipids,
sterols and their derivatives. Organic solvents used in this method
are any of the variety of solvents useful in dissolving lipids
during the course of liposome preparation; these include, without
limitation, methanol, ethanol, dimethylsulfoxide, chloroform, and
mixtures thereof Preferably, the organic solvent is chloroform or
methylene chloride.
[0047] Still further provided herein is a method of transfecting
the cells of an animal with a targeted fusogenic liposome
comprising one or more bioactive agents such as, but not limited
to, a nucleic acid. The method comprises the steps of preparing a
fusogenic liposome encapsulating a bioactive agent; preparing a
targeting moiety covalently bound to a linking agent said linking
agent being capable of electrostatically binding to said fusogenic
liposome at normal physiological pH, mixing the loaded fusogenic
liposome with the targeting conjugate and contacting the cells with
the composition comprised of the loaded fusogenic liposome
electrostatically bound to the targeting agent. Such contact is
either in vitro, in which case, a composition comprising the
liposome is added to the culture medium surrounding the cells, or
in vivo, in which case the liposome is administered in a
pharmaceutical composition also comprising a pharmaceutically
acceptable carrier, and is administered to the animal by any of the
standard means of administering such compositions to animals.
[0048] The targeting moiety of the invention can be any chemical
composition that favors the positioning of a liposome to a specific
site or sites. More than one targeting moiety may be utilized on a
single liposome. Preferably, the targeting moiety is selected from
the group consisting of a vitamin such as folate; transferrin; an
antibody such as OVB-3, anti-CAl.sub.25, anti-CEA, and others;
sialyl Lewis X antigen, hyaluronic acid, mannose derivatives,
glucose derivatives, cell specific lectins, galaptin, galectin,
lactosylceramide, a steroid derivative, an RGD sequence, a ligand
for a cell surface receptor such as epidermal growth factor (EGF),
EGF-binding peptide, urokinase receptor binding peptide, a
thrombospondin-derived peptide, an albumin derivative and/or a
combinatorial molecule directed against various cells.
[0049] The linking moiety can be any chemical composition that is
capable of simultaneously binding to the lipid bilayer of a
liposome electrostatically and binding to a targeting or
stabilizing moiety covalently, such that the electrostatic bond can
be so weakened in reduced pH environment (relative to the
extra-cellular plasma not in the immediate vicinity of the cell) so
that at least some of the linking moiety at least temporarily
dissociates from the liposome, thereby exposing at least a portion
of the outer lipid bilayer for at least some period of time. The
linking moiety is preferably a small molecule that does not hinder
the interaction between the targeting moiety and targeted cell.
More than one linking moiety can be used on a single liposome, and
the linking moieties used to bind the targeting and stabilizing
moieties may be the same or different. Preferably, the linking
moiety is selected from the group consisting of polylysine,
protamine, polyethyleneimine, polyarginine, polyacrylate, a
spermine derivative, cytochrome c, an annexin, heparin sulfate, an
aminodextran, polyaspartate, polyglutamate, a polysialic acid,
and/or poly(2-ethylacrylic acid).
[0050] The stabilizing moiety of the invention is any chemical
composition that inhibits or prevents a liposome from fusing with
other liposomes or non-target cells, and/or protects the lipid
bilayer of the liposome from the disruptive, degrading or
interfering action of detrimental compounds (e.g. serum proteins).
More than one stabilizing moiety may be used on a single liposome.
Preferably, the stabilizing moiety is selected from the group
consisting of polyethylene glycol, polyvinylpyrolidone, a dextran,
a polyamino acid, methyl-polyoxazoline, polyglycerol, poly(acryloyl
morpholine), and/or polyacrylamide.
[0051] Preferred embodiments of the invention will now be described
in the Examples below. The Examples are intended to be illustrative
of the invention and are not intended to limit the scope of the
invention defined in the appended claims.
EXAMPLE 1
Formulations
Preparation of Liposomes for Delivery of Nucleic Acids:
[0052] Preferably, for the delivery of nucleic acids to cells,
liposomes are prepared according to the method described in U.S.
Provisional Application Ser. No. 60/122,365 entitled "Encapsulation
of Bioactive Complexes in Liposomes", filed on Mar. 2.1999. One
embodiment is described below.
[0053] Plasmid Purification: Two plasmids were used in this study:
the pZeoSVLacZ plasmid which is 6.5 kb, and expresses the IacZ gene
for .beta.-galactosidase in mammalian cells from the SV40 early
enhancer-promoter, allowing selection in mammalian cells and E.
coli using the antibiotic zeocin; and, the pEGFP-Cl plasmid, which
is 4.7 kb and expresses enhanced green fluorescent protein (EGFP)
from a human cytomegalovirus immediate early promoter, allowing
selection in E. coil using kanamycin, and in mammalian cells using
G418. Plasmids were purified from E. Coli (Baumann and Bloomfield,
1995; or by Qiagen kit per manufacturer's instructions (Qiagen,
Valencia, Calif.))--the final ratio of O.D. at 260 nm to O.D. at
280 nm was greater than 1.9 for all preparations; agarose gel
electrophoresis indicated DNA in the expected size range.
Preparation of Liposomal-DNA Formulations (Fusogenic N-Acyl-PE
Liposomes: Two Step Emulsion Method):
[0054] Samples were prepared by diluting 200 Ag of DNA into 125 Al
of low salt buffer (LSB; 10 mM TrisHCl, 1 mM NaCl, pH 7.0, all LSB
used in these preparations also contained 200 mM sucrose), and then
combining the resulting suspension with 1 ml CHCl.sub.3 containing
30 gmole of 70:30 molar ratio of N-Cl.sub.2 DOPE and DOPC, in a
13.times.100 Pyrex tube while vortexing. The sample was immediately
sonicated for 12 seconds in a bath sonicator (Laboratory Supplies
Co. Hicksville, N.Y.) under maximum power, to form an emulsion with
plasmid DNA first. Subsequently, a 125 .mu. aliquot of LSB
containing spermine (preferably 16 to 40 millimolar) was added to
this emulsion with vortexing and sonication.
[0055] Resulting emulsions were placed, within a few minutes, in a
flask on a Rotovap (Buchi Laboratoriums-Technik AG, Switzerland).
Organic solvent was removed while rotating the flask at its maximum
rate, while the vacuum was modulated with a pin valve. Initially a
vacuum of approximately 600-650 mm was established, this being
subsequently increased, as rapidly as possible without excessive
bubbling, until the maximum vacuum was reached (approximately 730
mm); the flask was then evacuated for another 25 minutes. The film
left on the flask was resuspended in 1 ml of 300 mM sucrose in LSB,
and the sample was extruded five times through 0.4 lam
polycarbonate membrane filters (Poretics, Livemore, Calif.). The
sample was then dialyzed against Hank's balanced salt buffer (HBSS)
without Ca.sup.2+/Mg.sup.2+, overnight at 4.degree. C.
[0056] After preparation of the nucleic acid-containing liposomes
described above, the free plasmid DNA was separated from
liposome-encapsulated DNA by centrifugation. The liposomes were
pelleted and washed prior to forming the targeting complex. The
preparations may be scaled-up to produce larger amounts of product
by methods known in the art.
Preparation of Charge Reversal Liposomal DNA Formulations:
[0057] In another embodiment, liposomes, heretofore referred to as
charge reversal liposomes, were made by first mixing the lipids in
chloroform (DOPC:POPE:cholesterol:cholesteryl
hemisuccinate:DOPAP:oleyl actetate, 12:50:2.5:12.5:10.5:10.5).
Next, plasmids were condensed with spermine using the two-step
lipid emulsion protocol previously described in the embodiment
above. One difference is that the final sucrose concentration in
the DNA solution to be encapsulated was 300 mM. Solvent was removed
by either rotary evaporation or sparging with nitrogen. The
liposomes were suspended from the resulting paste in 300 mM
sucrose, extruded through 0.4 .mu.m filters, then dialyzed against
PBS or Hanks balanced salt solution (HBSS). Unencapsulated plasmid
was removed by centrifuging the sample at 10,000 g. The
liposome/plasmid pellet was washed, respun and resuspended in PBS
or HBSS. The size distribution of the liposomes was determined by
light scattering using a NICOMP model 370 submicron particle sizer
(NICOMP, Santa Barbara, Calif.). The amount of plasmid present in
the liposomal pellet was quantitated using a PicoGreen fluorescent
assay (Molecular Probes, Inc., Eugene, Oreg.) as described in
Example 3 "Assays".
[0058] Liposomes of this composition were characterized by
transmission electron microscopy of a liposome/plasmid preparation
with polylysine-anti-OVB3 ligand attached. The sample was placed on
a carbon coated EM grid and negatively stained with 1% uranyl
acetate. Liposomes of a heterogenous size were observed with
varying amounts of stain penetration. The final liposome/plasmid
preparations gave a Gaussian size distribution by light scattering
analysis. The number weighted mean diameter of 7 different
preparations was 140 nm.+-.20 nm (standard deviation of the mean
for any single preparation ranged between 40-55%). Plasmid content
in the washed liposome/plasmid samples ranged from 1.25-2.45 .mu.g
DNA/gmol lipid, with a mean of 1.6 .mu.g DNA/.mu.mol lipid. This
plasmid fraction was protected from digestion with DNAse I
suggesting either encapsulation or very tight association with the
liposomes.
EXAMPLE 2
Synthesis of Targetingistabilizing Moiety--Linker Conjugates
[0059] Targeting/stabilizing modules were synthesized to use with
liposomes prepared as in example 1. Targeting/stabilizing moieties
may be covalently coupled to a linker for electrostatic
interaction, such as polylysine (pK), by any method known in the
art. Examples of processes for coupling polylysine to targeting
agents such as folic acid, transferrin and antibodies are provided
herein for exemplary purposes only. Methods for coupling a linking
agent such as polylysine to a stablizing agent such as PEG or to a
stabilizing agent and a targeting moiety are also provided
herein.
a. Preparation of Folic acid, PEG and Polylysine Without Glutaric
Acid (FA-NH-PEG-CO-PL or FPK)
Preparation of FAA
[0060] Folic acid (50 mg, 0.11 mmole) was dissolved in 6 ml of
DMF:PY (5:1; v/v). To this solution dicylohexylcarbodiimide (DCC,
140 mg, 0.68 mmoles) was added and the reaction mixture was stirred
at room temperature for 2-3 h. Dicyclohexylurea (DCU) precipitate
developed with time.
Deprotection of Boc Group From BocNH-PEG-CO.sub.2NHS
[0061] BocNH-PEG-CO.sub.2NHS (3400 m.w., Shearwater Polymers,
Huntsville, Al.) was dissolved in 5 ml of CH.sub.2Cl.sub.2:TFA
(4:1,v/v) solution. This mixture was stirred at room temperature
for 2 hours. TLC analysis revealed that the reaction had gone to
completion. The product gave a positive ninhydrin test. The
solvents were removed under reduced pressure and the sample dried
under high vacuum.
Coupling FA to NH.sub.2-PEG-CO.sub.2,NHS
[0062] To the FAA reaction mixture, H.sub.2N-PEG-CO.sub.2NHS (186
mg, 0.057 mmole) was added in 2.5 ml of DMF:triethylamine (100:1
v/v) and the reaction mixture was stirred at room temperature
overnight. TLC analysis showed that the reaction had gone to
completion. The product was UV and ninhydrin positive. Solvents
were removed from the reaction mixture under reduced pressure and
the sample dried under high vacuum.
Coupling FA-NH-PEG-CO.sub.2NHS to Polylysine
[0063] To a solution of FA-NH-PEG-CO.sub.2NHS (100 mg, 0.027 mmole)
in 5 ml of DMF:Py (4:1 v/v), were added PLL (62.15 mg, 0.0224
mmole) and E.sub.t3N (3.4 mg, 4.7 ul, 0.0336 mmole). The reaction
mixture was stirred at room temperature overnight. At this time
point TLC analysis revealed that a new polar spot was formed which
was UV positive and gave a positive ninhydrin test. Solvents were
removed from the reaction mixture under reduced pressure and the
sample dried under high vacuum. The residual material was suspended
in CHCl.sub.3 and spun at 4000 rpm for 30 min. The supernatant
solid material was separated and washed with CHCl.sub.3. Pure
Sample weight 55.4 mg (37.5% Yield).
[0064] Conjugates of folate or PEG directly linked to polylysine or
other charged molecules may also be prepared by similar
procedures.
b. Preparation of Folate-PEG-Polylysine (FA-PEG-GA-PL or FPGK)
Using Glutaric Acid
Preparation of Folic Acid Anhydride (FAA)
[0065] Folic acid (50 mg, 0.11 mmole) was dissolved in 6 mL of
N,N-dimethylformamide:pyridine (DMF:PY) (4:1 v/v). To this solution
was added dicyclohexocarbodiimide (DCC) (140 mg, 0.68 mmole) and
the reaction mixture was stirred at room temperature for .about.3
h. With time dicyclohexylurea (DCU) precipitate was observed in
reaction mixture.
Coupling FA to PEG (3400)
[0066] To the FAA reaction mixture prepared above,
H.sub.2N-PEG-NHBoc (3400 m.w. Shearwater Polymers, Huntsville, Al.)
(192 mg, 0.057 mmole) was added in 1 ml of NN-dimethylformamide.
This reaction mixture was stirred at room temperature overnight. At
this point TLC analysis revealed that the reaction had gone to
completion. The product formed was UV positive and gave a negative
ninhydrin test. Solvents were removed under reduced pressure.
Deprotection of Boc Group From FA-NH-PEG-NHBoc
[0067] The residual material was dissolved in 5 ml of
CH.sub.2Cl.sub.2:TFA (4:1 v/v) and the reaction mixture was stirred
at room temperature for 2-3 hr. At this time point, TLC analysis
revealed that the reaction had gone to completion. The product was
UV positive and gave a positive ninhydrin test. Solvents were
removed at reduced pressure and the residual material dried under
high vacuum.
Introduction of GA Molecule (FA-NH-PEG-NH-CO(CH.sub.2);3000 H)
[0068] The residual material was dissolved in 5 ml of DMF:Py (4:1
v/v). To this solution Et.sub.3N (15.7 .mu.l, 11.41 mg, 0.113
mmole) and Glutaric anhydride (9.7 mg; 0.085 mmole) were added and
the reaction mixture was stirred at room temperature overnight. TLC
analysis showed that the reaction had gone to completion. TLC
analysis was performed in CHCl.sub.3:CH.sub.3OH:H.sub.2O (65:30:5;
v/v/v). The product gave a negative ninhydrin test. The residual
weight of the sample was 649 mg.
Coupling Polylysine (PL) (FA-NH-PEG-NH-CO(CH.sub.2).sub.3
CO-PL):
[0069] DCC (44 mg, 0.21 mmole) was added to a solution-of
FA-NH-PEG-NHCO(CH.sub.2)3000 H (residual material) (162 mg, 0.043
mmole) in 4 ml of DMSO:Py (4:1 v/v). This mixture was stirred at
room temperature for 2-3 hrs. With time DCU precipitate developed.
At this point polylysine HBr (32 mg, 0.011 mmole) was added to the
reaction mixture in 1.4 ml of DMSO:Py:Et.sub.3N (1000:200:200
.mu.l) solution. This reaction mixture was stirred at room
temperature overnight. At this time point TLC analysis in
CHCl.sub.3:CH.sub.3OH:H.sub.2O (60:35:5 v/v/v) revealed that the
reaction had gone to completion. TLC analysis gave positive UV and
ninhydrin tests. Solvents were removed under reduced pressure. The
residual material was dissolved in CHCl.sub.3 and the precipitate
separated by spinning at 3000-4000 rpm for 30 minutes. The solid
mixture was separated and washed with CHCl.sub.3. The pure sample
weighed 19.1 mg (27% Yield).
c. Preparation of Folate-PEG-Protamine (FA-PEG-GA-Pro or FPGPr)
Using Glutaric Acid
[0070] FA-PEG-GA was prepared as described in part b in the
Folate-PEGPolylysine synthesis scheme.
Cougling Protamine to
(FA-NH-PEG-NH-CO(CH.sub.2).sub.3CO-Protamine):
[0071] DCC (8.03 mg, 0.039 mmole) was added to a solution of
FA-NH-PEG-NH-CO(CH.sub.2).sub.3OOOH (residual material) (50 mg,
0.013 mmole) in 3.0 ml of CHCl.sub.3. This mixture was stirred at
room temperature for 2-3 hrs. At this point protamine free base
(13.3 mg , 0.0033 mmole) was added to the reaction mixture in 1.5
ml of 1,1,1,3,3,3-hexafluoroisopropanol. This reaction mixture was
stirred at room temperature overnight. At this time point TLC
analysis in CHCl.sub.3:CH.sub.3OH:H.sub.2O (60:35:5 v/v/v) revealed
that the reaction had gone to completion. TLC analysis gave
positive UV and ninhydrin tests. Solvents were removed under
reduced pressure. The residual material was dissolved in CHCl.sub.3
and the precipitate separated by spinning at 3000-4000 rpm for 30
minutes. The solid mixture was separated and washed with
CHCl.sub.3. The pure sample weighed 5.5 mg (22% Yield).
[0072] Conjugates of folate alone or PEG alone directly linked to
polylysine or other charged molecules may also be prepared by minor
modifications of this method.
d. Preparation of Acetyl-PEG-Protamine (Ac-PEG-GA-Pro or AcPGPr)
Using Glutaric Acid
[0073] Several variations of the PEG conjugates were prepared as
controls to compare with the above conjugates. These were missing
one of more component of the active conjugate. The Ac-PEG-GA-Pro
conjugate, or AcPGPr, was prepared by substituting an acetyl group
for folate on the conjugate. It was, therefore, not targeted to a
cell receptor.
[0074] In addition to control experiments, it may be desirable to
use such a nontargeted protecting moiety to modulate the liposomal
surface for optimal targeting in vivo. For instance, a non-targeted
conjugate may be used in addition to a targeting conjugate on the
liposomal surface.
Acetylation of NH2-PEG-NHBOC:
[0075] To a solution of H.sub.2N-PEG-NHBOC (100 mg, 0.0294 mmole)
in 3.0 mL of CHCl.sub.3 were added acetic anhydride (9.0 mg, 7.85
.mu.L, 0.088 mmole) and excess amount of TEA. This reaction mixture
was sealed under nitrogen and stirred at room temperature for 34 h.
At this time point TLC analysis in CHCl.sub.3:CH.sub.3OH (9:1 v/v)
revealed that the reaction had gone to the completion. TLC analysis
gave negative Ninhydrin test. Solvents were removed under reduced
pressure and residual material was used in next step without
purification.
Degrotection of BOC gp from Ac-NH-PEG-NHBOC:
[0076] To a solution of Ac-NH-PEG-NHBOC in 2.2 mL of
CH.sub.2Cl.sub.2 was added 0.25 mL of TFA. This reaction mixture
was stirred at room temperature for 2 h. At this time point TLC
analysis in CHCl.sub.3:CH.sub.3OH (9:1 v/v) revealed that the
reaction had gone to the completion. TLC analysis gave positive
ninhydrin test. Solvents were removed under reduced pressure and
residual material used in the next step without purification.
Introduction of GA Molecule on Ac-NH-PEG-NH,:
[0077] The residual material was dissolved in 3.0 mL of CHCl.sub.3.
To this solution excess amount of Et.sub.3N and Glutaric anhydride
(5 mg; 0.044 mmole) were added and the reaction mixture was stirred
at room temperature overnight. TLC analysis showed that the
reaction had gone to completion. TLC analysis was performed in
CHCl.sub.3:CH.sub.3OH (9:1 v/v). The product gave a negative
ninhydrin test. The residual material was used in the next step
without further purification.
Coupling Ac-NH-PEG-NH-CO-(CH.sub.2).sub.3-OOOH to Protamine
(Pr):
[0078] To a solution of Ac-NH-PEG-NH-CO-(CH.sub.2).sub.3OOOH in 2.0
mL of CHCl.sub.3 was added DCC (18.2 mg, 0.088 mmole) and the
reaction mixture was stirred at room temperature for 2-3 h. At this
time point white ppte was developed. Protamine (30.0 mg, 0.0074
mmol) in 1.6 mL of 1,1,1,3,3,3 Hexafluoro isopropyl alcohol was
added to the reaction mixture and the reaction mixture was stirred
at room temperature over night. At this time point, TLC analysis
gave a polar positive spot. Solvents were removed under reduced
pressure and residual material was suspended in CHCl.sub.3. The
suspension was centrifuged at 3500 rpm for 15 min. The solid
material was separated from clear solution to give 23 mg, yielding
35%.
e. Characterization of the Amine Content of Protamine and
Polylysine Conjugates With PEG and Folate or an Acetyl Group
[0079] The ratio of protamine or polylysine to the remaining
molecular mass in the final products was characterized by
fluorescent derivatization of free amino groups with fluorescamine,
a reagent that becomes fluorescent upon reaction with amines. As a
calibration of fluorescence response, weighed amounts of free
polylysine or protamine were reacted with fluorescamine to yield a
fluorescent product. Standard curves were prepared for either
protamine or the 3,000 molecular weight polylysine molecule used
for conjugate formation. The concentration of amines in the
conjugates was then determined by reaction with fluorescamine. The
assay was done in a 96 well Cytofluor 4000 fluorescence plate
reader (PerSeptive Biosystems, Cambridge, Mass.). After dilution of
the sample into 50 .mu.L total volume of water, 200 .mu.l of 130 mM
NaBH.sub.2O.sub.3 buffer at pH 9.5 and 50 pl 0.2 mg/ml
fluorescamine which was dissolved in acetone were added. The
samples were incubated at room temperature for 20 minutes after
which the fluorescence was measured at 490.+-.20 nm with 395.+-.12
nm excitation. The fluorescence readings were compared to the
standard curves to determine the amount of protamine or polylysine
in a given weight of conjugate and the ratio of this amount to the
total mass was calculated. For the protamine conjugates with PEG
and either folate or an acetyl group the expected ratio of a 1:1
conjugate is 0.52 and 0.54, respectively, while determined ratios
were 0.50.+-.0.05 and 0.56.+-.0.05, respectively. For the
polylysine conjugates FPK and FPGK, the calculated ratios are 0.45
and 0.44, respectively, while the determined ratios were
0.64.+-.0.17 and 0.35 t 0.05, respectively. Because of the
polydispersity of the polylysine used for this purpose, these
ratios are only approximate guidelines for the 1:1 complexes.
[0080] The protamine conjugates clearly contained a single
protamine per PEG group, while the polylysine conjugates were close
to the desired ratios.
f. Preparation of a Phospholipid Derivative of Folate and PEG,
Folate-PEGDOPE (FA-PEG-DOPE or FPPE)
[0081] A phospholipid conjugate of folate and PEG was prepared to
compare to the conjugates of folate prepared above. The
phospholipid conjugate would insert hydrophobically into the
liposomal membrane, while the other conjugates would interact with
the liposomal membrane via an electrostatic attraction. The results
are further discusses in Example 8.
Coupling DOPE to t-BOCNH-PEG-CO.sub.2NHS:
[0082] To a solution of DOPE (20 mg, 0.026 mmole) in 3.0 mL of
CHCl.sub.3 were added t-BocNH-PEG-CO.sub.2NHS (91.5 mg, 0.026
mmole) and TEA (2.7 mg, 0.026 mmole). This reaction mixture was
sealed under nitrogen and stirred at room temperature overnight. At
this time point TLC analysis in CHCl.sub.3:CH.sub.3OH:H.sub.2O
(65:25:4 v./v) revealed that the reaction had gone to the
completion. Solvents were removed under reduced pressure and
residual material was purified on column chromatography to give 168
mg (53%). Some of characteristic 'H NMR (CDCl.sub.3) signals are: 5
0.86 (t, 6H, J=6.35 Hz, Cl1.sub.3), 1.25-1.27 (br, (CH.sub.2),, for
DOPE), 1.43 (s, t-BOC), 3.63 (br, CH.sub.2's for PEG) and 5.32 (br
signal for olefinic protons).
Deprotection of t-BOC from t-BOCNH-PEG-DOPE Conjugate:
[0083] To a solution of t-BOCNH-PEG-NH-DOPE conjugate in 4.0 mL of
CH.sub.2Cl.sub.2 was added 1.0 mL of TFA. This reaction mixture was
stirred at room temperature for 2 h. At this time point TLC
analysis in CHCl.sub.3:CH.sub.3OH:H.sub.2O (65:25:4 v/v) revealed
that the reaction had gone to the completion. Solvents were removed
under reduced pressure and residual material was purified on column
chromatography to give 44 mg. Some of characteristic 'H NMR
(CDCl.sub.3) signals are: 8 0.86 (t, 6H, J=6.11 Hz, CH.sub.3),
1.25-1.27 (br, (CH.sub.2)r, for DOPE), 3.63 (br, CH.sub.2's for
PEG) and 5.32 (br signal for olefinic protons).
Coupling FA to NH.sub.2-PEG-DOPE:
[0084] To a solution of FA (10.58, 0.026 mmole) in 3.0 mL of
DMSO:Py (2:1 v/v) were added DCC (14.8 mg, 0.072 mmole) and
NH.sub.2-PEG-DOPE (45 mg, 0.012 mmole). This reaction mixture was
sealed under nitrogen and stirred at room temperature for
overnight. At this time point TLC analysis in
CHCl.sub.3CH.sub.3OH:H.sub.2O (75:35:6 v/v) revealed that the
reaction had gone to the completion. TLC analysis gave positive UV,
ninhydrin and molybdate tests. Solvents were removed under reduced
pressure and residual material was purified on column
chromatography to give 21.5 mg (43%). Some of characteristic 'H NMR
(CDCl.sub.3:CD.sub.3OD 8:1 v/v) signals are: 8 0.79 (t, 6H, J=6.62
Hz, CH.sub.3), 1.18-1.21 (br, (CH.sub.2).sub.n for DOPE), 3.56 (br,
CH.sub.2's for PEG), 5.25 (br signal for olefinic protons) and
6.60-8.6 (3 signals for FA).
g. Coupling Antibody to Polylysine: Carbohydrate-Specific
Polylysine Coupling to the Monoclonal IgG OVB-3
[0085] An antibody conjugate was also prepared for electrostatic
linkage to liposomes to target such liposomes to the appropriate
cells.
OVB-3 Isolation:
[0086] OVB-3 is a monoclonal (mab) IgG that binds specifically to
human ovarian carcinoma cells. The hybridoma cell line was obtained
from ATCC (HB-9147). Hybridoma cells were injected into Balb/c mice
and ascites was collected a couple weeks later. The mab was
isolated from the ascites using a Protein A column and stored in a
phosphate buffer (150 mM NaCl, 20 mM NaP, , pH 7.5)
Preparation of Thiolated Aolylysine:
[0087] Free sulfhydryl groups were introduced on to polylysine
polymers by modifying the amines with 2-iminothiolane (Traut's
reagent). Hydrobromide polylysine in the 1,000 to 4,000 molecular
weight range was obtained from SIGMA. The polylysine (45 mg) was
dissolved in 1.0 ml of borate buffer (100 mM NaBH.sub.2O.sub.3 pH
8.0). To this solution, 1 ml of 95.9 mg/ml 2-iminothiolane
dissolved in water was added. The mixture was incubated for 1 hour
in the dark with gentle shaking. After incubation, the sample was
transferred to a 30 ml glass centrifuge tube and 18 ml of
isopropanol was added. The sample was centrifuged for 20 minutes at
9K RPM. The supemate was removed and the pellet was dried with a
gentle stream of N.sub.2. The pellet was redissolved in 2 ml of the
pH 8.0 borate buffer.
Labeling the Polylysine with Alexa 350:
[0088] In order to provide a quantitative measure of the extent of
mab to polylysine coupling, the thiolated polylysine was labeled
with the fluorescent probe Alexa 350 carboxylic acid succinimidyl
ester which was obtained from Molecular Probes. A 10 mg/ml Alexa
350 DMSO stock solution was prepared. Seventy-five 0 of Alexa 350
stock solution was added to 2 ml of previously prepared thiolated
polylysine sample. The sample was incubated in the dark at room
temperature for 1 hour with gentle shaking. After incubation, the
sample was transferred to a 30 ml glass centrifuge tube and 18 ml
of isopropanol was added. The sample was centrifuged for 20 minutes
at 9K RPM. The supemate was removed and the pellet was dried with a
gentle stream of N.sub.2. The sample was redissolved in 100 to 200
gl of water. The sample was freeze-dried in order to obtain the
sample weight. Following the determination of the sample weight,
the sample was dissolved in NaP, buffer (100 mM NaP, , pH 7.0) at a
concentration of 20 mg/ml.
Oxidation of the Mab by Periodate:
[0089] The polylysine coupling to OVB-3 was done at the
carbohydrate region of the mab. Two hundred p,l of 300 mM Na104 in
water was added to 2 ml of 5 mg/ml OVB-3 in 150 mM NaCl, 20 mM NaP,
pH 7.5. The sample was incubated for 1 hour at room temperature in
the dark. Glycerol ( 200 g,I ) was added to stop the oxidation
reaction. The sample was then subjected to a PD-10 Sephadex G25
desalting column which was equilibrated with a pH 5.0 acetate
buffer (100 mM Na Acetate, pH 5.0). The sample was reconcentrated
back to 2 ml using an Amicon stirred ultrafiltration cell with 30K
MWCO filter.
Coupling the Thiolated Polylysine to the Oxidized OVB-3:
[0090] The thiolated polylysine was linked to the carbohydrate
oxidized OVB-3 using the heterobifunctional cross-linker MPBH
(4-(4-N-maleimidophenyl)butyric acid hydrazide) which was obtained
from Pierce. One hundred p,l of an 80 mM DMSO MPBH stock solution
was added to the previously prepared 2 ml oxidized OVB-3 solution
(100 mM Na Acetate, pH 5.0). The final MPBH concentration was 4 mM.
The sample was incubated for 2 hours at room temperature in the
dark with gentle shaking. The sample was then run down a PD-10
Sephadex G25 desalting column which was equilibrated with a pH 7.0
P; buffer ( 100 mM NaP1, pH 7.0). The OVB-3 column fractions were
pooled and 15 mg of previously prepared thiolated polylysine (100
mM NaP,, pH 7.0) was added. The sample was incubated at room
temperature for at least 3 hours. The unreacted thiolated
polylysine was separated from the OVB-3 polylysine conjugate by gel
filtration on (Sephacryl-200 HR) or a Protein A column ( Pharmacia
Biotech HiTrap Protein A column) using a pH 7.4 TES buffer (150 mM
NaCl, 10 mM TES, 0.1 mM EDTA, pH 7.4). The OBV-3 polylysine
conjugate was pooled, reconcentrated by stirred ultrafiltration to
a stock solution concentration of about 1 mg/ml, and stored in the
refrigerator.
EXAMPLE 3
Assay Methods and Materials:
a. Cell Cultures for Measurements of Liposome Binding and
Transfection
[0091] OVCAR-3 cells were plated at 2.times.105 cells per ml in
96-well plates in 0.1 ml per well of RPMI 1640 with 10% heat
inactivated fetal bovine serum. Cells were allowed to grow for two
days (approximately 4048 hours) before transfections were
performed; at this point the cells were at confluency. In the case
of pHdependent liposomes described below, the cells were allowed to
grow for only one day before transfection.
b. Measure of Cell Number (CBAM, Calcein Blue Acetoxy Methyl
Ester)
[0092] Cell number in terms of total intracellular esterase
activity was determined by washing the cells with phosphate
buffered saline (PBS), staining with calcein blue acetoxy methyl
ester (CBAM-5 .mu.M in PBS for approximately 3045 minutes at room
temperature), and then rinsing the plates with PBS). 100 .mu./well
of detergent solution (1% Cl.sub.2E8, TE, pH 8) was added to each
well. Cell number was determined in a Cytofluor 2 instrument by
determining the calcein blue fluorescence (excitation at 360 nm,
emission at 460 nm, gain usually at 80). Plates were again washed 2
times with detergent solution and read again in the same detergent
solution to correct for background. A series of unlabeled control
wells were also read as an internal blank. All tansfection (EGFP)
and liposome binding (rhodamine) fluorescent readings were
corrected by dividing by the CBAM readings.
c. Measurement of Binding of Liposomes to Cells
[0093] Liposomes comprising about 0.1 to about 1 mole % N-lissamine
rhodamine B sulfonyl) phosphatidylethanolamine were incubated with
the cells and the rhodamine fluorescence was observed visually and
by measurement on a Cytofluor II plate reader using 560 nm
excitation and 620 nm emission.
d. Transfection Efficiency Assay
[0094] Transfection success, and expression of the transfected
nucleic acid in a cell, can be detected in a number of ways, these
generally depending upon either detection of the physical presence
of the nucleic acid in the cell, e.g., by incorporation of
radionucleotides in the nucleic acid, or by detecting expression of
the protein encoded by the nucleic acid. This can be accomplished
in a number of ways, including, without limitation, where the
protein is a detectable, e.g., fluorescent, marker, or where the
protein is a selectable, e.g., cytotoxic agent-resistance,
marker.
[0095] For example, the plasmid pEGFP-1 contains a DNA sequence
encoding the enhanced green fluorescence protein, whose presence is
detected by fluorescence microscopy or fluorescence plate reader.
Accordingly, successful transfection of cells with this plasmid is
readily determined by assessing the quantity of fluorescence
exhibited by the cells. The transfection activity of the liposomal
preparations encapsulating pEGFP-Cl plasmid DNA was tested as
follows. Transfection solutions were prepared by dilution of
appropriate liposome or DNA samples into the desired buffer or
medium. The plates were aspirated to remove medium.
[0096] Transfection solutions (0.1 ml per well for 96-well plates)
were prepared by dilution of dialyzed samples containing the
pEGFP-Cl plasmid into medium or buffer (approximately 2 mM total
lipid unless indicated otherwise) containing 10% heat inactivated
fetal bovine serum (unless otherwise designated), and were then
added to the wells and incubated at 37 degrees C. for 3 hours. The
wells were aspirated, and medium containing 10% heat inactivated
fetal bovine serum was added to each well. Because of the
previously demonstrated silencing of transgenes under the CMV
promoter (Tang et al., 1997; Dion et al., 1997) a histone
deacetylase inhibitor, 5 mM sodium butyrate, included in the medium
to enhance expression.
[0097] After incubation at 37 degrees C. in a cell culture
incubator for 18-22 hours, the medium was aspirated and a 0.5 ml
wash of Dulbecco's PBS was added. At this point or just before the
wash, photomicrographs were taken of the samples still on tissue
culture plates with an Olympus IMT-2 inverted microscope using the
10.times. objective. The samples were then dissolved in detergent
and readings were taken for corrected total EGFP fluorescence, in
terms of the total number of live cells, using the calcein blue AM
assay described above.
e. Beta Galactosidase Assay
[0098] Cells are assayed for .beta.-galactosidase activity using
the chemiluminescent .beta.-galactosidase detection kit from
Clontech according to the manufacturer's directions. Briefly, 200
NI washed IP cells are spun at 1200 rpm for two minutes. The
supernatant is removed and the cell pellet lysed in 300 .mu. lysis
buffer (100 mM potassium phosphate, pH 7.8 with 0.2% Trition X-100,
prepared as described by Clontech) by vortexing for -30 seconds and
shaking gently for 15 min. Cell debris is then removed by
centrifugation at 14000 rpm for 2 min. 200 pl reaction buffer is
added to 45 pl cell lysate in 96 well plates, mixed and incubated
for 60 min at room temperature. Luminescence is read on a
microplate luminometer (EG&G Berthold), recording light signals
at 5 second intregrals.
f. Sources of Materials
[0099] N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine
(transesterified from egg PC), DOPC, EPC and N-Cl.sub.2-DOPE were
purchased from Avanti Polar Lipids (Alabaster, AL). OVCAR3 ovarian
carcinoma cells were purchased from NCI-Frederick Cancer Research
Laboratory (Frederick, Md.). The pEGFP-Cl plasmid, and E. coli
DH5.alpha. competent cells were purchased from Clontech
Laboratories (Palo Alto, Calif.). pZeoSVLacZ plasmid, competent
cells and Hanahan's S.O.C. were purchased from Invitrogen (San
Diego, Calif.). Hanks Balanced Salt Solution (HESS), RPMI 1640 and
heat inactivated fetal bovine serum were purchased from Gibco/BRL
(Grand Island, N.Y.). DNase-free RNase and RNase-free DNase I were
purchased from Boehringer Mannheim (GmbH, Germany). Agarose was
purchased from FMC Bioproducts (Rockland, Md.). Bacto agar, Bacto
tryptone and yeast extract were purchased from DIFCO Laboratories
(Detroit, Mich.). Calcein blue acetoxy methyl ester (CBAM),
PicoGreen and SybrGreen I dyes were from Molecular Probes (Eugene,
Org.). A polylysine-transferrin (pKT) and free polylysines were
obtained from Sigma (St. Louis, Mo.). pKT consists of a 30-70 kDa
poly-L-lysine coupled to human holotransferrin such that there are
approximately 3 transferrin molecules per polylysine. There is also
an fluorescein isothiocyanate (FITC) group on each conjugate.
EXAMPLE 4
Charge Reversal Liposomes: Electrostatic Linking of Target!
ng/Protecting Modules to Charge Reversal Liposomes is
pH-Dependent.
[0100] In one embodiment of the modular delivery invention,
liposomes were designed to reverse charge on a change from pH 7 to
a lower pH found in cell organelles such as endosomes. To determine
whether the targeting/protecting conjugates would stably bind to
liposomes under conditions of physiological pH and be released from
such liposomes under conditions such as lower pH, the following
experiment was carried out. Liposome were made by first mixing
POPE: DOMDODAP:Cholesterol:Cholesteryl hemisuccinate:Oleoyl acteate
(51:12.2:10.7:10.7:2.6:12.8:10.7) in chloroform. EGFP plasmid,
condensed with spermine, was encapsulated during liposome
preparation according to the twostep emulsion method (example 1).
300 mM sucrose was used as plasmid and spermine dilution buffer.
300 mM sucrose was used in the initial hydration buffer to
facilitate separation of the liposomes from bulk buffer by
centrifugation. The final liposome preparation was dialyzed against
PBS before use. The final lipid concentration was .about.20 mM.
[0101] Cell-targeting protein complexes were bound to the external
surface of the plasmid-containing liposomes through non-covalent
interactions. At pH 7.4, the liposomes have a net negative charge
that provides a binding surface for positively charged proteins or
moieties linked to positively charged polypeptides (pKT,
pK-OVB3-Ab). The number of liposomes in a 200 NJ aliquot was
estimated assuming a 20 mM total lipid concentration and a liposome
diameter of 150 nm. Ratios of 1-5 protein complexes per liposome
were tested. Protein stock solutions (FITC-labeled pKT, pK-OVB3)
were made at a concentration of 0.5 mg/ml in PBS, pH 7.4. An
aliquot containing the desired amount of protein was then diluted
in 200 .mu.PBS. For a standard protein binding assay, the protein
solution was added dropwise to the liposome solution with continual
mixing. Equal volumes of protein solution were mixed with the
liposomes at mole ratios such that the overall charge of the
liposome-protein assembly remained negative. The protein/liposome
mixture was incubated at room temp for 15-30 min. and then
separated into two equal volume aliquots. The pH of one aliquot s
was lowered to pH 5 by addition of HCl, followed by incubation at
room temp for an additional 15 min. Liposomes and bound protein
were separated from free protein by centrifugation at 14,000 g for
30 min. The liposome pellets were resuspended in PBS and the pH of
all supernatants returned to pH 7.4 with addition of NaOH before
analysis. Protein levels in the supernatant were measured using the
Biorad Coomassie protein assay for pK-OBV3-Ab. For the FITC-labeled
pKT, fluorescence was measure for each fraction (Ex:485 Em:530).
Liposomes without added ligand and protein solutions without added
liposomes were used as controls.
[0102] Table 1 shows the results of the pH-dependent binding of pKT
at two concentrations, 2 or 5 pKT complexes per liposome. (Note:
half neutralization of the liposomal negative charge would occur
with -3 pKT bound.) At pH 7.4 virtually all of the protein pelleted
with the liposome fraction during centrifugation at both pKT
concentrations. When the pH was lowered to 5, a difference was
found between the samples. At a pKT/liposome mole ratio of 2, all
of the pKT remained associated with the liposome even at low pH. At
the higher pKT/liposome ratio, a fraction of the pKT completely
dissociated from the liposome at pH 5 and remained in the
supernatant after centrifugation. These results suggest that there
may be two types of binding sites for pKT on the liposome: A higher
affinity site that is not pH-dependent and binds up to two ligand
complexes per liposome and a lower affinity site where the protein
complex can be toggled on or off the liposome surface by a change
in pH.
[0103] Most of the pKT (-80%) remains bound to these liposomes in
20% serum, and a significant amount remains bound up to 40% serum.
This amount of targeting ligand is sufficient to provide specific
cell binding through a transferrin receptor.
[0104] Table 1 also shows the pH-Dependent binding of 3K-polylysine
conjugated to an antibody to the OVB3 antigen, which is found on
ovarian cancer cells (pK-OVB3). Analogous to the pKT study, 2 or 5
pK-OVB3-Ab complexes were bound through electrostatic interactions
per negatively charged liposome. The liposomes and bound targeting
ligand were separated from unbound soluble protein by
centrifugation. The amount of unbound pK-OVB3-Ab in the
supernatants increased significantly when the pH was lowered from
7.4 to 5 (.about.30% increase at a pK-OVB3-Ab/liposome mol ratio of
2, and .about.50% increase at a mol ratio of 5).
[0105] Taken together, these results demonstrate that these
liposomes bind a variety of targeting ligands via a cationic linker
at neutral pH. At low pH, the affinity of the ligand complex may
vary with the polyvalency of the cation, and at least some of the
targeting complex should dissociate from the liposome at low pH. At
pH 5, as in the endosomal or lysosomal compartment, the charge on
cholesterol hemisuccinate should change from negative to neutral,
and DODAP would become fully positively charged, resulting in a net
positively charged membrane surface. This surface would have less
affinity for the positively charged polylysine. TABLE-US-00001
TABLE 1 pH-Dependent binding of targeting modules to
charge-reversal liposomes of total protein added found free in bulk
buffer' Ligand polylysine-transferrin.sup.b
polylysine-OVB3-IgG{grave over ( )} Ligand polylysine- polylysine-
mol ligand per transferrin.sup.b OVB3-IgG.sup.c mol liposomes pH
7.4 pH 5 pH 7.4 pH 5 2 1.5 0 0 30 5 1 18 7 33 .sup.aFree and
liposome-associated ligand complexes separated by centrifugation.
.sup.bFluorescence of FITC-labeled ligand measured for isolated
liposome pellets and supernatants. .sup.cProtein level measured in
isolated supernatants. .sup.dHCI added to sample at 7.4 to reduce
the pH
EXAMPLE 5
Charge Reversal Liposomes: Binding of Serum Proteins is Minimal
Effect of Ligand Complexes on C3 Acitvation by Liposomes:
[0106] There are many factors that influence the circulation
lifetime of liposomes in vivo. One critical aspect is the
binding/activation of complement factor C3 by the liposomes (see
Semple, et al. (1998) Advanced Drug Delivery Reviews 32:3 17 and
Devine and Bradley (1998) Advanced Drug Delivery Reviews 32:19-29
for reviews); activation of C3 increased greatly liposome clearance
from blood. The effect of modular targeting complexes on the
activation of C3 by liposomes was tested using the pH-sensitive
formulation.
Binding of C3Complement Component to Liposomes:
[0107] The ability of the liposomes to bind the C3 component of
complement was tested by a modification of the in vitro hemolysis
assay of Devine et al. (1994) Biochim. Biophys. Acta 1191:43-51 and
AN et al. (1997) Biochim. Biophys. Acta 1329:370-382. Briefly,
antibody-sensitized sheep erythrocytes (all reagents were from
Sigma Chemical Co., St. Louis, Mo., unless noted) were washed and
resuspended as directed by the manufacturer. Rat sera complement
was hydrated in water then diluted 2-fold with GVB.sup.2+buffer.
200 .mu. liposomes, with or without bound targeting ligand
(polylysine-transferrin, polylysine-anti-OVB3-IgG or
polylysine-PEG-folate) were mixed with 100 ml diluted rat sera and
incubated at 37.degree. C. for 30 min with continuous shaking. 300
.mu.GVB.sup.2+ buffer was added and any liposome aggregates
pelleted by centrifugation. Eight successive 2-fold dilutions, into
GVB 2+ buffer, were then done for each sample. 100 .mu. of the
sheep erythrocytes were added to an equal volume of each
liposome/sera dilution and incubated for 30 min at 37.degree. C.
with shaking. Further hemolysis was stopped by adding GVB-EDTA
buffer. Intact red cells and membrane fragments were then pelleted,
and the absorbance of the supernatant was measured at 415 nm.
GVB.sup.2+ buffer served as a negative control for C3 binding, and
the absorbance of an equal volume of osmotically lysed red cells
taken to be 100% hemolysis.
[0108] C3-mediated hemolysis curves were generated for the charge
reversal liposomes with and without bound targeting ligand. The
charge reversal liposomes alone gave a hemolysis profile very
similar to the buffer control, suggesting little or no activation
of C3 by these liposomes. When targeting complex was added, the
hemolysis profile was similar to the liposome-only curve for all
three targeting modules tested. The decrease in CH50 relative to
buffer was determined from linear fits of the middle portion of the
hemolysis curves by the method described by AM et al. These results
are shown in Table 2. Very minor decreases (3.3-7%) in CH50 values
were found for the pH-sensitive liposomes with and without bound
targeting ligand. These values are significantly less than those
observed by Ahl, et al. (1997) for liposomes that were rapidly
cleared from the circulation of rats. They are also less than
values found by those investigators for liposomes not cleared
completely from the circulation in the first hour.
[0109] These results suggest that the presence of bound targeting
molecules does not significantly increase the amount of
liposome-mediated C3 activation. This lack of C3 activation may
result in increased circulation time for these liposome vectors.
TABLE-US-00002 TABLE 2 Effect of targeting ligand on complement
fixation by pH-sensitive liposomes Targeting ligand % decrease in
CH50.sup.a no ligand 3.7 .+-. 1.2 (3) polylysine-transferrin.sup.b
3.3 .+-. 1.7 (4) polylysine-antiOVB3.sup.c 4.5 .+-. 1.3 (4)
polylysine-PEG-folate.sup.d 7 (1) .sup.a% decrease relative to
buffer (100% CH50, 0% decrease) calculated as described by Ahl et
al. The number of experiments is listed in parentheses.
.sup.bcombined data for samples with 2-5 mol pK-transferrin per mol
liposomes .sup.ccombined data for samples with 2-5 mol pK-anti-OBV3
per mol liposomes .sup.dsingle sample with 5 mol pK-PEG-folate per
mol liposome
EXAMPLE 6
Charge Reversal Liposomes: Enhancement of Transfection Efficiency
by Targeting
[0110] NIH Ovcar-3 cells were grown as in Example 3 for 24h before
transfection assays were performed. Aliquots of pH-sensitive
liposomes containing pEGFP-C1 plasmid were mixed with a desired
amount of targeting ligand in PBS and incubated at room temperature
for 15 min. The sample was diluted 5-fold into RPMI with or without
heat inactivated FBS and 50 of a 50 mM CaCl.sub.2/20 mM MgCl.sub.2
solution added per 0.5 ml liposomes and mixed. 90 .mu. liposome
solution was added to PBS-washed cells in each well and incubated
for .about.5 h at 37.degree. C. The transfection solution was
removed, the cells washed with PBS, then allowed to incubate for
24-36 h in RPMI supplemented with 10% heat inactivated FIBS and 5
.mu.M sodium butyrate.
[0111] EGFP transgene expression was determined qualitatively by
viewing the cells still in the culture plate by epi-fluorescence
microscopy and quantitatively by reading the fluorescence intensity
of each well after lysing the cells in 1% Cl.sub.2E8 detergent
using a PE Biosystems cytofluor4000 platereader (Ex:485 Em:530) as
in Example 3. Cell viability was measured by an enzymatic
fluorescent assay. Prior to lysis, the cells were incubated for 40
min in 25 p,M calcein blue AM (Molecular Probes). Fluorescence
intensities of the calcein blue hydrolysis product (Ex:360, Em:460)
were measured during the same run as the EGFP readings. For
comparison between conditions, transgene expression was corrected
for cell number by dividing the EGFP reading by the calcein blue
reading for each well.
[0112] The ability of the pH-triggerable liposomes to transfect
cells was tested in Ovcar3 cells in vitro. Three targeting ligands
were tested (pKT, pK-OVB3 and FPK) at 1, 3 or 5 protein complexes
per liposome. The liposome formulation, alone or in combination
with the targeting ligands, showed a small amount of cell toxicity.
Calcein blue levels were approximately 20% lower for wells
incubated with the plasmid-containing liposomes than for untreated
cells or cells incubated overnight in media with sodium butyrate
but without liposomes. The amount or type of targeting moiety did
not significantly alter the level of calcein blue measured.
[0113] Transgene expression was first assessed qualitatively by
epifluorescence microscopy. FIG. 1 shows representative
photomicrographs of Ovcar3 cells transfected with the pH-sensitive
charge reversal liposomes with pEGFP-Cl plasmid encapsulated.
Without bound targeting ligand, low, but clearly visible cells
filled with EGFP were observed (FIG. 1a). With the addition of pKT,
the number of cells showing green fluorescence increased (FIG. 1b).
The absolute number of cells transfected varied somewhat from one
transfection experiment to another. However, the relative amount of
transfection, when comparing different targeting moieties, was
consistent between experiments. Similar results were obtained with
pK-OVB3 and FPK ligands.
[0114] The effects of increasing amounts of cell targeting moiety
were determined quantitatively for pKTor FPK ligands. The results
are shown in FIG. 2. The amount of transgene expression was
corrected for cell number by dividing the EGFP fluorescence reading
by the calcein blue reading for each well after background
subtraction. This value was then multiplied by 1000 for plotting.
An increase in transgene expression was found with increasing
ligand concentration for all three binding targeting agents. The
higher transgene expression for the pKT series, may simply reflect
the increased number of targeting ligands per protein complex. pKT
has 3 transferrins per polylysine chain where polylyssne-PEG-folate
has 1 ligand per polylysine chain. The titration of targeting
complex was stopped at a 5:1 protein: liposome mol ratio to keep
the overall charge of the liposomes with pKT and pK-folate negative
at pH 7.4. As a control, pure 30K polylysine was used as targeting
ligand at a 3:1 protein: liposome ratio. The amount of transfection
was similar to that for no ligand.
EXAMPLE 7
Fusogenic AfLacyl-PE Liposomes: Demonstration of Enhancement of
Transfection Efficiency Mediated by Folate Targeting.
[0115] Fusogenic N-acyl-PE liposomes are negatively charged at
neutral pH as are the charge neutralization liposomes. The charge
density of the N-acyl-PE liposomes decreases at lower pH, but does
not completely reverse. However, partial neutralization can also
allow some loosening or exchange of electrostatically bound
conjugates at low pH. Results and further discussion are provided
in Example 8.
[0116] Liposomes composed of 70% NCl.sub.2DOPE and 30% DOPC
encapsulating spermine and the pEGFP-Cl plasmid were prepared as in
example using the two step emulsion method followed by pelleting
and washing of the liposomes to remove external DNA. A stock of
liposomes 20 mM in total lipid concentration was mixed with the
appropriate amount of a 340 p,M stock of
folate-PEG-glutarylprotamine (FPGPr) conjugate or a control
derivative in which the folate group was replaced by an acetyl
group (AcPGPr). The mixture was diluted with Hanks buffered salt
solution (HBSS) without Ca.sup.2+, or Mg.sup.2+ to reach a total
lipid concentration of 4 mM and heat inactivated fetal bovine serum
was added to a final concentration of 10% (v/v). To adjust the
Ca.sup.t+ and Mg.sup.2+ concentrations to near the expected
physiological levels a stock of 60 mM CaCl.sub.2 and 40 mM
MgCl.sub.2 was added at 20 .mu. per ml of liposome solution just
before placing the liposomes into the empty wells of the 96-well
tissue culture plates. Plates were prepared and transfection
analysis carried out as in Example 3.
[0117] The data are shown in FIG. 3. The folate conjugates greatly
enhanced transfection efficiency of the liposomes, while a control
conjugate bearing only an acetyl group did not. These data
demonstrate that transfection by these liposomes is enhanced by
electrostatically bound targeting/stabilizing conjugates and that
the effect is dependent on the presence of folate in the conjugate.
This suggests that a folate receptor is utilized.
EXAMPLE 8
Fusogenic WLacyl-PE Liposomes with Folate Targeting:
Electrostatically Bound Targeting/Stabilizing Modules Versus
Covalent Coupling to Lipids.
[0118] Liposomes as described in Example 7 were prepared for
association with several conjugates containing folate, PEG and a
positively charged group for interaction with the membrane. These
conjugates were folate-PEG-protamine with a glutaryl linker
(FPGPr), folate-PEG-polylysine with a glutaryl linker (FPGK) and
folate-PEG-polylysine (FPK) synthesized as described in the Assays
and Methods section. A folate-PEG-phosphatidylethanolamine (FPPE)
derivative was also synthesized for comparison to the conjugates
that interact by electrostatic interaction. The FPPE derivative
inserts into the liposomal membrane and is held there by strong
hydrophobic interactions. The other derivatives bind to the
liposomes via an electrostatic interaction and can potentially
dissociate in the endosome so that the fusogenic membrane can be
exposed. The FPPE cannot dissociate as easily. The dissociation is
important to remove the large PEG-containing targeting moiety for
membrane fusion to occur.
[0119] Liposomes were prepared as described in Example 3,
containing 200 mM sucrose with spermine and DNA. After pelleting
and washing to remove external DNA, the various conjugates were
associated with the liposomes. For the FPPEcontaining liposomes,
FPPE was incorporated into the liposomes at 2.5 mole % of the total
phospholipid. The FPGK, FPK and FPGPr conjugates was added to the
external leaflet of the liposomes at a concentration that also was
2.5 mole % of the external phospholipid. Buffer, serum and divalent
cations were added to the samples as described in example 7 before
addition to OVCAR-3 cells in 96-well plates. The final total lipid
concentration was 4 mM. After incubations as described Example 7,
the green fluorescence due to the expression of EGFP was determined
as described in Example 3.
[0120] FIG. 4 shows the comparison between the various conjugates
in that the electrostatically-linked conjugates were much more
active than the covalentlylinked FPPE conjugate. Similar results
were obtained in which three separate preparations of the
FPPE-containing liposomes were compared to the FPGPr conjugate. The
FPGPr conjugate was also compared to the FPPE conjugate at a lower
mole percentage in the membrane (0.4 mole %). In this case a
similar result was obtained, i.e. that the FPPE conjugate showed no
transfection activity, while the electrostatic conjugate showed
substantial activity. The FPPE conjugate was not entirely inactive
as it did show some enhancement of transfection activity with the
charge reversal liposomal system (examples 4-6) at a very low mole
percentage (less than 0.1 mole %).
EXAMPLE 9
Fusogenic Af-acyl-PE Liposomes: Targeting Via the pK-OVB-3 Antibody
Conjugate
[0121] Another embodiment of the modular targeting/stabilizing
concept involves an antibody to ovarian cancer cells called OVB-3
coupled to linkers as in Example 2.
[0122] The pK-OVB-3 conjugate was found to bind to anionic
liposomes, but not to zwitterionic liposomes, indicating the
electrostatic nature of binding. This was demonstrated with more
than one type of anionic liposome (data not shown).
Stability of Ligosomal Membrane
[0123] The effect of pK-OVB-3 targeting module binding on liposome
integrity was examined by fluorescent NBD-phospholipids. NBD
labeled phosphatidylethanolamine (headgroup label, transesterified
from egg PC) was obtained from Avanti Polar Lipids (Alabaster, Al.)
and incorporated into /V-acylPE liposomes (example 1) at 0.5 mole %
of total lipid. The membrane impermeant ion dithionite has been
shown to rapidly reduce the NBDphospholipid in the outer lipid
monolayer to a non-fluorescent form. The remaining NBD-phospholipid
in a sealed liposome can only be reduced following the addition of
a detergent. The addition of 20 mM sodium dithionite to
NBDphospholipid labeled N-Cl.sub.2-DOPE /DOPC (7:3) liposomes
containing encapsulated DNA (prepared as in example 1) rapidly
decreased the level of NBD fluorescence from the outer monolayer as
shown in FIG. 5. An approximate reduction of 60% indicates that a
single bilayer membrane is found in most of the liposomes. Addition
of detergent reduced the remaining NBDphospholipid on the inner
monolayer of the liposomes by dissolving the liposomes. pK-OVB3
conjugate binding to these liposomes had no significant effect on
the magnitude or time course of the NBD-phospholipid reduction by
sodium dithionite. This indicates that conjugate binding to the
liposome surface did not significantly destabilize or alter the
basic structure of these fusogenic liposomes.
Enhanced Uptake of Liposomes by gK-OVB-3:
[0124] Liposomes with pK-OVB-3 targeting modules were prepared by
incubating negatively-charged liposomes with the pK-OVB-3 conjugate
for 15 minutes at room temperature. During this incubation
essentially all the pK-OVB-3 targeting modules are
electrostatically bound to the surface of the anionic liposomes.
Liposomes with pK-OVB-3 targeting modules were prepared at
conjugate to lipid ratios ranging from 0 to 100 .mu. protein per
.mu.mole lipid. These pK-OVB-3 coated liposomes were then
immediately incubated with OVCAR-3 cells at a lipid concentration
of 1 mM for 11/2 hours at 37.degree. C. in the presence of 10% heat
inactivated fetal calf serum. The OVCAR-3 cells were grown in
Costar 96 well plates for approximately 24 hours starting at a cell
concentration of 2.times.10.sup.4 cells per well. Following this
incubation the OVCAR-3 cells were extensively washed by aspiration
to remove unassociated liposomes. The liposomes were labeled with
0.2 mole percent of the fluorescent lipid rhodamine-DOPE to follow
the liposome uptake in a fluorescent plate reader (560.+-.10 nm
excitation, 520 t 20 nm emission). FIG. 6 shows that increasing the
amount of pK-OVB-3 targeting module bound to the surface of
N-Cl.sub.2-DOPE/DOPC (7:3 mole ratio) liposomes significantly
increase the level of OVCAR-3 cell uptake of these liposomes in the
presence of 10% serum.
Enhancement of Transfection by OVB-3 Targeting:
[0125] A series of transfection experiments were performed to test
the effect of the antibody conjugates. The pK-OVB-3 conjugates were
prepared as described above. A stock of polylysine-OVB-3 antibody
conjugate (conc. 0.5 mg/ml), a conjugate of a 3K polylysine to the
OVB-3 antibody, was prepared as described above.
[0126] N-Cl.sub.2-DOPE/DOPC ( 70/30) liposomes were prepared to
encapsulate the plasmid pEGFP-Cl, spermine and sucrose as described
in Example 1 and pelleted and washed as described in Example 1.
OVCAR-3 cells were grown in 96 well plates as described in Example
3.
[0127] All transfection experiments were with OVCAR-3 cells in
tissue culture, as described above. Varying amounts of the OVB-3
conjugate were added directly to a 40 mM liposome stock and diluted
into HBSS without calcium or magnesium. The final total lipid
concentration was 4 mM for all samples. 10% heat inactivated fetal
bovine serum was added to each sample. 20 p,l of a 60/40 mM Ca/Mg
stock was added to every ml of liposome solution just before
addition to tissue culture plates. Experiments were performed in
quadruplicate.
[0128] Liposomes were incubated for transfection of OVCAR-3 cells
as described above. Total cell number and transfection activity
were assessed by fluorescence measurements as described above.
[0129] The results are shown in FIG. 7. In FIG. 7, the fluorescence
of EGFP corrected for total cell number by the calcein blue reading
was plotted against the concentration of the OVB-3-polylysine
conjugate. The corresponding fluoresence photomicrographs are shown
in FIG. 8. As can be seen in the Figures, the antibody-polylysine
conjugate greatly enhances transfection efficiency in 10%
serum.
Specificity of Enhancement of Transgene Expression for the OVB-3
Antibody:
[0130] The monoclonal antibody OVB-3 had a relatively high level of
binding to OVCAR-3 cells, while non-specific mouse serum IgG
binding to OVCAR-3 cells was relatively insignificant. Plasmid
encapsulated N-Cl.sub.2-DOPE/DOPC (7:3 mole ratio) liposomes were
prepared with pK-OVB3 or mouse serum pK-IgG targeting modules as
desceibed above. There was no significant difference in the level
of polylysine incorporation for the two antibody conjugates. The
conjugate to lipid ratio for both liposome preparations was 12.5
.mu.g protein per gmole lipid. The fusogenic liposomes were then
incubated with OVCAR-3 cells for 3 hours at 37.degree. C. at 4 mM
lipid in 10% heat inactivated serum. This medium was replaced by
growth medium and the cells were allowed to grow overnight. GFP
transgene expression was determined by GFP fluorescence (485.+-.10
nm excitation, 530.+-.12 nm emission) and was normalized to the
total number of living cells using a calcein blue AM fluorescence
assay. The targeting module made with OVCAR-3 specific monoclonal
antibody OVB-3 significantly increased GFP transgene expression of
N-Cl.sub.2-DOPE/DOPC (7:3 mole ratio) liposomes relative to control
non-targeted liposomes, while the non-specific mouse IgG-pK
conjugate did not produce any significant increase in transgene
expression. The s relative amounts of transgene expression
(normalized to 100 for the OVB-3 results) were 100.+-.21 for the
OVB-3 targeted system versus 23 t 5 for no targeting and 20.+-.15
for the non-specific IgG targeted liposomes. This demonstrates the
target specific characteristic of the OVB3-pK targeting module.
EXAMPLE 10
Fusogenic 1VLacyl-PE Liiposomes: Targeting via
Polylysine-Transferrin (pKT)-Enhancement of Binding to OVCAR-3
cells
[0131] Liposomes were prepared with approximately 1 mole %
Lissamine rhodamine-phosphatidylethanolamine as a fluorescent
membrane probe. Liposomes prepared as described in Example 1 (
70/30 NCl.sub.2DOPE/DOPC) were incubated with varying
concentrations of polylysine transferrin (pKT). The pKT was made up
as a 1 mg/ml solution in Hanks balanced Salt solution (HBSS)
without Ca or Mg. Since aggregation of the liposomes occurred at a
ratio of approximately 0.3 g conjugate (pKT)/mmole of total
phospholipid (the expected range for charge neutralization at the
outer liposome surface for liposomes of this composition),
pKT/liposome ratios were kept below this range for the
investigations.
[0132] In order to determine if the pKT conjugate affected the
binding of liposomes to OVCAR-3 ovarian cancer tumor cells, cells
grown in 96 well plates were incubated with liposomes with and
without the pKT conjugate electrostatically bound to the liposome.
A ratio of approximately 0.2 g pKT/mmol lipid was used in all
samples. Cells were incubated with liposomes at 0.1 mM, 1.0 mM, 5
mM and 15 mM total lipid in the presence and absence of serum
(heatinactivated FBS at 10%) to determine the effect of serum on
binding of the liposomes to the cells. The binding solutions were
prepared by adding the materials in the following order: liposomes,
pKT, buffer, serum where the buffer is HBSS without without
Ca.sup.2+, or Mg.sup.2+. Just before addition of the solutions to
the cells, Ca.sup.2+ and Mg.sup.2+ were added to adjust the overall
concentrations to 1.2 and 0.8 mM respectively. At the termination
of the incubation period, the wells were aspirated, rinsed to
remove unbound liposomes and binding was determined by the amount
of rhodamine fluorescence bound to the cells. The results are shown
in FIG. 9. Liposomes having no polylysine transferrin bound to
their bilayer showed limited binding to the cell surface when
incubated in the presence of serum. However, when the liposomes had
polylysine transferrin bound to their surface, the binding to the
cells was significantly increased. In the absence of serum, the
polylysine transferrin conjugate had little effect on the liposome
binding to OVCAR 3 cells. The serum proteins significantly inhibit
the binding to liposomes to the cells. Transferrin pK conjugate
attached to the liposomes reduced or eliminated the serum induced
inhibition of liposome binding to the cells.
[0133] It is clear from the results that the addition of pKT
greatly enhances binding of liposomes to these cells. Further, the
pKT appears to reduce or eliminate the serum inhibition of liposome
binding.
EXAMPLE 11
[0134] Fusogenic N-Acyl-PE Liposomes: Enhancement of Binding and
Transfection by Polylysine-Transferrin Targeting
[0135] N-Cl.sub.2-DOPE/DOPC liposomes containing spermine-condensed
pEGFP plasmid DNA-liposomes and Labeled by 0.5 mole % lissamine
rhodaminephosphatidylethanolamine were prepared. In one experiment,
sucrose-loaded liposomes were prepared by inclusion of 200 mM
sucrose in the buffer with DNA and the buffer with spermine. After
removal of organic solvent and formation of liposomes and dilution
into 300 mM sucrose buffer, the liposomes were extruded and
dialyzed into Hanks Buffered Salt Solution without Ca.sup.2+ and
Mg.sup.2+ (HBSS). The liposomes were then pelleted, washed and
resuspended at twice the normal concentration in HBSS, i.e., to a
final lipid concentration of approximately 40 mM. This stock was
diluted to 20 mM and was used for experiments in 96 well plates
[0136] As described in Example 3, 96 well plates were seeded with
OVCAR-3 cells at 2.times.10.sup.5 cells/ml of medium (RPMI 1640
with 10% fetal bovine serum) two days prior to transfection
experiments. Transfections were performed, as described in Example
3, by evacuating the wells and adding 100 Al/well of the desired
premixed transfection solutions and incubating at 37.degree. C. for
3 hours. At this point, the transfection solutions were removed and
100 .mu./well of RPMI 1640 medium with 10% FBS and 5 mM sodium
butyrate was added. After overnight incubation at 37.degree. C.,
the plates were treated for fluorescent assay of transfection as
described in Example 1. Plates were washed 2.times. with the same
detergent solution and read again in the same detergent solution.
All EGFP readings were corrected by dividing by the CBAM readings
for total cell esterase activity.
[0137] In parallel, a set of experiments was performed using empty
liposomes labeled with a lissamine rhodamine-PE derivative (ex. 560
nm, em. 620 nm),. These were the same liposomes as described in
Example 1. The liposomes served as indicators of liposomal binding
under various conditions and as controls, especially in the cases
where pKT was used. In the latter case, the fluorescein labeling of
the pKT is a potential interference in the EGFP readings for
transfection, and appropriate corrections were made in some cases.
In comparison to the EGFP plasmid-containing liposomes, the
rhodamine labeling level was higher, and the diameter was slightly
smaller for these liposomes.
[0138] All experiments, including binding experiments, were
performed in the manner of a transfection experiment, i.e. a 3 hour
incubation with liposomes, followed by overnight incubation with
serum and butyrate-containing medium.
[0139] Comparisons were made at a single liposome concentration and
in 10% FBS, while other components varied. The order of addition of
materials was as listed below. The added "buffer" was HBSS without
Ca/Mg in all cases. Ca.sup.2+and Mg.sup.2+were added at the end as
described above. Liposome stocks were approximately 20 mM total
lipid.
[0140] Results are shown in FIG. 10. FIG. 10 demonstrates that pKT
enhances the binding of liposomes to cells and transfection in a
dose dependent manner. In FIG. 11, photos of the same experiments
also demonstrate the pKT effect, where red fluorescence indicates
liposomal lipid and green fluorescence the expression of the
delivered gene.
EXAMPLE 12
Fusogenic IVLacyl-PE Liposomes: Inhibition of Polylysine
Transferrin Mediated Transfection by an Anti-Transferrin
Antibody
[0141] The effect of an anti-transferrin antibody was further
investigated in this example. Liposomes and assays were prepared as
described in Examples 1 and 3. Comparisons were made at 1 mM total
lipid, 0.2 mg/ml pkT and in 10% FBS (always heat inactivated as in
all previous examples). The samples also contained either 1.4 mg/ml
anti-transferrin antibody (Sigma, St. Louis) or 1.4 mg/ml
nonspecific bovine IgG (Sigma, St. Louis). Materials were added in
the order described in Example 7 to prepare stocks for addition to
tissue culture wells. Ca.sup.2, and Mg.sup.2+ were added at the end
as in Example 7.
[0142] Photos were taken the day after the transfection incubation,
and the results are shown in FIG. 12.
[0143] FIG. 12 clearly shows that the anti-transferrin inhibited
transfection by the liposomes. It therefore appears that this
antibody inhibits transfection mediated by the
polylysine-transferrin conjugate, although binding of the liposome
to cells is not strongly inhibited.
[0144] Notably, polylysine alone at a concentration approximately
equal to the pKT concentration did not mediate either liposome
binding or transfection (see Example 13). Therefore, it appears
that the transferrin molecule plays some role in the binding of the
liposomes to the cells, and undoubtedly plays a role in enhancing
transfection.
Example 13
Fusogenic AfLacyl-PE Liposomes: Polylysines Alone Do Not Mediate
Liposomal Delivery and Transfection.
[0145] The OVB-3-polylysine conjugates were prepared as described
above. The pK-OVB-3 was added as a 0.5 mg/ ml stock to a 40 mM
lipid liposome solution and then diluted with HBSS without Ca/Mg
such that the final total lipid concentration was 4 mM and the
pK-OVB-3 concentration of 0.1 mg/ml. The weight percent of
polylysine (pK) in the conjugates was estimated to be 3%. Based on
this estimate, a stock of 3 kDa pK at approximately 0.015 mg/ml was
used at equal volume as the pK-OVB-3 solutions to prepare liposome
conjugates, i.e. the final pK concentration was 3% of the pK-OVB-3
or 0.003 mg/ml.
[0146] The pKT sample was obtained from Sigma (St. Louis) and is
described above. It contains 30-70 kDa polylysine group. For
comparison to this conjugate, a 30-70 kDa polylysine (not
conjugated) was obtained from Sigma (St. Louis). Based on the
manufacturer's determination of approximately 0.3 polylysine (pK)
units per transferrin in the conjugates, the appropriate amount of
free pK was added to the liposomes in the comparison experiments.
In the pKT experiments, the liposome concentration was at 2 mM
total lipid and the pKT concentration 0.1 mg/ml. The liposome-pKT
complexes were prepared by adding the appropriate amount of the 1
mg/ml pKT stock to a concentrated (20 mM total lipid) liposome
solution and then diluting with the appropriate amount of HBSS
without calcium or magnesium.
[0147] For the FPK conjugate experiments, 40 .mu.l of a 340 .mu./ml
stock of FPK was added to 40 .mu. of 40 mM liposomes and then
diluted to give a final lipid concentration of 4 mM and a final FPK
concentration of 34 .mu.g/ml.
[0148] For FPGK conjugate experiments, 80 gl of a 340 .mu.g/ml
stock of FPGK was added to 40 p.1 of 40 mM liposomes and then
diluted to give a final lipid concentration of 4 mM and a final FPK
concentration of 68 .mu./ml.
[0149] The FPK and FPGK conjugates also contain 3 kDa polylysine
(pK) units. For comparison of pK to the FPK and FPGK conjugates, a
stock of 3 kDa pK equivalent to the FPK and FPGK solutions was
used. Since the pK is approximately 40 weight % in these
conjugates, a 136 .mu./ml stock was used. The final lipid
concentration in the transfection experiments was 4 mM and the
final FPK and FPGK concentrations were 34 and 68 mg/ml
respectively, while the final free pK concentration for comparison
was 27 p.g/ml.
[0150] N-Cl.sub.2-DOPE/DOPC ( 70/30) liposomes were prepared to
encapsulate the plasmid pEGFP-Cl, spermine and sucrose as described
above and pelleted and washed as described above. OVCAR-3 cells
were grown in 96 well plates as described above.
[0151] All transfection experiments were with OVCAR-3 cells in
tissue culture, as described above. The OVB-3 conjugate were added
directly to a 40 mM liposome stock and diluted into HBSS without
calcium or magnesium. The final total lipid concentration was 4 mM
for all samples. 10% heat inactivated fetal bovine serum was added
to each sample. 20 gl of a 60/40 mM Ca/Mg stock was added to every
ml of liposome solution just before addition to tissue culture
plates. Experiments were performed in quadruplicate.
[0152] Liposomes were incubated for transfection of OVCAR-3 cells
as described above. Total cell number and transfection activity
were assessed by fluorescence measurements as described above.
[0153] The results are shown in FIG. 13. The fluorescence of EGFP
corrected for total cell number by the calcein blue reading was
plotted against various conditions for transfection. FIG. 13 shows
that the conjugates are always much more active than the
corresponding free polylysines in terms of transfection, suggesting
that the activity is not mediated by the polylysine portion of the
conjugates alone. The data also show that the folate conjugates
enhance transfection.
EXAMPLE 14
Fusogenic Ahacyl-PE Liposomes: Enhancement of Ex Vivo Binding of
Liposomes to OVCAR-3 Ascites Cells by Polylysine Transferrin
[0154] As the first step to testing in vivo transfection, the
binding of liposomes to human OVCAR-3 ascites cells removed from a
mouse xenograft was examined. Liposomes were prepared as described
in Example 1. As in Example 1, both DNA-containing and empty
liposomes were labeled with a rhodamine-PE fluorophore.
[0155] A series of experiments was performed using OVCAR-3 cells
recovered from the ascites fluid of SCID mice. The lavage was
accomplished by injection of 6 ml of PBS into the peritoneal cavity
of a mouse that had been injected with 1.times.10.sup.7 cells 40
days earlier. The recovered cells were pelleted and washed with PBS
and resuspended at an estimated density of approximately
1-2.times.10.sup.6/ml (from hemacytometer) in HBSS without Ca and
Mg. Aliquots of 10, 20 or 50 p,l were placed in the round-bottom
wells of a polypropylene 96-well plate. HBSS was added to adjust
the total volume of each well to 100 p,l. At this point the plate
was spun in a centrifuge, then a fine needle aspirator was used to
collect the fluid. After a wash in HBSS, 50 gl of a concentrated
(about 10.times.) cell-free intraperitoneal fluid collected from
OVCAR-3-bearing SCID mice and adjusted with 20 p,l/ml of a 60/40 mM
Ca.sup.2+/Mg.sup.2+ stock was added to each well. Then 50 gl of
each liposome sample with added Ca.sup.2+/Mg.sup.2+ was added to
each well, and the wells incubated at 37.degree. C. for 3 hours.
After incubation, the wells were washed (by centrifugation) 3 times
with HBSS (without Ca and Mg) and resuspended in RPMI 1640 with 10%
heat inactivated fetal bovine serum and 5 mM sodium butyrate. The
cells were monitored by fluorescence microscopy.
[0156] The results are shown in FIG. 14. FIG. 14 reveals that pKT
substantially enhanced binding of the liposomes to the OVCAR3
cells. There may have been some transfection as well. The
dramatically enhanced binding seen here ex vivo would be expected
to ultimately greatly enhance in vivo transfection.
EXAMPLE 15
In Vivo Transfection of Intraperitoneal OVCAR-3 Cells by
Polylysinetransferrin Targeted Liposomes
[0157] In this example we treated a mouse bearing OVCAR3 tumor in a
manner similar to the model presented in Son,. Cancer Gene Therapy
4, 391; 1997, incorporated herein by reference. The treatment
involved injecting a very high tumor cell number into the mouse and
pretreating with cisplatin before transfection.
[0158] Two high cell number OVCAR-3 SCID mice were set up as in
Example 6 on day 1. On day 42, 0.2 ml of 0.5 mg/ml cisplatin was
injected IP. On day 46, each mouse received an IP injection of a
liposome preparation as follows: 0.5 ml of pEGFP-Cl-containing
liposomes (NC1.sub.2-DOPE/DOPC, 70/30) were injected IP (one mouse
received liposomes with the pZeoLacZ plasmid encapsulated rather
than the pEGFP-Cl plasmid). The liposome stock was 40 mM total
lipid, was diluted 1:1 with 1 mg/ml pKT, and was constituted in
Hanks Balance Salt .Solution (HBSS) without Ca.sup.2+ or Mg.sup.2+.
Just before injection, 20 p/ml of a stock of 60 mM and 40 mM
Ca+.sup.2 and Mg.sup.2+, respectively, was added, which brought
Ca.sup.2+ and Mg.sup.2+ to 1.2 and 0.8 mM respectively. The same
liposome injections followed on days 49 and 52. On day 53, 0.5 ml
of the same 20 mM butyrate solution in HBSS without Ca/Mg was
injected IP. On day 54, both mice were sacrified and peritoneal 6
ml PBS lavage was performed on each mouse to obtain ascites cells.
A fairly low number of cells was obtained, probably as a result of
the platinum treatment. Some peritoneal wall and mesentery tissue
was also dissected and frozen for future analysis. The ascites
cells were pelleted and washed 2 times with PBS, collected into 0.5
ml and diluted 10.times. for observation and photographs. The
pZeoLacZ-treated cells were resuspended in 5 ml of cold PBS and the
pEGFP-treated cells were resuspended in 10 ml of cold PBS. A 0.3 ml
aliquot of each was diuted to 1 ml in a plastic cuvette containing
PBS and O.D. read at 650 nm against buffer blank. The readings were
pZeoLacZ-0.836; pEGFP-0.618. A rough estimate of the pEGFP sample
was that it contained 1.sub.2e6 cells/ml. The pZeoLacZ sample was
diluted by adding 1.76 ml PBS to approximate the cell concentration
of the other sample. Aliquots were taken from these for
chemiluminescent .beta.-galactosidase assays, and the results are
shown in FIG. 15. The EGFP sample serves as a control for the
pZeoLacZ.
[0159] FIG. 15 shows that significant .beta.-galactosidase activity
occurred in the pZeoLacZ transfected cells, but only at the
background level in the pEGFP control. The data confirms that in
vivo transfection occurred.
EXAMPLE 16
In Vivo Transfection of OVCAR-3 Cells Under Different Conditions
Mediated by Polylysine-Transferrin Targeted Liposomes
[0160] Previous examples provided evidence for pKT-mediated
transfection in vivo using a high cell number OVCAR-3 IP system in
the SCID mice. We also utilized a lower cell number model where
more mice could be treated.
[0161] 11 SCID mice (female CB17 from Taconic) were injected on day
1 with 1.times.10.sup.7 OVCAR-3 cells from tissue culture. The
cells were allowed to grow until day 73. On day 73 the mice were
injected intraperitoneally (IP) with 0.2 ml of 0.5 mg/ml cisplatin
in PBS. On day 77, 80 and 83, the mice received IP injections of
liposomes or lipid complexes. Four types of DNA-containing systems
were injected (see below). On day 84, 0.5 ml of 20 mM sodium
butyrate in HBSS w/o Ca/Mg was injected IP. On day 85, peritoneal
lavage was performed as in Example 4. Cells from lavage samples
were collected for analysis as described above. Assays for
.beta.-galactosidase were performed with a chemiluminescent
substrate as in Example 3
[0162] Group 1: Liposomes encapsulating pCMVR plasmid (for
.beta.-galactosidase expression) at a total lipid concentration of
40 mM were mixed in equal volume with a stock of 2 mg/ml
polylysine-transferrin conjugate (pKT). The liposome mixture
received 12 .mu. per ml of a stock of 60 mM Ca.sup.2+ and 40 mM
Mg.sup.2+ just before injection of 0.8 ml of this mixture into the
peritoneal cavity of each of 4 mice.
[0163] Group 2: Exactly the same protocol except that liposomes
containing the pEGFP-C1 plasmid were used.
[0164] Group 3 DC-cholesterol/DOPE (2/3) liposomes were complexed
with pCMV.beta. plasmid DNA. A 4mM stock of the above liposomes
(prepared by sonication in a bath sonicator from a rehydrated film)
in 20 mM HEPES, pH 7.0, was diluted 1:1 in distilled deionized
water to give a 2 mM stock. A stock of pCMV(3 plasmid DNA was
diluted in water to 1 mg/ml. Equal volumes of the stocks were mixed
approximately 15 minutes before intraperitoneal injection as
described in examples above. 0.3 ml were injected IP into each of 2
mice.
[0165] Group 4: Exactly the same as the above DC-cholesterol
complexes, , except that pEGFP-Cl plasmid DNA was used. 0.3 ml was
injected IP into 1 mouse.
[0166] The liposomes comprised /V-Cl.sub.2-DOPE/DOPC ( 70/30)
prepared sterile and as in the Assays and Methods section,
including pelleting and washing. The pKT conjugate is the same as
discussed above. The data are shown in FIG. 8.
[0167] FIG. 16 reveals that the pKT-mediated liposomal transfection
of the the IacZ gene clearly worked in vivo in this IP system.
[0168] Though the invention has been described with reference to
specific embodiments, those of ordinary skill in the art will
recognize that various modifications, omissions, changes and/or
substitutions may be made without departing from the spirit and
scope of the invention defined in the appended claims.
* * * * *