U.S. patent application number 12/197794 was filed with the patent office on 2009-03-12 for encapsulation of bioactive complexes in liposomes.
Invention is credited to Patrick Ahl, Donna Cabral-Lilly, Andrew Janoff, Paul Meers, Tong Shangguan.
Application Number | 20090068256 12/197794 |
Document ID | / |
Family ID | 22402271 |
Filed Date | 2009-03-12 |
United States Patent
Application |
20090068256 |
Kind Code |
A1 |
Meers; Paul ; et
al. |
March 12, 2009 |
Encapsulation of Bioactive Complexes in Liposomes
Abstract
This invention provides a method to prepare
liposome-encapsulated bioactive agents, such as nucleic acids,
comprising complexation of the bioactive agents to reverse micelles
prior to forming liposomes, as well as methods of using the
liposomes so formed and formulations to deliver nucleic acids to
cells.
Inventors: |
Meers; Paul; (Princeton
Junction, NJ) ; Shangguan; Tong; (Princeton, NJ)
; Cabral-Lilly; Donna; (Princeton, NJ) ; Janoff;
Andrew; (Yardley, PA) ; Ahl; Patrick;
(Princeton, NJ) |
Correspondence
Address: |
FOLEY HOAG, LLP;PATENT GROUP, WORLD TRADE CENTER WEST
155 SEAPORT BLVD
BOSTON
MA
02110
US
|
Family ID: |
22402271 |
Appl. No.: |
12/197794 |
Filed: |
August 25, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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09914615 |
Dec 26, 2001 |
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PCT/US00/05395 |
Mar 1, 2000 |
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12197794 |
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60122365 |
Mar 2, 1999 |
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Current U.S.
Class: |
424/450 ;
514/44R |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 9/1272 20130101; A61K 9/127 20130101; A61P 43/00 20180101 |
Class at
Publication: |
424/450 ;
514/44 |
International
Class: |
A61K 9/127 20060101
A61K009/127; A61K 31/7052 20060101 A61K031/7052; A61P 43/00
20060101 A61P043/00 |
Claims
1. A method of encapsulating a bioactive complex in a liposome
which comprises the steps of: (a) dissolving at least one
amphipathic lipid in one or more organic solvents (b) combining a
first aqueous suspension comprising a bioactive agent with the
lipid containing organic solution of step (a) so as to form an
emulsion comprising the bioactive agent and the lipid; (c) adding a
second aqueous suspension comprising a complexing agent to the
emulsion of step (b), (d) incubate the emulsion of step (c) to
allow the complexing agent to contact the bioactive agent thereby
forming a complex of the bioactive agent with the complexing agent
within the lipid stabilized water droplets; wherein said complex is
no greater in diameter than the diameter of the droplet and, (e)
removing the organic solvent from the suspension of step (d), so as
to form liposomes comprising the complexed bioactive agent and the
lipid.
2. A method of encapsulating a bioactive complex in a liposome
which comprises the steps of: (a) dissolving at least one
amphipathic lipid in one or more organic-solvents (b) combining a
first aqueous suspension comprising a complexing agent with the
lipid containing organic solution of step (a) so as to form an
emulsion comprising the complexing agent and the lipid; (c) adding
a second aqueous suspension comprising a bioactive agent to the
emulsion of step (b), (d) incubate the emulsion of step (c) to
allow the complexing agent to contact the bioactive agent thereby
forming a complex of the bioactive agent with the complexing agent
within the lipid stabilized water droplets; wherein said complex is
no greater in diameter than the diameter of the droplet and, (e)
removing the organic solvent from the suspension of step (d), so as
to form liposomes comprising the complexed bioactive agent and the
lipid.
3. The method of claim 1, wherein the bioactive agent is a nucleic
acid
4. The method of claim 1, wherein the nucleic acid is DNA.
Description
FIELD OF THE INVENTION
[0001] This invention is directed to a method for the encapsulation
of complexes, such as polycation-condensed nucleic acids, in
liposomes, using an emulsion stabilized by amphipathic lipids as an
intermediary within which the complex forms. This invention is also
directed to the liposome encapsulated complexes so formed. The
method of this invention is applicable for providing liposomes
loaded with a variety of compounds which heretofore have been
difficult to load into liposomes at high compound to lipid
ratios.
BACKGROUND OF THE INVENTION
[0002] In order to be useful as pharmaceutical preparations,
bioactive agents must be able to reach the therapeutic site in an
adequate therapeutic effective amount. While many bioactive agents
and drugs are stable in vivo, others are often degraded. When such
degradation occurs prior to the drug or bioactive agent reaching
its target site, a non-therapeutic amount of drug will reach the
target site. Other drugs or bioactive agents are taken up by
non-target systems, once again resulting in the lack of a
therapeutic amount of a drug or bioactive agent reaching the target
site at therapeutically effective amounts. Certain polar drugs can
not enter cells at all because of their inability to cross the
target cell membrane. The only way that these polar drugs may enter
a cell is by uptake by the process of endocytosis, exposing them to
degradative lysosomal enzymes in the cell. Yet another problem in
the therapeutic delivery of drugs or bioactive agents is the
inability to administer a high enough concentration of the drug or
bioactive agent to be therapeutic, while avoiding toxicities often
associated with some drugs or bioactive agents. These problems have
been approached by a number of different methods. When a drug or
bioactive agent has no toxicity associated with it, it may be
administered in high enough doses to account for degradation,
removal by non-target organs and lack of targeting to the site
where the therapeutic drug or bioactive agent is required. However,
many drugs or bioactive agents are either too expensive to allow
such waste or have toxicities that prevent administration of such
high dosages. Numerous methods have been used to overcome some of
the problems encountered in administering therapeutic amounts of
drugs or bioactive agents.
[0003] One such method is the encapsulation of drugs or bioactive
agents in liposomes. While some drugs or bioactive agents can be
encapsulated in liposomes at therapeutically effective doses by
passive loading or by gradient loading, these methods are limited
either to drugs or bioactive agents with specific chemical
properties or to drugs or bioactive agents that can be administered
in relatively low concentrations. Some bioactive compounds such as
weak bases or weak acids can be loaded remotely into preformed
liposomes to form highly concentrated complexes. This type of
loading, referred to as remote or gradient loading, requires that
the drug or bioactive agent be temporarily able to pass through the
lipid bilayer of the liposome. However, this is not the case for
all bioactive molecules, many of which cannot pass through the
liposomal bilayer.
[0004] One area in which attempts to administer therapeutic levels
of drugs or bioactive agents have been only partially successful is
the area of gene therapy. Gene therapy involves the introduction of
an exogenous gene into an appropriate cell type, followed by
enablement of the gene's expression within the cell at
therapeutically relevant levels. Such therapy has progressed, in a
relatively short period of time, from basic research to the
introduction into cells of a variety of genes, including those
useful for treating cancers (Duque et al., Histol Histopathol, 13:
231-242 (1998); Runnebaum et al., Anticancer Res., 17: 2887-2890
(1997)). While naked DNA, in some cases has been taken up into
cells (Wolff et al., Science, 247: 1465-1468 (1990)), it generally
cannot be, due to its large size and high degree of negative
charge; moreover, naked DNA cannot be designed so as to be targeted
to specific cells. Accordingly, successful gene therapy generally
is reliant upon the availability of "vectors" for introducing DNA
and other nucleic acids into cells.
[0005] Presently, there are two major groups of DNA delivery
systems, viral and non-viral. Viral vectors, including
replication-deficient viruses, such as retroviruses, adenoviruses,
and adeno-associated viruses, have thus far been the most widely
described gene delivery vehicles (Robbins et al., Trends in
Biotech, 16: 35-40 (1998)). However, their use has been hampered by
the immunogenicity of their viral components, potential risk of
reversion to a replication-competent state, potential introduction
of tumorgenic mutations, lack of targeting mechanisms, limitations
in DNA capacity, difficulty in large scale production and other
factors (see, e.g., Lee and Huang, J Biol Chem, 271: 8481-8487
(1996)).
[0006] Two major types of nonviral vehicles have been developed as
alternatives to viral vectors. Cationic liposome-DNA complexes (or
"lipoplexes," Feigner et al., Proc Nat/Acad Sci USA, 84: 7413-7417
(1987)), consisting of cationic lipids and DNA have thus far been
the most widely described alternative to viral vectors for gene
delivery. However, such lipoplexes suffer from several major
drawbacks when used in gene therapy, including low stability, high
cytotoxicity, non-biodegradability, poor condensation and
protection of DNA, serum sensitivity, large size and lack of tissue
specificity. Moreover, as the lipoplexes are positively charged,
they generally interact nonspecifically with the negatively charged
surfaces of most cells; accordingly, it is generally not possible
to target such lipoplexes to specific sites in vivo.
[0007] Another variation of lipoplexes and DNA involves
polylysine-condensed DNA bound to anionic liposomes (Lee and Huang,
J Biol Chem, 271: 8481-8487 (1996)). These require certain anionic
lipids to form the active structure. The lipoplexes formed either
do not completely encapsulate the DNA or must form two or more
bilayers around the condensed DNA. In the latter case delivery to
the cytoplasm would require the DNA to cross at least three
membranes. This would be expected to inhibit transfection
efficiency. In the former case, stability may be compromised by
exposure of the DNA in physiological salt solutions.
[0008] Liposomes are an additional type of nonviral vector
alternative, and offer several advantages for such use in
comparison to the lipoplexes. For example, liposomal bilayers form
around encapsulated nucleic acids, thereby protecting the nucleic
acids from degradation by environmental nucleases; lipoplexes, by
contrast, do not encapsulate nucleic acids, and hence, cannot
completely sequester them away from environmental nucleases.
Moreover, liposomes can encapsulate, in their aqueous compartments,
other bioactive agents in addition to nucleic acids; lipoplexes, by
contrast, cannot because they do not encapsulate aqueous volume.
Furthermore, liposomes can be made to be neutrally charged or
anionic, as opposed to the restricted ionic nature of the
aforementioned lipoplexes. Thus, liposomes can be designed so as to
avoid cytotoxicities induced by the delivery vehicle itself and to
enhance their accumulation at specific sites of interest.
[0009] While the concept of encapsulating bioactive agents in
liposomes is not new, many agents have been difficult to
encapsulate in liposomes at any level and others have proven
difficult to encapsulate in liposomes at levels that would be
therapeutically effective. Many small molecules can be encapsulated
in liposomes but leak out. Thus, it has also been difficult to
encapsulate some bioactive agents and have them retained within the
liposomes at a therapeutically effective dose for a therapeutically
effective time. For instance, it has been difficult to encapsulate
particularly large molecules into a complex within a liposome. It
has also been difficult to use many water soluble molecules as
therapeutic agents because they are unable to penetrate the cell
membrane. When encapsulated stably into liposomes that can fuse to
cell membranes, it is possible to deliver these drugs at
therapeutically effective doses into the target cells. The method
of the present invention enables formation of liposomes containing
such drugs or bioactive agents in a therapeutically useful
form.
[0010] Several attempts have been made to encapsulate nucleic acids
in liposomes, these including use of the reverse-phase evaporation
(Fraley et al., J Biol Chem, 255: 10431-10435 (1980)),
dehydration-rehydration (Alizo et al., J Microencap, 7: 497-503
(1990)) and freeze-thaw (Monnard et al., Biochem Biophys Acta,
1329: 39-50 (1997)) methods of liposome formation. However, each of
these methods has several limitations, including requirements for
low starting concentrations of nucleic acid, resulting in
significant percentages of empty vesicles in the product liposomes,
inability to reproducibly encapsulate sufficient quantity of DNA in
liposomes to be therapeutically effective at the desired target
site and difficulties in optimizing the vehicles for protection of
their encapsulated nucleic acids from nuclease-mediated
degradation.
[0011] Attempts have also been made to complex DNA with complexing
agents and subsequently encapsulate the complexed DNA in liposomes.
Complexing agents are agents that react with other molecules
causing the precipitation or condensation of the molecules.
Complexing agents useful in the practice of the present invention
are selected from the group consisting of charged molecules that
have a charge opposite to the charge on the bioactive agent. The
complexing agent may be selected from the group of charged
molecules consisting of spermine, spermidine, hexammine cobalt,
calcium ions, magnesium ions, polylysines, polyhistidines,
protamines, polyanions such as heparin and dextran sulfate, citrate
ions, or sulfate ions. For instance, polycations of charge +3 or
higher, e.g., polyamines, polylysine and hexammine cobalt (III) are
known (see Chattoraj et al., J Mol Biol, 121: 327-337 (1978);
Gosule L C and Schellman J A. Nature 259: 333-335 (1976); Vitello
et al., Gene Therapy, 3: 396-404 (1996); Widom et al. J. Mol.
Biol., 144: 431-453 (1980); Arscott et al., Biopolymers, 30:
619-630 (1990); Wilson et al., Biochem, 18: 2192-2196 (1979)) to be
able to condense DNA molecules, through interaction with multiple
negative charges on the DNA. Polyamines, e.g., spermidine (3+) and
spermine (4+), have, unlike other types of polycations, been found
to occur naturally in all living cells (see, e.g., Ames and Dubin,
J Biol Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev
Biochem, 53: 749-790 (1984)). High polyamine levels are known to
exist in actively proliferating animal cells, and are believed to
be essential therein for maintaining normal cell growth (Ames and
Dubin, J Biol Chem, 253: 769-775 (1960); Tabor and Tabor, Annu Rev
Biochem, 53: 749-790 (1984); Hafner et al., J Biol Chem, 254:
12419-12426 (1979); Pegg, Biochem J, 234: 249-262 (1986)).
[0012] Liposome encapsulation of spermine-condensed linear DNA in
liposomes has been attempted by Tikchonenko et al., Gene, 63:
321-330 (1988). However, the starting DNA concentration therein was
low, with the consequence that the resulting liposomes also had a
low ratio of encapsulated DNA to liposomal lipid (0.02-0.2
micrograms DNA per micromole lipid). Moreover, such condensation of
linear DNA molecules in the absence of intermolecular DNA
aggregation required control over spermine concentrations to an
impracticable degree of precision. Additionally, Baeza et al., Ori
Life Evol Biosphere, 21: 225-252 (1992) and Ibanez et al., Biochem
Cell Biol, 74: 633-643 (1996) both report encapsulation of 1-4
micrograms per micromole of spermine-condensed SV40 plasmid DNA in
liposomes. However, neither of their preparations were dialyzed
against high salt buffers subsequent to liposome formation, the
reported amounts of encapsulated DNA actually may include a
significant percentage of unencapsulated DNA. Since these liposomal
formulations were not exposed to DNAase degradation to determine
the percentage of DNA actually sequestered in the liposomes, the
high reported amounts probably do not reflect actually encapsulated
DNA.
[0013] Efficient preparation and use of liposomal encapsulated
nucleic acids requires the use of high-concentration suspensions of
nucleic acids, in order to minimize the percentage of empty
liposomes resulting from the process and to maximize the
DNA:liposomal lipid ratios. However, condensation of DNA at high
concentrations during known methods of liposome formation generally
results in intermolecular aggregation, leading to the formation of
nucleic acid-based structures unsuitable for gene delivery. Large
aggregates formed by condensation of DNA directly with a complexing
agent cannot be easily encapsulated in liposome and such large
aggregate structures (on the order of the size of cells) can not
efficiently deliver materials to target cells. For instance, if the
aggregates are larger than 500 nm, they are rapidly cleared from
the circulation because of their size after intravenous
administration. On the other hand, larger aggregates may be
administered to cells in vitro. However, sometimes the aggregates
as too large too be taken up by cells.
[0014] Thus, in order to deliver a variety of drugs in
therapeutically effective amounts into target cells, it was
necessary to provide a method of making liposomes that contain
bioactive agents complexed so as to decrease their permeability
through the lipid bilayer, while providing a method that also
limits the size of the complex to be encapsulated in the liposome
so that the resultant therapeutic product is in a therapeutic size
range.
SUMMARY OF THE INVENTION
[0015] The present invention provides a method of encapsulating a
bioactive complex in a liposome which comprises the steps of:
[0016] (a) dissolving at least one amphipathic lipid in one or more
organic solvents [0017] (b) combining at least one aqueous
suspension comprising a solution containing a first molecule
selected from the group consisting of a bioactive agent and a
complexing agent with the lipid-containing organic solution of step
(a) so as to form an emulsion in the form of a reverse micelle
comprising the first molecule and the lipid; [0018] (c) adding a
second aqueous suspension comprising a second molecule selected
from the group consisting of a bioactive agent and a complexing
agent wherein if the first molecule is a bioactive agent, the
second molecule is a complexing agent and vice versa, to the
emulsion of step (b), [0019] (d) incubating the emulsion of step
(c) to allow the complexing agent to contact the bioactive agent
thereby forming a complex of the bioactive agent with the
complexing agent within lipid stabilized water droplets; wherein
said complex is no greater in diameter than the diameter of the
droplet and, [0020] (e) removing the organic solvent from the
suspension of step (d), so as to form liposomes comprising the
complexed bioactive agent and the lipid.
[0021] The method of the present invention is useful for the
preparation of therapeutically useful liposomes containing a wide
range of bioactive molecules complexed with complexing agent within
the liposome. Preferably, the liposomes are fusogenic liposomes
which by the method of the present invention can encapsulate a
variety of molecules. These fusogenic liposomes are able to fuse
with cell membranes and enable the delivery of bioactive agents in
therapeutically effective amounts to cells and organs. In addition,
the method of the present invention also allows more than one
bioactive agent to be encapsulated in a liposome. One or more
bioactive agents may be encapsulated in the same liposomes at the
same time by the method of the present invention. If more than one
bioactive agent is encapsulated in a liposome by the method of the
present invention, it is not necessary for each of the bioactive
agents to be in the form of complexes.
[0022] Some bioactive agents easily pass through the lipid bilayer
and therefore, are not stably sequestered in liposomes. By forming
complexes of the bioactive agents with a complexing agent, the
bioactive agent remains in the liposomes. A major hurdle has been
the problem of encapsulating complexed bioactive agents into
liposomes. When the bioactive agent and complexing agent are mixed
in solution prior to encapsulation in liposomes, many complexes
that are uncontrollably large are formed at the concentrations
necessary for efficient loading of liposomes. The term bioactive
complex is any bioactive agent bound to a complexing agent such
that the complex thus formed results in a change in the physical
properties such as decreasing the size of the bioactive molecule,
decreasing the solubility of the bioactive agent, precipitating the
bioactive agents, condensing the bioactive agent, or increasing the
size of the complex. Liposomes that fuse with cell membranes are
able to deliver a vast category of molecules to the inside of
cells. One advantage of the invention is that, by forming the
complex of the bioactive agent in the reverse micelles, the
formation of unsuitable large complexes incapable of being
encapsulated in therapeutically useful liposomes is prevented.
[0023] The formation of complexes comprising a bioactive compound
within liposomes has the advantage that such complexes are less
likely to leak out of the liposome before delivery to the desired
target cell. Furthermore, the formation of a complex can
concentrate a large amount of the bioactive agent within the
liposome such that the ratio of bioactive agent-to-lipid is high
and delivery is efficacious. The disclosed method provides for
complexation of bioactive materials with complexing agents within
an emulsion followed by encapsulation within a liposome in a manner
that prevents the formation of extremely large, detrimental
aggregates, greater than several microns, of the bioactive agent
and complexing agent.
[0024] In one embodiment, the method of the present invention has
provided a method to encapsulate nucleic acid complexes. For
instance, nucleic acids, such as DNA, are complexed with a
condensing agent within reverse (inverted) micelles, followed by
formation of liposomes from the micelles. While, as described
above, previous attempts have been made to encapsulate DNA in
liposomes, none of said methods were successful at efficiently
preparing therapeutically useful liposomal DNA.
[0025] This invention provides a method to prepare a liposome
comprising a condensed nucleic acid, in amounts of at least about
0.5 micrograms nucleic acid per micromole of liposomal lipid.
[0026] The liposomes' lipid component preferably comprises a
derivatized phospholipid and an additional lipid, generally in
proportions of about 20-80 mole % derivatized phospholipid to about
80-20 mole % additional lipid. Preferred derivatized phospholipids
include: phosphatidylethanolamine (PE)-biotin conjugates;
N-acylated phosphatidylethanolamines (NAPEs), such as N--C12 DOPE;
and, peptide-phosphatidylethanolamine conjugates, such as
Ala-Ala-Pro-Val DOPE. The additional lipid can be any of the
variety of lipids commonly incorporated into liposomes; however,
where the derivatized phospholipid is a NAPE, the additional lipid
is preferably a phosphatidylcholine (e.g., DOPC). Preferably, the
nucleic acid is DNA.
[0027] Also provided herein is a method to prepare a pharmaceutical
composition comprising the liposome and a pharmaceutically
acceptable carrier; said composition can be used to deliver the
nucleic acid to the cells of an animal.
[0028] Other and further objects, features and advantages will be
apparent from the following description of the preferred
embodiments of the invention given for the purpose of disclosure
when taken in conjunction with the following drawings.
BRIEF DESCRIPTION OF THE DRAWINGS
[0029] FIG. 1. Micrographs of spermine-mediated plasmid DNA
aggregation (200 micrograms plasmid DNA in 125 microliters LSB was
mixed gently with 7 mM spermine in 125 microliters LSB). (A) Light
microscope observation after 15 minutes incubation at room
temperature (bar represents 10 microns). (B) Cryo TEM observation
(bar represents 100 nm).
[0030] FIG. 2. Schematic representation of method of DNA
encapsulation. Condensation of DNA occurs (I) within
phospholipid-stabilized water droplets that have formed around the
DNA in a bulk organic solvent. Separate spermine-containing
droplets transfer (II) spermine into the DNA containing droplets by
transient (III) contact and exchange. After condensation within the
emulsion (IV), vesicles are formed by solvent evaporation and
further extruded to smaller sizes (V).
[0031] FIG. 3. Effect of liposomal N--C12 DOPE on spermine-mediated
aggregation of plasmid DNA. Equilibrium dialysis was performed in a
three-chamber dialysis device (see Example 4). The curve on the
left is from dialyses without liposomes, while the curve shifted to
the right is from dialyses that included a chamber with liposomes.
X-axis: spermine concentration (mM); y-axis: turbidity (O.D. 400
nm).
[0032] FIG. 4. Agarose gel analysis of plasmid DNA protection in
N--C12 DOPE/DOPC (70:30) formulations--an aliquot from each
preparation after extrusion and dialysis was divided, and one part
digested with DNase I (see Example 9). Lane 1. Preparation without
spermine. Lane 2. Same as lane 1 but digested with DNase I. Lane 3.
Preparation with spermine. Lane 4. Same as lane 3, but digested
with DNase I.
[0033] FIG. 5. Light micrographs of the particles in N--C12
DOPE/DOPC (70:30) sample prepared as described in Example 3 with
pZeoLacZ plasmid and spermine (A) versus polystyrene beads with an
average diameter of 269.+-.7 nm (B) (bars represent 10 nm).
[0034] FIG. 6. Freeze-fracture TEM micrographs (see Example 7) of
N--C12 DOPE/DOPC (70:30) samples prepared with plasmid and
spermine, as described in Example 3. Arrow points to the particle
with apparently encapsulated material (bar represents 400 nm).
[0035] FIG. 7. Cryo TEM micrographs (see Example 8) of liposomes
with N--C12 DOPE/DOPC and the pZeoLacZ plasmid without spermine
(a), or with spermine (b), said liposomes being prepared as
described in Example 3. In (a) fiber-like structures are seen
outside (star) and apparently inside (arrow) liposomes. In (b), an
arrow points to a toroid that resembles polycation condensed
plasmid DNA (bars in (a) and (b) represent 100 nm). Photomicrograph
(c) represents an EPC sample made with spermine. Toroid (arrow) and
bent rod (star) structures are compared with multilamellar
liposomes (pound sign) [bar represents 50 nm].
[0036] FIG. 8. Fluorescence photomicrographs of confluent OVCAR3
cells after transfection (see Example 11) with the N--C12 DOPE/DOPC
(70:30) preparations. Liposomal samples were prepared (see Example
3) with pEGFP-C1 plasmid DNA (a) with spermine or (b) without
spermine; a sample (c) of empty N--C12 DOPE/DOPC (70:30) liposomes
without spermine plus free pEGFP-C1 plasmid DNA added outside the
preformed liposomes was also tested. The amount of plasmid DNA
added to the empty liposomes in sample c was equal to the total
amount in each of the other preparations. Equal liposome
concentrations were used in the experiments.
[0037] FIG. 9. Quantitation of EGFP expression in OVCAR 3 cells
transfected with pEGFP-C1, as measured by the EGFP fluorescence
level. Transfection experiments (a, b and c, see Example 11) were
the same as in the previous figure legend. In addition,
formulations tested were: d) egg PC liposomes prepared with
spermine and pEGFP-C1 plasmid (see Example 3); and, e) no
additions. The cells were washed and labeled with CBAM, and then
dissolved in detergent to measure the fluorescence of EGFP and
calcein blue (see Example 10; error bars are .+-.s.d).
[0038] FIG. 10. Association of transfection activity with the lipid
pellet of N--C12-DOPE/DOPC (70:30) prepared with spermine and
pEGFP-C1 plasmid DNA (see Example 3); the initial plasmid DNA and
spermine solutions contained 200 mM sucrose. After extrusion and
dialysis, half of the sample was used for transfection without
further handling (a), and the lipid particles from the rest of the
sample were pelleted by centrifugation and washed once with HBSS
before being used for transfection (b). An N--C12-DOPE/DOPC (70:30)
sample with only the 200 mM sucrose was also prepared, and plasmid
DNA and spermine were both added externally just before dialysis at
an amount equal to that used in the other samples. The pellet of
this empty sample (c) was prepared the same way, then, an equal
lipid amount of each of the samples was used for transfection under
the conditions described in the previous figure legends. After
overnight incubation, the cells were labeled with CBAM and the
fluorescence of EGFP and calcein blue were measured (error bars are
.+-.s.d).
[0039] FIG. 11. Transfection via N--C12 DOPE/DOPC (70:30) liposomes
in mouse ascites fluid compared to buffer. Ascites was obtained
from the lavage of a tumor-bearing SCID mouse as described in
Example 13. Cells were incubated with plasmid DNA-containing
liposomes (not a pellet) at a final concentration 10 mM total lipid
in HBSS or HBSS with ascites fluid, at a final protein
concentration of approximately 3.5 mg/ml (see Example 11). After 3
hr. of incubation, the transfection solution was replaced with
serum- and butyrate-containing medium for approximately 20 hr.
Expression of EGFP was measured via its fluorescence (error bars
are .+-.s.d).
[0040] FIG. 12. Fluorescent photomicrographs of OVCAR-3 cells
transfected (see Example 11) with to N--C12 DOPE/DOPC (70:30)
liposomes in buffer or mouse ascites fluid. Cells treated as
described in the legend to FIG. 12 were photographed. Photograph A
represents transfection without peritoneal ascites fluid and
photograph B with peritoneal ascites fluid; cells are confluent in
these views.
[0041] FIG. 13. Fluorescent probe determination of liposome
lamellarity.
[0042] FIG. 14. Fluorescent photomicrographs of OVCAR-3 tumor
transfected in vivo with N--C12 DOPE/DOPC (70:30) liposomes
containing pEGFP-C1. Panel A depicts the expression of EGFP. Panel
B depicts the red fluorescence from rhodamine--labeled
liposomes.
[0043] FIG. 15. Fluorescent photomicrographs of OVCAR-3 tumor taken
from a different site than FIG. 14 transfected in vivo with N--C12
DOPE/DOPC (70:30) liposomes containing pEGFP-C1. Panel A depicts
the expression of EGFP. Panel B depicts the red fluorescence from
rhodamine-labeled liposomes.
[0044] FIG. 16. Fluorescent photomicrographs of control tumor
tissue. Panel A depicts difuse green fluorescense. Panel B depicts
the lack of red fluorescence from rhodamine-labeled liposomes
[0045] FIG. 17. Graph depicting expression of .beta.-galactosidase
activity in muscle tissue after transfection in vivo.
DETAILED DESCRIPTION OF THE INVENTION
[0046] Following are abbreviations, and the corresponding terms,
used throughout this application: PE, phosphatidylethanolamine; PC,
phosphatidylcholine; EPC, egg phosphatidylcholine; DO-, dioleoyl-;
DOPC, dioleoyl phosphatidyloholine; DOPE, dioleoyl
phosphatidylethanolamine; NAPE, N-acylated
phosphatidylethanolamine; N--C12 DOPE, N-dodecanoyl dioleoyl
phosphatidylethanolamine; AAPV-DOPE, Ala-Ala-Pro-Val-dioleoyl
phosphatidylethanolamine; CBAM, calcein blue acetoxy methyl ester;
PBS, phosphate buffered saline; LSB, low salt buffer; HBSS, Hank's
balanced salt solution; EGFP, enhanced green fluorescence protein;
SPLV, stable plurilamellar liposomes; MLVs, multilamellar
liposomes; ULVs, unilamellar liposomes; LUVs, large unilamellar
liposomes; SUVs, small unilamellar liposomes; ds DNA, double
stranded DNA; TEM, transmission electron microscopy.
[0047] The present invention provides a method of encapsulating a
bioactive complex in a liposome which comprises the steps of:
[0048] (a) dissolving at least one amphipathic lipid in one or more
organic solvents [0049] (b) combining at least one aqueous
suspension comprising a solution containing a first molecule
selected from the group consisting of a bioactive agent and a
complexing agent with the lipid-containing organic solution of step
(a) so as to form an emulsion in the form of a reverse micelle
comprising the first molecule and the lipid; [0050] (c) adding a
second aqueous suspension comprising a second molecule selected
from the group consisting of a bioactive agent and a complexing
agent wherein if the first molecule is a bioactive agent, the
second molecule is a complexing agent or vice versa, to the
emulsion of step (b), [0051] (d) incubating the emulsion of step
(c) to allow the complexing agent to contact the bioactive agent
thereby forming a complex of the bioactive agent with the
complexing agent within lipid stabilized water droplets; wherein
said complex is no greater in diameter than the diameter of the
droplet and, [0052] (e) removing the organic solvent from the
suspension of step (d), so as to form liposomes comprising the
complexed bioactive agent and the lipid.
[0053] The method of the present invention is useful for the
preparation of therapeutically useful liposomes containing a wide
range of bioactive molecules complexed with complexing agent within
the liposome. Preferably, the liposomes are fusogenic liposomes
which by the method of the present invention can encapsulate a
variety of molecules. These fusogenic liposomes are able to fuse
with cell membranes and enable the delivery of bioactive agents in
therapeutically effective amounts to cells and organs. In addition,
the method of the present invention also allows more than one
bioactive agent to be encapsulated in a liposome. One or more
bioactive agents may be encapsulated in the same liposomes at the
same time by the method of the present invention. If more than one
bioactive agent is encapsulated in a liposome by the method of the
present invention, it is not necessary for each of the bioactive
agents to be in the form of complexes.
[0054] The term "Bioactive agents" means any compound or
composition of matter that can be administered to animals,
preferably humans, for therapeutic or diagnostic purposes. The
method of the present invention is useful for encapsulating
bioactive agents including but not limited to water-soluble
membrane-impermeant agents such as nucleic acids, nucleotide or
nucleoside analogs such as cytosine .beta.-D-arabinofuranoside
5'-triphosphate (araCTP), proteins such as cytochrome c, polar
anticancer agents such as cisplatin, N-phosphono-acetyl-L-aspartic
acid or 5-fluoroorotic acid, polar or charged derivatives of
anticancer agents, polar peptides, histone deacetylase inhibitors
such as butyrate, etc. Bioactive agents also include, but are not
limited to, agents selected from the group consisting of nucleic
acids such as DNA and RNA, 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; 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.
[0055] The term bioactive complex is any bioactive agent bound to a
complexing agent such that the complex thus formed results in a
change in the physical properties such as decreasing the size of
the bioactive molecule, decreasing the solubility of the bioactive
agent, precipitating the bioactive agents, condensing the bioactive
agent, or increasing the size of the complex.
[0056] Water-in-oil emulsions containing reverse micelles have been
used previously to study enzyme kinetics (e.g. Bru et al., Biochem
J, 310: 721-739 (1995)) and to form liposomes (e.g. Szoka et al.,
Proc Nat Acad Sci USA, 75: 4194-4198 (1978); Gruner et al.,
Biochem, 24: 2833-2842 (1984)), but the use of such emulsions to
modulate complexation of two compounds for the purpose of loading
liposomes has not been previously reported.
[0057] Emulsions can be formed by various methodologies, well
within the purview of ordinarily skilled artisans. Sonication,
vortexing, mechanical stirring, static mixing, homogenization,
injection, microfluidization, colloid mills, pressure emulsifiers
and/or Kady mills can be used to prepare emulsions of various types
including various orders of addition of materials. The emulsions of
the present invention are formed in two steps so that at least one
component, the bioactive agent or the complexing agent is
pre-sequestered within the water droplets of the lipid-stabilized
emulsion before addition of the aqueous dispersion of the other
agent.
[0058] Upon removal of solvent from the lipid-stabilized emulsion,
"liposomes" are formed. Solvent can be removed by any number of
methods including but not limited to rotary evaporation and
streaming of nitrogen.
[0059] "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.
[0060] Liposomes (see, e.g., Cullis et al., Biochim. Biophys Acta,
559: 399-420 (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 degradative effects
of factors, e.g., nuclease enzymes, present in the external
environment. Such protection of encapsulated content is, in the
case of nucleic acid molecules, demonstrated, for example, by the
type of agarose gel analysis set forth in Example 9, the results of
which are presented in FIG. 4.
[0061] 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.
[0062] 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.
[0063] 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 50-300
nm.
[0064] 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.
[0065] 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 (WO98/16240), 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.
[0066] Accordingly, in the most preferred embodiments of this
invention, the liposomes prepared by the method of the present
invention 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. Preferably, liposomes of
this invention have a lipid component which comprises a derivatized
phospholipid and an additional lipid. The derivatized phospholipid
has the formula:
##STR00001##
wherein: Z is selected from the group consisting of biotin, a
maleimide moiety, a group designated R.sup.3 and a group having the
formula X-Y; X is a linker selected from the group consisting of a
single bond and the group R.sup.4; and, Y is an enzyme cleavable
peptide comprising an amino acid sequence which is the substrate of
a cell-secreted peptidase. Each of R.sup.1, R.sup.2, R.sup.3 and
R.sup.4 is a group having the formula
--OC(O)(CH.sub.2).sub.n1(CH.dbd.CH).sub.n2(CH.sub.2).sub.n3(CH.dbd.CH).su-
b.n4(CH.sub.2).sub.n5(CH.dbd.CH).sub.n6(CH.sub.2).sub.n7(CH.dbd.CH).sub.n8-
(CH.sub.2).sub.n9CH.sub.3, wherein: n1 is zero or an integer of
from 1 to 22; n3 is zero or an integer of from 1 to 19; n5 is zero
or an integer of from 1 to 16; n7 is zero or an integer of from 1
to 13; n9 is zero or an integer of from 1 to 10; and, each of n2,
n4, n6 and n8 is zero or 1. Each of n1, n2, n3, n4, n5, n6, n7, n8
and n9 is the same or different at each occurrence.
[0067] For R.sup.1 and R.sup.2, the sum of
n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is independently an integer of from
12 to 22, whereas for R.sup.3 and R.sup.4, the sum of
n1+2n2+n3+2n4+n5+2n6+n7+2n8+n9 is independently an integer of from
2 to 22. Said derivatized phospholipid preferably comprises about
20 to 80 mole percent of the liposomal lipid.
[0068] Where R.sup.3 is
--C(O)(CH.sub.2).sub.n1(CH.dbd.CH).sub.n2(CH.sub.2).sub.n3(CH.dbd.CH).sub-
.n4(CH.sub.2).sub.n5(CH.dbd.CH).sub.n6(CH.sub.2).sub.n7(CH.dbd.CH).sub.n8(-
CH.sub.2).sub.n9CH.sub.3, the derivatized phospholipid is an
N-acylated phosphatidylethanolamine ("NAPE," see WO98/16199).
Preferably, R.sup.3 is then --OC(O)(CH.sub.2).sub.n1CH.sub.3, more
preferably --OC(O)(CH.sub.2).sub.10CH.sub.3.
[0069] 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.
[0070] NAPE-induced bilayer destabilization induces the bilayers to
fuse to biological membranes in the vicinity and hence, enhances
the bilayers' fusogenicity (Shangguan et al., Biochim Biophys Acta,
1368: 171-183 (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.2+ 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.
[0071] Where R.sup.3 is the acyl chain or the peptide, and hence,
where the derivatized phospholipid is a NAPE or peptide-lipid
conjugate, at least one of R.sup.1 and R.sup.2 is preferably an
unsaturated acyl chain, i.e., at least one of n2, n4, n6 and n8
therein is equal to 1. Unsaturated acyl chains include, without
limitation, palmitoleate, oleate, linoleate, linolenate, and
arachidonate chains. Preferably, the unsaturated acyl chain is an
oleate chain
("--OC(O)(CH.sub.2).sub.7CH.dbd.CH(CH.sub.2).sub.7CH.sub.3"). More
preferably, both R.sup.1 and R.sup.2 are oleate chains, i.e., the
derivatized phospholipid then is:
##STR00002##
wherein Z is R.sup.3 or X-Y. Most preferably, the derivatized
phospholipid is then:
##STR00003##
i.e., "N--C12 DOPE".
[0072] Where the derivatized phospholipid is N--C12 DOPE, the
liposomal lipid preferably also comprises a phosphatidylcholine,
preferably a PC having at least one unsaturated acyl chain, and
most preferably dioleoyl phosphatidylcholine. Preferably, the
liposomal lipid comprises about 70 mole % N--C12 DOPE and about 30
mole % DOPC (i.e., is a "70:30" formulation of N--C12 DOPE and
DOPC, wherein liposomal lipid concentrations are referred to herein
by ratio, and wherein such ratios are an indication of the relative
percentages in the liposomal lipid of the particular lipids
referred to).
[0073] 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., Biochim. Biophys.
Acta., 1149:180 (1993); Gabizon et al., Pharm. Res., 10(5):703
(1993); Park et al., Biochim, Biophys Acta., 257: 1108 (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).
[0074] Headgroup-modified lipids are typically
phosphatidylethanolamines (PE's), for example dipalmitoyl
phosphatidylethanolamine ("DPPE"), palmitoyloleoyl
phosphatidylethanolamine ("POPE") and dioleoyl
phosphatidylethanolamine ("DOPE"), amongst others. Such lipids have
headgroups generally derivatized with a polyethylene glycol, or
with an organic dicarboxylic acid, such as succinic or glutaric
acid ("GA"), or their corresponding anhydrides. The amount of the
headgroup-modified lipid incorporated into the lipid carrier
generally depends upon a number of factors well known to the
ordinarily skilled artisan, or within his purview to determine
without undue experimentation. These include, but are not limited
to: the type of lipid and the type of headgroup modification; the
type and size of the carrier; and the intended therapeutic use of
the formulation. Typically, from about 5 mole percent to about 20
mole percent of the lipid in a headgroup-modified lipid-containing
lipid carrier is headgroup-modified lipid.
[0075] Complexing agents generally including but are not limited to
a group oppositely charged to the bioactive agent including
spermine, spermidine, cobalt hexamine, calcium ions, magnesium
ions, polylysines, polyhistidines, protamines, polyanions such as
heparin and dextran sulfate, citrate ions and sulfate ions. One of
skill in the art will also recognize other useful complexing agents
useful in the method of the present invention.
[0076] Condensed nucleic acids 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. Condensed nucleic acids are
encapsulated in the liposomes at a level of at least about 0.5
micrograms per micromole liposomal lipid, or at least about 0.75,
1.0, 1.25, 1.5, 1.75 or 2 micrograms per micromole. More
preferably, the liposomes contain about 2 micrograms nucleic acid
per micromole lipid to about 20 micrograms per micromole.
"Condensed" as used herein in connection with nucleic acids refers
to nucleic acids which have been combined with one or more
polycations such that the nucleic acid strands are more tightly
packed than would be the case in the absence of the polycations.
Such packing allows nucleic acids to be encapsulated in liposomes,
yet leaves the nucleic acids in a transfectable,
transcription-ready conformation.
[0077] Accordingly, in preferred embodiments of this invention, the
method prepares liposomes comprising a condensed DNA and liposomal
lipid which comprises about 70 mole % N--C12 DOPE and about 30 mole
% DOPC. Such liposomes contain at least about 0.5 micrograms
condensed DNA per micromole of lipid.
[0078] Liposomes provided by the method of the present invention
can contain one or more bioactive agents in addition to the
complexed bioactive agent. 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.
[0079] 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.
[0080] The liposome of this invention can 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.
[0081] 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, Nairn (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.
[0082] Further provided herein is a method of encapsulating a
nucleic acid, e.g., DNA, in a liposome which comprises the steps
of: (a) combining an aqueous suspension of the nucleic acid with an
organic solution comprising a lipid, e.g., a derivatized
phospholipid and an additional lipid, so as to form a suspension of
reverse (inverted) micelles comprising the nucleic acid and the
lipid; (b) adding a polycation to the micellar suspension, so as to
condense the nucleic acid within the reverse micelles; and, (c)
removing the organic solvent from the suspension of step (b), so as
to form liposomes comprising the nucleic acid and the lipid from
the reverse micelles. The ratio of nucleic acid to liposomal lipid
achieved by the encapsulation method is at least about 0.5
micrograms nucleic acid per micromole lipid.
[0083] 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.
[0084] Polycations useful in the method of the present invention to
condense nucleic acids are any of the chemical compounds having
three or more ionizable groups which can be used to condense
nucleic acids, other bioactive agents or drugs; these include,
without limitation, polylysine, polyamines (e.g., spermine and
spermidine), hexammine cobalt (III), polyhistidine,
polyethyleneimine and the like. Preferably, the polycation is
spermine. Nucleic acids useful in the practice of this invention
include, DNA, e.g., genomic DNA, cDNA and plasmid DNA, linear or
closed, as well as RNA. Nucleic acids are suspended in aqueous
media by commonly understood, and readily practiced, methods, e.g.,
vortexing, of suspending macromolecules. Suitable aqueous media are
aqueous solutions of various additives, such as buffering agents,
and are substantially free of certain ingredients, such as salts
and nuclease enzymes; such media include, without limitation low
salt buffer ("LSB," see Example 3 hereinbelow).
[0085] Water-in-oil emulsions stabilized by phospholipids contain
reverse micelles. Reverse micelles (see Bru et al., Biochem J, 310:
721-739 (1995)) are amphipathic lipid-based structures in which the
lipids' hydrophilic domains are sequestered inside the micelles'
surfaces, while the lipids' hydrophobic domains are arrayed around
the exterior.
[0086] Emulsions with reverse micelles are formed, as described
above and in FIG. 2 hereinbelow, shield bioactive agents including
nucleic acids sequestered therein from the intermolecular contacts
which would otherwise lead to their aggregation in the presence of
complexing agent and unsuitability for incorporation into
liposomes. Said process is conducted so as to maximize the
percentage of the resulting liposomes containing the desired
complexes.
[0087] Within the emulsion, complexes are formed by way of the
exchange of added complexing agents, such as polycations, or
bioactive agents between the aqueous compartment of the reverse
micelles in the emulsion (see, e.g., Bru et al., FEBS, 282: 170-174
(1991); Fletcher et al., J Chem Soc Faraday Trans 1, 83: 985-1006
(1987)). In the case of encapsulation of DNA complexes, suitable
polycations are any of those polycations useful to condense nucleic
acids. For example, spermine and spermidine have both been used
(see, e.g., Chattoraj et al., J Mol Biol, 121: 327-337 (1978) and
Gosule et al., Nature, 259: 333-335 (1976), the contents of which
are incorporated herein by reference), in vitro to condense
individual plasmids, but only at low DNA concentrations, in order
to avoid aggregation of the condensed plasmids. Such concentrations
were minimal enough that, had liposome encapsulation of the
condensed nucleic acids been attempted, there would have been a
significant number of empty, i.e., non-DNA containing, liposomes.
Polylysine and hexamine cobalt (III) are also available for nucleic
acid condensation.
[0088] Concentrations of polycations suitable for condensing
nucleic acids are those concentrations which result in the
neutralization of a sufficient number of nucleic acid negative
charges, e.g., about 90% or more of the negative charges in the
case of DNA (Wilson et al., Biochem, 18: 2192-2196 (1979)).
Ordinarily skilled artisans are well able to determine suitable or
optimal polycation concentrations given the nucleic acid to be
condensed, the polycation used, nucleic acid concentrations and
polycation valency.
[0089] Moreover, additional factors well within the purview of
ordinarily skilled artisans to determine and account for can affect
the concentrations of polycations suitable for condensation of
bioactive agents such as nucleic acids. For example, NAPEs such as
N--C12 DOPE bear a net negative charge, by way of the additional
acyl chain; hence, such lipids can interact with positively charged
molecules, thereby diminishing the pool of polycations available
for nucleic acid condensation.
[0090] Accordingly, in such cases, it may be necessary to add an
amount of polycation above that otherwise required for nucleic acid
condensation. Such sufficient additional amounts of polycations can
be determined by a number of means, including, for example, the
type of partitioning experiments set forth in Example 4. Such
experiments provide data (see FIG. 3) showing the additional
polycation concentrations required for nucleic acid condensation.
For example, with the concentrations of nucleic acid and lipid used
in Example 3, 0.6 mM spermine was sufficient for plasmid DNA
condensation, but this amount increased to 0.85 mM in the presence
of the NAPE N--C12 DOPE, in the concentration set forth. However,
polycation concentrations greater than these, i.e., greater than
minimally necessary, can be used--for instance, again looking to
the conditions of Example 3 as exemplary, a final spermine
concentration of 8-20 mM in the emulsion was found to be optimal
for nucleic acid and lipid charge neutralization.
[0091] Ordinarily skilled artisans are well able to determine lipid
and nucleic acid concentrations suitable for the practice of this
invention. For example (see Example 3, hereinbelow), in order to
encapsulate condensed plasmid DNA within 200 nm spherical
liposomes, 200 micrograms of pZeoLacZ plasmid DNA in 125
microliters of LSB were combined with 30 micromoles of a 70:30 mole
ratio combination of N--C12 DOPE and DOPC.
[0092] Accordingly, preferred embodiments of this invention are
practiced with a condensed nucleic acid which is plasmid DNA, a
lipid comprising a derivatized phospholipid, e.g., N--C12 DOPE,
chloroform and spermine, e.g., at a concentration of about 1 mM or
greater.
[0093] Still further provided herein is a method of transfecting
the cells of an animal with a bioactive agent such as a nucleic
acid, said method comprising the step of contacting the cells with
a liposome of this invention containing the complexed nucleic acid.
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.
[0094] In vivo contact, especially where specificity or targeting
is desired, is aided by incorporating in the liposome a means of
either directing the liposome to a specific site, e.g., by
conjugating an antibody to the liposomes via streptavidin, causing
the liposome's contents to be preferentially released at a certain
site, e.g., by the incorporation of NAPEs or peptide-lipid
conjugates into the liposomes, 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. Accordingly, successful
transfection of cells with this plasmid (see Examples 10-12) is
readily determined by assessing the quantity of fluorescence
exhibited by the cells. Results of these experiments (see FIGS.
8-12), both the successful transfection of OVCAR-3 cells with the
pEGFP-1 plasmid, as well as the high level of expression of the
transfected plasmid in a significant percentage of the transfected
cells.
[0096] Such successful expression was observed only where the
transfected DNA had been polycation condensed; samples not
processed with spermine exhibited none, or almost no, fluorescence
(see FIG. 8). Quantification of fluorescent protein expression
(FIG. 9) demonstrated that transfection with polycation-condensed
DNA resulted in significant levels of expression, while
transfection of samples processed without spermine did not result
in quantifiable fluorescence. Moreover, transfection with free,
i.e., unencapsulated, DNA also resulted in no observable or
quantifiable fluorescence (FIGS. 8c and 9c).
[0097] This invention will be better understood from the following
examples, which are merely exemplary of the invention as defined in
the claims following thereafter.
EXAMPLES
Example 1
Materials
[0098] N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine
(transesterified from egg PC), DOPC, EPC and N--C12-DOPE were
purchased from Avanti Polar Lipids (Alabaster, Ala.). OVCAR3
ovarian carcinoma cells were purchased from NCI-Frederick Cancer
Research Laboratory (Frederick, Md.). The pEGFP-C1 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 (HBSS), RPMI 1640 and
heat inactivated fetal bovine serum and Lipofectin 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,
Me.). 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, Oreg.).
Example 2
Plasmid Purification
[0099] Two plasmids were used in this study: the pZeoSVLacZ plasmid
which is 6.5 kb, and expresses the lacZ 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-C1 plasmid, which
is 4.7 kb and expresses enhanced green fluorescent protein (EGFP)
from a human cytomegalovirus immediate early promoter, allowing
selection in E. coli using kanamycin, and in mammalian cells using
G418. Plasmids were purified from E. Coli (Baumann and Bloomfield,
Biotechniques, 19: 884-890 (1995))--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.
Example 3
Liposomal-DNA Formulations
[0100] Samples were prepared by diluting 200 .mu.g of DNA into 125
.mu.l of LSB, and then combining the resulting suspension with 1 ml
CHCl.sub.3 containing 30 .mu.mole of 70:30 molar ratio of N--C12
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.l
aliquot of LSB containing various concentrations of spermine (16 to
40 millimolar) was added to this emulsion with vortexing and
sonication. Samples without spermine were prepared in the same way,
except that spermine was omitted from the second 125 .mu.l aliquot.
Preparation of the samples with EPC was also identical, except that
7 mM spermine used.
[0101] 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 .mu.m
polycarbonate membrane filters (Poretics, Livermore, 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.
[0102] Other lipid compositions were used to encapsulate condensed
DNA according to the present invention. Plasmids were condensed and
encapsulated into liposomes as described in this example above and
sedimented as in Example 12. The lipid composition of the liposomes
was cholesterol hemisuccinate: cholesterol:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine:
1,2-dioleoyl-sn-glycero-3-phosphoethanolamine: dioleoyl
dimethylammonium propane:oleoyl acetate in a ratio of
12.5:2.5:50:12:10.5:10.5. After pelleting and washing the DNA/lipid
ratio for these liposomes was determined as in Example 10. Typical
DNA/lipid ratios were 1.4-2.1 .mu.g DNA per micromole of lipid.
Example 4
Spermine Partitioning
[0103] N--C12-DOPE bears a net negative charge which could
potentially interact with positively charged spermine and affect
the condensation process. Therefore, it was necessary to test the
spermine partitioning between DNA and liposomes of this composition
in a low salt buffer dialysis experiment. Experiments designed to
measure the partitioning of spermine between negatively charged
phospholipids and DNA were performed with a three chamber dialysis
device (Sialomed, Md.)--each chamber contained 250 .mu.l of liquid.
The desired amount of spermine was diluted into LSB and placed in
the center chamber, which was flanked by two 100,000 m.w. cutoff
dialysis membranes. The chamber on one side of the spermine chamber
contained 400 .mu.g of pZeoLacZ plasmid DNA in a total volume of
250 .mu.l LSB. The chamber on the other side contained either 250
.mu.l of LSB alone, or empty N--C12 DOPE/DOPC (70:30) liposomes,
prepared as described in Example 3 at a total lipid concentration
of 30 mM, in 250 W of LSB--in this arrangement, only spermine has
access to all three chambers. Since it is known that neutralization
of plasmid DNA by spermine leads to aggregation (FIG. 1), the
turbidity of the solution in the DNA-containing chamber was used as
a means of monitoring spermine partitioning. If the liposomes
completely sequestered spermine away from the DNA, the DNA would
not aggregate. The amount of available negatively charged lipid was
approximately twice the amount of negative charge on the DNA in
these experiments. Each dialysis device was rotated on a 12 inch
motorized wheel overnight (approximately 20 hours). The
DNA-containing chamber was then withdrawn with repeated pipetting
to mix the sample, and it was placed in a 250 .mu.l volume cuvette.
The absorbance at 400 nm was used to monitor turbidity against the
buffer background.
[0104] Spermine titration curves of DNA turbidity were constructed
for dialyses with, and without, liposomes present (FIG. 3). The
approximate shift in the curve due to the presence of liposomes was
used to calculate the relative binding constants for the lipids and
the DNA, assuming that each spermine molecule binds to four
nucleotide phosphate groups or four phospholipids, in simple
equilibria with association constants K.sub.DNA and K.sub.lipid,
respectively. In low salt, the dissociation constant for spermine
from DNA is know to be in the micromolar range (Wilson et al.,
Biochem, 18: 2192-2196 (1979); Gosule et al., J Mol Biol, 121:
327-337 (1978)). Therefore, the free concentration of spermine was
taken as negligible at the millimolar spermine concentrations
necessary for DNA aggregation in these experiments.
[0105] Fractional neutralization of the DNA phosphate groups by
spermine required for DNA aggregation, .gamma., was taken as 0.9,
based on the data obtained in the absence of liposomes. This is the
same value reported to be required for DNA condensation, agreeing
with the previous observation that aggregation accompanies
condensation at high DNA concentrations (Wilson et al., Biochem,
18: 2192-2196 (1979); Gosule et al., J Mol Biol, 121: 327-337
(1978)). Assuming [DNA-spm]=.gamma.[DNA].sub.total at the point of
aggregation and [lipid-spm]=the shift in the curve, one can use the
equation
K.sub.DNA/K.sub.lipid=[.gamma./(1-.gamma.)].times.[(Lipid.sub.total-shif-
t)/(shift)]
When the Lipid.sub.total is taken as the total concentration of
negatively charged lipid exposed on the outside of the liposomes
divided by four, the ratio of apparent equilibrium constants is
178, i.e. the spermine binding to DNA is much more avid than
binding to the lipids. The ratio of binding constants and the first
factor on the right are constants. Therefore the last factor on the
right can be used to calculate the shift in the spermine titration
curve for DNA condensation for any total lipid concentration,
including the higher effective concentration used in the
emulsions.
[0106] The data presented in FIG. 3 demonstrates that the presence
of the liposomes only slightly shifts the curve for DNA
aggregation. Thus, approximately 0.6 mM spermine in the initial 250
.mu.l emulsion was sufficient to condense the plasmid DNA, while a
total of 0.85 mM would be sufficient to condense the DNA in the
presence of the amount of N--C12-DOPE used. Therefore, it would be
expected that the plasmid DNA can be truly condensed in these
preparations without the complication of neutralization of the
negatively charged lipids which could destabilize the
liposomes.
Example 5
Light Microscopy of Liposome Samples
[0107] The precondensation of the DNA for potential encapsulation
into liposomes was tested. Massive aggregation occurred as judged
by a large turbidity change of the solution. This was not
unexpected since similar problems have been reported. Light
microscopy of plasmid aggregates (FIG. 1) was performed using 200
.mu.g of pZeoLacZ plasmid in 125 .mu.l LSB, mixed gently with 7 mM
spermine in 125 .mu.l LSB and incubated for 15 min at room
temperature.
[0108] Microscopic observation (FIG. 1A) demonstrated that the
aggregates were generally much larger than 1 .mu.m and often as
large as 5-10 .mu.m. The large size of these aggregates was further
confirmed by cryo-electron microscopy (FIG. 1B). Of particular note
at this magnification are the regular arrays of fibers, perhaps as
a result of spermine-induced condensation to a partially ordered
structure. There were also some curved rods suggestive of the
beginnings of toroidal structures, but no complete toroids.
Aggregates formed in this way were too large to be useful for a
delivery system.
[0109] For estimation of the size of N--C12-DOPE/DOPC (70:30)
liposomes containing DNA (FIG. 5), polystyrene beads with an
average diameter of 269.+-.7 nm (Duke Scientific Corp., Palo Alto,
Calif.) were diluted with H.sub.2O to a concentration appropriate
for microscopy, and samples of the N--C12-DOPE/DOPC (70:30))
liposomes containing DNA were used after extrusion and dialysis
without further dilution (approximately 20 mM lipid). The samples
were examined under an Olympus BH-2 fluorescence microscope
(Olympus, Lake Success, N.Y.) at 1000.times..
[0110] Results are presented in FIG. 5. The DNA-containing liposome
particles appeared relatively uniform in size and shape at this
magnification, and the approximate size of sample particles
appeared very similar to those obtained from dynamic light
scattering studies. Comparison of this DNA-containing liposome
particles sample to the spermine-aggregated DNA in FIG. 1A
demonstrates the benefit of condensing the DNA in the reverse
micelles according to the present invention prior to forming the
liposomes. There was no evidence of the very large aggregates
observed when spermine interacted directly with DNA in aqueous
solution, indicating that the emulsion condensation method may
greatly inhibit such aggregate formation.
Example 6
Particle Analysis by Light Scattering
[0111] The N--C12DOPE/DOPC preparations were characterized by
quasi-elastic light scattering. Particle size analysis was
performed using a Nicomp 370 particle size analyzer (Particle
Sizing Systems, Santa Barbara, Calif.). Samples were diluted
approximately ten-fold for analysis. A Gaussian analysis was
performed in the vesicle mode, and number weighted averages are
reported. The data for spermine-condensed pZeoLacZ plasmid DNA
prepared as in Example 3 could be fit by a Gaussian size
distribution with a number average particle size of 222.6 nm.
Example 7
Freeze-Fracture TEM
[0112] The fusogenic N--C12DOPE/DOPC preparations with encapsulated
DNA were further characterized by freeze fracture TEM. About 2
.mu.l of sample was deposited between two Balzers copper double
replicating holders and frozen in liquid propane. The sample was
fractured at -100.degree. C., 10.sup.-6-10.sup.-7 barr and shadowed
with platinum (<45.degree. C.) and carbon in a Balzers BAF 400
freeze-fracture device. Replicas were digested with 5% bleach
overnight, washed with distilled water and mounted on 300 mesh
grids. The images were obtained with a Philips 300 TEM.
[0113] Results are presented in FIG. 6. Most of the particles were
small in size (less than 400 nm), consistent with NICOMP results.
Because of the prevalent fracture plane though lipid bilayers,
observation of internal contents is rare with this technique.
However, a small number of the particles appeared to have some
encapsulated structures which could represent condensed DNA.
Example 8
Cryo Transmission Electron Microscopy
[0114] Cryo-EM was used to confirm the liposomal nature of the
preparations and to possibly visualize any encapsulated materials
For EPC sample and spermine-aggregated DNA, copper grids coated
with a holey carbon support were used without further treatment.
For N--C12-DOPE/DOPC (70:30) DNA-containing liposome samples, EM
grids with a holey carbon film were rendered positively charged by
placing a drop of a 0.1 mM polylysine solution on the grid surface,
and allowing it to sit for one min. The polylysine was blotted off
and the grid rinsed with several drops of distilled water, followed
by several drops of sample buffer. A 5 .mu.l aliquot of sample was
then placed on the grid, blotted to a thin film and immediately
plunged into liquid ethane. The grids were stored under liquid
nitrogen until used. They were viewed on a Philips CM12
transmission electron microscope (Mahwah, N.J.), operating at an
accelerating voltage of 120 kV. A 626 cryoholder (Warrendale, Pa.)
was used to maintain sample temperature between -177.degree. C. to
-175.degree. C. during imaging. Electron micrographs were recorded
of areas suspended over holes under low electron dose conditions.
Magnifications of 35,000.times. or 60,000.times., and underfocus
values of 1.8-2.5 .mu.m were used.
[0115] Results are presented in FIG. 7. When spermine was omitted
from the procedure for the N--C12DOPE/DOPC liposomes, primarily
unilamellar, relatively small but structurally heterogeneous
liposomes were observed (FIG. 7a), consistent with the Nicomp
analysis. A number of liposomes appeared tubular, probably as a
result of the osmotic gradient generated during the preparation
procedure. Some liposomes showed interior fiber-like structures
possibly representative of uncondensed DNA (left arrow).
Unencapsulated free fibers could also be seen (right arrow).
[0116] DNA containing N--C12DOPE/DOPC liposome samples prepared
with spermine (FIG. 7b,c) were also heterogeneous in size, shape
and lamellarity. Some particles were normal-looking liposomes with
no visible encapsulated material. However, others contained
electronically dense, well defined toroidal structures (FIG. 7b,
arrows) that were not seen in the samples without spermine. Such
structures were not related to the particular lipid used, as
toroidal (FIG. 7c, right arrow) and bent rod structures (FIG. 7c,
left arrow) were also observed in egg PC preparations, which tended
to be more stable under the cryo-EM sample preparation conditions.
The spacing between the fine lines within the rods and toroids were
uniform and significantly smaller than the spacing between two
membranes in multilamellar liposomes (star). These toroidal
structures bear great resemblance to the toroids and rods observed
when free DNA is condensed by spermine (Chattoraj et al., J Mol
Biol, 121: 327-337 (1978)) or other condensing agents (Arscott et
al., Biopolymers, 30: 619-630 (1990); Gosule and Schellman, J Mol
Biol, 121: 327-337 (1978)) in dilute solutions. The parallel and
concentric fine lines visible within the rods and toroids also
resembled the lines seen within the plasmid aggregates (FIG.
1b).
[0117] Membranes could be clearly observed around some of the
toroidal structures (e.g. FIG. 7b). It is likely that all of the
observed toroids are encapsulated within an ion-impermeable
barrier, since condensed DNA toroids cannot exist in the high salt
buffer in which the liposomes were ultimately suspended. Therefore,
it would appear that a major portion of these preparations consists
of liposomally encapsulated plasmid DNA.
Example 9
Agarose Gel Analysis
[0118] Protection of plasmid DNA from DNase digestion was evaluated
by agarose gel electrophoresis for liposomally encapsulated plasmid
DNA prepared with spermine and a control sample prepared without
spermine. A 50-.mu.l aliquot of the desired preparation was diluted
into 145 .mu.l HBSS without Ca2+/Mg2+, and 1 .mu.l of 0.2 M MgCl2
plus 2 .mu.l DNase I (20 units) were added with mixing. After a 6
hr incubation at room temperature, 2 .mu.l of 0.5 M EDTA was added
to stop the reaction. For undigested controls, a 50 .mu.l aliquot
of each sample was mixed with 150 .mu.l HBSS (w/o Ca2+/Mg2+).
Samples were then extracted with phenol/CHCl3/isoamyl alcohol and
precipitated with ethanol as described (Sambrook et al., Molecular
cloning: A laboratory manual, 2nd ed. Cold Spring Harbor Laboratory
Cold Spring Harbor, N.Y., pp B4-B5 (1989)). The pellet was
dissolved in 20 .mu.l TE (pH 8.0), 5 .mu.l of which was loaded on a
0.8% agarose gel. The gels were stained with a 1:10,000 dilution of
stock SYBR Green I nucleic acid gel stain (Molecular Probes) for 30
minutes, and visualized on a FotoSpectrum.RTM. ultraviolet
transilluminator (light box). Photographs were taken on the light
box with a Polaroid MP 4+ camera system. These photographs were
then scanned on a ScanJet IIC.RTM. (Hewlett Packard, Palo Alto,
Calif.) and digitized with Aldus Photostyler.RTM. (U-Lead Systems,
Torrance, Calif.).
[0119] Results are presented in FIG. 4 demonstrate that both
preparations allowed significant DNA protection or apparent
encapsulation.
Example 10
Quantitation Analysis
[0120] To quantitate DNA protection, DNA was extracted from each
aliquot and measured by a fluorescent assay. PicoGreen fluorescent
assays (Haugland, Handbook of fluorescent probes and research
chemicals, 6th ed. Molecular Probes, Inc., pp 161-162 (1996)) were
used to quantitate DNA that had been extracted by the
phenol/chloroform procedure set forth in Example 9. A working
solution was prepared by adding 100 .mu.l PicoGreen stock
(Molecular Probes) to 20 ml TE (pH 7.5). The extracted sample was
first diluted 100.times. with TE (pH 7.5). Then a 14 .mu.l aliquot
of the diluted sample was mixed with 986 .mu.l TE (pH 7.5) and 1 ml
PicoGreen working solution. The mixture was incubated in the dark
at room temperature for 4 minutes. The PicoGreen fluorescence was
recorded at room temperature on a PTI Alphascan fluorometer (South
Brunswick, N.J.), with excitation wavelength of 480 nm, and
emission wavelength of 520 nm, with a >500 nm highpass filter
(Schott Glass Technologies, Duryea, Pa.). The fluorescence of 1 ml
TE (pH 7.5) and 1 ml PicoGreen working solution mixture was used as
blank. The percent DNA being protected from DNase I digestion was
calculated by subtracting the blank, and taking the undigested
sample as 100%. Under our experimental conditions, the fluorescent
signal from digested DNA was insignificant.
[0121] The sample with spermine displayed 10.1.+-.5.6 percent
plasmid protection, while 19.0.+-.4.5 was protected in the sample
without spermine.
Example 11
Transfection Assays
[0122] The transfection activity of the liposomal preparations
encapsulating pEGFP-C1 plasmid DNA was then tested. OVCAR3 cells
were plated at 1.times.10.sup.5 cells per ml in 24-well plates, or
at 2.times.10.sup.5 cells per ml in 96-well plates in 1 ml or 0.1
ml per well, respectively, of RPMI 1640 with 10% heat inactivated
fetal bovine serum. Cells were allowed to grow for two days
(approximately 40-48 hours) before transfections were performed; at
this point the cells were at confluency. Transfection solutions
were prepared by dilution of appropriate liposome or DNA samples
into serum-free medium. The plates were aspirated to remove medium
and washed once with Dulbecco's phosphate buffered saline followed
by aspiration.
[0123] Transfection solutions (0.5 ml per well for 24-well plates,
0.1 ml per well for 96-well plates) were prepared by dilution of
dialyzed samples containing the pEGFP-C1 plasmid 10-fold into
serum-free medium (approximately 2 mM total lipid unless indicated
otherwise), 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., Human Gene Therapy,
8: 2117-2124 (1997); Dion et al., Virology, 231: 201-209 (1997)) 5
.mu.M of the histone deacetylase inhibitor, trichostatin A, was
added to each well to enhance expression. In the experiments
presented in the last 2 figures, another histone deacetylase
inhibitor, 5 mM sodium butyrate, was used instead.
[0124] After incubation at 37 degrees C. in a cell culture
incubator for 18-22 hours, the medium was aspirated and washed with
0.5 ml aliquots of Dulbecco's PBS. Photomicrographs were taken of
the samples still on tissue culture plates with an Olympus IMT-2
inverted microscope using the 10.times. objective. The PBS was
aspirated and 0.5 ml (0.1 ml for 96 well plates) of 5 .mu.M calcein
blue acetoxy methyl ester (CBAM) in PBS was added to each well and
incubated for 40 minutes at room temperature. Cells were washed
again with PBS, aspirated, and 0.5 ml (0.1 ml for 96 well plates)
of 1% C.sub.12E.sub.8 in TE buffer (pH 8.0) was added to each well.
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. Fluorescence of the plates was measured in a
Cytofluor II fluorescent plate reader (PerSeptive Biosystems,
Framingham, Mass.). Readings for calcein blue loaded in live cells
were made at excitation 360 nm and emission of 460 nm with a gain
of 80. These readings were verified to be linear with the number of
cells originally plated up to a level where confluence was
observed. For the data shown in FIG. 10, a liposomal pellet
separated from external DNA was used (Example 12). Because the
Ca.sup.2+ and Mg.sup.2+ levels in RPMI 1640 are significantly lower
than in serum, the data in FIGS. 11 and 12 were obtained after
supplementing the serum-free medium with Ca.sup.2+ and Mg.sup.2+ to
attain 1.2 and 0.8 mM, respectively, during the transfection.
[0125] An approximate conversion to EGFP fluorescence per unit
cellular protein could be estimated by measuring the average
protein concentrations of 48 hour cultures of the OVCAR-3 cells in
24 and 96 well experiments extracted with 1% Triton-100 detergent.
A bicinchoninic acid assay (Pierce Chemical, Rockford, Ill.) was
used with bovine serum albumin as a standard. For FIG. 9, bar "a,"
the total average background-corrected fluorescence reading per
well was 670 units. From a separate plate, the average total
cellular protein per well at the time of transfection (48 hr.) was
approximately 88.4 .mu.g/well giving 7.6 fluorescence units per
.mu.g of total cellular protein in a volume of 0.5 ml for the 24
well plate experiments. In FIG. 10, the data for bar "a" (96 well
experiment), represents an average background-corrected EGFP
fluorescence of approximately 420 units per well with an average
total cellular protein concentration of 27 .mu.g per well, giving
15.5 fluorescence units per .mu.g of total cellular protein in a
total volume of 0.1 ml. In FIG. 11, (96 well experiment), the bar
"a" fluorescence reading was 103 fluorescence units per .mu.g of
cellular protein.
[0126] To model intraperitoneal delivery (data in FIGS. 11 and 12),
the transfection was performed by first adding 50 .mu.l of a
concentrated cell-free lavage fluid from the peritoneal cavity of
tumor-bearing SCID mice (Example 13) to each aspirated well of a
96-well plate with OVCAR-3 cells grown as described above. To each
well was added 50 .mu.l of an N--C12-DOPE/DOPC liposome-DNA
formulation, prepared as described in Example 3, and resulting in a
final lipid concentration of approximately 10 mM and a final
encapsulated DNA concentration of approximately 7-14 .mu.g/ml
(total DNA of 67 .mu.g/ml). Incubations were performed as described
above. In this case, the peritoneal lavage fluid was adjusted to
approximately serum levels of Ca.sup.2+ and Mg.sup.2+ (1.2 mM and
0.8 mM, respectively) by adding a concentrated stock. The
liposomal-DNA solution was also adjusted to the same levels of
Ca.sup.2+ and Mg.sup.2+ by addition of the concentrated stock just
before addition of the liposomes to the cells.
[0127] The data in FIGS. 8 and 9 demonstrate that the formulation
of NC12-DOPE/DOPC (70/30) liposomes encapsulating
spermine-condensed plasmid DNA was active in transfection of
OVCAR-3 cells. The data show that the activity was dependent on the
presence of the spermine condensing agent and on the encapsulation
of the plasmid DNA within the liposomes. The data in FIG. 10
demonstrate again that the transfection activity is associated with
lipid-encapsulated DNA and not free external DNA. The data in FIGS.
11 and 12 show that transfection can also occur in the presence of
the potential interfering substances (e.g. serum proteins) found at
the intraperitoneal site of the OVCAR-3 tumors.
Example 12
Sedimentation of Plasmid DNA and Lipid Particles
[0128] In order to demonstrate the transfection activity of the
encapsulated plasmid DNA, it was necessary to separate free plasmid
DNA from liposome-encapsulated DNA. The following preparation
method was utilized. Liposomes, prepared by sedimentation to
removed external DNA, were used in the experiments, the results of
which are shown in FIG. 10. For sedimentation experiments,
N--C12-DOPE/DOPC (70:30) liposome samples were prepared by the
method of Example 3 with spermine, except that 200 mM sucrose was
included in the LSB. Headgroup labeled lissamine rhodamine
B-phosphatidylethanolamine (Rh-PE) was also added as a lipid probe
at 10 .mu.g/ml. A 500 .mu.l aliquot of the preparation was then
centrifuged at 16,000.times.g for 3 hours. After the supernatant
was removed, the pellet was resuspended in HBSS without
Ca.sup.2+/Mg.sup.2+. The suspension was centrifuged at
16,000.times.g for 3 hours. The pellet was resuspended in 500 .mu.l
HBSS without Ca.sup.2+/Mg.sup.2+. Aliquots of 50 .mu.l of each
fraction were taken for DNase I digestion (Example 9). After
phenol/CHCl.sub.3 extraction and ethanol precipitation, the plasmid
DNA in each aliquot was measured by PicoGreen assay (Example 10)
and used to calculate the percentage of protected plasmid, and the
percentage of total plasmid DNA, in each fraction.
[0129] For measurement of the distribution of lipids, a 40-.mu.l
aliquot of each fraction was dissolved in 0.2% C.sub.12E.sub.8 in a
total volume of 2 ml and the fluorescence monitored with an
excitation wavelength of 560 nm with a 550.+-.20 nm bandpass filter
(Melles Griot, Irvine, Calif.), and an emission wavelength of 590
nm. As a control, empty N--C12-DOPE/DOPC (70:30) liposomes were
prepared as above. After dialysis, 100 .mu.g of EGFP plasmid was
added to 500 .mu.l of the sample. The sample was then centrifuged
and quantitated for lipids and plasmid DNA.
[0130] Approximately 80% of the lipid pelleted under these
conditions, while only about 14% of the total DNA pelleted. The
transfection activity of the pelleted material is shown in FIG. 10.
Transfection activity was clearly associated with the lipid pellet,
i.e., with the liposome encapsulated DNA.
Example 13
Lavage Fluid
[0131] In order to test the effect of the intraperitoneal proteins
on the transfection activity of the preparations described herein,
a lavage fluid was prepared as described below. A cell-free 6 ml
HBSS lavage was taken from a SCID mouse 7 weeks after injection of
OVCAR-3 cells and was concentrated to 1 ml with a 10,000 mw cutoff
spin concentrator. Protein recovery is approximately 60%. This
fluid comprising approximately 10 mg/ml protein in HBSS was
supplemented with Ca.sup.2+ and Mg.sup.2+ to within the normal
serum range, added to cultured OVCAR-3 cells and gently mixed in
equal volume with liposomes in HBSS at the same Ca/Mg level, to
give a final lipid concentration of 10 mM.
[0132] The results of these transfection experiments are shown in
FIGS. 11 and 12. Despite the known inhibitory effects of serum
proteins on transfection efficiency, substantial activity remained
under these conditions using the formulation prepared by the method
of the present invention.
Example 14
Liposome Loading Efficiency
[0133] The efficiency of the loading of liposomes using a
precondensed DNA method was compared to the method described
herein. Liposomes were prepared as described by Ibanez et al.,
Biochem Cell Biol, 74: 633-643 (1996). pEGFP plasmid DNA was
dissolved at 66 .mu.g/ml in TS buffer (10 mM Tris, 1 mM NaCl, pH
7.0). 2 ml of this solution was mixed with 2 ml of 23 mM spermidine
in TS buffer to precondense the DNA yielding a very cloudy
solution. This was stored overnight at 4.degree. C. The next day, a
total of 9 .mu.mol of lipid was dissolved in 1 ml of diethyl ether.
To this was added 330 .mu.l of the DNA and spermidine solution with
vortexing. The mixture was then immediately sonicated three times
for 5 seconds each (Laboratory Supply sonicator #G112SOI). The
diethyl ether was then removed using a rotary evaporator at
37.degree. C. to form liposomes. Four such samples were prepared
for each lipid composition. The liposomes were pelleted at
200,000.times.g for 30 minutes. The supernatant was removed and 500
.mu.l more TS buffer added and the centrifugation repeated. After
three total cycles, the liposomes were extruded through MF
membranes with 0.45 .mu.m pores. Liposomes were used for
determination of encapsulation at this point or a portion of them
were dialyzed against Hanks balanced salt solution without
Ca.sup.2+ or Mg.sup.2+. For comparison liposomes were also prepared
as described in example 3. DNA encapsulation was measured as
described in examples 9 and 10. All digestions were for 6 hours.
Lipid concentration was measured by HPLC. All components but
cholesterol were quantitated using a Waters Sherisorb silica column
(3 .mu.m) with a mobile phase of
acetonitrile:methanol:H.sub.2SO.sub.4, 100:3:0.05, and detected by
UV absorbance. Cholesterol was measured on a Phenomenex Luna C18
column (5 .mu.m) with a mobile phase of 96:4 methanol:water and
detected by an elastic light scattering detector. Lipid
compositions tested are given in the table below.
TABLE-US-00001 TABLE 1 DNA/lipid ratio: (.mu.g DNA/.mu.mol total
lipid)* post Formulation Pre-digestion digestion Literature
values:** EPC:CHOL (1:1) 1.00 EPC:Brain PS:CHOL (4:1:5) 2.87
EPC:EPA:CHOL (4:1:5) 2.64 EPC:cardiolipin:CHOL (5:1:4) 2.53 Low
salt (10 mM Tris): EPC:CHOL (1:1) 0.52 0.07 EPC:Brain PS:CHOL
(4:1:5) 0.70 0.01 EPC:EPA:CHOL (4:1:5) 0.48 0.04
EPC:cardiolipin:CHOL (5:1:4) 0.78 0.09 Isotonic salt after
preparation: EPC:CHOL (1:1) 0.42 0.10 EPC:Brain PS:CHOL (4:1:5)
0.78 0.08 EPC:EPA:CHOL (4:1:5) 1.63 0.16 EPC:cardiolipin:CHOL
(5:1:4) 0.65 0.20 TLC 70:30 formulation 8.53 0.59 *Lipid recovery
estimated by HPLC. DNA quantitated by extraction and PicoGreen
assay. **Ibanez, M., Gariglio, P., Chavez, P., Santiago, C. W. and
Baeza, I. (1996) "Spermidine-condensed DNA and cone shaped lipids
improve delivery and expression of exogenous DNA transfer by
liposomes," Biochem Cell Biol, 74: 633-643 (1996).
[0134] Liposomes prepared by condensation of DNA within an emulsion
as described herein resulted in much higher DNA:lipid ratios than
liposomes prepared with precondensated DNA.
Example 15
Determination of Lamellarity of Liposomes
[0135] Liposomes composed of 70:30 N--C12-DOPE/DOPC and
encapsulating plasmid DNA were prepared as described in Example 3
and sedimented and washed as in Example 12. These liposomes also
contained an NBD probe at 0.5 mole % of the total lipid. Liposomes
were diluted to 80 .mu.M total lipid concentration in phosphate
buffered saline in a stirred fluorometer cuvette. NBD fluorescence
was measured with excitation at 450 nm and emission at 530 nm. A
final concentration of 20 mM sodium dithionite was injected into
the cuvette with the liposomes to reduce exposed NBD probe. FIG. 13
demonstrates that approximately 50-55% of the NBD signal
disappeared indicating that the liposomes in the preparation were
primarily unilamellar, i.e. about half the lipid probes were
exposed to the membrane-impermeable reducing agent, sodium
dithionite.
Example 16
Transfection of Subcutaneous Human Tumor in SCID Mice by
Intratumoral Injection
[0136] Human OVCAR-3 cells (2.times.10.sup.6) were injected
subcutaneously into SCID mice and allowed to grow for several weeks
until an average diameter of approximately 4-7 mm was reached.
[0137] Liposomes containing spermine, the pEGFP-C1 plasmid and 70
mole % of N--C12-DOPE and 30 mole % of DOPC were prepared as in
Example 3. The liposomal membranes also contained 0.5 mole %
Rhodamine-PE as a fluorescent liposome marker.
[0138] 0.11 ml of a liposome solution in Hanks Balanced Salt
Solution without calcium or magnesium (HBSS) at a total lipid
concentration of approximately 40 mM was injected directly into the
center of the tumors after adjustment of Ca.sup.2+ and Mg.sup.2+
levels to 1.2 and 0.8 mM, respectively. One day later, 0.11 ml of
20 mM sodium butyrate in HBSS was injected at the same sites. After
24 hours, tumors were excised and frozen. Later, 14-30 .mu.m thick
sections were obtained on a cryostat instrument at -20.degree. C.
and mounted frozen onto glass cover slides and secured with a cover
slip. Frozen tumor samples were mounted in O.C.T. embedding
medium.
[0139] Transgene expression of the pEGFP-C1 plasmid was assessed by
confocal microscopy of 20 .mu.m cryosections of the fixed frozen
tissue. The frozen sections were examined using the Olympus
BX50/Biorad MRC 1000 confocal microscope with an Argon/Krypton
laser. (Ex 488 nm, Em 515 for EGFP; Ex 568, Em 585 for rhodamine).
Areas of tissue sections were imaged at 20.times. magnification. No
image enhancements were used, but a color scale was applied for
figure preparation. FIG. 14 shows a pair of fluorescent images from
a tissue section taken from a pEGFP-C1 plasmid treated tumor. The
lower panel shows red fluorescence from the rhodamine-labeled
liposomes. The very high lipid signal (yellow) suggests that this
section was near the sight of liposome injection. The top panel
shows green fluorescence due to transgene expression. The signal
from the expressed plasmid is near, but not coincidental to, the
lipid signal, and appears to represent true expression of the
plasmid in the tumor. FIG. 15 shows another pair of fluorescent
images from a different tumor section with expressed EGFP. A
fluorescent image pair from a cryosection of control tumor tissue
is shown in FIG. 16. Weak, diffuse green fluoresence inherent in
the tissue is visible in the top panel, but no area of intense
fluorescence was found in any control tissue section. There was no
red fluorescence in any control tumor section.
Example 17
Transfection of Mouse Muscle In Vivo
[0140] The transfection activity of NC12-DOPE/DOPC (70:30)
liposomes encapsulating the pZeoSVLacZ plasmid was tested in vivo
in mouse leg muscle. The liposomes were prepared as described in
Example 3. Female DBA mice housed under standard conditions were
used for this experiment. 50 .mu.l liposomes containing the
pZeoSVLacZ plasmid was injected directly into one rear leg muscle
on day one. The injection site was near the fore thigh of the leg.
The opposite leg received either 50 .mu.l liposomes with pEGFP-C1
plasmid or no treatment. On day 2, 50 .mu.l 20 mM sodium butryate
in HBSS was injected into the liposome treated legs. The mice were
sacrificed on day 3 and the leg muscle excised as four sections:
fore leg, hind leg, fore thigh and hind thigh. One half of the
tissue from each section was frozen immediately in liquid propane,
while the other half was fixed in 4% paraformaldehyde,
cryoprotected with 30% sucrose then flash frozen in propane.
[0141] Transgene expression of the pZeoSVLacZ plasmid delivered via
the N12-DOPE/DOPC (70:30) liposomes was assayed using the Clontech
luminescent .beta.-gal kit. Unfixed muscle was defrosted, cut into
sections and homogenized as follows. 15 ml lysis buffer (9.15 ml
K.sub.2HPO.sub.4, 0.85 ml KH.sub.2PO.sub.4, 20 .mu.l Triton X100,
10 .mu.l DTT) was added for each 1 mg wet tissue and hand
homogenized for 5 min, then incubated at room temp for 20 min. The
samples were then spun for 2 min at 14,000 rpm to pellet tissue
debris. Aliquots of the supernatants were assayed for
.beta.-galactosidase activity as directed by Clontech. Light
readings were measured after 60 min using a Berthold plate
luminometer. Readings were averaged for different muscle sections
of the same type. The results are shown in FIG. 17. A significant
increase in .beta.-galactosidase activity over control was found in
muscle sections of the fore thigh. Slight increases were also noted
in hind leg and hind thigh tissue.
[0142] These results demonstrate the in vivo transfection and
transgene expression of pZeoSVLacZ plasmids when delivered to mouse
muscle using the N12-DOPE/DOPC (70:30) liposome vector.
Example 18
Comparison of Transfection with Liposomal Condensed DNA and
Cationic Lipoplex
Cationic Lipoplex Preparation:
[0143] Complexes of cationic lipids and helper lipids with plasmid
DNA were prepared shortly before use. Lipofectin was purchased from
Gibco BRL (Grand Island, N.Y.). For Lipofectin, the lipid alone was
incubated in serum-free medium for approximately 45 minutes before
complexation with DNA, as suggested by the manufacturer
(Invitrogen). Equal volumes of 4 .mu.g/ml DNA and 40 .mu.g/ml lipid
or equal volumes of 20 .mu.g/ml DNA and 200 .mu.g/ml lipid, all in
serum free RPMI 1640 medium, were mixed and allowed to incubate for
approximately 10-15 minutes before addition to wells of the tissue
culture plates. Ca.sup.2+ and Mg.sup.2+ were adjusted to 1.2 mM and
0.8 mM final concentration, respectively, by addition of a
concentrated stock just before addition of the lipoplexes to the
cells. The ratio of lipid/DNA used for Lipofectin was based on an
optimization comparing several ratios.
[0144] DC-Cholesterol/DOPE (4/6) complexes were formed essentially
as previously described (Muldoon et al., Biotechniques 22, 162-167
(1997)) and used within 15 minutes. The optimized DNA/lipid ratio
was used in all experiments, i.e. 4 .mu.g/ml DNA was mixed with
equal volume of 20 .mu.g/ml lipid or 20 .mu.g/ml DNA was mixed with
equal volume of 100 .mu.g/ml lipid.
[0145] All other complexes were formed using a set of cationic
lipids or lipid mixtures from a single manufacturer (Invitrogen).
These were prepared as suggested by the manufacturer at the
1.times. concentration and at the suggested lipid/DNA ratios.
Transfection assays were performed as described in Example 11.
Comparison to Cationic Lipoplexes:
[0146] The pelleted liposomes free of external DNA was used for
direct comparison of transfection to cationic lipoplexes at equal
DNA concentrations. These data are presented in Table 2 relative to
the liposomal treatment (all data after incubation in sodium
butyrate, a nontoxic activator of transgene expression (Tang et
al., Human Gene Therapy, 8: 2117-2124 (1997); Wheeler et al.,
Biochim Biophys Acta 1280 (1996); Gruner et al., Biochem 24:
2833-2842 (1984)) and at physiological Ca.sup.2+ and Mg.sup.2+
levels; all data was normalized in terms of total intracellular
esterase activity). In Table 2, the cell viability and transfection
are taken as 1.0 for the N--C12-DOPE/DOPC liposomes, i.e numbers
greater than 1.0 represent the factor by which either of these
parameters is higher in the test system. The transfection activity
of the N--C12-DOPE/DOPC (70:30) liposomes was generally in the
range of that found for cationic lipoplexes under these conditions.
Some lipoplexes gave considerably lower and some considerably
higher activity. Lipoplexes containing
3.beta.[N-(dimethylaminoethane)-carbamoyl]cholesterol and
dioleoylphosphatidylethanolamine (DC-chol/DOPE) were particularly
active. However, like all cationic lipoplexes, they were
considerably more toxic than the liposomes to the particular cells
used in these experiments, especially at the higher concentration.
This could be observed in the lower calcein blue fluorescence after
treatment with the lipoplexes (Table 2 data) as well as the
microscopic observation of rounded and disrupted cells after
treatment (data not shown). In several cases, the transfection
efficiency of cationic lipoplexes actually decreased relative to
liposomes at the higher concentration, probably as a result of
their toxicity. No toxicity was observed with the liposomally
encapsulated DNA. Interestingly, treatment with the liposomes
commonly caused an increase in the final calcein blue fluorescence
between 10 and 30 percent, possibly as a result of protection from
the effects of the incubation in serum-free medium.
[0147] The importance of the relatively low toxicity of this
liposomal plasmid DNA delivery system is not completely apparent in
the tissue culture systems because the transfection efficiency
reaches saturation at the relatively low levels of DNA used in the
experiments above. However, the situation in vivo is expected to be
much different. The large excess of nonspecific binding sites in
vivo may necessitate the use of high levels of DNA and/or multiple
injections for efficient expression in the target cells. There may
be a limit to the use of cationic lipoplexes in this situation
because of their toxicity.
[0148] Ovcar-3 cells were incubated with washed liposomal pellets
(as in FIG. 10) or lipid complexes with equal amounts of pEGFP-C1
plasmid DNA for 3 hours in serum-free medium. All transfection
procedures were as described in Example 11 and include adjustment
of Ca.sup.2+ and Mg.sup.2+ levels to 1.2 and 0.8 mM,
respectively.
TABLE-US-00002 TABLE 2 relative relative cell relative relative
cell transfection survival transfection Lipid/ survival 2 .mu.g/ml
10 .mu.g/ml 10 .mu.g/ml Mixture.sup.a 2 .mu.g/ml DNA.sup.b s.d.
DNA.sup.b,c s.d. DNA.sup.b s.d. DNA.sup.b,c s.d. 1 0.631 0.078
0.619 0.203 0.313 0.011 0.942 0.222 2 0.651 0.084 0.987 0.216 0.401
0.009 0.794 0.207 3 0.639 0.059 0.833 0.272 0.352 0.006 1.186 0.267
4 0.739 0.078 1.655 0.382 0.297 0.017 1.327 0.395 5 0.618 0.070
0.096 0.064 0.460 0.011 0.091 0.024 6 0.704 0.077 1.050 0.224 0.366
0.008 1.182 0.266 7 0.660 0.069 0.096 0.030 0.431 0.008 0.802 0.231
8 0.708 0.089 1.651 0.381 0.139 0.014 0.325 0.077 (lipofectin) 9
1.095 0.101 4.930 0.991 0.383 0.032 2.451 0.627 (DC-chol/ DOPE)
.sup.aCationic lipid complexes were prepared with the following
lipids: #1- 1:1 mixture of Tris-((2-glutaroyl-4-amino-N-dioctadecyl
amine)-4'-(2,5-diaminopentanoyl-(2'',5''-diaminopropylethyl))
amine, trifluoroacetate and
2-Amino-(2',2'-dimethyl)ethyl-methylphosphonic
acid-O-octadecyl-(1'-heptadecyl) ester, trifluoroacetate (Pfx-1);
#2- 2,5-Diaminopentanoyl-glycyl-glycyl-N-octadecyl-(1'-heptadecyl)
amide, trifluoroacetate (Pfx-2); #3- 1:1 mixture of
2,5-Diaminopentanoyl-(2',3'-di-3-aminopropyl)-2-aminoacetyl-2-aminoacetyl-
-N-octadecyl-(1'-heptadecyl) amide, trifluoroacetate and DOPE
(Pfx-3); #4- 1:1 mixture of
2-Amino-(2',2'-dimethyl)ethyl-methylphosphonic
acid-O-octadecyl-(1'-heptadecyl) ester, trifluoroacetate and
2,5-Diaminopentanoyl-2-aminoacetyl-N-dioctadecyl amide, trifluoride
(Pfx-4); #5- 1:1 mixture of
2,5-Diaminopentanoyl-(2,5-di-3-aminopropyl)-glutaroyl-N-octadecyl-(1'-hep-
tadecyl) amide, trifluoroacetate and
2,5-Diaminopentanoyl-(2,2,5,5-tetra-3-aminopropyl)-glycyl-N-dioctadecyl
amine, trifluoroacetate (Pfx-5); #6- 1:1 mixture of
2,5-Diaminopentanoyl-(2,5-di-3-aminopropyl)-1,2-diaminoehtyl-O-octadecyl--
(1'-heptadecyl)carbamic acid, trifluoride and DOPE (Pfx-7); #7-
Bis-(2,5-diaminopentanoyl-(2,5-di-3-aminopropyl)-cystyl-N-dioctadecyl
amine))disulfide, trifluoroacetate (Pfx-8); #8- lipofectin; #9-
DC-cholesterol/DOPE 4/6. .sup.bData is expressed relative to the
N-acyl-PE-containing liposomes, taken as 1.0, i.e., the numbers
represent the factor by which each lipoplex is more or less toxic
or active. Data from more than one series of experiments was
compared using lipid #2 as a standard. .sup.cTransfection
efficiency was measured by EGFP fluorescence as in FIG. 11 and
corrected for total cell esterase activity as reflected in the
total fluorescence of calcein blue (see Example 11).
[0149] One skilled in the art will readily appreciate the present
invention is well adapted to carry out the objects and obtain the
ends and advantages mentioned, as well as those inherent therein.
The compounds, compositions, methods, procedures and techniques
described herein are presented as representative of the preferred
embodiments, or intended to be exemplary and not intended as
limitations on the scope of the present invention. Changes therein
and other uses will occur to those of skill in the art that are
encompassed within the spirit of the appended claims.
* * * * *