(Received for publication, August 29, 1995; and in revised form, December 7, 1995)
From the
We have developed a lipidic gene transfer vector, LPDII, where
DNA was first complexed to polylysine at a ratio of 1:0.75 (w/w) and
then entrapped into folate-targeted pH-sensitive anionic liposomes
composed of dioleoyl phosphatidylethanolamine (DOPE)/cholesteryl
hemisuccinate/folate-polyethlene glycol-DOPE (6:4:0.01 mol/mol) via charge interaction. LPDII transfection of KB cells, a cell line
overexpressing the tumor marker folate receptor, was affected by both
the lipid to DNA ratio and the lipid composition. At low lipid to DNA
ratios (e.g. 4 and 6), LPDII particles were positively
charged; transfection and cellular uptake levels were independent of
the folate receptor and did not require a pH-sensitive lipid
composition. Meanwhile, transfection and uptake of negatively charged
LPDII particles, i.e. those with high lipid to DNA ratios (e.g. 10 and 12), were folate receptor-dependent and required
a pH-sensitive lipid composition. The transfection activity of LPDII
was lost when the inverted cone-shaped DOPE was replaced by dioleoyl
phosphatidylcholine. LPDII particles with lipid to DNA ratios of 4, 6,
10, and 12 were 20-30 times more active than
DNA
3-
-[N-(N`,N`-dimethylethane)carbamoyl]cholesterol
cationic liposome complexes in KB cells and were much less cytotoxic.
On the sucrose gradient, LPDII particles had a migration rate in
between those of the free DNA and the DNA
polylysine complex. An
electron micrograph of LPDII showed a structure of spherical particles
with a positively stained core enclosed in a lipidic envelope with a
mean diameter of 74 ± 14 nm. This novel gene transfer vector may
potentially be useful in gene therapy for tumor-specific delivery.
The prospect of correcting human disorders through gene therapy has recently created much excitement in the scientific community(1) . The development of suitable delivery vectors for in vivo gene transfer is a prerequisite for the clinical application of therapeutic genes. Although several highly efficient biological (viral) vectors including recombinant retroviruses(2) , adenoviruses(3) , and adeno-associated viruses (4) are available, they suffer from immunogenicity, toxicity, lack of tissue specificity, difficulty in large scale production, and potential risk of inducing tumorigenic mutations and/or generating active viral particles through recombination(5) . Synthetic vectors such as liposomes, therefore, have become an attractive alternative due to their lack of immunogenicity and restraints on the DNA they can carry, the potential for tissue-specific targeting, relative safety, and relative ease of large scale production(6, 7) .
Both cationic and
anionic liposomal vectors have been evaluated for DNA delivery.
Cationic liposomes, first developed by Felgner et al. in 1987,
form slightly cationic complexes with the negatively charged DNA
molecules which can then be taken up by cells via charge
interaction(8, 9) . The disadvantages of cationic
liposomal vectors are the requirement for unnatural cationic lipids,
suboptimal DNA condensation, cytotoxicity, limited efficiency, lack of
tissue specificity, relatively large particle size, and potential
incompatibility with the biological environment, which is rich in
negatively charged macromolecules, extracellular matrix components,
lipoproteins, and cells. To improve DNA condensation, Gao and Huang ()recently designed a cationic
liposome
polylysine
DNA ternary complex (LPDI) consisting of
a condensed polylysine/DNA core and a cationic lipidic envelope. These
particles had a higher transfection activity than DNA
cationic
liposome complexes but required the removal of excess cationic lipids
for optimal activity.
pH-sensitive anionic liposomes have also been shown to mediate gene transfer but suffer from poor encapsulation efficiency due to the large size and the negative charge of the uncondensed DNA(11) . Passive encapsulation of DNA into anionic liposomes usually requires the use of a high concentration of lipids to increase the ``entrapped volume'' which leads to the generation of excessive amounts of empty liposomes. The entrapment efficiency, however, still rarely exceeds 20%. Moreover, treatments commonly used for improving liposome encapsulation or reducing particle size such as repeated freeze-thaw cycles, polycarbonate membrane extrusion, and sonication may lead to severe DNA damage(12) .
Another major focus in gene therapy vector development has been
tissue-specific targeting. A variety of targeting ligands have been
examined for liposome targeting including
folate(13, 14, 15) . The receptor for folate
has recently been identified as a tumor marker, especially among
ovarian carcinomas(16, 17, 18) . Folate
retains its receptor affinity when derivatized via its -carboxyl
group. Folate-conjugated macromolecules and liposomes have been shown
to be specifically taken up by cultured receptor-bearing tumor cells as
well as by implanted xenographs derived from human tumor cells in
vivo(19, 20, 21, 22) . As a low
molecular weight ligand, folic acid (M
=
441) has the advantages of being stable and nonimmunogenic compared to
their proteinacious counterparts, such as monoclonal antibodies (23) , and still having a relatively high receptor affinity (K
1 nM). Recently,
folate-targeted liposomes have been successfully used in the tumor cell
specific delivery of anticancer drugs and antisense
oligodeoxynucleotides in vitro(24) . Also,
folate-derivatized polylysine
DNA complexes combined with
inactivate adenoviruses have been shown to mediate tumor cell-specific
transfection(25) .
In this article, we describe a vector
where polylysine-condensed DNA is entrapped into folate-targeted
anionic liposomes via charge interaction. Due to its structural
similarity to LPDI, we name this new vector LPDII (liposome-entrapped polycation-condensed DNA). ()LPDII differs from LPDI in that anionic
lipids instead of cationic lipids are used. The description
``liposome-entrapped'' is based on structural information
derived from electron micrographs of LPDII presented in this article.
We find this novel DNA vector to be more efficient and less cytotoxic
compared to conventional cationic liposomal vectors. We evaluated the
effects of lipid/DNA ratio, lipid composition, excess free ligand, and
folate receptor expression on the transfection efficiency of LPDII. We
also examined the structure of LPDII by electron microscopy. The
potential advantages of LPDII as a gene therapy vector are discussed.
Folic acid, poly-L-lysine (hydrobromide, molecular
mass = 25.6 kDa), and cholesteryl hemisuccinate (CHEMS)
were obtained from Sigma. Dioleoyl phosphatidylethanolamine (DOPE),
dioleoyl phosphatidylcholine (DOPC), and dioleoyl phosphatidylserine
(DOPS) were purchased from Avanti Polar Lipids, Inc.
[
H]-Labeled cholesteryl hexadecyl ester was
purchased from DuPont;
3-
-[N-(N`,N`-dimethylethane)carbamoyl]cholesterol
(DC-Chol) and folate-PEG-DOPE were synthesized as described previously (9, 21) . Luciferase assay kits were purchased from
Promega. Folate-deficient modified Eagle's medium and F-12
nutrient mixture were purchased from Life Technologies, Inc.
Figure 1:
Effect of polylysine/DNA ratio on the
size of polylysineDNA complexes. (Inset: time-dependent
size increase of ``isoelectric'' complexes formed at the
polylysine/DNA ratio of 0.47:1 w/w.) The complexes were prepared by
mixing dilute solutions of polylysine and DNA in deionized water, as
described under ``Materials and Methods.'' Apparent sizes
were determined by light scattering. Because of the nonspherical shape
of the polylysine
DNA complexes, the size values should only be
used as a reference.
Folate-targeted LPDII particles were generated by mixing anionic
liposomes composed of DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) and
the cationic DNApolylysine (1:0.75 w/w) complexes. As shown in Fig. 2,, the apparent size of the resulting LPDII particles
varied with the lipid/DNA ratio. A large particle size was observed at
the lipid/DNA ratio of 8 presumably due to the formation of near
``isoelectric'' particles. At either higher or lower
lipid/DNA ratios, however, smaller particles were formed possibly due
to the formation of particles carrying either net positive or negative
charge, the size of which remained stable at 4 °C for at least one
week.
Figure 2:
Effect of lipid/DNA ratio on the size of
LPDII particles. LPDII particles were prepared by mixing
polylysineDNA (0.75:1 w/w) complexes with anionic liposomes
composed of DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol). Particle
size was determined by light scattering. Due to some degree of sample
heterogeneity, the size values should only be used as a
reference.
Figure 3:
Effect of lipid/DNA ratio on the
transfection activity of LPDII particles. LPDII particles containing
the pRSVL plasmid carrying the luciferase reporter gene were prepared
by mixing polylysineDNA (0.75:1 w/w) complexes with anionic
liposomes. KB cells were transfected and assayed for luciferase
activity, as described under ``Materials and Methods.''
Formulations: folate-targeted LPDII with the lipid composition
DOPE/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol) (
); folate-targeted
LPDII plus 1 mM free folic acid (
); nontargeted LPDII
with the lipid composition DOPE/CHEMS (6:4 mol/mol) (
). The
transfection activity of DNA
DC-Chol cationic liposome complexes
was 2382 ± 729 relative light units (RLU)/µg protein. Error bar = 1 S.D. (n = 3). Luciferase
activity unit: 2343 RLU is equal to the light emission of 0.02 µCi
of [
C]chloramphenicol measured by the
luminometer in the Cytoscint
mixture (purchased from
ICN), which in turn is equivalent to 1 luciferase light unit measured
under standard conditions.
The transfection
activity of LPDII particles was compared to that of the
DNADOPE
DC-Chol cationic liposome complexes. At optimal
lipid/DNA ratios (e.g. 4, 6, 10, and 12), the transfection
activity of LPDII particles was
20-30 times higher. This may
be the result of a much higher cytotoxicity of the DNA
cationic
liposome complexes. When more confluent (
70%) cells were used in
the transfection, less differences in transfection activity were
observed between LPDII and DNA
cationic liposome complexes (data
not shown).
We also analyzed the relative toxicity of LPDII
particles based on the amount of extractable cellular protein in the
lysate (Fig. 4). DNApolylysine complexes without lipid or
LPDII particles with lipid/DNA ratios of 4, 6, 10, and 12 showed only
low levels of cytotoxicity. Meanwhile, LPDII at the lipid/DNA ratios of
8 and 16, as well as the DNA
cationic liposome complexes, showed
relatively high levels of cytotoxicity (Fig. 4). Control LPDII
prepared from nontargeted liposomes showed unexpectedly high
cytotoxicity, especially at high lipid/DNA ratios.
Figure 4:
Effect of lipid/DNA ratio on the KB cell
cytotoxicity of LPDII particles. Cytotoxicity was evaluated by protein
content in the cellular extract following transfection and cell lysis
(see Fig. 3legend) using untreated cells as a reference.
Formulations: folate-targeted LPDII (); folate-targeted LPDII
plus 1 mM free folic acid (
); nontargeted LPDII (
).
For cells transfected with DNA
DC-Chol cationic liposome
complexes, the protein content was 32 ± 11% of the untreated
control.
To further examine the role of folate receptor in transfection, CHO cells, a cell line that does not overexpress the folate receptor, were transfected with folate-targeted LPDII particles. Efficient transfection was observed only with cationic particles with lipid/DNA ratios of 4 and 6 but not with anionic particles with higher lipid/DNA ratios (Fig. 5). This reaffirmed the role of folate receptor in the transfection activity of anionic LPDII particles in KB cells.
Figure 5:
LPDII transfection of CHO cells. CHO cells
in 24-well plates were transfected with LPDII containing pRSVL and
assayed for luciferase activity, as described under ``Materials
and Methods.'' Formulations: folate-targeted LPDII ();
folate-targeted LPDII plus 1 mM free folic acid
(
).
In
summary, transfection with LPDII particles was independent of the
folate receptor at cationic charge ratios and receptor-dependent at
anionic charge ratios. This result was in agreement with previous
studies by Plank et al.(14) on a liver cell targeted
lipidic DNA vector. In addition, LPDII particles also had a greater
transfection activity and a lower cytotoxicity compared to
DNAcationic liposome complexes.
Figure 6:
Effect of lipid/DNA ratio on the KB cell
DNA uptake. KB cells were incubated with LPDII particles containing I-labeled pRSVL plasmid and assayed for DNA uptake by
cell-associated radioactivity after washing twice with
phosphate-buffered saline, as described under ``Materials and
Methods.'' Formulations: folate-targeted LPDII(
);
folate-targeted LPDII plus 1 mM free folic acid (
);
nontargeted LPDII (
). Uptake of DNA
DC-Chol cationic
complexes was 0.0045 ± 0.0014 µg/µg protein which
corresponds to
8.7% of the total DNA
added.
Figure 7:
Effect of lipid composition on the KB cell
transfection activity of LPDII. Formulations: folate-targeted LPDII
composed of DOPE/DOPS/folate-PEG-DOPE (8:2:0.01 mol/mol) ();
folate-targeted LPDII composed of DOPE/DOPS/folate-PEG-DOPE (8:2:0.01
mol/mol) plus 1 mM free folic acid (
); folate-targeted
LPDII composed of DOPC/CHEMS/folate-PEG-DOPE (6:4:0.01 mol/mol)
(
).
Figure 8:
Electron micrographs of
DNApolylysine complexes LPDII particles. Samples were stained
with 1% uranyl acetate and examined under electron microscope as
described under ``Materials and Methods.'' Panels: A, DNA
polylysine (1:0.75 w/w) complexes; B,
LPDII, unpurified; C, LPDII, fraction 6 on the sucrose
gradient (see Fig. 10); D, LPDII, fraction 7 on the
sucrose gradient. Scale bar = 200 nm (Panel
A).
Figure 10:
Fractionation of LPDII on a sucrose
gradient. A, LPDII particles containing I-labeled DNA and
H-labeled lipids were
fractionated on a 0/20/40% (0.5/1/1 ml) discontinuous sucrose gradient
at 100,000
g for 35 min. Ten 250-µl fractions were
collected starting from the top of the gradient and counted for DNA and
lipid radioactivity.
I-labeled DNA (
);
H-labeled lipids (
). B, KB cell transfection
activity of the major DNA-containing fractions. C, sucrose
gradient profile of free DNA (
), free liposomes (
), and
DNA
polylysine complexes (
).
One possible mechanism by which LPDII particles may have formed is illustrated in Fig. 9. In this model, DNA is first condensed into a cationically charged complex with polylysine. The cationic complex is then entrapped into anionic liposomes by spontaneous charge interaction.
Figure 9: Possible mechanism for the formation of LPDII. The targeting ligand in the current study is folate.
In Fig. 8B, we observed some very small
(<20 nm), possibly lipidic, particles (arrows). In an
attempt to remove these, LPDII was fractionated on a 0/20/40%
discontinuous sucrose gradient. As shown in Fig. 10A,
the majority of the LPDII particles migrated as an intermediate density
peak at fractions 6 and 7. Free DNA and free liposomes remained mostly
in fractions 1 and 2, whereas DNApolylysine complexes migrated to
the last fraction in the controls (Fig. 10C). Electron
micrographs of fractions 6 and 7 of the LPDII particles (Fig. 8, C and D) looked the same as those of the unpurified
particles (Fig. 8B) except for the absence of the small
low electron density particles. The sucrose gradient profile of LPDII (Fig. 10A) also showed a strong correlation in the
distribution of
I-labeled DNA and
H-labeled
liposomes. The fractions at the top of the gradient may consist of a
small amount of empty liposomes and free DNA, since the total lipid was
in excess in terms of charge in these LPDII particles. The differences
in the migration rate of the remaining fractions may be due to the
varying degree of DNA condensation by polylysine resulting in different
particles densities. Major DNA-containing fractions including 1, 2, 6,
7, and 10 were tested for KB cell transfection. As shown in Fig. 10B, very little transfection activity was
observed with fractions 1 and 2, which probably consist of mostly free
DNA and free liposomes. Meanwhile, fraction 6 had approximately the
same transfection activity as the unpurified LPDII particles (Fig. 3). Fractions 7 and 10 had less transfection activity,
possibly due to the high sucrose concentration in the transfection
medium derived from the gradient. These results suggested that the
small amount of contaminating particles found in unpurified LPDII
particles had little or no inhibitory effects on KB cell transfection,
and that purification is not required for LPDII particles prepared at a
proper lipid/DNA ratio. This is in contrast with LPDI particles
described by Gao and Huang,
which required the use of
excess amounts of cationic lipids in its preparation and, therefore,
the subsequent removal of excess lipids by sucrose gradient for optimal
activity.
We have developed a self-assembled transfection vector in
which highly condensed DNApolylysine complexes were incorporated
into folate-targeted anionic liposomes. We found that these vectors
were more active in transfection and less cytotoxic. Unlike
DNA
cationic liposome complexes which usually undergo slow
aggregation upon storage, our novel vectors were small in size
(
70-80 nm) and were not prone to aggregation when carrying a
net charge. Depending on the lipid/DNA ratio, either cationic (at low
lipid/DNA ratios) or anionic (at high lipid/DNA ratios) LPDII particles
could be generated. The cationic particles were highly active in
transfection but were not tissue specific, therefore, may be useful as
a high efficiency single-vial nonspecific transfection vector.
Meanwhile, the efficient receptor-dependent transfection activity of
anionic LPDII particles makes them possible candidates for
tissue-specific gene delivery. The requirements for folate receptor
expression and pH-sensitive lipid composition strongly suggested the
roles of the receptor and endocytosis in LPDII transfection.
Similar
to the LPDI particles developed by Gao and Huang, LPDIIs
are compact spherical particles with a highly condensed DNA core. The
lipidic coating on these particles may not only further stabilize the
structure of the particle, protect the DNA from the nucleases, but also
confer endosomal disruption activity and a site for ligand attachment.
Unlike LPDI, negatively charged LPDII is targetable and do not require
complicated steps of purification or the use of cationic lipids in its
composition.
The design of LPDII particles eliminated potential problems associate with traditional anionic liposomal DNA vectors such as low encapsulation efficiency and generation of excessive empty liposomes. Since no unnatural cationic lipids are required, and at high lipid/DNA ratios the overall charge of the particles were anionic, LPDII particles may be somewhat more biocompatible in vivo compared to the positively charged DNA vectors. Taken together with their compact size, stability and high transfection activity, we believe that LPDII particles may potentially be useful for tissue-specific DNA delivery in vivo.