Linear DNA low efficiency transfection by liposome can be improved by the use of cationic lipid as charge neutralizer.

Abstract

A plasmid expressing the beta-galactosidase enzyme was used to transfect Vero cells in order to evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore whether the structure of the complexes was interfering with the transfection, atomic force microscopy (AFM) was used. It has confirmed the difference between the linear and circular condensates: whereas the circular DNA:liposome complexes presented compact spherical or cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures, with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al. (1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA charge in order to improve the transfection efficiency of the linear DNA. Using this method, we were able to obtain the expression of the transgene without an associated toxicity observed with the linear DNA liposome delivery.

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Linear DNA Low Efficiency Transfection by Liposome Can Be Improved by the
Use of Cationic Lipid as Charge Neutralizer
Andrea von Groll,†Yan Levin,‡Marcia C. Barbosa,‡and Ana P. Ravazzolo*,†
Departamento de Patologia Clı ´nica Veterina ´ria, Faculdade de Veterina ´ria, and Departamento de Fı ´sica Teo ´rica, Instituto de
Fı ´sica, Universidade Federal do Rio Grande do Sul, Campus do Vale, Porto Alegre, RS, Brazil
A plasmid expressing the ?-galactosidase enzyme was used to transfect Vero cells in order to
evaluate the efficiency of a liposome-mediated transfection by circular and linear DNA. The
results obtained showed a low rate of transfection by linear DNA:liposome complexes. To explore
whether the structure of the complexes was interfering with the transfection, atomic force
microscopy (AFM) was used. It has confirmed the difference between the linear and circular
condensates: whereas the circular DNA:liposome complexes presented compact spherical or
cylindrical structures of about 100-800 nm, the linear DNA showed pearl necklace-like structures,
with pearls varying from 250 to 400 nm. On the basis of the theory proposed by Kuhn et al.
(1999), low concentrations of cationic amphihile were used to neutralize or reverse the DNA
charge in order to improve the transfection efficiency of the linear DNA. Using this method,
we were able to obtain the expression of the transgene without an associated toxicity observed
with the linear DNA liposome delivery.
Introduction
Transfection of DNA into eukaryotic cells has been used for
different purposes. Gene therapy (1) and DNA immunization
(2) are some of the clinical applications of this technology.
Delivery of DNA to the target cells could be mediated by
different vehicles: viral vectors, liposomes, cationic lipids, and
other reagents are used to overcome the membrane barrier. The
DNA transfection methods must take into account the negative
charge present on the cell membrane, the stability of the DNA
in the cytoplasm, the DNA transport to the cell nucleus, and
the expression of the target gene (3).
Liposomes are one of the strategies proposed to facilitate the
DNA delivery (4). Several studies have demonstrated that
endocytosis is the major pathway through which the DNA enters
the cell when associated with liposomes (5, 6). These results
are contrary to the mechanism originally proposed by Felgner
(7), fusion of the lipoplex with the cellular membrane followed
by the release of the DNA into the cytoplasm. The use of
liposomes in genetic immunization has recently been reviewed
in ref 8. The main advantages of this approach are the induction
of humoral and cellular immune response greater than by the
injection of DNA alone, the DNA protection from nucleases,
and the priming of professional antigen presenting cells (APCs).
Besides the vehicle used for transfection, the DNA topology
can influence the gene delivery and protein expression. Com-
parisons between circular and linear DNA have been performed
demonstrating differences of the transfection efficiency (9, 10),
as well as of the sustained protein expression using viral (11)
and nonviral (12) gene delivery methods. Although circular
supercoiled DNA has demonstrated a higher efficiency of
transfection when analyzed by protein expressing cell number,
linear DNA has been described as more stable in vivo (12) and
in vitro (10) experiments. Moreover, minimal linear DNA
transfection has been proposed as a method of choice to
vaccinate large animals, avoiding plasmid antibiotic resistance
genes (13).
Two main aspects of transfection were evaluated in this
work: the analysis of liposome:DNA complexes, comparing
linearized and circular forms, and the use of an alternative
method of transfection based on minimal concentration of
cationic amphiphile.
Our results indicate that under the same experimental
conditions, the linear DNA-liposome mediated transfection
exhibits a much lower efficiency than the complex in which
DNA appears in a circular form. To evaluate the differences
observed, atomic force microscopy (AFM) was used to analyze
the DNA complexes. Next, following the work of Kuhn et al.
(14), we employed a new strategy based on charge neutralization
or reversal by a minimal concentration of cationic amphiphile.
This approach demonstrated itself to be quite efficient for
transfection of the linear DNA with a very low toxicity.
Materials and Methods
Cells. Vero cells (ATCC/CCL-81) used for plasmid trans-
fection were cultured and maintained in Dulbecco’s modified
Eagle’s medium (D-MEM, SIGMA). Cultivation was performed
with 10% supplementation of fetal bovine serum (FBS) and 2%
was used to maintenance of cells in D-MEM.
Plasmid. The pCH110 (Pharmacia) plasmid was used for
transfection. It is a eukaryotic vector that expresses the
?-galactosidase enzyme under the control of SV40 promoter.
To linearize the plasmid, BamHI digestion was performed. The
DNA quantitation was obtained spectrophotometrically and
confirmed by the comparison with a high DNA mass ladder
(GIBCO BRL). This measurement was done using the Scion
Image software (Scion Corporation). Briefly, a standard curve
was generated with the ladder, and the bands observed were
*To whom correspondence should be addressed. Phone: (55) 5133166141.
Fax: (55) 5133167305. E-mail: ana.ravazzolo@ufrgs.br.
†Faculdade de Veterina ´ria.
‡Instituto de Fı ´sica.
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10.1021/bp060029s CCC: $33.50© 2006 American Chemical Society and American Institute of Chemical Engineers
Published on Web 07/12/2006

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measured in pixels. Finally, the pixel value obtained with the
linearized plasmid was then compared to the values of the
standard curve and determined by linear interpolation.
Transfection. Circular and linearized (see above) pCH110
were used to transfect Vero cells in 25 cm2flasks. Vero cells
(5 × 105/25 cm2) were trypsinized 15 h before transfection to
obtain around 80% of confluence. Transfections were carried
out with Lipofectamine (Invitrogen), as recommended by the
manufacturer; we shall refer to this as the manufacturer-
recommended concentration (MRC). Briefly, 2 µg of circular
or linearized plasmid was incubated with 15 µL of Lipo-
fectamine for 45 min at room temperature in a total volume of
200 µL of DMEM without serum. The complex, in a total
volume of 2 mL, was then incubated with the cells at 37 °C
during 5 h, when 10% FCS DMEM was added. Twenty-four
hours after transfection the medium was replaced by freshly
prepared 10% FCS DMEM. To evaluate transfection efficiency,
the cells were submitted to detection of ?-galactosidase activity
by staining with 5-bromo-4-chloro-3-indolyl-?-D-galactopyra-
noside (X-gal), as described previously (15). The percentage
of blue cells expressing the enzyme was estimated by inverted
optical microscopy visualization.
To overcome linearized plasmid transfection inefficiency by
Lipofectamine, we used an alternative method to transfect the
linearized pCH110. The theory of this method was described
elsewhere (14). Basically, a cationic lipid, dimethyldioctade-
cylammonium bromide (DDAB) (Sigma), at low concentrations
is used to neutralize the DNA charge. Both circular and
linearized plasmid DNA were submitted separately to transfec-
tion in a proportion of 2 µg of DNA to 35 µg of DDAB. DNA
and DDAB were incubated at room temperature in 1 mL of a
154 mM NaCl solution for 10 min. The next steps were the
same as those described for liposome transfection.
Atomic Force Microscopy. The complex DNA:liposome was
analyzed by atomic force microscopy (AFM) in a NanoScope
III (Digital Instruments). Complex formation was performed in
a final volume of 1 mL of Milli Q water. The proportion of
DNA:Lipofectamine was the same as that used to transfect Vero
cells, as well as the incubation period before analysis. The
mixture was deposited in a mica support and air-dried. The D
scanner with a range of 1-100 µm was used for all samples.
The scanning rate was 0.5-1.0 Hz.
Gel Electrophoresis. The formation of DNA:Lipofectamine
complex was also analyzed by gel electrophoresis (16). Linear-
ized and circular plasmids were incubated with Lipofectamine
in the proportion indicated by the manufacturer (MRC) and the
conditions established for the transfection experiment. Briefly,
350 ng of DNA was incubated with 2.6 µL of Lipofectamine
in D-MEM for 45 min at room temperature in a final volume
of 30 µL. Different linear DNA:Lipofectamine ratios (Table 1)
were also submitted to incubation to evaluate complex forma-
tion: two to five times more Lipofectamine than the MRC was
used. Finally, the samples were submitted to electrophoresis on
a 1% agarose gel and staining by ethidium bromide and were
visualized by UV transillumination.
Results
Liposome Transfection Efficiency of Circular and Linear-
ized pCH110. Linearized DNA has been described as more
stable than a circular plasmid when used to transfect eukaryotic
cells, leading to a more sustained expression of the target gene.
To evaluate the transfection efficiency of the liposome-mediated
methods, the two topologies were transfected using Lipo-
fectamine. Under the same conditions, no ?-galactosidase
enzyme activity was observed in Vero cells transfected with
the linear DNA:liposome complexes. On the other hand, using
a light microscope, the presence of transfected blue cells was
estimated to be around 40% for circular DNA. A vacuolization
of the cells was also observed, but not with the linear DNA
complex (data not shown). The toxicity associated with the
circular DNA transfection provides another motivation for
improving the efficiency of the methods based on the linear
DNA topology.
Atomic Force Microscopy Analysis of the DNA:Liposome
Complex. To evaluate whether the inefficiency of the linear
DNA transfection is due to an impaired complex formation, the
DNA:liposome complexes were analyzed by the AFM (Figure
1). The images obtained have demonstrated different patterns
of association between the lipofectamine and the circular and
the linear DNA. The circular complexes were found to be
composed of compact structures with variable sizes: spheres
of about 100-500 nm in diameter and cylinders of 300-800
nm long (Figure 1, A and B). On the other hand, linear DNA
showed pearl necklace-like structures with pearls varying from
250 to 400 nm (Figure 1, C and D).
Eletrophoretic Mobility of Linear and Circular DNA:
Liposome Complexes at Different Ratios. When associated
with liposomes, the circular DNA was very efficient at trans-
fecting the eukaryotic cells. Under the same conditions, the
transfection efficiency of the linear complexes dropped all the
way down to zero. To better understand this dramatic difference,
an eletrophoretic mobility analysis of the two types of complexes
was performed. It was found that during the electrophoresis,
the circular DNA:liposome complexes were retained inside the
gel. There was also no, or very little, incorporation of the
ethidium bromide, signifying that the condensed DNA is fully
protected (dressed) by the associated liposomes (Figure 2, lane
3).
In the case of the linear DNA, no transfection was observed
when using the same amount of Lipofectamine. The AFM
showed the condensate structures to be very different from those
of the circular plasmid. Furthermore, during the electrophoresis
there was no or very little retention of the linear DNA inside
the agarose gel (Figure 2, lane 4). The incorporation of the
ethidium bromide was signaled by a strong fluorescence,
comparable in its intensity to that of pure linear DNA without
any Lipofectamine (see Figure 2, compare lanes 2 and 4). This
suggests that there is only a very partial blockage of the DNA
by the associated Lipofectamine. This, again, is consistent with
our earlier AFM observations that showed that linear DNA:
Lipofectamine complexes retained their extended conformation.
We next used two, three, and five times the manufactured
recommended concentration (MRC) of Lipofectamine to try to
transfect the linearized plasmid. The DNA retention started only
when the concentration of the Lipofectamine was at least three
times the MRC (Figure 2, lane 6). Furthermore, even at five
times the MRC there was still some fluorescence, signifying
only a partial blockage of the linear DNA by the associated
Lipofectamine. The fraction of the DNA that was retained in
the gel (at five times the MRC; Figure 2, lane 7) was then used
Table 1. DNA and Lipofectamine Ratios Used for Analysis
DNA
topology
DNA (µg):Lipofectamine (µL)
ratiotransfectiona
circular
linear
2:15
2:15
2:30
2:45
2:75
yes
no
no
no
yes
aDetection of blue cells.
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to transfect the Vero cells. In this case, it was possible to
visualize some transfected blue cells (less than 5%), however,
with a very high toxicity to the cell monolayer (Figure 3A).
DDAB-Mediated Transfection. On the basis of the theory
proposed by Kuhn and colleagues (14), Vero cells were also
transfected with the linear DNA and the amphiphile molecule
DDAB. Ten to fifteen percent of the cells were expressing
?-galactosidase activity, as detected by incubation with the
substrate. Moreover, the pattern of expression was different
when compared to the one observed with the Lipofectamine
transfected cells (using circular plasmid), for which the blue
intensity and the distribution in the cytoplasm were higher
(Figure 3B). The DDAB transfected cells presented a less intense
color with a more granular distribution (Figure 3C).
Discussion
The low efficiency of the DNA uptake and its stability in
the cytoplasm of the transfected eukaryotic cells are some of
the major causes of low protein expression, despite the amount
of injected DNA (17). Endocytosis has been proposed as the
mechanism through which the DNA:liposome complexes enter
the cells. The lipid composition and the vesicle size are both
important for the transfection efficiency (18). A combination
of neutral and cationic lipids has been used to avoid the DNA
degradation (19) as, for example, in the case of Lipofectamine,
the commercial liposome used in this work.
The complex size is also an important feature for an efficient
cell transfection. The estimated size of the liposome:DNA
condensates is around 400 nm (20). Our AFM analysis, however,
showed very different patterns of the DNA complexation when
comparing circular and linear topologies: large compact ag-
gregates were observed with circular DNA, and necklace-like
structures with the linear plasmids. The different geometry of
the condensates might also account for the dramatic disparity
Figure 1. Analysis of DNA:Lipofectamine complexes by atomic force microscopy. Circular DNA:Lipofectamine complex images (A and B)
showed irregular compact structures of 100-800 nm, and the linear DNA:Lipofectamine complex images (C and D) presented pearl necklace-like
structures with pearls varying from 250 to 400 nm. The bars in the left lower corners represent 500 nm.
Figure 2. Eletrophoretic mobility of the linear DNA:Lipofectamine
complex. Agar gel electrophoresis of the linear DNA in association
with an increasing concentration of Lipofectamine (as compared to the
manufacturer recommended proportions (MRC, 2 µg DNA:15 µL
Lipofectamine)): 1× (lane 4), 2× (lane 5), 3× (lane 6), and 5× (lane
7). λ ) DNA marker (EcoRI and HindIII digested); lane 1 ) circular
DNA; lane 2 ) linear DNA; lane 3 ) circular DNA:Lipofectamine
complex (1×, 350 ng:2.6 µL).
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in the transfection efficiency between the two topologies. While
the transfection of the more compact circular plasmids is likely
to go through endocytosis, the pathway of entry of extended
linearized DNA aggregates might be quite different.
To improve the linear DNA transfection efficiency, we have
tried to increase the concentration of the Lipofectamine by as
much as 5-fold the MRC value. Except for a higher toxicity,
no improvement in the transfection efficiency was observed.
Considering the minimal linear DNA as a better choice for
the purpose of the DNA vaccination (12, 13) and a very high
toxicity of the liposome formulations for its transfection, the
use of other vehicles must be explored. One such possibility
has been studied in this work: use of the minimal concentration
of the cationic lipid. It should be noted that the cationic lipid
chosen by us, DDAB, also takes part of the liposome formula-
tion (16), however, at concentrations 2 orders of magnitude
higher than the ones used by us, 55 µM as compared to 13.4
mM for the same quantity of the DNA. Therefore, one advantage
of using amphiphile molecules at low concentrations is the
reduced toxicity and increased efficiency in the case of linear
DNA transfection. Again, we would like to stress that to
transfect linear DNA using Lipofectamine was practically
impossible.
When comparing liposome and DDAB transfected cells with
circular DNA, a different pattern of ?-galactosidase expression
was observed with light microscopy visualization. The blue
staining was more intense with the liposome than with the
DDAB-mediated transfection. This and the inefficacy of Lipo-
fectamine in transfecting linear DNA suggests different path-
ways of DNA cellular uptake between the two methods. In
addition, the analysis of ethidium bromide incorporation and
the electrophoresis of the linear DNA:DDAB complexes showed
no differences compared to DNA alone (data not shown),
suggesting that the linear DNA:DDAB complexes retain their
noncompact structure. More studies are necessary to elucidate
the mechanism by which the linear DNA:DDAB complexes
enter the cells.
To conclude, use of amphiphile molecules at low concentra-
tions can be a viable alternative for transfecting linear DNA
without a high toxicity associated with the liposome-mediated
delivery.
Acknowledgment
A.v.G. had a scholarship from CAPES, Ministe ´rio da Edu-
cac ¸a ˜o, Brasil. This research was partially supported by grants
from Ministe ´rio da Cie ˆncia e Tecnologia, PRONEX em Viro-
logia Veterina ´ria and by the CNPq, Conselho Nacional de
Desenvolvimento Cientı ´fico e Tecnolo ´gico. We thank Lizandre ´ia
Brombatti for technical assistance with atomic force microscopy.
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Accepted for publication June 6, 2006.
BP060029S
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