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INTRODUCTION |
Despite their low efficiency in comparison to viral vectors
(1-3), cationic vectors retain high attractiveness in gene therapy due
to their theoretically excellent safety profile. Many research efforts
are currently dedicated to optimizing transfection efficiency of
nonviral vectors, and in this context, several novel chemical entities
have recently being disclosed (4-6). However, rational (as opposed to
empirical) chemical design of improved vectors requires a better
knowledge of the multistage process by which nonviral vectors promote
transgene expression in eucaryotic cells. Initial interaction of the
synthetic vector with cDNA to form a complex (7-9) as well as
delivery of the complex into the cell via an endocytosis
pathway (9-13) have been studied in detail. By comparison, much less
is known about intracellular trafficking of the transgene to the
nucleus where it is transcribed. Release of cDNA from the endosomes
is suggested to proceed by destabilization of the endosomal membrane
(14). Zabner et al. (9) postulated that the cDNA must
then dissociate from the cationic lipid vector before naked cDNA
enters the nucleus. To this end, anionic lipids normally found on the
cytoplasmic-facing monolayer of cell membrane can very potently
displace cDNA from the cationic liposome·cDNA complex
(14).
In this report, we demonstrate that synthetic polymers such as
polyethylenimine (4, 15) and polylysine differ from cationic lipids
inasmuch as (i) polymers promote gene delivery from the cytoplasm to
the nucleus and (ii) transgene expression in the nucleus is prevented
by complexation with cationic lipids but not with cationic polymers.
Our findings provide new rationale for future developments targeted to
improve the efficiency of nonviral vectors and highlight the in
vitro superiority of cationic polymer vectors over cationic
lipids.
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EXPERIMENTAL PROCEDURES |
Zeta Potential Measurements--
Polyethylenimine
(PEI)/DNA1 zeta potential was
determined using a Zetamaster 3000 (Malvern Instrument, Orsay, France)
with the following specifications: sampling time, 30 s; three
measurements per sample; viscosity, 1.014 centipoise; dielectric
constant, 79; temperature, 20 °C.
Electron Microscopy--
Carbon films were prepared by
sublimation on freshly cleaved mica and recovered by flotation on
Cu2+/Rh2+ grids (300 mesh, Touzard & Matignon,
Courtaboeuf, France). After overnight drying, grids were kept on a
blotting paper in a Petri dish. Immediately before sample addition,
grids were glow-discharged (110 mV, 25 s). A drop (5 µl) of
sample solution was left on the grid for 1 min. Complexes were
negatively stained with 30 µl of aqueous uranylacetate (1% w/w) for
20 s, and excess liquid was removed with blotting paper.
Observations were performed at 80 kV with a Philips EM 410 transmission
electron microscope.
Cell Cultures--
COS-7 cells, pancreatic epithelioid CFPAC-1
from a
F5808/
F508 cystic fibrosis patient and human A549 lung
carcinoma cells obtained from the ATCC (Rockville, MD) were cultured as
previously indicated (16).
Polycations and Plasmids--
PEI (25 kDa) and polylysine were
used as 10 mM monomer aqueous stock solution.
Dioctadecylamidoglycyl spermine (Transfectam®, Promega,
Madison, WI) was used as a 2 mM ethanolic solution. N-(1-(2,3-dioleolyloxy)propyl)-N,N,N-trimethylammonium
methylsulfate (DOTAP, Boehringer Mannheim) was used in aqueous solution
at 1 mg/ml. A pCMV-LacZ plasmid in which the
-galactosidase cDNA
was established under the control of the CMV promoter was used as a
reporter gene. Alternatively, we used a pCMV-cystic fibrosis transmembrane conductance regulator (CFTR) plasmid (a kind gift from J. Ricardo, Lisbon, Portugal) in which a CMV promoter drove expression of
the CFTR protein.
Cell Transfection--
Adherent cells were seeded in 24-well
plates (Life Technologies, Inc.). Amounts and volumes given below refer
to a single well. Two µg pf pCMV-LacZ were diluted into 50 µl of a
150 mM NaCl solution. The desired amount of 25-kDa PEI was
diluted into 50 µl of 150 mM NaCl, gently vortexed, spun
down (500 rpm for 5 s), and then incubated for 10 min. PEI was
used at PEI/cDNA ratios between 2 and 10 equivalents (PEI/cDNA
ratio calculated on the basis of PEI nitrogen/cDNA phosphate; see
Ref. 15). The cationic vector was added to the plasmid solution, mixed,
vortexed, spun down, and incubated for 10 min. Twenty-four h
post-transfection, cells were fixed for 15 min with 0.5% formaldehyde
and then analyzed histochemically using X-gal (17). All experiments
were done in duplicate.
Intracellular Plasmid Microinjections--
Cells were
microinjected at day 1 post-plating. In this procedure, we used the
Eppendorf microinjector 5246 system, the micromanipulator 5171 system,
and a Nikon Diaphot inverted microscope. Nuclear or intracytoplasmic
injections were performed with the Z (depth) limit option using a 0.3-s
injection time and 30-80-hectopascal injection pressure. Injection
pipettes (internal diameter 0.5 ± 0.2 µm) were pulled from
borosilicate glass capillaries. Plasmids were diluted at a final
concentration ranging from 0.01 to 100 µg/ml in an injection buffer
made of 50 mM HEPES, 50 mM NaOH, 40 mM NaCl, pH 7.4. 0.5% fluorescein isothiocyanate-dextran
(150 kDa) was added to the injection medium. The plasmid solution was injected into the cells, and the exact number of nuclearly or cytoplasmically injected cells was counted. Twenty-four h
post-injection, cells were fixed and analyzed for exogenous
-galactosidase expression. Dioctadecylamidoglycyl spermine was used
at six charge equivalents (six cationic amino groups per phosphate
group). For polylysine, we used 0.6 µl of polylysine 10 mM/µg of plasmid. This mixture yields a
polylysine/cDNA ratio of 2, corresponding to a PEI/cDNA ratio
of 5 on the basis of the ionic charges of the complexes. In a separate
set of experiments, we observed that polylysine transfection efficacy
peaked at 2 equivalents.2 For
DOTAP, we used 6 µl of DOTAP/µg of plasmid.
Determination of Injected Volume--
The volume of the solution
intracellularly microinjected was determined using radiolabeled probes.
0.5% fluorescein isothiocyanate-dextran supplemented with
99Tc (80 µCi/µl) or 111In (50 µCi/µl)
was injected into the nucleus of 100 cells. Immediately after
injection, cells were washed in phosphate-buffered saline containing
137 mM NaCl, 2.7 mM KCl, 8 mM
Na2HPO4, 1.5 mM
KH2PO4, 1 mM CaCl2, 1 mM MgCl2, pH 7.4. Cells were trypsinized and
suspended into 1.5 ml of culture medium, and the radioactivity of the
solution in cpm was determined. Different solutions of known volumes
and radioactive concentrations were used as internal standards. In COS-7 cells, 8735 ± 823 cpm/ml was measured in the cell lysate (n = four experiments) using 99Tc. This
value corresponded to an estimated injection volume of 923 ± 191 fl/cell (range: 587 to 1402 fl/cell). A comparable estimate was also
obtained using 111In. Thus, for a pCMV-LacZ plasmid
concentration of 100 µg/ml, the number of injected cDNA copies
was in the order of 10,000/cell. This value is 1 log lower than the
number of cDNA copies entering a cell during transfection with PEI,
as evaluated with electronic microscopy imaging (18). The large
variability we observed in injection volume estimate is consistent with
previous reports (19).
6-Methoxy-1-(3-sulfonatopropyl) quinolinium (SPQ) Fluorescence
Assay--
Our method has been reported in detail elsewhere (16).
Cells placed on glass coverslips were loaded with the intracellular dye
SPQ (Molecular Probes) by incubation in hypotonic (50% dilution) medium. The coverslip was mounted on the stage of a Nikon Diaphot inverted microscope equipped for fluorescence and illuminated at 370 nm. The emitted light was collected by a high resolution image
intensifier coupled to a video camera (Extended ISIS camera system,
Photonic Science) connected to a digital image processing board
controlled by FLUO software (Imstar, Paris, France). To standardize the
fluorescence response to solution changes, the initial fluorescence
level in the presence of I
was taken as zero. The control
Tyrode's solution for SPQ experiments contained 145 mM
NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 5 mM HEPES, 5 mM glucose, and 10 µM bumetanide, and the pH
was adjusted to 7.4 with NaOH. I
and
NO3
media were identical to the control
Tyrode solution except that I
or NO3
replaced Cl
as the dominant extracellular anion.
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RESULTS |
Efficiency of cDNA Injection in COS-7 Cells--
pCMV-LacZ
plasmids complexed or not with PEI were microinjected into COS-7 cells,
which were analyzed for exogenous
-galactosidase expression 24 h post-injection. Control experiments demonstrated that nuclear or
cytoplasmic injection with naked cDNA or with PEI·cDNA
complexes was not associated with cell toxicity. As illustrated in Fig.
1A, epifluorescence microscopy
discriminated cells that were correctly injected into the nucleus from
those injected into the cytoplasm. The efficiency of our injection
procedure was defined as the ratio between the number of cells per
Petri dish exhibiting X-gal blue staining (Fig. 1B) divided
by the number of injected cells exhibiting fluorescein
isothiocyanate-dextran fluorescence. Fig.
2A summarizes data obtained
after intranuclear injection and shows that the injection efficiency
increased according to a sigmoidal relation as the number of injected
cDNA copies increased in the range
10
1-104 copies/cell. Fig. 2A,
(right panel) shows that transgene expression was comparable
in the absence or presence of PEI, irrespective of the PEI/cDNA
ratio used, i.e. of the ionic charge of the PEI·cDNA complexes. Data obtained with intracytoplasmic microinjections are
summarized on Fig. 2B. As previously reported (20-22),
intracytoplasmic injection of naked cDNA led to a low expression of
the reporter gene (<15% of the cells), yet only at injected cDNA
copies/cell greater than 103 (Fig. 2B).
Remarkably, cDNA complexation with PEI increased transgene expression (Fig. 2B). Comparable expressions (40-50%) were
observed after injection of 104 copies in the cytoplasm and
102 copies in the nucleus. Increased expression when
cDNA was complexed with PEI was noticed only for PEI/DNA ratio
higher or equal to 2 (Fig. 2B) and may be a consequence of
the electrostatic charge and/or of the morphology of the complexed DNA.
Zeta potential (an indication of surface charge) measurements of
PEI·cDNA complexes performed with increasing amounts of PEI
showed that particles become neutral at a PEI/cDNA ratio around
3.5. The morphology of PEI·cDNA complexes may be inferred from a
combination of centrifugation and electron microscopy experiments.
Complexes formed at a PEI/cDNA ratio of 1 cannot be pelleted
(<5%, 10 min, 11,000 × g), presumably because of
extensive DNA looping out (Fig. 3),
whereas for PEI/DNA ratio 2-10, complexes were compact and
centrifugable (>80%). We next explored whether in cytoplasmic
injection experiments, the reporter gene was necessarily reaching the
nucleus at the time of cell division. To this goal, isolated cells
(<10% confluency) were injected in the cytoplasm with pCMV-LacZ
(104 cDNA copies/cell) complexed with 5 equivalents of
PEI (n = 4 assays; 107 cells). If plasmid cDNA only
had access to the nucleus during the transient breakdown of the nuclear
membrane that is associated with mitosis, then one would expect to see
only clusters of at least 2 cells expressing the transgene. We observed
that 20 cells out of 40 expressing the transgene were isolated cells (Fig. 4), indicating that cell mitosis
was not an absolute requirement for transgene expression.

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Fig. 1.
A, fluorescence microscopy of COS-7
cells after intranuclear (left) or intracytoplasmic
injections (right) with fluorescein isothiocyanate-dextran
containing solution. B, fluorescence microscopy and X-gal
staining in the same cells intranuclearly injected with pCMV-LacZ
(injected cDNA copies, 10,000/cell).
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Fig. 2.
Efficiency of intranuclear (A)
and cytoplasmic (B) cDNA microinjections in COS-7
cells. In A and B (left panels)
dose-effect relations with naked pCMV-LacZ cDNA (open
symbols) or with PEI·cDNA complexes at 5 equivalents
(filled symbols) are shown. A variable number of injected
plasmid copies as indicated on the x axis was used. Data are
mean ± S.E., with n between 178 and 650. Right panels show the effects of varying the PEI/cDNA ratio on
injection efficiency. Cells were injected with 10,000 plasmid copies.
Stars indicate significant difference, with
p < 0.05 (*) or p < 0.001 (***)
between cells injected with naked DNA and cells injected with DNA
complexed with PEI.
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Fig. 3.
Electron micrographs of PEI·DNA
complexes. Particles prepared at 1 equivalent of PEI (a
and b) show incomplete DNA condensation, with nucleic acid
looping out of the particle core. Higher input of PEI (10 equivalents
(c)) allows the formation of an homogenous population of
spherical complexes.
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Fig. 4.
Light microscopy of an isolated COS-7 cell
after intracytoplasmic injection of PEI·pCMV-LacZ complexes at 5 equivalents. X-gal staining.
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Expression Kinetics in Transfected and in Injected
Cells--
COS-7 were transfected with pCMV-LacZ complexed with
PEI. Eight h post-transfection, a low percentage of cells showed
-galactosidase activity (Fig. 5,
left panel). At 12 and 24 h post-transfection,
20
and 60% of the cells expressed the transgene, respectively. By
contrast, 4 h post-injection of naked or complexed cDNA into the nucleus, a high percentage of cells already expressed the transgene. In cells injected into the cytoplasm with naked cDNA, at
least 12 h of incubation were needed to detect transgene
expression. Eight h post-injection in the cytoplasm of PEI·cDNA
complexes, a detectable proportion of cells stained blue. Prolonging
the incubation period from 24 to 48 h only slightly increased
expression efficiency. Expression kinetics were also followed using a
pCMV-CFTR plasmid and a SPQ fluorescence assay. Measurements of
membrane permeability to halide under cAMP stimulation was used to
detect functional CFTR proteins in relation to post-injection time
(Fig. 5, right panel). Direct intranuclear PEI/cDNA
injection in COS-7 cells led to the expression of a CFTR-related
conductance within 4 h, i.e. within the same time frame
as
-galactosidase expression. Comparable kinetics were observed with
nuclear injection of naked cDNA. Microinjection of PEI·pCMV-CFTR
in the cytoplasm markedly delayed transgene expression in comparison to
nuclear injections, with detectable transgene expression 12 h
post-injection. We concluded that (i) exogene transcription and
translation are fast processes and (ii) transgene expression kinetics
after intracytoplasmic injection and transfection were comparable,
pointing at plasmid intracellular trafficking to the nucleus as the
rate-limiting step.

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Fig. 5.
Transgene expression kinetics after
transfection or microinjection in COS-7 cells. In the left
panel, COS-7 cells were either injected into the nucleus
(circles) or the cytoplasm (triangles) with naked
pCMV-LacZ cDNA (injected cDNA copies, 10,000 per cell (open symbols)) or with pCMV-LacZ complexed with 5 equivalents of PEI (filled symbols) or transfected with
pCMV-LacZ (2 µg) complexed with 5 equivalents of PEI (crossed
symbols). After various durations of incubation as indicated on
the x axis, cells were revealed for -galactosidase
activity and counted. * denotes p < 0.05; **
p < 0.01; and *** p < 0.001 versus cells injected with naked cDNA. , nuclear
naked; , nuclear PEI; , transfected; , cytoplasm naked; ,
cytoplasm PEI. The right panel shows pCMV-CFTR expression kinetics after direct microinjection of pCMV-CFTR in COS-7 cells assayed with SPQ fluorescence microscopy. The rate of SPQ dequenching under nitrate application in the presence of forskolin (10 µM), which reflects the cell permeability to halide upon
cAMP stimulation, is plotted against time elapsed after nuclear or
cytoplasmic injections with pCMV-CFTR complexed with PEI (filled
symbols) or nuclear injections with naked pCMV-CFTR (open
circles). *** denotes p < 0.001 versus
cells injected in the cytoplasm with cDNA·PEI complexes. Injected
copy number: 10,000 in all cases. Data are mean ± S.E. of 6-12
different cells.
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Transfection and Injection Efficiencies in COS-7, CFPAC-1, and A549
Epithelial Cell Lines--
Transfection efficiency was markedly
variable between different cell lines, since it was much greater in
COS-7 than in CFPAC-1 or A549 cells (Fig.
6A). Consistent with previous
studies using PEI (4), transfection efficiency was optimal at a
PEI/cDNA ratio of at least 5 equivalents, i.e. when
complexes were positively charged. Nuclear injection of cDNA·PEI
led to comparable expression for all cells (Fig. 6B),
suggesting similar transcription levels. By contrast, the efficiency
after cytoplasmic injections markedly varied with the cell line, with
the highest efficiency in COS-7 cells and the lowest in A549 cells.

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Fig. 6.
Transfection and injection efficiencies in
COS-7 (black bars), CFPAC-1 (gray bars), or
A549 cells (empty bars). A, cells were
transfected with pCMV-LacZ complexed with PEI at variable equivalents.
B, comparison of injection efficiencies in different cell
lines after intranuclear or intracytoplasmic injections of cDNA·PEI complexes at 5 equivalents. Injected copy number: 10,000 in all cases. ** denotes p < 0.01 versus
intracytoplasmically injected COS-7 cells.
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Expression Efficiency after Injection with Various Cationic
Vectors--
Finally, we investigated the influence of the vector's
molecular nature on transgene expression. In agreement with previous reports (9, 22), neither cytoplasmic nor nuclear injection of cDNA
complexed with cationic lipids such as dioctadecylamidoglycyl spermine
or DOTAP produced blue stained cells (Fig.
7). By contrast, nuclear and cytoplasmic
injections of cDNA complexed with polylysine produced consistent
expression of the reporter gene albeit with a lower efficacy than with
PEI (p < 0.001 in both cases).

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Fig. 7.
Percentage of X-gal-positive cells after
injection of cDNA pCMV-LacZ plasmid complexed with different
vectors. Intranuclear (filled bars) or intracytoplasmic
(empty bars) injections of COS-7 cells were performed with
pCMV-LacZ (copy number: 10,000/cell) complexed with 5 equivalents of
PEI or 6 equivalents of dioctadecylamidoglycyl spermine. Polylysine and
DOTAP were complexed to the plasmid as described under "Experimental
Procedures." Data are mean ± S.E. n, the number of
injected cell per experimental condition, varied from 325 to 470. DOGS, dioctadecyl amido glycyl spermine.
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DISCUSSION |
Our results demonstrate that cDNA complexation with cationic
polymers promotes gene accessibility to the nucleus, a property that is
not observed with cationic lipids. When 10,000 naked cDNA copies
were injected in the cytoplasm, only 13% of the cells expressed the
transgene. A comparable expression efficiency was obtained when less
than 10 copies of naked cDNA were directly injected into the
nucleus. This suggests that less than 1/1000 naked cDNA copies
injected in the cytoplasm were effectively trafficked to the nucleus,
an estimate that is in agreement with previous observations (9, 18). A
similar calculation shows that 1/100 cDNA·PEI complexes were
trafficked to the nucleus and thus that cDNA complexation increased
10-fold the access of the transgene to the nucleus. Our results also
show that in opposition to cationic lipids, cationic polymers when
injected in the nucleus do not prevent gene expression and thus likely
disassemble from cDNA in the nucleus, a process that is
supposedly fast since expression kinetics were very similar with coated or uncoated cDNA. We have also showed that
rupture of the nuclear envelope as occurs during cell division
was not an absolute requirement for penetration of
cDNA·PEI complexes into the nucleus. This observation is
in agreement with efficient in vitro (23) and in
vivo (24) PEI-mediated transfections of post-mitotic
cells such as neurons. In addition, we found that compaction
of plasmid cDNA into spherical particles rather than the
ionic strength of the cDNA·PEI complexes appeared as a
critical factor for transgene trafficking to the nucleus.
Finally, expression kinetics after intracytoplasmic injection
of cDNA·PEI complexes or classical PEI-mediated cell
transfection were comparable, suggesting trafficking through
the nuclear envelope as the rate-limiting step. The simplest
explanation to account for our results as a whole is that
plasmids coated with polymers but not with cationic lipids are
targeted to the nucleus. However, alternative mechanisms could
also very well explain our observations, including improved intracytoplasmic mobility or polymer-related protection of
cDNA from endogenous nuclease degradation. Current
programs in our laboratory are dedicated to evaluating this
latter hypothesis. Transfection efficiency with PEI markedly
varied depending on the cell line, with low levels of
expression in CFPAC-1 or A549 epithelial cells.
Intracytoplasmic injection of cDNA·PEI complexes in
CFPAC-1 and in A549 cells led a lower level of expression as compared with COS-7 cells, although this did not reach
significance in CFPAC-1 cells. These results suggest that intracellular
barriers to plasmid trafficking quantitatively varies with cell
type.
Nuclear transport in mammalian cells is a critical limiting step in
gene transfer using nonviral vectors (9, 18). Our data are in line with
a reasonable scheme proposed by Xu and Szoka (14) postulating that
during transfection, cDNA is released from cationic lipids in the
cytoplasm and is then trafficked uncoated by an inefficient mechanism
into the nucleus. The present work suggests that the sequence leading
to transfection with cationic polymers differs from the latter scheme
inasmuch as coated cDNA may penetrate post-mitotic nuclei where it
is most likely released by competitive interaction with genomic DNA
(25). Our data also suggest that cDNA compaction rather than the
ionic charge of cDNA complexes improves nuclear targeting.
Altogether these findings provide a novel mechanistic basis for
rational vector design.
We thank Dr. A. Faivre for his kind help
with injection volume measurement. We thank B. Le Ray for her expertise
with cell culture and M. J. Louerat for plasmid amplification.