Biomaterials 22 (2001) 471}480
Poly(ethylenimine)-mediated gene delivery a!ects endothelial cell
function and viability
W.T. Godbey , Kenneth K. Wu, Antonios G. Mikos *
Department of Bioengineering, Rice University, P.O. Box 1892, MS 142 Houston, TX 77251-1892, USA
Division of Hematology and Vascular Biology Research Center, The University of Texas Health Science Center at Houston,
6431 Fannin Street, Houston, TX 77030, USA
Received 4 April 2000; accepted 9 June 2000
Abstract
Poly(ethylenimine) (PEI) was used to transfect the endothelial cell line EA.hy 926, and the secreted levels of three gene products,
tissue-type plasminogen activator (tPA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand Factor (vWF), were
assessed via ELISA. We found that the levels of these gene products in cell supernatants increased by factors up to 16.3 (tPA), 8.3
(PAI-1), or 6.7 (vWF) times the levels recorded for untreated cells, and roughly correlated with the percentage of cells that expressed
the reporter plasmid. Transfections carried out using promotorless constructs of the same reporter plasmid also yielded increases in
tPA, PAI-1, and vWF to similar extents. Additionally, data regarding cell viability were gathered and found to inversely relate to both
the e!ectiveness of the PEI used for transfection and the secreted levels of the three mentioned products. There appeared to be two
distinct types of cell death, resulting from the use of either free PEI (which acts within 2 h) or PEI/DNA complexes (which cause death
7}9 h after transfection). Cells were also transfected by poly(L-lysine) and liposomal carriers, and increases in secreted tPA similar to
those seen with PEI-mediated transfection were observed for positively transfected cells. The results of these investigations indicate
that non-viral gene delivery can induce a state of endothelial cell dysfunction, and that PEI-mediated transfection can lead to two
distinct types of cell death. 2001 Elsevier Science Ltd. All rights reserved.
Keywords: Polyethylenimine; Polylysine; Liposomes; Transfection; Gene products; Endothelial cells
1. Introduction
Poly(ethylenimine) (PEI) has been demonstrated as an
e$cient gene delivery vehicle both in vitro and in vivo
[1]. However, questions as to the mechanism of PEImediated transfection remain largely unanswered. Recently, it was discovered that PEI/DNA complexes enter
cell nuclei intact during the transfection process [2]. This
"nding brings to light the question of what e!ect the
polycationic polymer has on cells after nuclear entry.
Polycations (such as PEI) act to spontaneously bind with
and condense plasmid DNA in the test tube [3,4], so it is
not unwarranted to predict that PEI in cell nuclei might
also interact with host DNA. Such an alteration of the
nuclear environment has the potential to alter host transcriptional processes and thereby a!ect the well being of
the cell (or organism) as a whole.
* Corresponding author. Tel.: #713-348-5355; fax: #713-348-5353.
E-mail address: [email protected] (A.G. Mikos).
With the rapid progress in gene delivery research, it is
very important to assess the global e!ects of transfection
on cells or organisms. Having genes delivered to and
expressed inside cells is not the only goal of gene therapy;
one must be able to do this without harming normal host
cells before a delivery system can be considered successful. PEI, being a relatively new transfection vector, must
be assessed in terms of its overall e!ects on cells before it
is considered for human testing. Alteration of endogenous gene expression resulting from any transfection
method should help guide the direction of research for
that given method. Additionally, a further understanding
of an agent's mechanism for gene delivery can help with
the design of novel vectors in the future.
The work described here addresses the e!ects of PEImediated transfection on endothelial cell function and
viability. We examined the levels of three human endothelial cell products*tissue-type plasminogen activator
(tPA), plasminogen activator inhibitor type 1 (PAI-1),
and von Willebrand Factor (vWF)*to determine
whether the extracellular levels of these products
0142-9612/01/$ - see front matter 2001 Elsevier Science Ltd. All rights reserved.
PII: S 0 1 4 2 - 9 6 1 2 ( 0 0 ) 0 0 2 0 3 - 9
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W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
remained una!ected by PEI-mediated transfection or were
otherwise altered. We also sought to discern whether any
e!ects on cell function were comparable for both free and
complexed PEI, as well as several other non-viral transfection methods. Finally, we wanted to know whether these
e!ects had any in#uence on cell viability.
The liposome formulation DOTMA/DOPE, at 1 : 1
(w/w), was purchased from Gibco (Grand Island, NY) as
Lipofectin2+, and DOSPA/DOPE, at 3 : 1 (w/w), was
purchased from the same supplier under the name
Lipofectamine2+.
2.4. Transfection
2. Materials and methods
2.1. Cells
The experiments described here were performed
in vitro on the EA.hy 926 cell line (passage 49}54). This is
a human endothelial cell line originally derived by
a fusion of human umbilical vein endothelial cells
(HUV-EC) and cells from a human lung carcinoma (cell
line A549, derived by Lieber et al. [5]) [6]. They were
chosen because they are immortal and they demonstrate
many characteristics of human endothelial cells, such as
human factor VIII-related antigen expression [6] and
prostacyclin production [7].
2.2. Plasmids
The expression plasmid used for transfection was
pEGFP-N1 (Clontech, Palo Alto, CA), which utilizes
a strong CMV-immediate early promoter to code for an
enhanced green #uorescent protein (GFP). A promotorless GFP plasmid, pEGFP-1 (Clontech), was also used
for some experiments. The plasmids were each ampli"ed
to su$cient quantities by standard molecular biology
techniques, including harvesting and puri"cation with
a Quiagen Maxi-Prep kit (Quiagen, Chatsworth, CA).
The purities of the DNA used were measured by spectrophotometry, with an A /A
ratio of at least 1.8.
2.3. Non-viral carriers
The PEI used in these experiments was branched, and
came from several suppliers. One product, purchased
from Sigma/Aldrich (St. Louis, MO), had a nominal
molecular weight of 25 000 Da. The remaining PEIs were
purchased from Polysciences (Warrington, PA) and had
nominal molecular weights of 70 000, 10 000, and 600 Da.
The molecular weight characterizations of these polymers were previously reported [1,8] and are summarized
in Table 1. Based on these data, the PEI with the nominal
molecular weight of 600 will henceforth be referred to as
low MW, and the PEI with the nominal molecular
weight of 10 000 will be referred to as intermediate MW.
The remaining two PEIs used will be designated by high
MW-S (Sigma/Aldrich) or high MW-P (Polysciences).
Poly(L-lysine)-HBr was purchased from Sigma-Aldrich
(St. Louis, MO), and had a reported molecular weight of
19 600 (LALLS) (29 300 by viscosity).
Cells were grown in 6-well plates and transfected as
previously described [8]. The number of cells initially
seeded into each well was 2L;(50 000), n3+0, 1, 2, 3, 4,,
depending on the experiment. For the purpose of seeding,
cell numbers were determined by hemocytometric analysis. Total transfection times were 2 h.
PEI-mediated transfection solutions were made at
a 7.5 : 1 PEI amine to DNA phosphate ratio, using 3.6 lg
of DNA per well. PLL-mediated transfections were made
at a 1 : 1 ratio of PLL to DNA basic units, also using
3.6 lg of DNA per well. Following 2 h of transfection,
transfection media were replaced with growth medium at
2 ll/mm of culture surface.
Liposome-mediated transfections followed the manufacturer's recommendations. Eighteen microlitres of liposome stock was diluted to 100 ll total volume with
OptiMEM2+ (Gibco) and allowed to stand at room
temperature for 30 min before adding the solution to
3.6 lg of DNA diluted to a total of 180 ll with OptiMEM2+. Complexes were allowed to form for at least
15 min before adding to cell wells that contained 2 ml of
OptiMEM2+ without serum or antibiotics. After 2 h of
transfection, the transfection medium was replaced with
the same amount of the same incubation medium described above for polycation-mediated transfections.
2.5. Analysis of cells and media
At three days post-transfection, cell media were
collected and "ltered through separate syringes with
0.45 lm "lters. (vWF samples were not "ltered). The
media were then frozen at !203C for later analysis. The
cells then received fresh medium before transport for
counting and transfection analysis via FACS.
Cells were prepared for FACS analysis by removal of
medium followed by addition of 0.3 ml of trypsin. After
detachment from their wells, the cells received 0.7 ml of
serum-free medium to slow the action of the trypsin. Cells
were then counted and #uorescence of the GFP reporter
was assessed by using a FACScan apparatus (Becton}Dickinson). Transfection e$ciencies were determined in the manner described in Ref. [9].
DOTMA refers to N-[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium chloride.
DOPE refers to dioleoylphosphatidylethanolamine.
DOSPA refers to 2,3-dioleyloxy-N-[2(spermine-carboxamido)
ethyl]-N, N-dimethyl-1-propanaminiumtri#uoroacetate.
W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
473
Table 1
Gel Fractionation Chromatography results (against poly(ethylene glycol) standards) for the PEIs used for transfection
(A) Ref. [8]
Low MW (Nominal MW 600)
Intermediate MW (Nominal MW 10 000)
High MW-P (Nominal MW 70 000)
(B) Ref. [1]
High MW-P (Nominal MW 70 000)
High MW-S (Nominal MW 25 000)
M
M
PI
N/A
5600$100
17 000$3800
N/A
7600$150
216 000$22 000
N/A
1.4$0.0
12.9$1.7
8400$2800
700$200
133 800$10 800
8000$2800
17.0$5.2
11.7$4.8
Sets of results were obtained with di!erent columns, and are referenced in respective publications cited in the upper left corner. M "number
average molecular weight, M "weight average molecular weight, PI"polydispersity index (equal to M /M ).
2.6. Analysis of host-gene products
Three endothelial cell products, PAI-1, tPA, and vWF,
released into the media, were measured by ELISA according to manufacturer directions. Kits for tPA and
vWF were purchased from American Bioproducts (Parsippany, NJ, Catalogue numbers 0240 and 0248, respectively). Kits for PAI-1 were purchased from American
Diagnostica Inc. (Greenwich, CT, Catalogue number
822). Samples were diluted with the manufacturers' blank
reagents (plus water, if needed). Dilution factors used
were vWF 1 : 1, PAI-1 1 : 20, and tPA 1 : 20 (1 : 5 when
experiments only involved an initial seeding of 50 000
cells/well).
ELISA results were collected using a Dynatech
MR5000 (Dynatech, Chantilly, VA) set at optical density
490. Each experiment was performed in duplicate, with
result pairs averaged to reduce error. Each ELISA average was then divided by the corresponding number of
cells counted at the end of the transfection experiment to
obtain a value proportional to the amount of product per
cell. The values representing product per transfected cell
were then normalized to those of non-transfected cells.
(The quotient for non-transfected cells was, by de"nition,
equal to 1.) This method was repeated separately for
every experiment to take into account uncontrollable
factors that a!ected cell growth rates between experiments. For "nal results re#ecting, say, n"3, three separately normalized data points went into determining the
averages and standard deviations for the data bar reported. Quotients for non-transfected cells would still be
equal to 1 (with a SD equal to zero) by de"nition.
2.7. Viability assays
Cell death was con"rmed through the use of the dyes
calcein AM, a cell permeant esterase substrate which
gains #uorescence after cleavage into calcein (E +
515 nm), and ethidium homodimer, a cell impermeant
marker which binds nucleic acids for a 40; increase in
#uorescence intensity (E +635 nm). The dyes were pur
chased as a kit (Molecular Probes, Cat. CL-3224) and
optimized/used according to supplier instructions for
analysis via #uorescence microscopy.
Cell numbers for all day 3 time points were determined
using FACS (see Section 2.5). Cell numbers for early time
points, i.e. 12 h post-transfection and sooner, were determined via hemacytometer following trypsinization.
2.8. Statistics
The normalized ELISA results were analyzed "rst by
F-test to determine whether a given pair of population
variances were equal (a"0.05). This information was
then used in designing appropriate t-tests for comparing
the means of population pairs. Single-factor ANOVA
was also used to analyze groups of ELISA data and
further support t-test statistics.
3. Results
The basis for using di!erent molecular weights of PEI
for these experiments comes from previous work performed in our laboratory that showed that the PEI
molecular weight makes an impact on the attainable
transfection e$ciency [8]. Those experiments produced
successful transfection only in cases where the PEI carrier was at or above the intermediate level of molecular
weight. In the experiments described here, transfection
with PEIs at and above the intermediate level of molecular weight signi"cantly increased the production and/or
release of tPA, PAI-1, and vWF (Fig. 1). The secreted
amounts of these products were not increased when cells
were transfected with DNA only, or with complexes
made with low MW PEI. Secreted amounts of tPA and
PAI-1 remained unchanged when cells were transfected
with PEI only (without plasmid). Transfection with free
PEI did, however, cause an increase in the detected level
of vWF.
For experiments using active enhanced green #uorescent protein (GFP) plasmids, the increase in tPA,
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W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
Fig. 1. Levels of tPA (a), PAI-1 (b), and vWF (c) secreted per cell, normalized to untreated controls. Groups of bars correspond to the number of cells
seeded into wells at the start of each experiment. Error bars indicate 1 SD. Bars signi"cantly di!erent from control values are marked with
* (p(0.05, n*3, except for 2 result sets for &PEI only' where n"2, marked with &2'): 䊏*No treatment, *DNA only, *PEI only (high MW-S),
*PEI/DNA complexes using low MW PEI, *PEI/DNA complexes using intermediate MW PEI, *PEI/DNA complexes using high MW-P
PEI, 䊐*yPEI/DNA complexes using high MW-S PEI.
PAI-1, and vWF levels roughly correlated with transfection e$ciency (Figs. 1 and 2). The two exceptions to this
were vWF levels after free PEI administration (addressed
later), and tPA levels when the high MW-S was used to
transfect cell populations initially seeded with 50 000
cells.
As the number of cells initially seeded into cell wells
was increased, the e!ects of transfection on the secretion
of the monitored endothelial cell products decreased
back toward control values. Because the amounts of PEI
and DNA used were constant, the ratio of transfecting
complexes per cell was therefore decreased as cell numbers were increased. Additionally, the degree of con#uence at the start of transfection was greater when a higher
number of cells was initially seeded. The lower ratio of
complexes per cell, in conjunction with the higher degree
W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
475
Fig. 2. Transfection e$ciencies (percentage of cells expressing GFP) resulting from PEI-mediated gene delivery. Error bars indicate 1 SD (n*4):
䊏*No treatment, *PEI/DNA complexes using low MW PEI, *PEI/DNA complexes using Intermediate MW PEI, *PEI/DNA complexes
using high MW-P PEI, 䊐*PEI/DNA complexes using high MW-S PEI.
Fig. 3. Cell numbers three days post-transfection, normalized to untreated controls. Groups of bars correspond to the number of cells seeded into wells
at the start of each experiment. Error bars indicate 1 SD. Results signi"cantly di!erent from controls are marked by * (p(0.05, n*5): 䊏*No
treatment, *DNA only, *PEI (high MW-S) only, *PEI/DNA complexes using low MW PEI, *PEI/DNA complexes using intermediate
MW PEI, *PEI/DNA complexes using high MW-P PEI, 䊐*PEI/DNA complexes using high MW-S PEI.
of con#uency, may explain why expression levels of GFP
decreased to zero as cell numbers increased. This also
explains the transfection procedure's lack of e!ect on
tPA, PAI-1, and vWF levels when seeded cell numbers
were increased.
Transfection also had an e!ect on cell viability (Fig. 3).
Control wells, wells transfected with DNA only, and
wells transfected with PEI/DNA complexes made with
low MW PEI all had roughly the same number of cells at
the end of each experiment, regardless of the number of
cells seeded. (Note that these wells did not express GFP.)
Wells transfected with PEI/DNA complexes made with
higher molecular weights of PEI all had fewer cells three
days post-transfection. Wells receiving free PEI also had
fewer viable cells at day 3 as compared to controls.
Considering only the cells receiving PEI/DNA complexes, there was an inverse relationship between the
percentage of cells expressing the GFP reporter and the
number of surviving cells.
Although the numbers of cells in wells transfected with
free PEI (high MW-S) or PEI/DNA complexes (made
with high MW-S and seeded at 50 000 cells/well) were
similar to three days post-transfection, the manner in
which cells died after exposure to transfection was di!erent. As shown in Fig. 4, most of the cells subjected to free
PEI appeared dead or otherwise altered at the end of the
2 h transfection period. Cells in the (high MW-S)
PEI/DNA wells demonstrated fewer visible toxic e!ects
at 2 h. However, by 7}9 h post-transfection, many of the
cells from these PEI/DNA transfected wells were dead
and detached from plate surfaces. Death was con"rmed
as described in Section 2.7 (viability assays), and it is
noted that the a!ected cells died (were made permeable)
before they detached (data not shown).
The levels of tPA were measured at early time points to
ascertain whether the sharp decrease in viable cells between 7 and 9 h time points corresponded with a sharp
change in tPA expression or secretion (Fig. 5). The results
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W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
Fig. 4. (A}F) Phase contrast images of cells at various time points following exposure to complexed or free PEI (High MW-S). 50 000 cells/well were
seeded: (A) untreated cells, 2 h post-transfection, (B) cells treated with PEI/DNA complexes, 2 h post-transfection, (C) cells treated with free PEI, 2 h
post-transfection, (D) untreated cells, 8 h post-transfection, (E) cells treated with PEI/DNA complexes, 8 h post-transfection, (F) cells treated with free
PEI, 8 h post-transfection, (G) numbers of cells, relative to untreated controls, at various times post-transfection: 䊏*No treatment, 䊐*PEI/DNA
complexes using high MW-S PEI, *PEI (high MW-S) only.
show no signi"cant increase in tPA levels over controls
for the time points shown. There does appear to be
a de"cit in tPA levels, relative to controls, prior to the
times of cell death for the high MW-S samples. Statistical
p-values for hour 5 of sample versus controls are 0.054
and 0.031, respectively.
A smaller set of experiments was repeated using a promotorless version of the pEGFP plasmid because there
appeared to be an inverse relationship between GFP
expression and cell viability in wells transfected with
PEI/DNA complexes. In these experiments, 50 000 cells
were seeded into each well, and tPA levels were noted at
three days (Fig. 6). For each pair of values shown in the
chart, transfecting complexes were made in the same
manner with the same reagents except for the speci"c
plasmid used. Although the number of cells for which
intact, promotorless plasmids entered the nucleus could
not be indicated via GFP expression, it is assumed that
the percentage was roughly the same as that shown, by
GFP expression, for the corresponding control where
identical PEIs were used with active reporter plasmids.
The results show that after three days, the levels of tPA in
the supernatants were roughly the same for each set of
transfection agents, regardless of whether the delivered
plasmid was expressed. For wells where the active plasmid was signi"cantly expressed, the amount of tPA was
once again elevated compared to controls.
Transfection e!ects on tPA secretion were also examined after using several di!erent established non-viral
methods for gene delivery, including both polycations
W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
477
Fig. 5. Amount of tPA secretion per cell normalized to controls, at time
points at or around 7}9 h cell death. Error bars indicate 1 SD (n"3).
The p-values from t-tests performed for the 5 h time point are 0.054 (no
treatment vs. complexes) and 0.031 (DNA only vs. complexes), respectively. There were no signi"cant di!erences between the samples for the
other time points: 䊏*No treatment, *DNA only, 䊐*PEI/DNA
complexes using high MW-S PEI.
Fig. 7. Examination of four non-viral delivery methods. 50 000 cells/
well were seeded. PEI-mediated transfection utilized PEI of molecular
weight 25 000. (A) Transfection e$ciencies (percentage of cells expressing GFP). Error bars indicate 1 SD (n*4). (B) Amount of tPA
secretion per cell, normalized to controls. Error bars indicate 1 SD
(n*4).
Fig. 6. Amount of tPA secretion per cell normalized to controls, three
days post-transfection. Each pair of bars represents one type of PEI
complexed with reporter plasmids. The reporter plasmids each coded
for an enhanced green #uorescent protein, either with or without
a promotor. Abscissa labels represent the type of PEI used in making
PEI/DNA complexes. Striped bars represent promotorless results. Error bars indicate 1 SD. Results signi"cantly di!erent from controls are
marked by * (p(0.05, n*5).
and liposomes. It was found that each method that
produced signi"cant levels of transfection e$ciency also
generated an increase in secreted tPA (Fig. 7). While
poly(L-lysine) (PLL) did not successfully transfect the cell
type used, it also did not produce any change in extracellular tPA levels nor did it kill cells for the amounts used
(data not shown).
4. Discussion
Because PEI has recently been shown to enter cell
nuclei during transfection [2], the question was immediately raised as to whether PEI's presence inside nuclei
a!ects cellular function, perhaps by altering host-gene
expression. If an alteration of expression pattern were to
occur, we expected the change to be a decrease in the
secreted amounts of the examined gene products due to
PEI binding to host DNA and blocking transcriptional
machinery. Surprisingly, however, we saw an increase in
the gene products concomitant with the e!ectiveness of
the PEIs used for transfection (Figs. 1 and 2). The increase is most likely due to endothelial cell activation
brought on by the stress of non-viral gene delivery.
In addition to any changes in the levels of endogenous
gene products, we were also interested in whether the
e!ects were the same regardless of whether cells were
exposed to free or complexed PEI. Additionally, we
wanted to know whether these e!ects had any in#uence
on cell viability. What we found was that transfection
with certain forms of PEI did have an e!ect on cell
viability (Fig. 3), and this e!ect was parallel to the transfection e$ciency. Finally, we examined the e!ects of
transfection on endothelial cell function using several
di!erent non-viral forms of gene delivery.
Transfections with free DNA and PEI/DNA complexes made with Low MW PEI were performed as
negative controls because these two agents have been
shown to be ine!ective in endothelial cell transfection
[8]. Two types of high MW PEIs were used because they
have been shown to aid in successful cell transfection, and
their e!ects on endothelial cell function were expected to
be similar to each other. The intermediate MW PEI was
selected because its level of transfection e$ciency, while
non-zero, is signi"cantly lower than what is attained by
the high MW PEIs [8]. Any function or viability e!ects
seen with the high MW PEIs were expected to be less
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W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
with the intermediate MW PEI. This expectation was
borne out, which supports the "ndings gathered for the
high MW agents.
There was no change in the media concentrations of
tPA and PAI-1 as compared to controls when the transfection reagent was free PEI (Fig. 1a and b). This brings
up the possibility that, since expression levels seemed tied
to GFP expression, the plasmid itself (or one of its components) might have something to do with the expression
of the endogenous genes. Transfections were therefore
repeated using a promotorless version of the GFP plasmid, and tPA levels were monitored (Fig. 6). Although
transfection e$ciencies could not be veri"ed via GFP
expression, they were assumed to be the same as in
adjacent wells (positive controls) where the active form of
the plasmid had been used for transfection. Even in the
absence of GFP expression we saw the same amount of
increase in tPA levels as noted before when the active
plasmids were used for transfection.
Two hours after transfection, most cells exposed to free
PEI were rounded and many had detached from the well
surface (indicating death). Cells exposed to the same PEI
complexed with DNA showed minimal morphological
e!ects at the 2 h time point. However, by 7}9 h posttransfection, over half of these latter cells had died
(Fig. 4). This indicated that there were two processes
occurring, each of which led to cell death. The "rst
process was relatively quick and was perhaps due to the
membrane destabilization e!ects of PEI. Free PEI has
been shown to destabilize the outer membranes of
Gram-negative bacteria [10,11] as well as liposomal
membranes made from phosphatidyl serine [12]. Additionally, it has been shown that the removal of free PEI
from DNA/PEI solutions by centrifugation and supernatant replacement increases cell viability [9]. We hypothesize that the cells that survived the 2 h free-PEI
challenge either did not take up as much of the polycation or were more able to neutralize, degrade, or
exocytose the polymer. Any of these reasons would account for the observed stasis in tPA or PAI-1 levels
following exposure to free PEI, because many cells that
theoretically could not prevent nuclear entry of the polymer would have been quickly killed by free-PEI's membrane destabilization e!ects before the polymer had
a chance to enter the nuclei. These cells, being dead,
would be unable to further produce either tPA or PAI-1.
The second, slower process that led to cell death is
linked to successful transfection, and shows characteristics of endothelial cell activation and dysfunction. It has
been shown that tPA, PAI-1, and vWF levels are increased in patients with cutaneous vasculitis [13], microand overt albuminuria [14], arterial thromboembolic
disease within 4 weeks after infarction [15], and various
carcinomas (with vWF levels depending on metastasis)
[15]. Additionally, hypercholesterolemia patients have
been noted to exhibit increased PAI-1 levels [16]. The
endothelial cell activation in patients with these conditions appears to be a form of cellular stress response.
PEI-mediated transfection could also stress endothelial
cells to the extent that a reaction similar to that seen in
the above conditions is elicited, which would explain the
increases in tPA, PAI-1, and vWF that were observed.
It is possible that the slower form of cell death, seen
when PEI is complexed with DNA, can be explained by
the kinetics which govern the release of DNA from PEI.
While complexed, there will be fewer free amines available for interaction with and disrupt of cell membranes.
It may take a certain amount of time, say 7}9 h, to free up
enough PEI for the disruption event. However, this restoration of PEI to its uncomplexed form is highly unlikely.
Following the nuclear entry of the complexes, the
stoichiometry of PEI to DNA will be vastly di!erent than
what was originally used to create the complexes in the
test tube. If PEI were to separate from plasmid DNA in
the nucleus then it is reasonable to expect the polycation
to bind to chromosomal DNA, which would prevent its
exposure to the plasma membrane. The PEI molecules
possibly could also bind some component involved with
nuclear export, but once in the cytoplasm there would
still be a lack of free polycation to interact with the
plasma membrane because the polycation would still be
bound to a carrier. It is more likely that the slower form
of cell death is due to something di!erent from simple
membrane destabilization.
There was an increase in vWF released upon exposure
of cells to free PEI (Fig. 1c). This is explained by the fact
that vWF is produced by cells before it is needed systemically, and stored in cytoplasmic Weibel}Palade bodies
(vesicles) [17,18]. Consistent with the "rst type of (quick)
cell death described above, the membrane disruption that
is occurring within the cells also applies to vesicular
membranes, thus causing the release of stored vWF into
the extracellular environment. Although tPA has also
been shown to be cytoplasmically stored, in granules, in
endothelial cells including HUV-EC, the storage granules di!er from Weibel}Palade bodies in terms of density,
morphology, and distribution within cells [19]. The differences might indicate resistance to PEI disruption by
the storage granules, and explain the absence of tPA
increase in cell media after cell exposure to free PEI.
Similarly, if PAI-1 (which is stored within a-granules of
platelets [20,21]) is also stored in endothelial cells, the
storage granules might resist PEI-mediated disruption in
the same manner as tPA storage granules.
In addition to PEI-mediated gene transfer, other forms
of non-viral gene delivery were examined for possible
e!ects on endothelial cell activation (Fig. 7). We found
that as long as the delivery method could produce successful transfection when an active plasmid was used,
there was also an increase in extracellular tPA levels.
This was true for gene delivery mediated by PEI,
DOTMA/DOPE and DOSPA/DOPE.
W.T. Godbey et al. / Biomaterials 22 (2001) 471}480
The results presented for liposomal transfection are
consistent with other published work. Liposomes made
with DOSPA/DOPE or DOSPER have been shown to
directly induce interferon-b gene expression [22]. Additionally, liposomes constructed with DOTMA or DOPE
have been observed to enter cell nuclei occasionally [23],
so the increases in tPA levels might be explained by the
nuclear entry of the gene carrier consistent with PEImediated transfection. In considering the fact that PLL is
not observed to enter nuclei in the cell type used in these
experiments [24], and that PLL did not induce an increase in extracellular tPA (Fig. 7), the possibility that the
nuclear entry of the gene carrier causes the noted e!ects is
further strengthened. This has serious implications for
the entire "eld of chemically mediated non-viral gene
delivery.
This research is important in that it demonstrates
clearly that non-viral gene delivery is not a simple,
straightforward process, there are many cellular events
that should be understood before gene delivery is used
for the treatment of disease. This work also helps to
explain why PEI-mediated transfection often leads to cell
death, and that there are at least two processes associated
with PEI toxicity. PEI has been used for years as a successful transfection agent, but a better understanding of
the mechanism behind the observed cell activation must
be obtained before this method of gene delivery can be
used in humans. A weakness of this work is the fact that
only secreted gene products were studied. Cytoplasmic or
nuclear gene products should also be examined*both
end products and mRNA intermediaries*to further ascertain whether the results seen are due to an increase in
transcription or translation rates (or both), increased
export of proteins, or some other factor. Such work is
already underway in our laboratories.
5. Conclusions
From the above results and discussion we conclude
that transfection with PEI alters endothelial cell function.
In the cases of tPA, PAI-1, and vWF, the alteration
occurs in the form of elevated production and/or secretion. Elevated tPA levels were also observed after liposome-mediated transfection. The elevated product levels
appear to be the result of endothelial cell activation, and
could be related to the presence of delivery vehicles in the
nucleus.
For the reporter plasmids used, the strength of the
promotor does not a!ect the increase in host-gene expression. Rather, it is the PEI used that determines the
e!ects on host-gene expression. PEIs that act as better
DOSPER refers to 1,3-dioleoyloxy-2-(6-carboxyspermyl)propylamide.
479
transfection agents have a greater perturbation e!ect
because PEI molecules that serve as successful vectors do
so by an increased ability to deliver carried genes, as well
as themselves, into cell nuclei. Likewise, it is the PEI used
that determines the e!ects on cell viability.
There exist at least two types of cell death associated
with PEI-mediated cell transfection. Chronologically,
the "rst is associated with free PEI and a!ects cells
almost immediately, while the second is associated with
cellular processing of PEI/DNA complexes and takes
more time (between 7 and 9 h). This second type of
cell death is possibly linked to PEI's presence in the
nucleus.
Finally, PEI's disruptive e!ect on vesicle membranes
was shown with the release of vWF. Free PEI caused the
release of stored vWF, but not tPA or PAI-1, from cells,
and thereby warrants further examination as a possible
agent in the treatment of von Willebrand's disease.
Acknowledgements
This material is based upon the work supported under
a National Science Foundation Graduate Fellowship
(WTG) and the National Institutes of Health (R29AR42639) (AGM), (PSO-NS-23327) (KKW), and (R01HL-50675) (KKW).
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