![Chemosensitization of Hey cells to docetaxel after exposure to YSA-targeted, siRNA loaded nanogels. A. Hey cells were plated onto 96-well plates (1 × 104 cells/well) and allowed to adhere overnight at 37°C and 5% CO2. The medium was removed, and the cells were washed with PBS followed by replacement of the medium. Wells were set up in triplicate to include several concentrations of YSA-targeted, siRNA-loaded nanogels (1 to 1000 μg/mL). The cells were incubated with the nanogels for four hours. The cells were washed with PBS, the medium replaced, and the cells incubated for an additional 48 hours before addition of docetaxel in order to allow reduction of EGFR expression. Docetaxel was then added, and cells were incubated with the taxane for an additional 96 hours. The percent cytotoxicity (Tox 8 assay) was assessed compared to untreated cells. B. Chemosensitization of Hey cells treated with the highest dose of YSA-targeted, siRNA-loaded nanogels (1000 μg/mL) and increasing concentrations of docetaxel are compared to controls: Unloaded YSA-conjugated nanogels (YSA-pNIPMAm), unloaded pNIPMAm nanogels (pNIPMAm), YSA peptide alone, and untreated cells. Results represent the mean of two independent experiments; error bars represent standard deviation. All cells treated with siRNA-loaded, targeted nanogels were significantly different from controls at all doses of docetaxel except where noted [see Additional file 3, Tables S1-S4].](https://www.researchgate.net/profile/William-Blackburn/publication/40906882/figure/fig5/AS:216393845874696@1428603721858/Chemosensitization-of-Hey-cells-to-docetaxel-after-exposure-to-YSA-targeted-siRNA-loaded_Q320.jpg)
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RESEA R C H ARTIC L E Open Access
Chemosensitization of cancer cells by siRNA
using targeted nanogel delivery
Erin B Dickerson
1,2,3,5
, William H Blackburn
2,4
, Michael H Smith
2,4
, Laura B Kapa
1,2,3,6
, L Andrew Lyon
2,4
,
John F McDonald
1,2,3*
Abstract
Background: Chemoresistance is a major obstacle in cancer treatment. Targeted therapies that enhance cancer
cell sensitivity to chemotherapeutic agents have the potential to increase drug efficacy while reducing toxic effects
on untargeted cells. Targeted cancer therapy by RNA interference (RNAi) is a relatively new approach that can be
used to reversibly silence genes in vivo by selectively targeting genes such as the epidermal growth factor receptor
(EGFR), which has been shown to increase the sensitivity of cancer cells to taxane chemotherapy. However,
delivery represents the main hurdle for the broad development of RNAi therapeutics.
Methods: We report here the use of core/shell hydrogel nanoparticles (nanogels) functionalized with peptides that
specially target the EphA2 receptor to deliver small interfering RNAs (siRNAs) targeting EGFR. Expression of EGFR
was determined by immunoblotting, and the effect of decreased EGFR expression on chemosensitization of
ovarian cancer cells after siRNA delivery was investigated.
Results: Treatment of EphA2 positive Hey cells with siRNA-loaded, peptide-targeted nanogels decreased EGFR
expression levels and significantly increased the sensitivity of this cell line to docetaxel (P < 0.05). Nanogel
treatment of SK-OV-3 cells, which are negative for EphA2 expression, failed to reduce EGFR levels and did not
increase docetaxel sensitivity (P > 0.05).
Conclusion: This study suggests that targeted delivery of siRNAs by nanogels may be a promising strategy to
increase the efficacy of chemotherapy drugs for the treatment of ovarian cancer. In addition, EphA2 is a viable
target for therapeutic delivery, and the siRNAs are effectively protected by the nanogel carrier, overcoming the
poor stability and uptake that has hindered clinical advancement of therapeutic siRNAs.
Background
Although a number of chemotherapeutic treatments
have been shown to be effective at inhibiting or elimi-
nating cancer cell growth in preclinical studies, clinical
applications are often limited due to the toxic side
effects associated with anticancer drugs. Patients are
often unable to tolerate the level of a drug needed to
effectively eliminate malignant cells while levels that can
be tolerated are insufficient therapeutically. As a result,
chemoresistance and subsequent tumor recurrence are
often the outcome of such therapies. An example of this
all too common event is the use of taxanes (paclitaxel
and its semi-synthetic analogue, docetaxel) in the
treatment of a variety of cancers including ovarian,
breast, prostate, and non-small cell lung cancers [1,2].
While surgery along with taxane- and platinum-based
chemotherapy for advanced ovarian cancer has allowed
up to 80% of women to achieve a clinical response [3],
cancers in most patients initially diagnosed with late
stage disease eventually recur.
Development of methods to circumvent resistance
may ultimately improve the impact of adjuvant therapy,
resulting in prolonged disease-free intervals and survival.
Novel targeted therapies that interfere with specific
molecular signaling pathways affecting cancer cell survi-
val are being developed as potential treatment options
to render cancer cells more sensitive to cytotoxic che-
motherapy. Targeted therapies that increase cancer cell
sensitivity to chemotherapies offer the benefits of lower-
ing unwanted side effects and increasing the likelihood
* Correspondence: john.mcdonald@biology.gatech.edu
1
School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta,
GA, 30332, USA
Dickerson et al.BMC Cancer 2010, 10:10
http://www.biomedcentral.com/1471-2407/10/10
© 2010 Dickerso n et al; licensee BioMed Cent ral Ltd. This is an Open Access article distributed und er the terms of the Creative
Commons Attri bution License (http://creativecommons.org /licenses/by/2.0), which permits unrestricted use, distribution, and
reproductio n in any medium, provided the original work is properly cited.
of destroying resistant cells while avoiding healthy cells
where there is little or no expression of the targeted
entity.
Recent studies have shown that sensitivity of ovarian
cancer cells to the taxane, paclitaxel, is enhanced when
the drug is administered in combination with an inhibi-
tor of EGFR. EGFR and its ligand, epidermal growth fac-
tor (EGF), play critical roles in the progression of
ovarian cancer through their effects on cellular prolifera-
tion, apoptosis, angiogenesis, and metastasis [4-6]. EGFR
is overexpressed or dysregulated in many solid tumors
[7-10], and high levels are expressed in 33-98% of all
epithelial ovarian cancers [11-14]. Their high expression
is believed to mitigate the effectiveness of taxane che-
motherapy by inhibiting cell division and apoptosis
[15-17]. Reports of inhibition of EGFR with tyrosine
kinase inhibitors (TKI) [e.g. gefitinib (Iressa)] and mono-
clonal antibodies (e.g. cetuximab) have demonstrated
that silencing of receptor activity increases chemosensi-
tization of tumor cells including ovarian cancer cells
[6,18-22]. While targeting EGFR as well as other mem-
bers of the human EGFR (HER) family has proven suc-
cessful, not all tumors that are expected to respond to
these agents do so. Often, emergence of drug resistance
occurs either by targeted mutation [23,24] or induction
of alternative signaling pathways [24,25]. These results
highlight the need for further targeted approaches.
Basedonthesefindings,wesoughttodetermineif
siRNA against EGFR could be selectively delivered to
ovarian cancer cells using a nanoparticle carrier. Tar-
geted cancer therapy by RNA interference (RNAi) is a
relatively new approach, and silencing EGFR by RNAi
has already shown promising results [26-30]. We report
here application of a novel and highly efficient method
for the targeted delivery of EGFR siRNA to ovarian can-
cer cells. The method is based on core/shell hydrogel
nanoparticle (nanogel) siRNA carriers, which represent
a convenient and versatile structure for targeted drug
delivery. The reader is referred to work from our groups
for more detailed information regarding the nanocarrier
[26,31,32]. These core/shell nanogels are composed
mainly of poly(alkylacrylamides), which can be easily
synthesized via multi-stage, free-radical initiated precipi-
tation polymerization [31]. In this fashion, a porous
hydrogel core appropriate for the entrapment of macro-
molecular therapeutics can be coated with a porous
hydrogel shell that displays the appropriate chemoliga-
tion sites for the attachment of targeting ligands. We
used a previously described 12 amino acid peptide
(YSAYPDSVPMMS or YSA) [33] coupled to the surface
of ~100-nm diameter core/shell nanogels [composed of
poly(N-isopropylmethacrylamide) (pNIPMAm) cross-
linked with N, N’-methylene(bisacrylamide)] [26,34] to
permit cell-specific targeting, and the subsequent
delivery of high concentrations of EGFR siRNA. The
YSA peptide mimics the ligand ephrin-A1, which binds
to the erythropoietin-producing hepatocellular (Eph) A2
receptor, while the core/shell nanogel offers an efficient
vehicle for cell entry, a protective environment for the
siRNA, and a depot for its controlled release. Delivery of
nanogel-loaded EGFR siRNA to EphA2 positive cells
resulted in the loss of EGFR expression followed by a
significant increase in the sensitivity of the targeted cells
to docetaxel. Our results indicate that this approach
may lead to considerable improvements in the treatment
of ovarian and other cancers by increasing the efficacy
of chemotherapy while simultaneously reducing the
associated negative side effects.
Methods
All materials were purchased from Sigma-Aldrich (St
Louis, MO) and used as received unless otherwise
noted.
Nanogel synthesis
For the present studies, we utilized a nanogel structure
that we have previously shown to have excellent siRNA
encapsulation and release properties in the context of in
vitro delivery [26]. The synthesis of the nanogels has
been described previously [26,35]. Briefly, nanogel core
particles were synthesized by free-radical precipitation
polymerization using a molar composition of 98% N-iso-
propylmethacrylamide (NIPMAm), 2% N, N’-methylene-
bis(acrylamide) (BIS) and a small amount (~0.1 mM)
acrylamidofluorescein (AFA) to render the nanogels
fluorescent for visualization. The core nanogels were
then used as seeds for the addition of a hydrogel shell
[31,35]. The shell composition was 97.5% NIPMAm, 2%
BIS, and 0.5% aminopropylmethacrylamide (APMA,
Polysciences, Warrington, PA). The APMA co-monomer
was included to provide chemoligation sites for peptide
immobilization.
Peptide conjugation
The YSA peptide (GenScript Corporation, Piscataway,
NJ) was conjugated to the nanogels via maleimide cou-
pling to the cysteine residue on the C-terminal end of
the peptides, as described [26]. Maleimide-functionalized
nanogels were prepared via EDC coupling of ε-maleimi-
docaproic acid (EMCA) to the primary amines in the
nanogel shell. Peptide coupling was performed by intro-
ducing the YSA peptide in a 1:1 molar ratio with amine
(YSA molecular weight = 1450.66 g/mol). The YSA pep-
tide was then conjugated to the nanogels via maleimide
coupling to the cysteine residue on the C-terminal end
of the peptides.
Cell culture
Hey cells were provided by Gordon W. Mills, Depart-
ment of Systems Biology, the University of Texas, M. D.
Anderson Cancer Center. Hey cells were cultured in
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RPMI 1640 (Mediatech, Manassas, VA) supplemented
with 10% v/v heat-inactivated fetal calf serum (Invitro-
gen, Carlsbad, CA), 2 mM L-glutamine (Mediatech), 10
mM HEPES buffer (Mediatech), penicillin (100 U/ml),
and streptomycin (100 μg/mL). SK-OV-3 cells were
from the National Cancer Institute and were propagated
in McCoy’s 5A with L-glutamine (Mediatech) supple-
mented with 10% v/v heat-inactivated fetal calf serum
(Atlanta Biologicals, Lawrenceville, GA), penicillin, and
streptomycin (Mediatech).
RNA encapsulation
Hydrogels were loaded with siRNA as previously
described [26]. Briefly, lyophilized nanogels were reswol-
len in the presence of the siRNA, thereby imbibing the
solute within the hydrogel network. In a typical proce-
dure, a 20 μM solution (250 μL) of EGFR siRNA (Dhar-
macon, Lafayette, CO) was prepared in phosphate
buffered saline (PBS). Nanogels were resuspended in
this mixture at a concentration of 4 mg per 250 μLof
siRNA solution and allowed to shake overnight at room
temperature. After the siRNA was encapsulated in the
nanogels, they were centrifuged and resuspended to a
final concentration of 10 mg/mL in cell culture medium
or PBS. Based on this procedure, the final concentration
of siRNA was determined to be 16.6 μgsiRNA/mgof
nanogels. For experiments using a non-specific siRNA,
siGLO (Dharmacon) was incorporated into nanogels at
the same concentration described for EGFR siRNA.
Immunoblotting
Hey or SK-OV-3 cells were plated into 6-well cell cul-
ture plates (5 × 10
5
cells/well), and the cells allowed to
adhere overnight at 37°C in a 5% CO
2
atmosphere.
After washing the wells with PBS and replacing the
medium, EGFR siRNA-loaded/YSA-conjugated nanogels
were added to the wells. Cells were incubated for four
hours, washed with PBS, and fresh medium was added
to the cells. The cells were incubated at 37°C and 5%
CO
2
in wells for 24, 48, 72, 96, and 120 hours. Control
wells were set up to include non-targeted/siRNA-encap-
sulated pNIPMAm particles, unloaded pNIPMAm parti-
cles, YSA alone, and untreated cells. Cells were lysed
after the designated time points, and immunoblotting
was carried out as described [26]. To determine the
optimal concentration of EGFR-siRNA needed for effi-
cient reduction of EGFR expression, the nanogel loading
procedure described above was used, but the concentra-
tion of particles delivered to each well was altered. The
initial concentration of siRNA-encapsulated particles (1
mg/mL of nanogels/5 × 10
5
cells) used for the time
point experiments was added to the first well. The con-
centration of subsequent wells was reduced by 10 fold
each, resulting in nanogel concentrations of 100, 10, and
1μg/mL per 5 × 10
5
cells. After four hours of incuba-
tion with the nanogels, the cells were washed with PBS,
and the medium was replaced. The cells were then incu-
bated for an additional 48 hours, and the samples pre-
pared for immunoblotting as described [26].
Treatment with docetaxel
Hey or SK-OV-3 cells were plated in 96-well cell culture
plates at a concentration of 1 × 10
4
cells/well. Hey or
SK-OV-3 cells were subjected to nanogel delivery of
siRNA at nanogel concentrations of 1000, 100, 10, and 1
μg/mL. Forty-eight hours after siRNA delivery, docetaxel
was added to Hey or SK-OV-3 cells at concentrations
ranging from 0.01-1000 nM. Treatment wells were set
up in triplicate, and the cells were incubated with doce-
taxel for an additional 4 days. After treatment, the cells
were washed with PBS, and 100 μL of medium was
addedbacktothewells.Tothis,10μLofTox8was
added to determine cell viability. The cells were incu-
bated with the Tox8 reagent according to the manufac-
turer’s instructions. The fluorescence was measured
(l
em
= 560 nm, l
ex
= 590 nm) by a Spectramax Gemini
Fluorescence Microplate Reader (Molecular Devices,
Sunnyvale, CA). Wells without cells but with Tox8 were
used as controls and subtracted from all treatments as
background. Each experiment was performed in
duplicate.
Statistical analysis
Statistical analysis of the immunoblot data was per-
formed using a non-parametric ANOVA (Kruskal
Wallis) test. If significance was indicated, a Dunn’spost
test was used to determine significance between groups.
Statistical analysis of siRNA-loaded nanogels plus doce-
taxel treated Hey or SK-OV-3 cells was compared to all
controls (pNIPMAm, YSA-pNIPMAm, YSA peptide
alone, and untreated cells). To determine significance
between groups, a one-way ANOVA test was performed.
If significance was indicated, a Tukey post test was per-
formed to determine significance between sample
groups. In all cases, significance was defined as P < 0.05.
Results
Down-regulation of EGFR in EphA2
+
ovarian cancer (Hey)
cells by targeted siRNA-loaded nanogels
By coupling a peptide-mimetic (YSAYPDSVPMMS) of
the EphA2 receptor’s ephrin-A1 ligand to core/shell
nanogels, we demonstrated previously the ability to tar-
get the delivery of siRNA to ovarian cancer (Hey) cells
expressing the EphA2 receptor [26]. Importantly, these
nanogels are nontoxic in both unmodified and targeted
forms, and enabled the delivery of siRNA in serum-con-
taining medium. To further establish the efficacy and
specificity of this targeting method, we established a
model using ovarian cancer cell lines either positive or
negative for expression of EphA2 and positive for
expression of EGFR. Figure 1A contrasts the high level
of EphA2 receptor expression by Hey cells with the lack
Dickerson et al.BMC Cancer 2010, 10:10
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of EphA2 expression in the SK-OV-3 cancer cell line.
Detection of EGFR was noted in both cell lines by
immunoblotting (Figure 1B). Because of the observed
differences in EphA2 expression levels, we hypothesized
that the level of EGFR siRNA delivery and the subse-
quent decrease in EGFR expression in the cell lines
would depend upon the presence of the EphA2 receptor
as well as the concentration of siRNA loaded-nanogels
added to the cells. Based upon this premise, reduction
of EGFR expression in SK-OV-3 cells (EphA2 negative)
should not be observed.
To test this hypothesis and measure the efficacy of the
siRNA loaded nanogels in our system, we determined
the time course of EGFR knockdown using EphA2 posi-
tive Hey cells. Lyophilized, YSA-targeted nanogels were
loaded with EGFR siRNA by reswelling the particles in a
concentrated solution of siRNA, as described [26]. This
method results in high efficiency siRNA encapsulation
(93 ± 1%) and approximately 70% retention of the
siRNA after the first 12 hours. Long retention times
may provide slow and continuous release of siRNA lead-
ing to prolonged reduction of the expressed target.
Following siRNA encapsulation, the loaded nanogels
were added to Hey cells and incubated at 37°C for four
hours. In all experiments, we maintained a constant
nanogel/cell ratio of 1 mg/mL of nanogels/5 × 10
5
cells,
unless noted. Unincorporated nanogels were removed
by washing and subsequent replacement of the cell cul-
ture medium. Treated cells were incubated for an addi-
tional 24, 48, 72, 96, and 120 hours to determine the
time course of EGFR reduction by the nanogel-delivered
siRNA. At each time point, the cells were lysed, and the
samples were prepared for immunoblotting to determine
the EGFR levels. Figure 2A shows the average (n = 3)
percent decrease in EGFR expression at each time point.
A significant decrease in EGFR expression (*P < 0.01)
was observed at both 48 and 72 hours when compared
to untreated (UT) controls. Significance (^P < 0.05) was
also observed at the 96-hour time point when compared
to untreated cells. These results indicate a maximum
reduction of EGFR expression at 48 hours, and reex-
pression of EGFR beginning at approximately 72 hours.
Expression gradually increased through 120 hours but
did not return to pretreatment levels. This may be due
to the slow but continuous release of siRNA from the
nanogels. A slight decrease in EGFR expression was
noted when the YSA peptide was used alone, which may
be due to cross-talk between the EGFR and the EphA2
receptors [36]. Changes in EGFR levels may be due to
loss of EphA2 as a result of YSA binding to the receptor
and subsequent degradation of EphA2 (Dickerson,
unpublished). Loss of EphA2 may disrupt EGFR expres-
sion through an as yet unknown mechanism. Note that
this result was not observed in all studies performed
(see Figure 3) and is under further investigation. In
addition, a small decrease in EGFR expression was
observed when cells were incubated with nanogels alone
(Ng), but these decreases were not significant (P > 0.05).
An immunoblot from one of three experiments is
shown in Figure 2B. An immunoblot from a second
experiment is presented in Additional file 1, Figure S1.
To determine the dose response for the delivery vector,
EGFR siRNA-loaded nanogels were incubated with Hey
cells using 10-fold serial dilutions of siRNA-loaded
nanogels so that the nanogel concentration ranged from
1μg/mL to 1000 μg/mL per 5 × 10
5
cells. Cells were
harvested 48 hours after nanogel addition, and the cell
lysates were analyzed by immunoblotting. In two out of
three experiments, decreased levels of EGFR were
observed at all concentrations. The average (n = 3)
reduction in EGFR expression from all experiments is
presented graphically in Figure 3A. A significant
decrease (*P < 0.01) in EGFR expression was observed
at the highest dose of delivered nanogels (1000 μg/mL)
when compared to untreated controls (UT), and com-
plete reduction of EGFR expression was observed with
Figure 1 Expression of EphA2 and EGFR in ovarian cancer cell
lines.A. Immunoblot analysis of EphA2 receptor expression in Hey
and SK-OV-3 ovarian cancer cell lines. High expression of EphA2 is
observed in Hey cells, but no expression is detected in the SK-OV-3
cell line. B. Expression of EGFR is observed in both the Hey and the
SK-OV-3 cell lines as shown by immunoblotting. In both cases, b-
actin expression was used to demonstrate equal loading of the
protein samples so that the relative levels of each receptor could be
compared between cell lines.
Dickerson et al.BMC Cancer 2010, 10:10
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as little as 10 μg/mL of siRNA-loaded nanogels in some
studies. An immunoblot demonstrating the reduction of
EGFR with as little as 10 μg/mL of siRNA-loaded nano-
gels is shown in Figure 3B. The data shown is from one
of three experiments. Data from a second experiment
are presented in Additional file 2, Figure S2. We pre-
viously used a nonspecific siRNA, siGLO, to observe
delivery of siRNA by YSA-targeted nanogels to Hey cells
[26]. As a further control, treatment of Hey cells with
YSA-targeted siGLO-loaded nanogels (siGLO) and ana-
lysis of EGFR expression levels did not result in loss of
EGFR expression when treated cells were compared
with the YSA peptide alone (YSA), untargeted nanogels
(Ng) or untreated cells (UT), indicating the specificity of
Figure 2 Down-regulation of EGFR by siRNA-loaded nanogels.
A. Hey cells were examined for EGFR expression by immunoblotting
of Hey cell lysates prepared from 24 to 120 hours after the addition
of EGFR siRNA-loaded nanogels. Nanogels were loaded with siRNA
at a concentration of 16.6 μg siRNA/mg of nanogels, and 1000 μg/
mL of the loaded particles was added to 5 × 10
5
cells. Untreated
(UT) cells were set at 100% expression of EGFR. Controls included
unloaded/untargeted nanogels (Ng), and YSA peptide alone (YSA).
Overall, treatment with EGFR siRNA significantly decreased receptor
expression at 48 and 72 hours (*P < 0.01) and also at 96 hours (^P
= 0.05). The error bars represent ± one standard deviation about
the average value (n = 3). B. An immunoblot from one experiment
out of three shows the decrease in EGFR expression over time.
Controls are the same as those in (A).
Figure 3 Down-regulation of EGFR by different concentrations
of siRNA-loaded nanogels.A. A dose curve was established for
EGFR expression by immunoblotting of Hey cell lysates. Nanogels
were loaded with siRNA at a concentration of 16.6 μg siRNA/mg of
nanogels, and 1000 μg/mL of the loaded particles was added to 5
×10
5
cells. For other concentrations, nanogels were diluted serially
by 10-fold (1000 μg/mL, 100 μg/mL, 10 μg/mL, and 1 μg/mL). All
cells were harvested at 48 hours after the addition of the siRNA
loaded nanogels. A significant decrease (*P < 0.01) in EGFR
expression was observed at the highest nanogel concentration
when compared to EGFR expression in untreated (UT) cells using
the averaged values from three experiments. YSA peptide alone
(YSA) was included as an additional control. B. An immunoblot from
one of three experiments demonstrating EGFR expression in Hey
cells after treatment with different concentrations of siRNA-loaded
nanogels is shown. Note in the study shown that complete
reduction in EGFR expression was observed when the concentration
of nanogels used was as little as 10 μg/mL. C. Hey cells were
treated with a non-specific siRNA, siGLO at a concentration of 1000
μg/mL of siGLO-loaded nanogels. Cells were harvested after 48
hours, and the levels of EGFR were examined by immunoblot.
Differences in EGFR expression were not observed in siGLO-nanogel
treated cells when compared with YSA peptide alone (YSA),
nanogels (Ng), or untreated (UT) controls.
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the EGFR siRNA (Figure 3C). The concentration of
siGLO-loaded nanogels used was 1000 μg/mL.
The role of the peptide-targeted receptor, EphA2, in
nanogel uptake, and the level of nonspecific nanogel
incorporation into cells were explored through the use
of an EphA2 negative cell line, SK-OV-3. Because these
cells lack EphA2 expression, we hypothesized that the
YSA-targeted nanogels would not be taken up by SK-
OV-3 cells through receptor-mediated endocytosis of
EphA2. Consequently, EGFR expression should not dif-
fer between targeted, siRNA-loaded nanogels and an
untargeted, but siRNA-loaded control (Ng+siRNA). Any
particle uptake could then be designated as nonspecific.
For these studies, siRNA (1000 μg/mL of nanogels) was
loaded into YSA-pNIPMAm nanogels and added to 5 ×
10
5
SK-OV-3 cells. Ten-fold serial dilutions of the nano-
gels were carried out to assess the affects of nanogel
concentration on the levels of EGFR. After 48 hours,
harvested samples were examined for receptor expres-
sion by immunoblotting. As expected, expression of
EGFR was not decreased after treatment with the loaded
nanogels regardless of the concentration of nanogels
used (Figure 4). Expression levels in SK-OV-3 cells trea-
tedwithsiRNA-loadednanogelsdidnotdifferfroman
untargeted control, demonstrating the high specificity of
the targeted nanogels for EphA2 positive cells but not
for EphA2 negative cells.
Epidermal growth factor receptor down-regulation in
siRNA-loaded nanogel treated cells sensitizes ovarian
cancer cells to docetaxel
Expression of EGFR is significantly related to chemosen-
sitivity in many cancers. The concept of
chemosensitization by EGFR blockade was provided by
studies utilizing EGFR-blocking antibodies in combina-
tion with cisplatin or doxorubicin in human tumor
xenografts [37,38]. Studies using a tyrosine kinase inhi-
bitor against EGFR showed an increased sensitivity of
ovarian cancer cell lines to paclitaxel after preincubation
with the inhibitor [22]. To determine if our targeted
delivery of EGFR siRNA to ovarian cancer cells could be
used to increase cell line sensitivity to taxanes, Hey cells
were incubated with EGFR siRNA-loaded nanogels for
48 hours to allow for maximum reduction in EGFR
expression (see Figures 2A and 2B). After 48 hours, cells
were treated with increasing concentrations of docetaxel
(0.1 to 1000 nM), and the percent cytotoxicity was
assessed. The results presented (Figure 5A) demonstrate
the docetaxel sensitivity of treated Hey cells was almost
8-fold greater than untreated controls. While Hey cells
treated with nanogel controls also showed increased
chemosensitivity (Figure 5B), these changes were signifi-
cantly less than those observed in cells treated with the
YSA-targeted, siRNA-loaded nanogels (P < 0.01). Excep-
tions included the pNIPMAM and YSA-pNIPMAm con-
trols where docetaxel concentrations were 0-0.1 (P >
0.05) at all nanogel concentrations examined, and for
pNIPMAm and YSA-pNIPMAm controls when 1 μg/
mL siRNA-loaded nanogels were delivered to cells fol-
lowed by incubation with 1 nM docetaxel (P > 0.05)
[see Additional file 3, Tables S1-S4]. Because SK-OV-3
cells lack expression of EphA2, and thereby lack the
means for receptor-mediated endocytosis of the targeted
nanogels, we did not expect the sensitivity of SK-OV-3
cells to docetaxel to be altered. Whereas an increase in
cytotoxicity of the siRNA-loaded nanogel treated SK-
OV-3 cells was noted as the concentration of docetaxel
was increased, unlike the effect observed in the Hey cell
line, sensitivity to the drug did not differ significantly
from controls (P > 0.05) (Figures 6A and 6B). These
results corroborate our earlier findings that EGFR levels
are not decreased in this cell line after treatment with
siRNA-loaded nanogels. It also substantiates the high
specificity of our peptide-targeted system, and demon-
strates little or no nonspecific uptake of nanogels by
SK-OV-3 cells as shown by the constant levels of EGFR
expression and unaltered chemosensitivity after nanogel
treatment.
Discussion
Novel therapies that interfere with specific molecular
signaling pathways have potential as treatment options
since they render cancer cells more sensitive to cyto-
toxic therapy. Although the role of EGFR in altering
tumor chemosensitivity has not yet been fully eluci-
dated, preclinical studies have suggested that blockade
of EGFR, and the resulting reversal of chemoresistance
Figure 4 Levels of EGFR in SK-OV-3 cells after treatment with
YSA-targeted, siRNA-loaded nanogels. EGFR expression in SK-OV-
3 cells was determined by immunoblotting after the addition of
several concentrations of YSA-targeted, siRNA-loaded nanogels (1 to
1000 μg/mL). All cells were harvested 48 hours after the addition of
the siRNA-loaded nanogels. Untargeted but siRNA-loaded nanogels
(Ng+siRNA) were included as a control. A change in EGFR
expression was not observed between treatment groups as
determined by immunoblotting.
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in many tumor types is a viable strategy for treatment of
cancers where frontline therapies have failed to induce a
cure. Chemosensitization by EGFR inhibition was
demonstrated in early studies using blocking antibodies
in combination with cisplatin or doxorubicin in human
tumor xenografts [37,38]. This same effect was later
observed using small TKIs such as gefitinib (Iressa)
[6,18,19,21]. Silencing of EGFR by RNAi is an alternative
to anti-EGFR therapy, and this approach has already
shown promising results [26-30]. However, further
advances must be made to overcome problems with
delivery and to insure specific delivery to the cells of
interest.
While we previously demonstrated the specificity of
YSA-targeted siRNA-loaded nanogels to cells expressing
EphA2 [26], the studies presented here serve as further
validation of EphA2 as a target for translatable thera-
peutic strategies. The EphA2 receptor is overexpressed
in a variety of cancers including ~75% of ovarian malig-
nancies, and expression of the receptor is associated
with poor prognosis, increased metastasis, and decreased
survival [39-41]. EphA2 shows limited expression in
adults, with expression restricted to a few epithelial tis-
sues [42]. Thus, due to its expression pattern, localiza-
tion, and functional importance in treatment outcome,
EphA2 is an attractive target for therapeutic agents in
ovarian as well as other cancers [43]. Several approaches
have been used to target EphA2 for cancer therapy
either by taking advantage of the tumor-promoting
function of EphA2 to modulate cell behavior and sup-
press tumor growth, or using EphA2 as a means to deli-
ver agents, such as exogenous drugs, to tumor cells and
the tumor microenvironment [44-47]. While these
results demonstrate reduced tumor growth and limited
metastatic spread, effectiveness of these treatments may
depend upon tumor type and whether a particular
tumor is dependent on EphA2-mediated pathways [48].
In this context, we noticed that treatment of Hey cells
with the YSA peptide alone showed diminished EGFR
expression when compared to untreated controls (Fig-
ures 2A and 2B) in some studies. Furthermore, Hey
cells treated with the YSA peptide alone also showed an
increased sensitivity to docetaxel when compared to
untreated controls (Figure 5B). These differences were
Figure 5 Chemosensitization of Hey cells to docetaxel after exposure to YSA-targeted, siRNA loaded nanogels.A. Hey cells were plated
onto 96-well plates (1 × 10
4
cells/well) and allowed to adhere overnight at 37°C and 5% CO
2
. The medium was removed, and the cells were
washed with PBS followed by replacement of the medium. Wells were set up in triplicate to include several concentrations of YSA-targeted,
siRNA-loaded nanogels (1 to 1000 μg/mL). The cells were incubated with the nanogels for four hours. The cells were washed with PBS, the
medium replaced, and the cells incubated for an additional 48 hours before addition of docetaxel in order to allow reduction of EGFR
expression. Docetaxel was then added, and cells were incubated with the taxane for an additional 96 hours. The percent cytotoxicity (Tox 8
assay) was assessed compared to untreated cells. B. Chemosensitization of Hey cells treated with the highest dose of YSA-targeted, siRNA-loaded
nanogels (1000 μg/mL) and increasing concentrations of docetaxel are compared to controls: Unloaded YSA-conjugated nanogels (YSA-
pNIPMAm), unloaded pNIPMAm nanogels (pNIPMAm), YSA peptide alone, and untreated cells. Results represent the mean of two independent
experiments; error bars represent standard deviation. All cells treated with siRNA-loaded, targeted nanogels were significantly different from
controls at all doses of docetaxel except where noted [see Additional file 3, Tables S1-S4].
Dickerson et al.BMC Cancer 2010, 10:10
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Page 7 of 11
significant(P<0.05)atdocetaxeldosesof1nMand
higher. Interestingly, although we demonstrated silen-
cing of EGFR with low doses of siRNA-loaded nanogels
(10 μg/mL), the doses of docetaxel needed for chemo-
sensitization in nanogel treated Hey cells were extremely
low (≥1 nM). In addition, differences in chemosensitivity
were not observed between low and high doses in our
study even though presented results (Figure 3A and 3B)
indicate that different levels of EGFR reduction were
achieved depending upon the concentration of siRNA-
loaded nanogels delivered. One possible explanation for
this may be oncogene addiction where cancer cells are
dependent upon or ‘addicted’to one or several genes for
maintenance of malignant phenotype and cell survival.
Evidence for oncogene addiction to EGFR and members
of the EGFR family has been described both at the cellu-
lar and clinical level [49]. The reader is referred to a
reviewbyWeinsteinandJoeforamoreindepth
description behind the mechanisms of oncogene addic-
tion [49].
Our results indicate that activation of EphA2 by the
YSA peptide [33] and subsequent EphA2 degradation
(Dickerson, unpublished) may lead to a reduction in
EGFR expression indicating cross-talk between the two
receptor signaling pathways. In fact, two recent studies
have shown that EphA2 interacts with members of the
EGFR receptor family, and these interactions may be
important for targeted therapies involving EphA2 and
EGFR [36,50]. Mice harboring ErbB2 (a member of the
EGFR family) in mammary epithelium were sensitive to
inhibition of EphA2 when compared to controls without
ErbB2. EphA2 formed a complex with ErbB2 in both
human and murine breast carcinoma cells, leading to
enhanced signaling through Ras-MAPK activation and
ultimately promoting tumor progression [50]. In addi-
tion, activated EGFR and the constitutively active EGFR
type III deletion mutant (EGFRvIII) were shown to
induce the expression of EphA2 in mammalian cell lines
[36]. Loss of EphA2 expression reduced cell motility of
EGFR-overexpressing cell lines. Miao et al [51] recently
presented evidence that EphA2 serves as a common
downstream effector molecule for growth factor signal-
ing, including signaling through EGF and EGFR provid-
ing further evidence of an EGFR-EphA2 interaction. As
a result, it is possible that loss of EphA2 may alter
EGFR expression levels. Thus, the interaction of EphA2
with members of the EGFR family indicates a functional
role for EphA2 in EGFR-expressing cancer cells. In our
system, loss or reduction of EphA2 through interaction
with YSA-functionalized nanogels may provide an
enhanced effect over delivery of EGFR siRNA
alone leading to a dual-targeting strategy for
Figure 6 Effects of increasing concentrations of docetaxel on SK-OV-3 cells treated with siRNA-loaded nanogels.A. Effects of docetaxel
on SK-OV-3 cells were tested alone or in combination with EGFR siRNA-loaded, YSA-targeted nanogels. The percent cytotoxicity (Tox 8 assay)
was assessed at increasing concentrations of docetaxel and compared to untreated controls. B. Chemosensitization of SK-OV-3 cells treated with
the highest dose of siRNA-loaded nanogels and increasing concentrations of docetaxel were compared to several controls. Controls included
unloaded YSA-conjugated nanogels (YSA-pNIPMAm), unloaded pNIPMAm nanogels (pNIPMAm), YSA peptide alone, and untreated cells. Results
are the mean of two independent experiments; error bars represent standard deviation. Chemosensitivity of nanogel treated SK-OV-3 cells did
not differ significantly from all the controls examined (P > 0.05) at all doses of docetaxel.
Dickerson et al.BMC Cancer 2010, 10:10
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Page 8 of 11
chemosensitization of ovarian tumors. While we did not
observe reduction of EGFR in all experiments presented,
the result is nonetheless intriguing and is under further
investigation.
The ability of siRNAs to potently but reversibly silence
genes in vivo has made them particularly well suited as a
drug therapeutic. However, poor stability under physio-
logical conditions limits the utility of systemic delivery
of siRNA, and its high molecular weight (~13 kDa) and
polyanionic nature prevent transport across the cell
membrane, further compounding the problem of thera-
peutic application. Thus, delivery represents the main
hurdle for broader development of siRNA therapeutics.
To our knowledge, the work presented here along with
our previous studies [26] provides the first description
using targeted, poly(alkylacrylamide)-based nanogels for
siRNA delivery. Furthermore, the core/shell nanogel
delivery system employed here is readily amendable to
selective surface functionalization by a variety of target-
ing molecules, offers a protective environment for sensi-
tive cargo, and shows excellent targeting uptake and
delivery in serum containing medium. The nanogel par-
ticles are also exceedingly simple to load, and extremely
high (>90%) degrees of siRNA incorporation are
observed. These properties and the low toxicity levels
indicated thus far by this formulation, along with the
low immunotoxcity demonstrated recently by Li et al
[52], indicate the promise of overcoming some of the
final obstacles hindering siRNA driven therapeutic stra-
tegies. Future studies investigating the in vivo delivery of
siRNAs to tumors using nanogels, and the effect on che-
mosensitization will aid in the refinement of targeted
siRNA delivery for treatment of ovarian cancer.
Conclusion
The results presented herein demonstrate the therapeu-
tic delivery of gene-specific siRNA cargo using peptide-
functionalized nanogels, with the subsequent reduction
of EGFR expression and increased chemosensitivity to
docetaxel as a highly effective strategy for the sensitiza-
tion of cancer cells to taxane chemotherapy. The
broader significance of this work lies in the establish-
ment of the YSA peptide and its EphA2 receptor target
for the specific and efficient delivery of siRNA directly
to cancer cells, and overcoming the main obstacle hin-
dering therapeutic viability of siRNA treatment, that of
delivery.
Additional file 1: Figure S1. Down-regulation of EGFR by siRNA-loaded
nanogels. An immunoblot from a separate experiment demonstrating
reduction of EGFR expression over time is shown. Note that untargeted
nanogels loaded with siRNA (Ng+siRNA) are used here as a further
control. A decrease in EGFR expression is noted with this control
indicating nonspecific uptake of the nanogels by the Hey cells.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2407-10-
10-S1.PDF ]
Additional file 2: Figure S2. Down-regulation of EGFR by different
concentrations of siRNA-loaded nanogels. An immunoblot from a
separate experiment demonstrating reduction of EGFR expression at the
1000 μg/mL dose of EGFR-siRNA loaded nanogels.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2407-10-
10-S2.PDF ]
Additional file 3: Tables S1-S4. Statistical analysis of siRNA-loaded
nanogels + docetaxel treated Hey cells compared to all controls
(pNIPMAm, YSA-pNIPMAm, YSA peptide alone, and untreated cells). To
determine significance between groups, a one-way ANOVA test was
performed. If significance was indicated, a Tukey post test was performed
to determine significance between sample groups. Significance was
defined as P< 0.05, and doses that were not significant are indicated as
ns.
Click here for file
[ http://www.biomedcentral.com/content/supplementary/1471-2407-10-
10-S3.PDF ]
Abbreviations
EGFR: epidermal growth factor receptor; EGF: epidermal growth factor;
EphA2: erythropoietin-producing hepatocellular (Eph) receptor A2; RNAi: RNA
interference; siRNA: small interfering RNA; YSA: 12 amino acid peptide
(YSAYPDSVPMMS); TKI: tyrosine kinase inhibitor; pNIPMAm: poly(N-
isopropylmethacrylamide).
Acknowledgements
This research was supported by grants from the Deborah Nash Harris
Endowment Fund (JFM), The Robinson Family Fund (JFM), and Ovarian Cycle
(JFM). EBD was supported by a grant from the Georgia Cancer Coalition. LAL
acknowledges support from Emory-Georgia Tech Nanotechnology Center for
Personalized and Predictive Oncology (5-40256-G11) and from DHHS (1 R21
EB006499-01).
Author details
1
School of Biology, Georgia Institute of Technology, 310 Ferst Drive, Atlanta,
GA, 30332, USA.
2
Petit Institute for Bioengineering and Bioscience, Georgia
Institute of Technology, 315 Ferst Drive, Atlanta, GA, 30332, USA.
3
Ovarian
Cancer Institute, Georgia Institute of Technology, 315 Ferst Drive, Atlanta,
GA, 30332, USA.
4
Department of Chemistry and Biochemistry, Georgia
Institute of Technology, 901 Atlantic Drive, Atlanta, GA, 30332, USA.
5
Veterinary Clinical Sciences Department, University of Minnesota, 1352 Boyd
Avenue, St. Paul, MN, 55108, USA.
6
Department of Cellular and Molecular
Medicine, Johns Hopkins School of Medicine, 1830 E. Monument Street,
Baltimore, MD, 21205, USA.
Authors’contributions
EBD, WHB, LAL, and JFM conceived the study. EBD, WHB, and MHS designed
and carried out the studies. WHB and MHS synthesized and characterized
the nanogels. LBK carried out experiments and assisted in data analysis. EBD
was responsible for statistical analysis. EBD, LAL, and JFM were responsible
for preparation of the manuscript. All authors read and approved the final
manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 28 August 2009
Accepted: 11 January 2010 Published: 11 January 2010
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