Selective inhibition of human brain tumor cells through multifunctional quantum-dot-based siRNA delivery

Article abstract of DOI:10.1002/anie.200905126

(Figure Presented) More than one Job: Quantum dots (QDs) conjugated with thiol-modified small Interfering RNA (siRNA) were functlonalized with thiol-modlfied RGD and HIV-Tat peptides. These multifunctional QDs were used for the targeted delivery and track

Full text of DOI:10.1002/anie.200905126

Angewandte
Chemie
DOI: 10.1002/anie.200905126
Bionanotechnology
Selective Inhibition of Human Brain Tumor Cells through
Multifunctional Quantum-Dot-Based siRNA Delivery**
Jongjin Jung, Aniruddh Solanki, Kevin A. Memoli, Ken-ichiro Kamei, Hiyun Kim,
Michael A. Drahl, Lawrence J. Williams, Hsian-Rong Tseng, and KiBum Lee*
One of the most promising new chemotherapeutic strategies
is the RNA interference (RNAi)-based approach, wherein
small double-stranded RNA molecules can sequence-specif-
ically inhibit the expression of targeted oncogenes.[1] In
principle, this method has high specificity and broad applic-
ability for chemotherapy. For example, the strategy of small
interfering RNA (siRNA) enables manipulation of key
oncogenes that modulate signaling pathways and thereby
regulate the behavior of malignant tumor cells. To harness the
full potential of this approach, the prime requirements are to
deliver the siRNA molecules with high selectivity and
efficiency into tumor cells and to monitor both siRNA
delivery and the resulting knockdown effects at the single-cell
level. Although several approaches such as polymer- and
nanomaterial-based methods[2] have been attempted, limited
success has been achieved for delivering siRNA into the
target tumor cells. Moreover, these types of approaches
mainly focus on the enhancement of transfection efficiency,
knockdown of non-oncogenes (e.g. the gene coding for green
fluorescent protein (GFP)), and the use of different nano-
materials such as quantum dots (QDs), iron oxide nano-
particles, and gold nanoparticles.[3,4] Therefore, to narrow the
gap between current nanomaterial-based siRNA delivery and
chemotherapies, there is a clear need to develop methods for
target-oriented delivery of siRNA,[5] for further monitoring
the effects of siRNA-mediated target-gene silencing by means
of molecular imaging probes,[4] and for investigating the
corresponding up/down-regulation of signaling cascades.[6]
Perhaps most importantly, to begin the development of the
necessary treatment modalities, the strategies for nanomate-
rial-based siRNA delivery must be demonstrated on onco-
genes involved in cancer pathogenesis.
Herein, we describe the synthesis and target-specific
delivery of multifunctional siRNA–QD constructs for selec-
tively inhibiting the expression of epidermal growth factor
receptor variant III (EGFRvIII) in target human U87 glio-
blastoma cells, and subsequently monitoring the resulting
down-regulated signaling pathway with high efficiency.[7]
Glioblastoma multiforme (GBM) is the most malignant,
invasive, and difficult-to-treat primary brain tumor. Success-
ful treatment of GBM is rare with a mean survival of only 10–
12 months.[8] EGFRvIII, the key growth factor receptor
triggering cancer cell proliferation in many cancer diseases
such as brain tumors and breast cancer, is a constitutively
active mutant of EGFR which is expressed in only human
GBM and several other malignant cancers, but not in normal
healthy cells (Figure 1A).[9] We targeted EGFRvIII, since it is
known that knockdown of this gene is one of the most
effective ways to down-regulate the PI3K/Akt signaling
pathway, a key signal cascade for cancer cell proliferation
and apoptosis.[6,10] Hence by targeting EGFRvIII, our siRNA
delivery strategy based on multifunctional nanoparticles
[*] J. Jung, A. Solanki, K. A. Memoli, H. Kim, M. A. Drahl,
Prof. L. J. Williams, Prof. K.-B. Lee
Department of Chemistry and Chemical Biology, Rutgers University
Piscataway, NJ 08854 (USA)
Fax: (+1)732-445-5312
E-mail: kblee@rutgers.edu
Homepage: http://rutchem.rutgers.edu/~kbleeweb/
Dr. K. Kamei, Prof. H.-R. Tseng
Department of Molecular and Medical Pharmacology
University of California, Los Angeles
Los Angeles, CA 90095 (USA)
Figure 1. A) Quantum dots as a multifunctional nanoplatform to
deliver siRNA and to elucidate the EGFRvIII-knockdown effect of PI3K
signaling pathway in U87-EGFRvIII B) Detailed structural information
of multifunctional siRNA–QDs. C) Two different strategies for the
siRNA–QD conjugate. L1 shows the linker for attaching siRNA to QDs
through a disulfide linkage which is easily reduced within the cells to
release the siRNA. L2 shows the linker for covalently conjugating
siRNA to QDs which enables the tracking of siRNA–QDs within the
cells.
[**] We thank V. Starovoytov and Dr. Y. Horibe for helping us with TEM,
and the New Jersey Biomaterial Center (Prof. Kohn) for allowing us
to use the cell culture facilities. K.-B.L. acknowledges the NIH
Directors’ Innovator Award (1DP20D006462-01) and is also grateful
to the NJ commission on Spinal Cord grant (09-3085-SCR-E-0).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.200905126.
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could potentially minimize the side effects caused by conven-
widely used to investigate siRNA-based silencing of EGFP,
since the suppression of EGFP expression does not compro-
mise cell viability. The transfection efficiency of three differ-
ent kinds of constructs were evaluated; constructs modified
with the RGD peptide only, those modified with the HIV-Tat
peptide only, and those with both HIV-Tat and RGD peptide.
Although the siRNA–QDs modified with only RGD showed
considerable selective internalization within U87-EGFP cells,
siRNA–QDs modified with a combination of RGD and HIV-
Tat peptides (the ratio of siRNA/RGD/HIV-Tat being 1:10:10
per QD) showed maximum internalization within U87-EGFP
cells, in close agreement with previous studies.[4] This optimal
condition was used for subsequent siRNA–QD experiments.
The U87-EGFP cell line was then treated with siRNA–
QDs (siRNA/QDs = 0.12 mm:0.011 mm), modified with HIV-
Tat [ ꢀ 1.2 mm] and RGD [ ꢀ 1.2 mm], and simultaneously
imaged using fluorescence microscopy (Figure 2). Cationic
lipids (X-tremeGENE, Roche) were used to further enhance
cellular uptake and prevent degradation of the siRNA within
the endosomal compartment of the cells. The siRNA–QDs
showed significant internalization into the cells. Knockdown
of the EGFP signal was observed after 48–72 h (Figure 2B).
Fluorescence intensity was influenced by other factors such as
exposure time, media conditions, and cell shrinkage. To
minimize the influence from these external factors, the
tional chemotherapies, specifically immune suppression,
while significantly improving the efficacy of chemotherapy
against GBM.
We prepared two types of siRNA–QD conjugates, one for
siRNA delivery and the other for siRNA tracking (Fig-
ure 1B,C). Core–shell CdSe/CdS/ZnS QDs with a diameter of
7 nm were synthesized[11] and coated with either trioctylphos-
phine oxide (TOPO) or hexadecylamine (HDA). In order to
make the QD constructs water-soluble and suitable for
conjugating with siRNA, we displaced these hydrophobic
ligands with a dihydrolipoic acid (DHLA) derivatized with an
amine-terminated poly(ethylene glycol) (PEG) spacer. The
expectation was that the dithiol moiety would provide strong
coordination to the QD surface and increase stability in
aqueous media, the PEG spacer would increase water
solubility and reduce nonspecific binding, and the amine
group would enable conjugation to the siRNA element.[12]
Two bifunctional linkers were synthesized and evaluated for
siRNA conjugation. The linker shown in L1, PTPPf [3-(2-
pyridyl)-dithiopropionic acid pentafluorophenyl ester], was
designed to release siRNA upon entering the cell by cleavage
of the disulfide linkage, through enzymatic reduction or
ligand exchange (e.g. glutathione).[13] The linker in L2, MPPF
(3-maleimidopropionic acid pentafluorophenyl ester), was
designed to be more robust, thereby enabling evaluation of
cellular uptake and localization of the siRNA construct within
the cellular compartments.[14] Details of the synthesis, char-
acterization and conjugation protocols are given in the
Supporting Information.
The final design component was functionalizing the
construct for tumor-cell-selective transfection. For this pur-
pose two functional peptides, thiol-modified RGD peptide
and thiol-modified HIV-Tat derived peptide, were attached to
the siRNA–QDs by the conjugation methods described
above. Brain tumor cells (U87 and U87-EGFRvIII) over-
express the integrin receptor protein avb3, which strongly
binds to the RGD binding domain.[15] RGD-functionalized
siRNA–QDs selectively accumulate in brain tumor cells in
vitro, and can be tracked by fluorescence microscopy.[16] In
addition, the HIV-Tat peptide enables efficient transfection of
siRNA–QDs in cells when it is directly attached to the QD
surface.[17] The density of siRNA on the QDs and the ratio
between siRNA strands and peptides were optimized for gene
knockdown. It was found that the density of 10 siRNAs per
nanoparticle and the ratio of 1:10 (siRNA for each peptide),
which was in close agreement with literature values,[4] was
optimal for knocking down the target genes (EGFP and
EGFRvIII) overexpressed in our U87 cell lines.
To optimize gene silencing with our siRNA–QD con-
structs and to assess the transfection efficiency and RNA
interference (RNAi) activity, we examined the suppression of
EGFP expressed in U87 cell lines that were genetically
modified to express EGFP. The cytotoxicity of the constructs
was determined by serial dilution studies. The range of
concentration causing minimal or negligible cytotoxicity was
identified, and subsequent experiments employed the con-
centrations within this range (see Figure S1 in the Supporting
Information).[18] Importantly, the EGFP cell line has been
Figure 2. Knockdown of EGFP in U87 cells using siRNA–QDs modified
with RGD and HIV-Tat peptides. (Note that yellow arrows mark U87-
EGFP cells transfected with the siRNA–QDs and the blue arrows
indicate PC-12 cells.) A) Control U87-EGFP cells without siRNA–QDs;
phase-contrast image (A1) and the corresponding fluorescence image
(A2). B) EGFP knockdown using multifunctional siRNA–QDs;
B1) Phase-contrast image shows that the morphology of U87-EGFP
cells has not changed relative to the control cells in (A). B2) Fluores-
cence image clearly shows the knockdown of EGFP in cells (marked by
yellow arrows) which have internalized the siRNA–QDs (red) after
48 h. C) U87-EGFP control cells (without siRNA–QDs) and U87-EGFP
cells transfected with siRNA–QDs were cocultured so as to investigate
them under the same conditions; C1) Phase-contrast image clearly
shows no difference in the morphology of the U87-EGFP control cells
and the siRNA–QDs transfected cells. C2) Fluorescence image clearly
shows the decrease in the EGFP signal in the U87-EGFP cells trans-
fected with siRNA–QDs as compared to the surrounding U87-EGFP
control cells. D) Phase-contrast image showing the target-oriented
delivery of siRNA–QDs in cocultures of the malignant U87-EGFP cells,
overexpressing the avb3 integrin receptors, and the less tumorigenic
PC-12 cells (blue arrows) incubated with the siRNA–QDs. It can be
clearly seen that most of the siRNA–QDs, owing to the presence of
RGD and HIV-Tat peptides, were taken up by the U87-EGFP cells and
not by the PC-12 cells. Scale bars: 100 mm.
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control U87-EGFP cells (without siRNA) were trypsinized
and co-cultured with U87-EGFP cells transfected with
siRNA–QDs in the same well. The U87 cells containing
siRNA–QDs were easily distinguishable from the control
cells owing to the bright fluorescence of the QDs (Fig-
ure 2C2). Cells with internalized siRNA–QDs showed con-
siderable knockdown of the EGFP protein relative to the
surrounding control U87-EGFP cells (Figure 2C).
To further demonstrate the target-specific delivery of the
siRNA–QDs, we incubated the siRNA–QDs modified with
Tat and RGD against EGFP in co-cultures of the U87-EGFP
cell line with other less-tumorigenic cell lines, such as PC-12
and SK-N-BE(2)C (see Figure S2 in the Supporting Informa-
tion), which have a considerably small number of integrin
receptors.[19] The presence of RGD tripeptide molecules on
the surface of the siRNA–QDs led to specific binding with
integrin receptors overexpressed in the U87 cells, resulting in
higher cellular uptake by the malignant U87 cells than by the
less tumorigenic PC-12 cells as seen by the selective
accumulation of the QDs within the U87-EGFP cells
(Figure 2D). These results confirmed our hypothesis that
the target-specific delivery of the siRNA–QDs into brain
cancer cells can be significantly enhanced by functionalizing
the QDs with targeting moieties like RGD tripeptide.
The intracellular delivery of the siRNA–QDs within the
U87-EGFP cells was also confirmed by transmission electron
microscopy (TEM), which clearly shows the presence of QDs
in the cytoplasm of the cells (Figure 3A). The knockdown
efficiency of the siRNA–QDs was similar to or slightly better
than that of the positive control consisting of U87-EGFP cells
transfected with only siRNA using X-tremeGENE (see
Figure S3 in the Supporting Information). This high trans-
fection efficiency appears to result from synergistic effects of
the two transfection peptides. Decrease in fluorescence
intensities (EGFP signal, green fluorescence) within cells
treated with the above-mentioned systems were then com-
pared with the intensity of U87-EGFP without siRNA. As
shown in (Figure 3B), the decrease in fluorescence intensity
of U87-EGFP incubated with siRNA–QDs and siRNA alone
was comparable, but drastically lower than that observed for
the control without siRNA. Cells containing siRNA–QDs
show a weaker green fluorescence (EGFP signal) than the
control. This data strongly suggests that siRNA–QDs can be
used simultaneously as delivery and imaging probes.
Having demonstrated the selective manipulation of the
U87-EGFP cell line, we then focused on the knockdown of
EGFRvIII with our siRNA–QD constructs. U87-EGFRvIII
cells were genetically modified to overexpress EGFRvIII, a
mutant-type epidermal growth factor receptor (EGFR) only
expressed within cancer cells.[20] This cell type was incubated
with our siRNA–QDs modified with Tat and RGD peptides
and armed with EGFRvIII-targeting siRNA. The cells were
simultaneously imaged for the internalization of siRNA–QDs
using fluorescence microscopy. Significant cell death was
observed in the wells loaded with siRNA–QDs against
EGFRvIII after 48 h (Figure 4A). Quantitative analysis
revealed that the number of viable U87-EGFRvIII cells, as
observed by fluorescence microscopy, decreased with increas-
ing incubation time. Relative to the control (U87-EGFRvIII
Figure 3. Knockdown efficiency of EGFP within U87-EGFP cells and
internalization of multifunctional siRNA–QDs. A) TEM analysis of the
internalization of the multifunctional siRNA–QDs into the U87-EGFP
cells; A1) Presence of multifunctional siRNA–QDs (yellow arrows)
within the cytoplasm and the endosome (scale bar: 5 mm).
A2) Enlarged image showing individual siRNA–QDs within the cyto-
plasm (scale bar: 2.5 mm). B) The bar graph represents the knockdown
of EGFP over 24 h, 48 h, and 96 h in U87-EGFP cells treated with
siRNA [0.12 mm] only (dark gray), and siRNA–QD [siRNA:QD=
0.12 mm:0.011 mm] (light gray). The EGFP knockdown data was nor-
malized with the expression levels of EGFP in the control U87-EGFP
cells (black).
without siRNA–QDs), there was a significant decrease in the
number of viable cells, thus demonstrating the effectiveness of
our nanoparticle-based siRNA delivery to knockdown the
oncogene. The result was confirmed using the MTT assay
which showed a decrease in the number of viable cells in the
well incubated with siRNA–QDs against EGFRvIII (Fig-
ure 4B). This assay further confirmed that the QDs them-
selves were noncytotoxic when used alone as they did not
result in any appreciable cell death (see Figure S1 in the
Supporting Information). The knockdown of EGFRvIII and
the inhibition of the downstream proteins in the PI3K
signaling pathway were confirmed using Western immuno-
blotting. The results (Figure 4C) confirm a considerable
decrease in the expression of EGFRvIII, and down-regulation
of phospho-Akt and phospho-S6 relative to the control. Thus,
these results demonstrate the specificity of the siRNA against
EGFRvIII, the inherent noncytotoxicity of the QDs, and the
facile evaluation and manipulation of cancer cell proliferation
with multifunctional QD constructs.
In summary, we have demonstrated an application of
multifunctional siRNA–QDs focusing on targeted delivery,
high transfection efficiency, and multimodal imaging/track-
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Communications
egies but also for dissecting signaling cascades triggered by
inhibiting specific proteins. Collectively, our strategy for
siRNA delivery based on multifunctional QDs has significant
potential for simultaneous prognosis, diagnosis, and therapy.
Received: September 13, 2009
Published online: November 30, 2009
Keywords: antitumor agents · gene knockdown · nanoparticles ·
.
nonviral siRNA delivery · target-specific delivery
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