Aptamer-functionalized, ultra-small, monodisperse silica nanoconjugates for targeted dual-modal imaging of lymph nodes with metastatic tumors

Article abstract of DOI:10.1002/anie.201205271

A dual-modal imaging probe based on size-controlled silica nanoconjugates was synthesized for targeted imaging of lymph nodes by means of both PET and near infrared fluorescence techniques. 20 nm nanoconjugates (see scheme) functionalized with an aptamer (green triangles) that targets 4T1 breast cancer cells improved the detection efficiency of sentinel lymph nodes with metastatic tumors. Copyright

Full text of DOI:10.1002/anie.201205271

Angewandte
Chemie
DOI: 10.1002/anie.201205271
PET/NIR Imaging
Aptamer-Functionalized, Ultra-Small, Monodisperse Silica
Nanoconjugates for Targeted Dual-Modal Imaging of Lymph Nodes
with Metastatic Tumors**
Li Tang, Xujuan Yang, Lawrence W. Dobrucki, Isthier Chaudhury, Qian Yin, Catherine Yao,
Stꢀphane Lezmi, William G. Helferich,* Timothy M. Fan,* and Jianjun Cheng*
Metastases are responsible for 90% of human cancer
deaths.[1] Most solid tumors metastasize through the circu-
lation system, and the sentinel lymph node (LN) is typically
the first site reached by the disseminating malignant cancer
cells.[2] The detection of LN metastases is therefore crucial for
accurate tumor staging and therapeutic decision making.[3]
The current standard method for LN assessment is lymphog-
raphy using a vital blue dye. However, this method is invasive,
involving extended nodal dissection, and can give a false
negative result if an LN is missed in surgery.[4] A non-invasive
LN imaging technique is urgently needed to improve the
accuracy of tumor staging.[5] Various techniques for sentinel
LN imaging have been investigated, such as near-infrared
(NIR) fluorescence imaging, computed tomography (CT),
magnetic resonance imaging (MRI), positron emission
tomography (PET), and ultrasound and photoacoustic imag-
ing.[4b,5b,6] However, each technique has its drawbacks, and
none is sufficient to provide all the necessary information for
LN assessment.[4b] PET is the most sensitive and specific
technique for in vivo molecular imaging,[7] but it suffers from
low spatial resolution. In contrast, fluorescence imaging has
high resolution and allows spatial visualization, which is
helpful for intraoperative guidance; but its application is
limited by poor tissue penetration. Therefore, combination of
both PET and fluorescence imaging together potentially
permit non-invasive assessment of LNs with high sensitivity
and excellent spatial resolution.
Silica nanoparticles (NPs) are widely used for biomedical
imaging applications because of the good biocompatibility
and optical transparency of silica.[8] We recently developed
a versatile, size-controlled, monodisperse, drug/dye silica
nanoconjugate (NC) that allows for conjugation with a variety
of functional moieties.[9] The robust silane chemistry and the
formulation strategy permit the construction of multifunc-
tional NCs, such as multi-modal imaging probes for in vivo
applications. It is generally accepted that the physicochemical
properties of NPs, especially their size, play a vital role in the
systemic and lymphatic biodistribution.[10] Because the size of
the silica NCs can be precisely controlled, they are ideal for
investigating size effects on their trafficking behavior in the
lymphatic system. The silica NCs are multifunctional and give
excellent size control for the preparation of nanoparticulate
probes with optimized properties for improved imaging of LN
metastases.[10c]
There have been many studies on the targeting of primary
tumors,[11] but very few attempts have been made to actively
target metastatic tumors specifically.[12] As an alternative to
antibodies for cancer targeting, aptamers, single-stranded
oligonucleotides that can bind to target molecules with high
specificity and affinity, have attracted much attention because
they are small, easy to synthesize, non-immunogenic, and can
be modified to resist denaturation and biodegradation.[13] The
capability of aptamers to target primary tumors has been
demonstrated in several studies in vivo.[13b,c] However, active
targeting of lymphatic metastases using aptamers has not
been reported.
Herein, we report a convenient, one-pot synthesis of
monodisperse, size-controlled silica NC probes for dual-
modal LN imaging using PET and NIR fluorescence. Mono-
disperse 20 nm silica NCs accumulated in sentinel LNs more
rapidly and to a greater extent than larger NCs (200 nm) and
were superior for efficient LN imaging. To further enhance
the targeting of LNs with metastatic tumors, we functional-
ized the 20 nm silica NCs with a 26-mer G-rich DNA aptamer
(Apt) derived from AS1411, which has high binding affinity
for nucleolin (NCL), a protein that is over-expressed in the
cytoplasm and on the plasma membrane of some cancer cells,
including breast cancer cells.[14] The NCL-Apt-functionalized
silica NCs showed markedly enhanced uptake in LNs with
metastatic tumors in a murine breast tumor model, and
[*] L. Tang, I. Chaudhury, Q. Yin, C. Yao, Prof. Dr. J. Cheng
Department of Materials Science and Engineering,
University of Illinois at Urbana-Champaign
1304 W. Green Street, Urbana, IL, 61801 (USA)
E-mail: jianjunc@illinois.edu
Dr. X. Yang, Prof. Dr. W. G. Helferich
Department of Food Science and Human Nutrition (USA)
E-mail: helferic@illinois.edu
Prof. Dr. L. W. Dobrucki
Department of Bioengineering (USA)
Prof. Dr. S. Lezmi
Department of Pathobiology (USA)
Prof. Dr. T. M. Fan
Department of Veterinary Clinical Medicine (USA)
E-mail: t-fan@illinois.edu
[**] J.C. acknowledges support from the NIH (Director’s New Innovator
Award 1DP2OD007246-01 and 1R21CA152627). L.T. was funded at
University of Illinois at Urbana-Champaign from NIH National
Cancer Institute Alliance for Nanotechnology in Cancer “Midwest
Cancer Nanotechnology Training Center” Grant R25 CA154015A.
Supporting information for this article (experimental details) is
available on the WWW under http://dx.doi.org/10.1002/anie.
201205271.
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improved the detection efficiency of
metastatic tumors in LNs.
Silica NCs for dual-modal imag-
ing were synthesized in a manner
similar to that reported recently.[9]
We first synthesized a silane-modi-
fied NIR dye (NIR-sil) and a silan-
ized chelating reagent (DOTA-sil)
that can bind a radionuclide (e.g.,
64Cu) for PET imaging. Because
silica is optically transparent and
its excitation and emission wave-
lengths can pass through the silica
matrix, NIR-sil was added immedi-
ately after the addition of tetraethyl
orthosilicate (TEOS) so that the
NIR-imaging ligand was stably
bound to and homogeneously dis-
tributed in the silica NCs (Sche-
me 1).[8b,15] DOTA-sil was added to
the NIR-dye-doped silica NCs for
conjugation of DOTA to their sur-
face for chelation of radionuclides.
Poly(ethylene
glycol)-containing
silane (PEG-sil) was then used to
conjugate PEG to the surface of the
silica NCs. Surface PEGylation of
NPs is routinely employed to pro-
long circulation, minimize nonspe-
cific absorption, and reduce particle
aggregation in vivo.[16] Finally, the
as-prepared PEGylated NCs were
labeled with 64Cu, by means of
a
chelation reaction (Scheme 1;
Scheme 1. Preparation of aptamer-functionalized dual-labeled silica NCs for both PET and NIR
fluorescence imaging.
Supporting Information, Figure S1).
We used the described proce-
dure to prepare NIR- and DOTA-
modified silica NCs with controlled sizes of 200 nm and 20 nm
(denoted NC200 and NC20, respectively; Figure 1a). NC200
and NC20 showed strong fluorescence emission at lem values
of 802.5 nm and 808.0 nm, respectively (Figure 1b).[17] Effec-
tive surface modification with DOTA was shown by the z-
potential measurement (Figure 1c). Both NC200 and NC20
had negatively charged surfaces (À34.8 mV and À136.3 mV,
respectively; Figure 1c) at pH 7.4, owing to the surface-bound
carboxy groups of DOTA (Scheme 1). These NCs chelated
64Cu cations (t1/2 = 12.7 h, b+ = 17.4%) with high labeling
efficiency (greater than 60%, Figure 1c and Figure S1) and
good stability in 50% reconstituted human serum (mimicking
physiological conditions; Figure S2).
As expected, the size of the as-prepared, dual-modal silica
NCs was well controlled.[9] Scanning electron microscopy
(SEM) indicated that the diameters of NC200 and NC20 were
198.7 Æ 11.8 and 23.1 Æ 2.3 nm, respectively (Figure 1a,c).
Remarkably narrow size distributions with coefficients of
variation (CV), less than 10%, were observed for both NCs.
Thus, these silica NCs, which we designed for preparing multi-
modal nanoparticulate imaging probes, feature synthetic
convenience, flexibility, excellent size control, and modularity
that allows for alteration of the functionality and surface
chemistry.
To explore the use of the silica NCs for non-invasive PET/
CT imaging of LNs in vivo and to identify the optimal NP size
for the most efficient LN accumulation, we investigated the
lymphatic trafficking of 64Cu-labeled NC200 and NC20 in
normal C57BL/6 mice (Figure 2a). Each mouse received
small-volume interstitial injections (a commonly used admin-
istration route for lymphatic distribution studies)[10f,g,18] of the
two NCs, one into each rear hock (left, NC20; right, NC200).
PET imaging was carried out to monitor the distribution of
the NCs on both sides (Figure 2b). The positions of popliteal
LNs (P-LNs), which are the closest LNs to the injection sites,
can be clearly identified in the CT images (yellow arrows,
Figure 2b). The overlaid CTand PET images show noticeable
radioactivity in the left P-LN as early as 12 minutes post
injection (p.i.) but not in the right P-LN. The signal increased
rapidly in the left P-LN from 3.5% injected dose per gram of
tissue (% I.D.gÀ1) at 12 minutes p.i. to 9.8% I.D.gÀ1 at
62 minutes p.i., suggesting fast and efficient lymphatic drain-
ing of NC20 (Figure 2d). The amount of accumulated NC20
in the left P-LN continued to increase slightly over time,
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Figure 1. a) SEM images of silica NCs. b) Fluorescence emission
spectrum of dual-labeled silica NCs. c) Characterization of silica NCs.
([a] Sizes of the hard cores were measured by SEM. D=average
diameter, SD=standard deviation. [b] CV=SD/D. [c] Conjugation effi-
ciencies (C.E.) of Ctrl- and Apt-DNA were determined by quantifying
the DNA concentration in the supernatant with a Nanodrop spectro-
photometer after the NCs were centrifuged down. [d] Number (No.) of
DNA molecules per NC was calculated from the DNA/NC feeding
ratio and the C.E. value. The silica NC density was set at 2.56 gcmÀ3
[e] 64Cu labeling efficiency (L.E.) was determined by quantifying the
radioactivity in the supernatant with a g-counter).
.
eventually reaching a plateau at about six hours p.i. (10.3%
I.D.gÀ1). This high level of lymphatic accumulation was
maintained for as long as 24 hours p.i. (10.4% I.D.gÀ1),
suggesting that NC20 particles were preferentially retained in
the LNs, presumably because they were taken up by
phagocytosis by resident nodal macrophages and dendritic
cells.[8a,10f,19] In contrast, the signal detected in the right P-LN,
on the side that had been injected with NC200, was negligible
during the first hour p.i. (0.66 and 1.9% I.D.gÀ1 at 12 minutes
and 62 minutes p.i., respectively); the majority of the NC200
particles remained highly localized at the injection site. The
radioactivity in the right P-LN remained low at 6 hours and
24 hours p.i. (1.3 and 2.7% I.D.gÀ1, respectively). These
observations suggest that NC20 gained entrance to and
traveled more easily in the lymphatic system than the
NC200. Three-dimensional reconstructed image and movie
(Figure 2c, Movie S1) also provide evidence for the signifi-
cantly enhanced accumulation of NC20 in the left P-LN at
6 hours p.i. At 24 hours p.i., accumulation of NC20 was
approximately 3.8 times that of NC200 (Figure 2d). These
results were confirmed by ex vivo measurement of the
Figure 2. a) Dual-labeled NC20 (left) and NC200 (right) were adminis-
tered to normal C57BL/6 mice by hock injection; the same amount of
radioactivity was injected on each side. b) In vivo whole-body dynamic
PET/CT imaging of mice was performed to assess the accumulation of
the silica NCs in the P-LNs (yellow arrows). c) Corresponding three-
dimensional PET/CT image at 6 h p.i. shows enhanced accumulation
of NC20 in the left P-LN (yellow arrow). d) Accumulation of 64Cu-
labeled NCs over time in the P-LNs was quantified by selecting the
regions of interest in the PET images and analyzing with the instru-
ment software. e) The radioactivity in the excised P-LNs (24 h p.i.) was
measured ex vivo with a g-counter (averageÆSD; n=3; *=p<0.05).
Figure 3. a) Dual-labeled NC20 (left) and NC200 (right) were adminis-
tered to normal C57BL/6 mice by hock injection. b) I-LNs were
collected and imaged for NIR fluorescence at lem =800 nm (green,
top) and autofluorescence from tissues (including the surrounding fat)
at lem =700 nm (red, top). The I-LNs are indicated by yellow circles
and arrows. The bottom row shows the pseudo-colored images of the
NIR fluorescence intensity in the I-LNs. c) NIR fluorescence intensity
in each I-LN was measured ex vivo at lem =800 nm with an Odyssey
NIR imaging system (averageÆSD; n=3; *=p<0.05).
radioactivity in the excised P-LNs with
a g-counter
(0.50 mCigÀ1 for NC20 and 0.13 mCigÀ1 for NC200; Fig-
ure 2e).
Because the silica NCs were also labeled with NIR dye,
they have the potential to be useful for intraoperative
guidance and high-resolution LN imaging. To demonstrate
the use of the integrated NIR fluorophore for fluorescence
imaging, we harvested the inguinal LNs (I-LNs) from both
sides 24 hours p.i. for ex vivo imaging with an Odyssey NIR
imaging system (Figure 3a,b). A strong fluorescence signal
(298.9) was observed in the left I-LN where NC20 was
injected into the ipsilateral hock; whereas a much weaker
fluorescence signal (50.5 a.u.) was detected in the right I-LN
after injection with NC200 (Figure 3c). The fluorescence
enhancement of NC20 in the left I-LN indicates that NC20
migrated approximately six times as efficiently to distant
regional LNs through the lymphatic vessels as did NC200.
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The preferential accumulation of NC20 in both the P-LN
and the I-LN demonstrates that NC20 was taken up by the
lymphatic system much more rapidly and was retained in the
LNs at a higher level and for a longer time than their larger
counterparts. Because of the sustained LN accumulation, use
of NC20 might overcome the problem with vital blue dyes
(e.g., Evans blue), which is that they diffuse away from the
LNs too rapidly to allow prolonged imaging.[4b,10d] Clearly, the
size of the nanoprobes played a vital role in controlling their
lymphatic trafficking. NC20 diffused rapidly from the inter-
stitial space into the lymphatic vessels, owing to their small
size, and they efficiently migrated to the draining LNs once
they reached the lymphatic vessels.[10f,20] NCs with diameters
greater than or equal to 100 nm are likely to be internalized
by peripheral dendritic cells first and then taken to the LNs by
these cells.[10f,20] This process usually takes more than
24 hours, which could explain the small increase in LN
accumulation of NC200 at 24 hours p.i. (Figure 2d). Our
observations agree with recent reports by Wang et al.[10g] and
Reddy et al.[10f] that 20–30 nm NPs are transported to LNs
much more efficiently than 100 nm NPs after interstitial
injection. The 20 nm NPs may also outperform the 30-50 nm
NPs with regard to lymphatic uptake.[10a,e,f,21] In contrast, NPs
with sizes less than 8 nm may preferentially migrate to the
blood circulation and be cleared rapidly by way of the renal
system.[10b,d] Thus, 20 nm silica NCs are likely in the optimum
size range (10–20 nm) for the most efficient passive targeting
of LNs.
Next, we functionalized the surface of NC20 with NCL-
Apts to assess the capability of aptamer-functionalized NCs to
target metastatic sentinel LNs. Scheme 1 illustrates the
conjugation of NCL-Apt to the surface-bound PEG of silica
NCs. Control DNA (Ctrl-DNA) with a random sequence was
also conjugated to NC20 for comparison. Conjugation of
DNA sequences was achieved with high conjugation effi-
ciency (C.E.; 73.2% for Ctrl-DNA- and 79.6% for NCL-Apt-
functionalized NC20s (designated as NC20-Ctrl and NC20-
Apt); Figure 1c). The DNA surface densities of NC20-Ctrl
and NC20-Apt were on average 8.6 and 9.4 DNA molecules
per NC, respectively (Figure 1c).
Figure 4. a) In vitro 4T1 cell targeting with NC20-Apt. Internalization of
NC20-Ctrl and NC20-Apt by 4T1 cells after 2 h incubation at 378C was
evaluated from the mean fluorescence of treated cells measured using
flow cytometry (**=p<0.01). b) Dual-labeled NC20-Ctrl (left) and
NC20-Apt (right) were administered to BALB/c mice with metastatic
LNs by hock injection. c) In vivo whole-body PET/CT imaging of BALB/
c mice was performed at 24 h p.i. to assess the accumulation of silica
NCs in mP-LNs (yellow arrows). d) Accumulation of the 64Cu-labeled
NCs in the mP-LNs was quantified by selecting the regions of interest
in the PET images and analyzing with the instrument software.
e) Accumulation of the 64Cu-labeled NCs was confirmed by ex vivo
measurement of excised mP-LNs with a g-counter (averageÆSD;
n=3; *=p<0.05).
legs of female BALB/c mice through interstitial injection.[8a,22]
After eight days, visible primary tumors had developed, and
strong localized bioluminescent signals from 4T1 tumors were
detected (Figure S4a). Tumor cells also metastasized to the P-
LNs, as evidenced by their enlarged sizes and histological
analysis of the excised metastatic P-LNs (mP-LNs, Fig-
ure S4b–d).[12c,23] Next, NC20-Ctrl and NC20-Apt were sub-
cutaneously injected into the interstitial space between the
primary tumors and the mP-LNs (Figure 4b) in a mouse with
mP-LNs on both sides. We acquired PET/CT images 24 hours
p.i. to compare the uptake of both NCs in the mP-LNs. A
much stronger PET signal was observed in the right mP-LN,
on the side injected with NC20-Apt (Figure 4c). The accu-
mulation of NC20-Apt in the mP-LN was approximately 2.3
times that of the NC20-Ctrl, as indicated by quantification of
the radioactivity in the PET images (6.2% I.D.gÀ1 for NC20-
Ctrl and 14.6% I.D.gÀ1 for NC20-Apt; Figure 4d). This result
was also confirmed by ex vivo measurement of the radio-
activity in the excised mP-LNs with a g-counter (6.5% I.D.gÀ1
for NC20-Ctrl and 14.8% I.D.gÀ1 for NC20-Apt; Figure 4e),
which correlates well with the in vivo data. In addition, mice
To evaluate the targeting capability of NC20-Apt in vitro,
we separately incubated Rhodamine
B isothiocyanate
(RITC)-labeled NC20-Ctrl and NC20-Apt with 4T1 murine
breast cancer cells. Flow-cytometric analysis showed that
there was a 1.6-fold increase in mean fluorescence intensity in
4T1 cells treated with NC20-Apt (20.8 a.u.) versus cells
treated with NC20-Ctrl (13.1 a.u.; Figure 4a). Of the cells
incubated with NC20-Apt for two hours, 70.7% became
fluorescently positive, whereas only 57.5% became positive
after incubation with NC20-Ctrl (Figure S3). These results
clearly demonstrate that NC20-Apt had the enhanced ability
to bind to 4T1 breast cancer cells in vitro.
Because NC20-Apt was selectively taken up by the 4T1
cancer cells, we expected that NC20-Apt would show
enhanced accumulation in LNs with metastatic 4T1 tumors.
Therefore, we evaluated the capability of NC20-Apt to target
metastatic tumors in sentinel LNs in vivo. First, we estab-
lished an LN metastasis tumor model by hock inoculation of
4T1 cells (stably transfected with firefly luciferase) in both
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[3] a) T. Aikou, Y. Kitagawa, M. Kitajima, Y. Uenosono, A. J.
with an mP-LN on one side (right) and a normal P-LN on the
other (left) received interstitial injections of NC20-Apt in
both rear hocks (Figure S5a). There was an approximately
2.1-fold increase in NC20-Apt accumulation in the mP-LN
compared to the normal P-LN (5.9% I.D.gÀ1 for P-LN and
12.1% I.D.gÀ1 for mP-LN; Figure S5b).
Bilchik, S. R. Martinez, S. Saha, Cancer Metastasis Rev. 2006, 25,
269 – 277; b) M. G. Harisinghani, J. Barentsz, P. F. Hahn, W. M.
Deserno, S. Tabatabaei, C. H. van de Kaa, J. de La Rosette, R.
Weissleder, N. Engl. J. Med. 2003, 348, 2491 – 2499.
[4] a) R. T. Lucarelli, M. Ogawa, N. Kosaka, B. Turkbey, H.
Kobayashi, P. L. Choyke, Lymphatic Res. Biol. 2009, 7, 205 –
214; b) F. Zhang, G. Niu, G. M. Lu, X. Y. Chen, Mol. Imaging
Biol. 2011, 13, 599 – 612.
[5] a) S. K. Nune, P. Gunda, B. K. Majeti, P. K. Thallapally, M. L.
Forrest, Adv. Drug Delivery Rev. 2011, 63, 876 – 885; b) S. M.
Cohen, B. G. Fishinghawk, M. S. Cohen, Adv. Drug Delivery
Rev. 2011, 63, 956 – 962.
[6] S. M. Moghimi, B. Bonnemain, Adv. Drug Delivery Rev. 1999,
37, 295 – 312.
[7] Y. Sun, M. X. Yu, S. Liang, Y. J. Zhang, C. G. Li, T. T. Mou, W. J.
Yang, X. Z. Zhang, B. A. Li, C. H. Huang, F. Y. Li, Biomaterials
2011, 32, 2999 – 3007.
The increased accumulation of NC20-Apt in the mP-LNs
was likely due to selective uptake by the metastatic 4T1 cells
in the mP-LNs.[12a] The enhanced uptake and retention of the
aptamer-functionalized dual-modal silica NCs in metastatic
LNs permits discrimination of metastatic and normal LNs and
thus improves the efficiency of tumor metastasis detection. A
widely used strategy for improving accumulation of NPs in
LNs is surface decoration of NPs with sugar molecules to
enhance uptake by macrophages, which home to the LNs.[24]
However, macrophages that take up the sugar-modified NPs
may also transport these NPs to the blood, resulting in non-
specific accumulation in other organs (e.g., liver and spleen).
Antibodies[12b,25] and peptides[8b,12a,c,22] are also employed as
active targeting ligands to modify the surface of NPs, to
improve the targeting efficiency for metastatic tumors in LNs.
However, the application of antibodies or peptides is limited
by their relative instability, high cost, and difficult large-scale
preparation. Highly specific aptamer-functionalized silica-NC
imaging probes may overcome these limitations and have
potential applications in the clinic.
In conclusion, we have developed monodisperse, size-
controlled silica NCs for dual-modal PET/NIR imaging of
sentinel LNs. Dual-modal imaging using this novel probe has
unique advantages over conventional lymphatic imaging
techniques. For example, PET imaging is a noninvasive
diagnostic method and overcomes the depth insensitivity of
optical imaging tools, and NIR fluorescence imaging can
compensate for the relatively low spatial resolution of PET
imaging and potentially provide convenient intraoperative
guidance for resection of metastatic LNs once they are
identified. When the size of the silica NCs was controlled as
20 nm, they accumulated rapidly and effectively in LNs, thus
allowing for improved LN imaging in vivo. For the first time,
aptamers were used to functionalize silica NCs for active
targeting of lymphatic metastases. Uptake and retention of
the aptamer-functionalized silica NCs in metastatic LNs were
significantly enhanced compared to the non-targeted silica
NCs. These dual-labeled silica NCs hold great potential for
improving the accuracy of clinical tumor staging by serving as
probes for efficient noninvasive targeted imaging of meta-
static LNs.
[8] a) X. L. Huang, F. Zhang, S. Lee, M. Swierczewska, D. O.
Kiesewetter, L. X. Lang, G. F. Zhang, L. Zhu, H. K. Gao, H. S.
Choi, G. Niu, X. Y. Chen, Biomaterials 2012, 33, 4370 – 4378;
b) M. Benezra, O. Penate-Medina, P. B. Zanzonico, D. Schaer,
H. Ow, A. Burns, E. DeStanchina, V. Longo, E. Herz, S. Iyer, J.
Wolchok, S. M. Larson, U. Wiesner, M. S. Bradbury, J. Clin.
Invest. 2011, 121, 2768 – 2780; c) R. Kumar, I. Roy, T. Y.
Ohulchanskky, L. A. Vathy, E. J. Bergey, M. Sajjad, P. N.
Prasad, ACS Nano 2010, 4, 699 – 708; d) L. Wang, K. M. Wang,
S. Santra, X. J. Zhao, L. R. Hilliard, J. E. Smith, J. R. Wu, W. H.
Tan, Anal. Chem. 2006, 78, 646 – 654; e) W. J. Rieter, J. S. Kim,
K. M. L. Taylor, H. Y. An, W. L. Lin, T. Tarrant, W. B. Lin,
Angew. Chem. 2007, 119, 3754 – 3756; Angew. Chem. Int. Ed.
2007, 46, 3680 – 3682; f) H. Meng, M. Xue, T. Xia, Z. Ji, D. Y.
Tarn, J. I. Zink, A. E. Nel, ACS Nano 2011, 5, 4131 – 4144.
[9] L. Tang, T. M. Fan, L. B. Borst, J. Cheng, ACS Nano 2012, 6,
3954 – 3966.
[10] a) X. Cai, W. Li, C.-H. Kim, Y. Yuan, L. V. Wang, Y. Xia, ACS
Nano 2011, 5, 9658 – 9667; b) L. M. Kaminskas, C. J. H. Porter,
Adv. Drug Delivery Rev. 2011, 63, 890 – 900; c) C. Oussoren, G.
Storm, Adv. Drug Delivery Rev. 2001, 50, 143 – 156; d) H.
Kobayashi, S. Kawamoto, M. Bernardo, M. W. Brechbiel, M. V.
Knopp, P. L. Choyke, J. Controlled Release 2006, 111, 343 – 351;
e) S. T. Reddy, A. Rehor, H. G. Schmoekel, J. A. Hubbell, M. A.
Swartz, J. Controlled Release 2006, 112, 26 – 34; f) S. T. Reddy,
A. J. van der Vlies, E. Simeoni, V. Angeli, G. J. Randolph, C. P.
OꢁNeil, L. K. Lee, M. A. Swartz, J. A. Hubbell, Nat. Biotechnol.
2007, 25, 1159 – 1164; g) H. Wang, S. T. Wang, H. Su, K. J. Chen,
A. L. Armijo, W. Y. Lin, Y. J. Wang, J. Sun, K. Kamei, J. Czernin,
C. G. Radu, H. R. Tseng, Angew. Chem. 2009, 121, 4408 – 4412;
Angew. Chem. Int. Ed. 2009, 48, 4344 – 4348; h) T. T. Goodman,
P. L. Olive, S. H. Pun, Int. J. Nanomed. 2007, 2, 265 – 274; i) S. D.
Perrault, C. Walkey, T. Jennings, H. C. Fischer, W. C. W. Chan,
Nano Lett. 2009, 9, 1909 – 1915.
[11] a) D. Peer, J. M. Karp, S. Hong, O. C. Farokhzad, R. Margalit, R.
Langer, Nat. Nanotechnol. 2007, 2, 751 – 760; b) M. E. Davis, Z.
Chen, D. M. Shin, Nat. Rev. Drug Discovery 2008, 7, 771 – 782.
[12] a) C. Leuschner, C. Kumar, W. Hansel, W. Soboyejo, J. K. Zhou,
J. Hormes, Breast Cancer Res. Treat. 2006, 99, 163 – 176; b) L.
Sampath, S. Kwon, M. A. Hall, R. E. Price, E. M. Sevick-
Muraca, Transl. Oncol. 2010, 3, 307 – 317; c) Z. Q. Yan, C. Y.
Zhan, Z. Y. Wen, L. L. Feng, F. Wang, Y. Liu, X. K. Yang, Q.
Dong, M. Liu, W. Y. Lu, Nanotechnology 2011, 22, 415103.
[13] a) D. Shangguan, Y. Li, Z. W. Tang, Z. H. C. Cao, H. W. Chen, P.
Mallikaratchy, K. Sefah, C. Y. J. Yang, W. H. Tan, Proc. Natl.
Acad. Sci. USA 2006, 103, 11838 – 11843; b) J. Cheng, B. A.
Teply, I. Sherifi, J. Sung, G. Luther, F. X. Gu, E. Levy-
Nissenbaum, A. F. Radovic-Moreno, R. Langer, O. C. Farokh-
zad, Biomaterials 2007, 28, 869 – 876; c) O. C. Farokhzad, J.
Received: July 4, 2012
Published online: November 7, 2012
Keywords: aptamers · breast cancer · dual-modal imaging ·
.
metastasis targeting · nanoparticles
[1] C. L. Chaffer, R. A. Weinberg, Science 2011, 331, 1559 – 1564.
[2] T. Tammela, A. Saaristo, T. Holopainen, S. Ylꢀ-Herttuala, L. C.
Andersson, S. Virolainen, I. Immonen, K. Alitalo, Sci. Transl.
Med. 2011, 3, 69ra11.
Angew. Chem. Int. Ed. 2012, 51, 12721 –12726
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 12725

.
Angewandte
Communications
Cheng, B. A. Teply, I. Sherifi, S. Jon, P. W. Kantoff, J. P. Richie,
R. Langer, Proc. Natl. Acad. Sci. USA 2006, 103, 6315 – 6320.
[14] a) Z. Cao, R. Tong, A. Mishra, W. Xu, G. C. L. Wong, J. Cheng,
Y. Lu, Angew. Chem. 2009, 121, 6616 – 6620; Angew. Chem. Int.
Ed. 2009, 48, 6494 – 6498; b) S. Christian, J. Pilch, M. E. Aker-
man, K. Porkka, P. Laakkonen, E. Ruoslahti, J. Cell Biol. 2003,
163, 871 – 878; c) S. Soundararajan, W. W. Chen, E. K. Spicer, N.
Courtenay-Luck, D. J. Fernandes, Cancer Res. 2008, 68, 2358 –
2365.
[20] M. F. Bachmann, G. T. Jennings, Nat. Rev. Immunol. 2010, 10,
787 – 796.
[21] S. M. Moghimi, A. C. Hunter, J. C. Murray, FASEB J. 2005, 19,
311 – 330.
[22] F. Zhang, G. Niu, X. Lin, O. Jacobson, Y. Ma, H. Eden, Y. He, G.
Lu, X. Chen, Amino Acids 2012, 42, 2343 – 2351.
[23] Z. Q. Yan, F. Wang, Z. Y. Wen, C. Y. Zhan, L. L. Feng, Y. Liu,
X. L. Wei, C. Xie, W. Y. Lu, J. Controlled Release 2012, 157, 118 –
125.
[15] S. W. Ha, C. E. Camalier, G. R. Beck, J. K. Lee, Chem. Commun.
2009, 2881 – 2883.
[16] P. Caliceti, F. M. Veronese, Adv. Drug Delivery Rev. 2003, 55,
1261 – 1277.
[17] N. Narayanan, G. Patonay, J. Org. Chem. 1995, 60, 2391 – 2395.
[18] E. J. Chaney, L. Tang, R. Tong, J. J. Cheng, S. A. Boppart, Mol.
Imaging 2010, 9, 153 – 162.
[24] a) V. Q. Hieu, M. K. Yoo, H. J. Jeong, H. J. Lee, M. Muthiah,
J. H. Rhee, J. H. Lee, C. S. Cho, Y. Y. Jeong, I. K. Park, Acta
Biomater. 2011, 7, 3935 – 3945; b) B. E. Ocampo-Garcꢂa, F. d. M.
Ramꢂrez, G. Ferro-Flores, L. M. De Leꢃn-Rodrꢂguez, C. L.
Santos-Cuevas, E. Morales-Avila, C. Arteaga de Murphy, M.
Pedraza-Lꢃpez, L. A. Medina, M. A. Camacho-Lꢃpez, Nucl.
Med. Biol. 2011, 38, 1 – 11.
[19] J. C. Alex, D. L. Weaver, J. T. Fairbank, B. S. Rankin, D. N. Krag,
Surg. Oncol. 1993, 2, 303 – 308.
[25] M. Takeda, H. Tada, H. Higuchi, Y. Kobayashi, M. Kobayashi, Y.
Sakurai, T. Ishida, N. Ohuchi, Breast Cancer 2008, 15, 145 – 152.
12726 www.angewandte.org
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2012, 51, 12721 –12726