2768?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
Multimodal silica nanoparticles are effective
cancer-targeted probes in a model
of human melanoma
Miriam Benezra,1 Oula Penate-Medina,1 Pat B. Zanzonico,2 David Schaer,3 Hooisweng Ow,4
Andrew Burns,5 Elisa DeStanchina,6 Valerie Longo,1 Erik Herz,7 Srikant Iyer,7 Jedd Wolchok,3,8
Steven M. Larson,1 Ulrich Wiesner,7 and Michelle S. Bradbury1
1Department of Radiology, 2Department of Medical Physics, and 3Immunology Program, Sloan-Kettering Institute for Cancer Research,
New York, New York, USA. 4Hybrid Silica Technologies, Ithaca, New York, USA. 5GE Global Research Center, Niskayuna, New York, USA.
6Antitumor Assessment Facility, Sloan-Kettering Institute for Cancer Research, New York, New York, USA.
7Department of Materials Science and Engineering, Cornell University, Ithaca, New York, USA.
8Department of Medicine, Sloan-Kettering Institute for Cancer Research, New York, New York, USA.
and comprehensively defined according to the following set of
diagnostic criteria: favorable distribution and targeting kinetics (1,
5–9), efficient renal clearance (10, 11), extended circulation (blood
residence) times (5, 12, 13), pronounced tissue signal amplifica-
tion (5, 6, 14), and improved tumoral penetration (15).
Recently reported design criteria have been proposed for advancing
targeted probes to the clinic (11). These have largely been restricted to
improving renal clearance properties of inorganic particles containing
inherently toxic components. The relative lack of in vivo toxicology
data associated with these probes has led to persistent and valid safety
concerns (16), prompting a number of particle-design modifications
(9, 17–19), including size, composition, and surface chemistry, that
promote more rapid clearance through kidney (renal) or liver (hepat-
ic) pathways (20). While such approaches may minimize toxicity, the
ultimate fate and impact of residual particles in the reticuloendothe-
lial system (RES; liver, spleen, lymph nodes, bone marrow) remains
unclear, particularly if toxicity is related to prolonged organ residence
times (16, 17). Accelerated particle clearance may also preclude cer-
tain clinical applications as a result of restrictions imposed upon the
available imaging period. Further, while rapid particle extravasation
may facilitate clearance and delivery to tumors, reduced receptor
binding (5) and increased nonspecific tissue dispersal (21, 22) may
also occur, increasing image background and, potentially, toxicity by
Only a relatively small percentage of new imaging agents under-
going comprehensive preclinical testing potentially satisfy diag-
nostic criteria for translation (2). Although a number of fluo-
Despite recent advances in nanoparticle probe development for
biomedicine, the translation of targeted diagnostic particle plat-
forms remains challenging (1–3). Nanoparticle-based materials
currently under evaluation in oncology clinical trials are largely
nontargeted drug delivery vehicles or devices for thermally treat-
ing tissue and not typically surface modified for direct detection
by clinical imaging tools. Tumor-selective diagnostic probes, in
addition to satisfying critical safety benchmarks, need to elucidate
targeted interactions with the microenvironment and their effects
on biological systems. Several key factors that limit the transla-
tion of such particle probes have been identified: suboptimum
pharmacokinetics, in vivo probe nanotoxicity (4), evolving regu-
latory considerations, reimbursement issues, and resource-inten-
sive scale-up of manufacturing for clinical trials. To address such
issues and comply with regulatory and clinical practice guidelines,
rigorous quantitative imaging approaches and analysis tools, such
as PET, will be essential for evaluating a variety of probes under-
going preclinical testing or transitioning into early-phase clinical
trials. By attaching a radiolabel to the particle surface, the clinical
potential of these molecularly targeted probes can be sensitively
Authorship?note: Miriam Benezra and Oula Penate-Medina contributed equally to
Conflict?of?interest: Hooisweng Ow and Ulrich Wiesner hold shares of the start-up
company Hybrid Silica Technologies.
Citation?for?this?article: J Clin Invest. 2011;121(7):2768–2780. doi:10.1172/JCI45600.
?The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
rescent particle platforms have been investigated (23–26), only
Cornell dots (C dots) meet the aforementioned criteria and have
received the first FDA-approved investigational new drug approv-
al of their class and properties for a first-in-human clinical trial.
Surface functionalized with small numbers of targeting peptides
(cyclic arginine–glycine–aspartic acid [cRGDY] peptides) and long-
lived radiolabels, this αvβ3 integrin–targeting particle tracer has a
unique combination of structural, optical, and biological proper-
ties: bulk renal clearance (10), favorable targeting kinetics, lack of
acute toxicity, superior photophysical features, and multimodal
(PET-optical) imaging capabilities. To the best of our knowledge,
these properties have not been collectively achieved or described
for any single inorganic particle platform. Importantly, the trans-
lation of this cancer-targeted, PET-optical platform represents a
significant clinical advance over our previous preclinical optical
imaging work using approximately 30-nm in diameter [i.d.] non-
PEGylated (24, 27) and approximately 3.3- and 6-nm i.d. PEGylat-
ed, nontargeting probes (10) in non-tumor-bearing animals.
We highlight the distinct advantages of using this renally excret-
ed, multimodal agent for tumor-selective targeting and nodal map-
ping studies in both small- (mouse) and large-animal (miniswine)
melanoma models. By combining key benefits of PET (depth pene-
tration, quantitation) with those of deep-red/NIR multiscale opti-
cal imaging technologies (28–30) (enhanced sensitivity/contrast,
multispectral capabilities), we initially monitored uptake, lym-
phatic drainage, and particle clearance in melanoma-bearing mice
(31–34). These methods were extended to a larger-animal sponta-
neous melanoma miniswine model (29) to assess the feasibility of
performing intraoperative image-guided metastatic disease detec-
tion, localization, and staging; to determine whether differences
in nodal tumor burden could be sensitively discriminated; and to
simulate more accurately the application of sentinel lymph node
(SLN) biopsy procedures in humans.
Nanoparticle design and characterization. From a clinical standpoint,
modifying an inherently nontoxic and biocompatible material,
amorphous silica, for targeted diagnostics and/or therapeutics
offers an attractive strategy for ultimately achieving a favorable
toxicologic profile in vivo, while, at the same time, preserving its
versatility in a variety of patient settings. To realize such a platform,
Cy5 dye-encapsulating core-shell silica nanoparticles (emission
maxima, >650 nm), coated with methoxy-terminated polyethylene
glycol (PEG) chains (PEG ~0.5 kDa), were prepared according to
previously published protocols (see Methods) (10, 27). The neutral
PEG coating prevented uptake by other cells (opsonization). The
use of bifunctional PEGs enabled attachment of small numbers of
ανβ3 integrin–targeting cRGDY peptide ligands (~6–7 ligands per
particle) (34) to maintain a small hydrodynamic size, facilitating
efficient renal clearance.
Peptide ligands were labeled with the positron-emitting radio-
nuclide 124I through the use of a tyrosine linker to provide a sig-
nal that can be quantitatively imaged in 3 dimensions by PET
(124I-cRGDY-PEG-ylated dots [124I-cRGDY-PEG-dots]; Figure
1A). An important practical advantage of relatively long-lived 124I
Multimodal C dot design for ανβ3 integrin targeting and characterization. (A) Schematic representation of the 124I-cRGDY-PEG-ylated core-shell
silica nanoparticle with surface-bearing radiolabels and peptides and core-containing reactive dye molecules (insets). (B) FCS results and single
exponential fits for measurements of Cy5 dyes in solution (black) and PEG-coated (PEG-dot, red) and PEG-coated, cRGDY-labeled dots (blue,
underneath red data set) showing diffusion time differences as a result of varying hydrodynamic sizes.
Photophysical characterization using FCSA
Free Cy5 dye
0.67 ± 0.008
3.53 ± 0.04
3.40 ± 0.04
3.25 ± 0.04
5.37 × 10–4
6.61 × 10–6
8.80 × 10–6
4.01 × 10–6
ATable 1 data were used in deriving the curves in Figure 1B.
2770? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
(physical half-life, 4.2 d) is that sufficient signal persists long
enough to allow radiodetection up to at least several days after
administration, when background activity has largely cleared, and
tumor-to-background contrast is maximized. PEG- and cRAD-
bound, PEG-coated particles containing tyrosine residues for 124I
labeling served as the control particle probes, the latter bearing
a scrambled peptide (i.e., cyclo-[Arg-Ala-Asp-Tyr]). Purification
of radiolabeled samples by size exclusion chromatography (Sup-
plemental Figure 1; supplemental material available online with
this article; doi:10.1172/JCI45600DS1) resulted in radiochemical
yields of more than 95%. Hydrodynamic diameters of approxi-
mately 7, 6.8, and 6.5 nm (mean ± SD, n = 15) were measured
for cRGDY-PEG-dots, PEG-dots, and cRADY-PEG-dots, respec-
tively, using fluorescence correlation spectroscopy (FCS) (Figure
1B and Table 1). The relative brightness of the cRGDY-PEG-dots
was determined to be approximately 200% greater than that of
the free dye (Table 1), consistent with earlier results (10, 35).
Based on these physicochemical properties, we anticipated
achieving a favorable balance between selective tumor uptake
and retention versus clearance of the targeted particle, thus max-
imizing target-tissue localization while minimizing toxicity and
normal-tissue radiation doses.
In vitro receptor-binding studies. To examine in vitro receptor-bind-
ing affinity and specificity of 124I-cRGDY-PEG-dots to melanoma
and vascular endothelial cell surfaces relative to those of particle
controls (124I-PEG-,124I-cRADY-PEG-dots), αvβ3 integrin–over-
expressing (M21, HUVEC) and –nonexpressing (M21L) lines
were used. Initially, M21 cells were incubated with increasing
124I-cRGDY-PEG-dot concentrations in the presence of excess non-
radiolabeled cRGD peptide, and γ-counting was performed (Fig-
ure 2A). Scatchard analysis of the binding data yielded a dissocia-
tion equilibrium constant, Kd, of 0.51 nM (Figure 2A, inset) and a
receptor concentration, Bmax, of 2.5 pM. Based on the Bmax value,
the αvβ3 integrin receptor density was estimated to be 1.0 × 104
Competitive integrin receptor binding studies with 124I-cRGDY-PEG-dots, cRGDY peptide, and anti–ανβ3 antibody using 2 cell types. (A) High-affin-
ity and specific binding of 124I-cRGDY-PEG-dots to M21 cells by γ-counting. The inset shows Scatchard analysis of binding data, plotting the ratio
of the concentration of receptor-bound (B) to unbound (or free [F]) radioligand or the bound-to-free ratio (B/F) versus the receptor-bound receptor
concentration; the slope corresponds to the dissociation constant, Kd. (B) ανβ3 Integrin receptor blocking of M21 cells using flow cytometry and
excess unradiolabeled cRGD or anti–ανβ3 antibody prior to incubation with cRGDY-PEG-dots and nonspecific binding with controls (RAD-PEG-dots,
PEG-dots). (C) Specific binding of cRGDY-PEG-dots to M21 cells as against M21L cells lacking surface integrin expression using flow cytometry.
Anti–ανβ3 integrin receptor antibody concentrations were used at 100 times (i.e., 100x) and 250 times (i.e., 250x) the particle (i.e., 124I-cRGDY-PEG-
dot) concentration. (D) Specific binding of cRGDY-PEG-dots to HUVECs by flow cytometry. Each bar represents mean ± SD of 3 replicates.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
integrin receptors per M21 cell, in reasonable agreement with the
previously published estimate (36) of 5.6 × 104 integrin receptors
per cell for this cell line. Incremental increases in integrin-specific
M21 cellular uptake were also observed over a temperature range
of 4°C to 37°C, suggesting that cellular internalization contrib-
uted to overall uptake (data not shown).
Binding specificity of cRGDY-PEG-dots was further demon-
strated with flow cytometry. Competitive binding assays showed
complete blocking of receptor-mediated binding (Figure 2B) using
anti–αvβ3 integrin antibody. No significant reduction was seen in
the magnitude of receptor binding (~10% of M21) with M21L cells
(Figure 2C) using either excess cRGDY peptide or anti–αvβ3 integ-
rin antibody. These results were confirmed by additional γ-count-
ing studies, and a 50% binding-inhibition concentration, IC50, of
1.2 nM was determined for the 124I-cRGDY-PEG-dot. This IC50
value suggests greater potency than that recently reported for engi-
neered, high-affinity 64Cu-DOTA-knottin peptides (IC50 ~20 nM)
(37). Potency of cRGDY-PEG-dots was additionally evaluated by an
anti-adhesion assay (5, 38) in order to assess particle multivalent
interactions with αvβ3 integrin receptors. A multivalent enhance-
ment factor of greater than 2.0 was determined for cRGDY-PEG-
dots relative to monomeric cRGD peptides after incubating each
with M21 cells (data not shown). A final set of studies showed that,
similar to M21 cells, excess antibody effectively blocked cRGDY-
PEG-dot receptor binding to HUVECs (Figure 2D).
Biodistribution and clearance studies. Biodistribution and blood,
renal, and hepatobiliary clearance were evaluated by i.v. admin-
istering tracer doses (~0.2 nanomoles) of 124I-cRGDY-PEG-dots
and 124I-PEG-dots to M21 tumor xenograft models. Although the
percentage of the injected dose (ID) per gram tissue (%ID/g) for
the targeted probe was measured over a 196-hour postinjection
(p.i.) time interval, comparison of the 124I-cRGDY-PEG-dot (Fig-
ure 3A) and 124I-PEG-dot tracers (Figure 3B) was restricted to a
96-hour window, as in vivo activity for the latter was not detect-
able at 1 week p.i. Statistically significant (P < 0.05) differences
in tracer uptake (%ID/g) values were observed for blood, tumor,
and major organs at 4 and 96 hours p.i. as well as at 24 hours p.i.
for tumor and several other tissues (Supplemental Table 1). The
targeted probe was almost entirely eliminated from the carcass at
1 week p.i. (~3% of the ID remained). Tracer clearance half times
Pharmacokinetics and excretion profiles of the targeted and nontargeted particle probes. (A) Biodistribution of 124I-cRGDY-PEG-dots (targeted)
in M21 tumor-bearing mice at various times from 4- to 168-hours p.i. The inset shows a representative plot of these data for blood to determine
the T1/2. Sm. int., small intestine; Lg. int., large intestine; conc., concentration. (B) Biodistribution of 124I-PEG-dots (untargeted) from 4- to 96-hours
p.i. (C) Clearance profile of urine samples collected up to 168-hours p.i. of unradiolabeled cRGDY-PEG-dots (n = 3 mice, mean ± SD). The inset
shows a strong correlation between the percentage of the injected particle dose excreted and the corresponding measured fluorescence signal. (D)
Corresponding cumulative %ID/g for feces at intervals up to 168-hours p.i. (n = 4 mice). For biodistribution studies, bars represent mean ± SD.
2772? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
(T1/2) and bioavailability for blood, tumor, and major organs are
shown in Table 2. For blood, the relatively long T1/2 value (mean
± SD) for the 124I-PEG-dot tracer (i.e., 7.3 ± 1.2 hours), which is
similar to that found for the nonradiolabeled particle control in
normal mice by fluorescence detection (10), decreased to 5.6 ± 0.15
hours (Figure 3A, inset) for the 124I-cRGDY-PEG-dot tracer.
By appropriate organ mass-adjusted translation of the forego-
ing biodistribution data to humans, human normal organ radia-
tion doses were derived and found to be comparable to those of
other commonly used diagnostic radiotracers (Table 2, columns
8 and 9). Along with the findings of formal toxicity testing that
the targeted probe was nontoxic at i.v. IDs 100 times the proposed
human dose equivalent (based on standard body mass-normalized
allometric scaling) and resulted in no tissue-specific pathologic
effects (Supplemental Figure 2 and Supplemental Table 2), first-
in-human targeted and nontargeted molecular imaging applica-
tions with these i.v.-injected agents are planned.
Efficient renal excretion was found for the approximately 7-nm
diameter targeted and nontargeted probes over a 168-hour time
period by fluorimetric analyses of urine samples. Fluorescence
signals were background corrected and converted to particle con-
centrations (%ID/μl) based on a serial dilution calibration scheme
(Figure 3C, inset; Supplemental Table 3, column 2; and ref. 10).
Concentration values, along with age-dependent conservative
estimates of the average urine excretion rate (39), permitted the
cumulative excreted fluorescence signal (%ID) to be computed
(Supplemental Table 3, column 4). Nearly half of the ID was
observed to be excreted over the first 24 hours p.i. and approxi-
Radiation dosimetry for i.v.-injected 124I-RGDY-PEG-dots and 124I-PEG-dots
? Mouse? HumanA
Small intestine wall
Large intestine wallC
Gall bladder wall
Urinary bladder wall
T1/2?(h)? Activity,?A? Absorbed?dose?
T1/2?(h)? Activity,?A? Absorbed?dose?
Effective dose equivalent
– – – – – – 0.86 0.26
– – – – – – 0.60 0.23
A70-kg standard human anatomic model. This is a “normal” (i.e., tumor-free) anatomic model and therefore does not yield tumor absorbed doses. Further,
the OLINDA dosimetry program for human dosimetry includes cross-organ and “remainder-of-body” dose contributions and therefore yields absorbed-dose
estimates even for organs for which time-activity data were not available. However, the OLINDA program does not include blood as a target region. In
contrast, the mouse absorbed-dose calculation ignored cross-organ doses and therefore yielded absorbed-dose estimates only for target regions (organs
and tumor) for which time-activity data were measured. B”%ID/G” indicates the percentage of the ID per gram of tissue. CThe OLINDA dosimetry program
for human dosimetry yields separate absorbed-dose estimates for the upper and lower large intestine walls, corresponding to the first and second entries,
respectively, for “large intestine wall.” DMouse melanoma model. “Activity, A,” for both the targeted (124I-cRGDY-PEG-dots) and nontargeted (124I-PEG-dots)
particles, refers to the zero-time intercept of the fitted time-activity concentration curve.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
mately 72% was excreted by 96 hours (Figure 3C), suggesting that
the bulk of excretion occurred in the first day p.i. No significant
particle fluorescence in urine could be detected 168 hours p.i.
Fluorimetric determinations correlated strongly with correspond-
ing γ-counter–derived %ID values (R2 = 0.965, Supplemental Table 3,
column 5), suggesting that no significant free 124I-cRGD was pres-
ent. In addition, as cumulative percentage dehalogenation val-
ues of urine samples by radio-TLC averaged less than 10% over a
24-hour period, RES trapping was not significantly underestimat-
ed (Supplemental Table 3, column 6). Fecal excretion profiles of
the 124I-cRGDY-PEG-dot indicated that, on average, 7% and 15%
of the ID was eliminated over 24 and 96 hours, respectively (Figure
3D). To assess particle integrity, FCS analysis of urine specimens
obtained prior to injection, and at 4 and 24 hours p.i. of the target-
ed probe, revealed that the particle was excreted intact and without
release of the encapsulated dye (Supplemental Table 4); minor dif-
ferences in solvent or surface chemistry likely contributed to the
Serial whole-body PET studies. PET imaging of integrin expres-
sion in M21 and M21L subcutaneous hind leg xenograft mouse
models was performed at multiple time points p.i. after i.v.
injection of either 124I-cRGDY-PEG-dots or control particles
(i.e., 124I-PEG-dots, 124I-cRAD-PEG-dots). Representative whole-
body coronal microPET images at 4-hours p.i. of M21 and M21L
tumors and at 24-hours p.i. of M21 tumor are shown in Figure
4A; a corresponding representative 24-hour fluorescence image
of the tumor is shown in Figure 4B. The specific targeting of the
αvβ3 integrin–overexpressing M21 tumor is clearly visible from
these images. Average tumor %ID/g and SDs are shown for the
following groups (Figure 4C): M21 (n = 7) and M21L (control)
tumors (n = 5) receiving the targeted 124I-cRGDY-PEG-dots;
M21 tumor mice (n = 5) receiving nontargeted 124I-PEG-dots;
and M21 mice (n = 3) receiving the 124I-cRAD-PEG-dots (Supple-
mental Figure 3A). At the time of maximum tumor uptake (~4
hours p.i.), up to 3-fold higher activity concentrations (%ID/g)
were seen in M21 tumors than in controls. Differences were
statistically significant at all time points p.i. (P < 0.05), except
at 1 hour (P = 0.27). In vivo integrin receptor blocking studies
of M21 tumors (n =3) additionally confirmed particle binding
specificity, with a ~6-fold decrease in tumor uptake in targeted
tracer uptake before versus after administration of excess cRGD
peptide (Supplemental Figure 3B).
Image-derived tumor-to-muscle uptake (%ID/g) ratios for the
124I-cRGDY-PEG-dots revealed enhanced tumor contrast at later
times (~24–72 hours p.i.), while those for 124I-PEG-dots declined
progressively with time (Figure 4D). This finding suggested that
124I-cRGDY-PEG-dots were tumor selective, which became more
apparent as blood activity was cleared during the initial 24-hour
period (compare Figure 4D with Figure 3A, inset). A statistically
significant correlation was found between PET-derived tumor
%ID/g values for both the targeted and nontargeted probes, and
the corresponding ex-vivo γ-counter–derived tumor %ID/g values
(correlation coefficient r = 0.94, P < 0.0016; Figure 4E), confirm-
ing the accuracy of PET for noninvasively deriving quantitative
Serial in vivo Cy5 fluorescence imaging and microscopy. In vivo fluo-
rescence imaging studies were conducted using our targeted and
nontargeted particle probes for mapping local/regional nodes and
Serial in vivo PET imaging of tumor-selective targeting. (A) Representative whole-body coronal microPET images at 4-hours p.i., demonstrating
M21 (left, arrow) and M21L (middle, arrow) tumor uptakes of 3.6 and 0.7 %ID/g, respectively, and enhanced M21 tumor contrast at 24 hours
(right). (B) Representative 24-hour fluorescence image of the tumor. (C) In vivo uptake of 124I-cRGDY-PEG-dots in αvβ3 integrin–overexpressing
M21 (black, n = 7 mice) and nonexpressing M21L (medium dark gray, n = 5 mice) tumors, 124I-PEG-dots in M21 tumors (dark gray, n = 5), and
124I-cRAD-PEG-dots in M21 tumors (light gray, n = 3). (D) M21 tumor-to-muscle ratios for 124I-cRGDY-PEG-dots (black) and 124I-PEG-dots (gray).
(E) Correlation of in vivo and ex vivo M21 tumor uptakes of cRGDY-labeled and unlabeled probes. Each bar represents mean ± SD.
2774? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
lymphatic channels in small-animal models, as the size of these
structures (1–2 mm) may not be adequately resolved by PET imag-
ing. Representative lymph node mapping was initially performed
across spatial scales using the targeted probe, in conjunction with
live-animal whole-body optical imaging (Figure 5A) and fluores-
cence microscopy techniques (Figure 5B), to visualize lymphatic
drainage from the peritumoral region to the inguinal and axillary
nodes in surgically exposed living animals. Peritumoral adminis-
tration of the targeted probe revealed drainage into and persistent
visualization of adjacent inguinal and popliteal nodes as well as
more remote axillary nodes over a 4-hour interval with less than
1-mm resolution. Higher-resolution (i.e., submillimeter) fluores-
cence images (Figure 5B, bottom row) permitted more detailed
intranodal architecture to be localized, including high endothelial
venules, known to facilitate passage of circulating naive lympho-
cytes into the node; this may have important implications for nodal
staging and the ability to detect micrometastases at earlier stages of
disease. Smaller, less intensely fluorescent lymphatic branches were
also visualized by fluorescence microscopy in the axillary region
(data not shown). Thus, the smaller size of the targeted probe per-
mitted the first draining, or sentinel, node proximal to the tumor
to be visualized and also enabled detection of more distant nodes
and visualization of the pattern of lymphatic drainage.
Differential rates of particle clearance were assessed for both
nontargeted (PEG-dots) and targeted probes in intact, living mice
bearing hind limb M21 xenografts versus those in controls. After
subdermal, 4-quadrant, peritumoral injection, relatively rapid dis-
persion of both particle probes in M21 mice was observed over
a 1-hour time period (measured against the initial 10-minute
time point), with representative PEG-dot images shown (Figure 5,
C and D). Using the fluorescence area increase (%) as a measure of
particle dispersion, no significant difference was seen between the
targeted and nontargeted probes over the first hour p.i. (Figure
5E). Further, in control animals, no percentage increase was found
for either probe (data not shown). Although no fluorescent nodes
were detected in intact mice, surgical exposure revealed lymphatic
drainage from the inguinal region to the axilla for both probes,
similar to that seen in Figure 5A.
Multimodal approaches were extended to a larger-animal spon-
taneous melanoma miniswine model (32) in order to assess the
feasibility of performing real-time, intraoperative image-guided
metastatic disease detection and staging and to determine whether
tumor burden could be sensitively discriminated with correlative
histology. After i.v. injection, whole-body dynamic 18F-fluorode-
oxyglucose (18F-FDG) PET-CT scanning identified an 18F-FDG-
avid melanomatous lesion adjacent to the spine on the upper back
Nodal mapping using multiscale near-infrared optical fluorescence imaging. (A) Whole-body fluorescence imaging of the tumor site (T) and
draining inguinal (ILN) and axillary (ALN) nodes and communicating lymphatics channels (LCs; bar) 1-hour p.i. in a surgically exposed living
animal. (B) Corresponding coregistered white-light and high-resolution fluorescence images (top row) and fluorescence images only (bottom
row), revealing nodal infrastructure of local and distant nodes, including high endothelial venules (HEVs). (C) Whole-body fluorescence image of
the tumor site 10 minutes after subdermal PEG-dot injection. (D) Delayed whole-body fluorescence image of the tumor site 1 hour after PEG-dot
injection. (E) Percentage increase in the area of fluorescence (fluor) relative to that measured at 10-minutes p.i. for targeted and nontargeted
probes. Scale bars: 1.0 cm (A); 500 microns (B); 3 mm (C and D).
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
as well as a hypermetabolic right-sided SLN in the posterior neck
(Figure 6A, white arrow). Subdermal, 4-quadrant peritumoral
injection of 124I-RGD-PEG-dots 48 hours later confirmed the 18F-
FDG imaging findings and identified 2 additional hypermetabolic
nodes, 1 within the left posterior neck (Figure 6B, arrowhead) and
the second immediately anterior to the SLN 5-minutes and 1-hour
p.i. by dynamic PET scanning. No additional PET-avid nodes or
other suspicious areas of increased uptake were seen on a 1-hour
whole-body PET scan. Corresponding optical imaging of the
excised right-sided SLN demonstrated fluorescence signal within
the black-pigmented (melanin-containing) gross specimen (Figure
6, C and D), which measured 1.3 × 1.0 × 1.5 cm3, as compared with
the smaller posterior left-sided hypermetabolic node, which mea-
sured 1.0 × 0.6 × 1.0 cm3 (Supplemental Figure 4A). Significantly
increased background-corrected SLN activity, detected with a
hand-held intraoperative positron probe, corroborated these opti-
cal findings (Figure 6D, annotation).
H&E-stained tissue sections from the SLN showed scattered,
dark melanomatous clusters on low-power views (Figure 6E),
which were found to comprise both melanoma cells and melanin-
containing macrophages (i.e., melanophages) on high-power views
(Figure 6F), similar to findings for excised primary lesions (data
not shown). Immunohistochemical staining of the SLN with a
known human melanoma marker, HMB45, demonstrated posi-
tive expression of this marker on low-power views (Figure 6G) and
high-power views (Figure 6H). Representative normal-appearing
porcine nodal tissue harvested from the neck revealed no meta-
static infiltration in representative low-power views (Figure 6I)
and high-power views (Figure 6J). By contrast, low-power views
(Supplemental Figure 4B and Figure 6K) of H&E-stained sections
from the posterior left-sided hypermetabolic node demonstrated
a few smaller-sized melanomatous clusters containing melanoma
cells and melanophages (Supplemental Figure 4C). Tumor burden
in this smaller node, estimated to be 10- to 20-fold less than in the
SLN by pathological analysis, was sensitively discriminated by the
targeted particle probe.
Here, we report on an ultrasmall, high-affinity, and efficiently
cleared silica nanoparticle probe, which has been recently approved
for first-in-human clinical trials and which successfully overcomes
a number of the limitations of other particle platforms. This mul-
timodal platform has advanced to the point of clinical translat-
ability. Its applications include real-time, intraoperative detection
and imaging of nodal metastases, differential tumor burden, and
lymphatic drainage patterns in melanoma. Although several inves-
tigators have synthesized radiolabeled fluorescent particle probes
(25, 30, 40), our multimodal agent has been radiolabeled with the
long-lived positron-emitter iodine-124, and, thus, we believe it
can provide unique longer-term pharmacokinetic clearance and
Imaging of metastatic disease in a spontaneous melanoma miniswine model. (A) Whole-body dynamic 18F-FDG PET-CT sagittal and axial views,
demonstrating primary tumor (green arrow) and single SLN (white arrow) posteriorly within the right (Rt) neck after i.v. injection. ant, anterior. (B)
High-resolution dynamic PET-CT scan 1 hour after subdermal, 4-quadrant, peritumoral injection of 124I-RGD-PEG-dots (SLN, arrow; left-sided
node, arrowhead). (C) Whole-body Cy5 fluorescence image of the excised SLN. (D) Gross image of the cut surface of the black-pigmented
SLN (asterisk), which measured 1.3 × 1.0 × 1.5 cm3, and annotated γ counted activity. (E) Low-power view of H&E-stained SLN, demonstrating
scattered melanomatous clusters. (F) Corresponding high-power view of H&E-stained SLN, revealing melanoma cells (yellow arrowheads) and
melanophages (white arrowhead). (G) Low-power image of a melanoma-specific marker, HMB-45, in representative SLN tissue. (H) High-power
image of HMB-45–stained SLN tissue. (I) Low-power image of representative normal porcine nodal tissue. (J) High-power image of representa-
tive normal porcine nodal tissue. (K) Low-power view of H&E-stained contralateral hypermetabolic lymph node, demonstrating scattered mela-
nomatous clusters (arrowhead). Tumor burden in this smaller node (1.0 × 0.6 × 1.0 cm3) was estimated to be 10- to 20-fold less than that in the
SLN by pathological analysis. Scale bars: 1 mm (E, G, I, and K); 20 μm (F, H, and J).
2776? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
targeting information over the course of days. The complemen-
tary nature of this platform, coupled with its small size (~7-nm
i.d.), may facilitate clinical assessments by enabling the seamless
integration of imaging data acquired at different spatial, tempo-
ral, and sensitivity scales, potentially providing new insights into
fundamental molecular processes governing tumor biology.
The results of this study underscore the clear-cut advantages
offered by PET. Using this quantitative and highly sensitive imag-
ing tool, we were able to noninvasively extract an accurate, repro-
ducible, and comprehensive body of data for the targeted probe: (a)
molecular information, including receptor expression levels, bind-
ing affinity, and specificity; (b) in vivo distribution and targeting
kinetics; (c) clearance and dehalogenation profiles; (d) blood/tissue
residence times and bioavailability; and (e) radiation dosimetry.
Our in vitro results show receptor-binding specificity of the
~7-nm–targeted particle probe to M21 cells and HUVECs. Similar
findings have been reported with receptor-binding assays using
the same cell types but with the monovalent form of the peptide
(36). Importantly, the multivalency enhancement of the cRGDY-
bound particle probe, along with the extended blood and tumor
T1/2 values, are key properties associated with the particle platform
that are not found with the monovalent form of the peptide.
The integrin-binding peptide, cRGD, a well-established integrin-
binding molecular marker, was selected to elucidate the biological
and kinetic properties of the peptide-bound particle and addition-
ally enable the biological performance of our multimodal platform
to be benchmarked against other cRGD agents (i.e., peptide trac-
ers). However, another more tumor-specific, high-affinity ligand
could be investigated as the targeting moiety in future studies. The
biological properties of particle-based systems are generally quite
different from those of simple (i.e., small monovalent) targeting
peptides, and the choice of the proper targeting agent (i.e., molecu-
lar versus particle-based probes) in the clinical setting will rest, in
part, on the application of interest, the tumor type/composition,
and standard-of-care considerations.
The relatively long blood T1/2 for the 124I-PEG-dot tracer may be a
consequence of the chemically neutral PEG-coated surface, render-
ing the probe biologically inert and significantly less susceptible to
phagocytosis by the RES. However, recognition of the 124I-cRGDY-
PEG-dot tracer by target integrins and/or more active macrophage
activity may have led to reductions in the T1/2 value. These values,
however, are substantially longer than published blood T1/2 values
of existing cRGDY peptide tracers (~13 minutes) (5), potentially
leading to increased probe bioavailability, facilitating tumor tar-
geting, and yielding higher tumor uptake over longer time peri-
ods. Moreover, the tumor T1/2 for the 124I-cRGDY-PEG-dot was
found to be 13-times greater than that for blood, compared with
only a 5-fold difference for the 124I-PEG-dot, suggesting substan-
tially greater target-tissue localization of the former than the lat-
ter. Such mechanistic interpretations of the in vivo data can be
exploited clinically to refine diagnostic, treatment-planning, and
The greater accumulation in and slower clearance from M21
tumors, relative to that of surrounding normal structures, allows
discrimination of specific tumor uptake mechanisms from non-
specific mechanisms (i.e., tissue perfusion, leakage) in normal tis-
sues. However, a small component of the M21 tumor uptake can
presumably be attributed to vascular permeability alterations (i.e.,
enhanced permeability and retention effects) (41), largely reflected
in the observed %ID/g increases for the control tracer (124I-PEG-
dots, Figure 4C) at earlier p.i. time points. At 1-hour p.i., no sig-
nificant %ID/g increases were seen in the M21 tumors over the
controls. This observation may represent the effects of differential
perfusion in the first hour, with tumor accumulation and reten-
tion primarily seen at later p.i. times (i.e., 24 hours). Further, in
comparison with those of the clinically approved peptide tracer,
18F-galacto RGD (42, 43), nearly 2-fold greater maximum uptake
values were found in M21 tumors for the targeted dots at 2-hours
p.i. (data not shown), while additionally offering advantages of
multivalent binding and extended circulation times.
The advantages of the combined optical-PET probe highlight
the versatility of the platform for in vivo applications. This is
particularly true in small-animal models, in which the ability to
assess anatomic structures having sizes at or well below the reso-
lution limit of the PET scanner (i.e., the so-called partial-volume
effect) may undermine detection and quantitation of activity in
lesions. In these cases, assessment of metastatic disease in small
local/regional nodes, important clinically for melanoma staging
and treatment, may not be adequately resolved by PET imaging,
given that the size of the nodes we typically observed was on the
order of the PET spatial resolution (1–2 mm). By exploiting the
significantly improved photophysical features of encapsulated
dyes, such as Cy5, as well as the enhanced detection sensitivity
and contrast achievable at longer emission wavelengths, detailed
information pertaining to the localization of superficial nodes,
lymphatic function, and clearance can be acquired using deep-
red/NIR fluorescence imaging strategies.
Larger-animal spontaneous melanoma models may more accu-
rately reflect human disease and enable improved simulation of
surgical procedures used in humans (i.e., SLN mapping). Locally
injected 124I-cRGDY-PEG-dot tracer and dynamic PET imaging
enabled superior detection sensitivity and discrimination of meta-
static tumor burden within hypermetabolic neck nodes compared
with the PET imaging agent, 18F-FDG. 18F-FDG is an indicator of
glucose metabolism, accumulating within metabolically active
tumors that use this substrate. Traditionally used to stage clini-
cal melanoma, it failed to accurately stage nodal disease in this
representative miniswine. A number of well-known limitations
are associated with whole-body 18F-FDG PET (44), particularly
when imaging patients with early-stage melanoma, including a
low mean sensitivity of 17.3% (0%–40%) for detecting SLNs and
an inability to detect micrometastases less than about 1-cm i.d.
In the present study, the 124I-cRGDY-PEG-dot tracer was able to
discriminate at least order of magnitude differences in metastatic
tumor burden (i.e., 10- to 20-times greater) between the bilat-
eral neck nodes, as determined by high-power microscopy, while
18F-FDG PET could not. Although metastatic disease was detected
within the SLN, tumor was missed within the smaller (1.0 × 0.6 ×
1.0 cm3) contralateral node.
Thus, for metastatic disease assessment, the use of dynamic PET
imaging as a depth-sensitive, volumetric imaging tool, in conjunc-
tion with 124I-cRGDY-PEG-dots, will offer distinct advantages in
large-animal models in the intraoperative setting. Superior detec-
tion sensitivity, metabolic nodal status, and enhanced penetration
for the mapping of deep-seated nodal and tissue activities may
complement extended real-time fluorescence imaging assessments
of metastatic tumor burden in the future. The use of PET will be
critical for confirming the findings of depth-insensitive optical
imaging tools, particularly in anatomic regions associated with
unpredictable patterns of metastatic disease spread.
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
Synthesis of cRGDY-PEG-dots, cRADY-PEG-dots, and PEG-dots. Particles were pre-
pared by a modified Stöber-type silica condensation (35, 45–47). Reactive
derivatives of the organic dye Cy5, exhibiting emission maxima about 650 nm,
were used to produce particles containing 2 dye equivalents within the parti-
cle core. Tyrosine residues were conjugated to PEG chains (48, 49) for attach-
ment of radioiodine or stable iodine moieties. All samples were optical density
matched at their peak absorption wavelength (640 nm) prior to radiolabeling.
cRGDY peptides containing the sequence cyclo-(Arg-Gly-Asp-Tyr) and cRADY
peptides containing the sequence cyclo-(Arg-Ala-Asp-Tyr) and bearing cys-
teine residues (Peptide International) were attached to functionalized PEG
chains via a cysteine-maleimide linkage. The number of cRGD ligands per
nanoparticle was empirically calculated from known FCS-derived nanopar-
ticle concentrations and starting concentrations of cRGD-PEG-silane deriva-
tives, assuming that the surface functionalization reaction completely con-
sumed all bifunctional PEGylation reagent as an upper bound.
Mechanism of PEG attachment to the C dot surface. Bifunctional PEGs, MAL-
dPEG12-NHS ester (Quanta Biodesigns Ltd.), were derivatized with silanes,
specifically 3-aminopropyl triethoxysilane (Gelest), for attachment to the silica
surface and for peptide coupling via reactions between the sulfhydryl groups
and maleimide moieties of the derivatized PEGs. In addition, methoxy-capped
PEG chains were added to the particle surface using functional organosilicon
compounds (Si compounds), specifically (MeO)3Si-PEG (Gelest), according to
modified protocols (40). Briefly, (MeO)3Si-PEG was added, at approximately
3 molar excess, to particles in a water/alcohol basic mixture (~1:5 v/v), and the
mixture was stirred overnight at room temperature.
Hydrodynamic size and relative brightness comparison measurements by FCS.
The hydrodynamic radius, brightness, and concentrations of cRGDY-PEG-,
cRADY-PEG-, and PEG-dots, as against free Cy5 dye, were initially deter-
mined by dialyzing these particle samples to water, diluting into physiolog-
ical saline (0.15 M NaCl in H2O), and analyzing the resulting specimens on
a Zeiss LSM 510 Confocor 2 FCS using HeNe 633-nm excitation (46). The
instrument was calibrated with respect to particle size prior to all measure-
ments. Average hydrodynamic sizes of the dye and particle species were
estimated based on diffusion time differences, while relative differences in
brightness were assessed using count rates per molecule per particle.
To assess particle integrity in urine specimens, size and brightness mea-
surements were determined prior to and at 4- and 24-hour after i.v. injec-
tion of 200 μl iodinated 127I-cRGDY-PEG-dots (n = 3 specimens per time
point). Three separate runs were performed per specimen to derive average
particle sizes and SDs. Controls for urine analyses were prepared by mixing
known volumes of particles with specimens collected from control mice.
Radiolabeling of C dot conjugates. Radiolabeling of the PEG- and cRGDY-
PEG-dots was performed using the IODOGEN method (Pierce) (10, 50).
Specific activities of the 124I-bound particle fractions eluted from PD-10
columns were ~300–1,000 mCi/μm, assessed using a dose calibrator (Cap-
intec) and radio-TLC. TLC plates were developed using a mixture of acetic
acid/methanol (80:20 v/v) and dried, and the absorbance was analyzed by
a TLC plate reader (Bioscan). Particle concentrations were based on fluo-
rescence spectrometer measurements (Varian Inc.) at maximum excitation/
emission wavelengths of 650 and 680 nm, respectively.
Cells and cell culture. Human melanoma M21 and M21 variant (M21-L,
αv negative) cell lines were obtained from D.A. Cheresh (University of
California San Diego, San Diego, California, USA). Cells were maintained
in RPMI 1640 media/10% fetal BSA, and 2 mM l-glutamine, penicillin,
and streptomycin (Core Media Preparation Facility, Memorial Sloan-
Kettering Cancer Center). HUVECs, cultured in M199 media/10% fetal
bovine serum, 20 μg/ml endothelial cell growth factor, and 50 μg/ml
heparin, penicillin, and streptomycin, were provided by S. Rafii, Weill
Cornell Medical Center, New York, New York, USA.
In vitro cell-binding and molecular specificity of 124I-cRGD-PEG-dots. To assay
particle binding and specificity for M21 cells, 24-well plates were coated
with 10 μg/ml collagen type I (BD Biosciences) in PBS and incubated
(37°C, 30 minutes). M21 cells (3.0 × 105 cells/well to 4.0 × 105 cells/well)
were grown to confluency and washed with RPMI 1640 media/0.5% BSA.
124I-cRGD-PEG-dots (0–4.0 ng/ml) were added to wells, and cells were incu-
bated (25°C, 4 hours), washed with RPMI 1640 media/0.5% BSA, and dis-
solved in 0.2 M NaOH. Radioactivity was assayed using a 1480 Automatic
Gamma Counter (Perkin Elmer) calibrated for iodine-124. Nonspecific
binding was determined in the presence of a 1,000-fold excess of cRGD
(Peptides International). Scatchard plots of the binding data were gener-
ated and analyzed using linear regression analyses (Microsoft Excel 2007)
to derive receptor-binding parameters (Kd, Bmax, IC50).
In vitro cell-binding studies using optical detection methods. Differential
binding of cRGDY-PEG-dots and PEG-dots to M21 cells was evaluat-
ed over a range of incubation times (up to 5 hours) and particle con-
centrations (0–8 ng/ml) using flow cytometry. After incubation, cells
(3.0 × 105 cells/well) were washed with RPMI 1640 media/0.5% BSA,
detached using 0.25% trypsin/EDTA, pelleted in a microcentrifuge tube
(5 minutes at 153 g, 25°C), resuspended in BD FACSFlow solution (BD
Biosciences), and analyzed in the Cy5 channel to determine the percent-
age of particle-bound probe (FACSCalibur, Becton Dickinson). The par-
ticle concentration (i.e., ~2.0 ng/ml) and incubation time (i.e., 4 hours)
yielding maximum differential binding were used for subsequent com-
petitive binding assays and specificity studies.
Competitive binding studies were performed after incubation of cRGDY-
PEG-dots with M21 cells, M21L cells, and HUVECs in the presence of
excess cRGD and/or mouse monoclonal anti-human integrin ανβ3 fluo-
rescein–conjugated antibody (Millipore) and analyzed by flow cytometry.
In addition, particle control binding studies (cRADY-PEG-dots and PEG-
dots) were conducted with M21 cells at concentrations similar to those
used for cRGDY-PEG-dots (~1 mM). To assess potency of the RGDY-PEG
dots relative to that of the cRGD peptide, an anti-adhesion assay was used
(5, 38). Ninety-six–well microtiter plates were coated with vitronectin in
PBS (5 μg/ml), followed by 200 μl RPMI/0.5% BSA (1 hour, 37°C). Cells
(3 × 104 cells/100 μl/well) were incubated in quadruplicate (30 minutes,
25°C), with various concentrations of cRGDY-PEG-dots or cRGD peptide
in RPMI/0.1% BSA, and added to vitronectin-coated plates (30 minutes,
37°C). Wells were rinsed with RPMI/0.1% BSA to remove nonadherent
cells. Adherent cells were fixed with 4% PFA (20 minutes, 25°C) and stained
with methylene blue (1 hour, 37°C) for determination of optical densi-
ties using a Tecan Safire plate reader (λex = 650 nm, λem = 680 nm). The
multivalent enhancement factor was computed as the ratio of the cRGD
peptide-to-cRGDY-PEG-dot IC50 values (5).
Animal models and tumor inoculation. All animal experiments were done in
accordance with protocols approved by the Institutional Animal Care and
Use Committee of Memorial Sloan-Kettering Cancer Center and followed
NIH guidelines for animal welfare. Male athymic nu/nu mice (6–8 weeks
old, Taconic Farms Inc.) were provided with water containing potassium
iodide solution to block thyroid gland uptake of free radioiodine and main-
tained on a Harlan Teklad Global Diet 2016, ad libitum (10). M21 or M21L
xenografts were generated by coinjecting equal volumes of cells (~5 × 106
cells/100 μl) and Matrigel subcutaneously into the hind legs of different
mice. Average tumor volumes of 200 mm3 were used for all studies.
In vivo pharmacokinetic and T1/2 measurements. The %ID/g values, corrected
for radioactive decay to the time of injection, were measured by sacrificing
groups of mice at specified times after i.v. injection of 124I-cRGDY-PEG-
dots or 124I-PEG-dots (~20 μCi/mouse) and harvesting, weighing, and
counting blood, tumor, and organs in a scintillation γ-counter. The result-
ing TAC data for each tissue were fit to a decreasing monoexponential
2778? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
function to estimate values of the tissue/organ T1/2 and the zero-time
intercept of the fitted time-activity concentration curve (determined for
both the targeted [124I-cRGDY-PEG-dots] and nontargeted [124I-PEG-dots]
particles), respectively, of the function.
In vivo excretion studies. The fraction of cRGDY-PEG-dots excreted in the
urine over time was estimated by fluorimetry (10) over a 168-hour period
(n = 3 mice/time point) and γ-counting. Briefly, mice were i.v. injected with
either 200 μl 127I-cRGDY-PEG-dots or 127I-PEG-dots for fluorimetric stud-
ies. Particle concentrations at each time point were determined on the basis
of serial dilution calibration curves (10). Concentration values, along with
estimates of average daily mouse urine volumes, were used to compute
cumulative %ID urine excreted. %ID values were correlated with the cor-
responding decay-corrected cumulative excreted radioactivity (%ID) at 1-,
4-, and 24- hours p.i. of 124I-cRGDY-PEG-dots. To assess cumulative fecal
excretion, feces were collected in metabolic cages over similar time intervals
after injection of 200 μl 124I-cRGDY-PEG-dots (n = 4 mice/time point), and
specimen activities were determined using a γ-counter.
Cumulative percentage dehalogenation was assessed by radio-TLC analy-
sis of urine samples using 10% acetic acid/methanol as a running buffer.
Plates were dried and analyzed with a Bioscan System 200 Imaging Scan-
ner; individual peaks were separated, and areas under the curve were calcu-
lated using system software.
Dosimetry. Time-activity functions derived for each tissue were analyti-
cally integrated (with inclusion of the effect of radioactive decay) to yield
the corresponding cumulative activity (i.e., the total number of radioac-
tive decays). 124I mouse organ absorbed doses were then calculated by
multiplying the cumulative activity by the 124I equilibrium dose constant
for nonpenetrating radiations (positrons) (51), assuming complete local
absorption of such radiations and ignoring the contribution of penetrat-
ing radiations (i.e., γ-rays). The mouse normal organ cumulated activities
were converted to human normal organ cumulated activities by adjust-
ment for the differences in total-body and organ masses between mice and
humans (assuming 70-kg standard human) (52). The human normal organ
cumulated activities calculated were entered into the OLINDA dosimetry
computer program to calculate, using the formalism of the Medical Inter-
nal Dosimetry Committee of the Society of Nuclear Medicine (53, 54), the
standard human organ absorbed doses.
Single-dose toxicity testing and histopathology of 127I-cRGDY-PEG-dots. Toxicity test-
ing was performed in 6 groups of male and female B6D2F1 mice (8 weeks old,
The Jackson Laboratory) at doses 100 times the proposed human dose equiv-
alent. The treatment group (n = 6 males, n = 6 females) received i.v.-injected
targeted probe (i.e.,127I-cRGDY-PEG-dots) at a dose of 1 × 10–9 moles/animal,
while the control group (n = 6 males, n = 6 females) received the same dose of
127I-PEG-dots (vehicle) in a single i.v. injection (200 μl). Untreated controls
(n = 2 males, n = 2 females) were additionally tested. Mice were observed daily
over 14 days p.i. for signs of morbidity/mortality and weight changes. Gross
necropsy, histopathology, and blood sampling for hematology and serum
chemistry evaluation were performed at 7 and 14 days p.i.
Serial PET imaging of tumor-specific targeting. Imaging was performed using a
dedicated small-animal PET scanner (Focus 120 microPET; Concorde Micro-
systems). Mice bearing M21 or M21L hind leg tumors were anesthetized
using 2% isoflurane anesthesia in oxygen during the scan period. One-hour
list-mode acquisitions were initiated after i.v. injection of 200 μCi 124I-cRGDY-
PEG-dots or 124I-PEG-dots in all mice, followed by serial 30-minute static
images over a 96-hour interval. 124I-cRADY-PEG-dot (200 μCi) static images
were additionally acquired at 4- and 24-hours after i.v. injection. Acquisition
of whole-body optical fluorescence images at 2-nm intervals from 630 nm to
850 nm, using a 575- to 605-bandpass excitation filter and a 645-nm long-
pass emission filter (Maestro, Cambridge Research Instruments), was per-
formed and spectrally deconvolved as previously described (10).
PET image reconstruction and analysis. Voxel count rates in the reconstruct-
ed images were decay corrected and converted to activity concentrations
(percentage %ID/g) by use of a system calibration factor derived from the
imaging of a mouse-size water equivalent. Three-dimensional region-of-
interest analysis of reconstructed images (ASIPro software, Concorde
Microsystems) yielded tumor and normal organ activity concentrations
(i.e., mean, maximum, and SD of the %ID/g). Tumor-to-muscle activity
concentration ratios were derived by dividing image-derived tumor %ID/g
values by γ-counter–derived muscle %ID/g values.
In vivo ανβ3 integrin receptor blocking studies. Average tumor uptake values
of 124I-cRGDY-PEG-dots (300 μCi) were determined before and 30 minutes
after i.v. injection of excess of cRGD peptide (6 μg/ml PBS). Initial uptake
values were determined 3 days prior to blocking.
Nodal mapping and clearance of particles using multiscale fluorescence imaging
techniques. Nude mice bearing hind leg tumors were injected by 4-quadrant,
peritumoral administration of a 50-μl PEG-dots or cRGDY-PEG-dot sample
and allowed to perambulate freely. Mice were anesthetized with a 2% isoflu-
rane/98% oxygen mixture, and serial whole-body optical imaging of intact
animals was performed at 10-, 20-, 30-, and 60-minutes p.i. and spectrally
deconvolved. Several non-tumor-bearing mice were additionally injected
with 50-μl PEG-dots or PEG-cRGDY-PEG-dots subdermally in the hind
leg. Superficial paramidline incisions were made along the ventral aspect of
mice, surgically exposing the region from the hind limb to axilla, in order
to enhance in situ optical detection of locoregional nodes (inguinal, axil-
lary) and draining lymphatics, using both the whole-body optical scan-
ner and a macroscopic fluorescence microscope fitted with 650 ± 20 nm
NIR excitation and 710-nm long-pass emission filters.
Imaging metastatic disease in spontaneous melanoma miniswine models. Spon-
taneous melanoma Sinclair miniature swine (10–12 kg, Sinclair Research
Center) were injected i.v. with 5 mCi 18F-FDG for dynamic whole-body
PET imaging detection of nodal and/or organ metastases, followed by CT
scan acquisition for anatomic colocalization. Additional dynamic PET-CT
scans were acquired after subdermal, 4-quadrant peritumoral injections of
124I-RGD-PEG-dots to detect and localize metastatic disease and for presur-
gical planning. Imaged nodes were confirmed intraoperatively within the
exposed surgical bed by visual inspection and γ-counting using hand-held
PET devices prior to excision. Harvested specimens, as well as nodal basins
bilaterally, were evaluated histologically with H&E staining and were addi-
tionally stained with specific human melanoma markers (HMB45, PNL2
Dako, 1:100) according to standard protocols to confirm the presence of
melanoma. Radioactivity levels were assessed within these tissue specimens
for correlative purposes with the hand-held PET probe. Whole-body opti-
cal fluorescence imaging of excised specimens was additionally performed
using the Maestro imaging system to identify nanoparticle fluorescence.
Statistics. Data are expressed as mean ± SD and compared, where indi-
cated, by 1-tailed Mann-Whitney U test. We assigned statistical signifi-
cance at P < 0.05. Statistical analyses comparing groups of tumor mice
receiving targeted/nontargeted probes or bearing M21/M21L tumors
were performed and demonstrated statistical significance with P < 0.05.
For biodistribution studies, the tissue-specific mean %ID/g values of
124I-cRGDY-PEG-dots (n = 7 mice) and 124I-PEG-dots (control, n = 5 mice)
were compared at each time point, with statistically significant differ-
ences in tracer activities observed in blood, tumor, and major organs at
4- and 96-hours p.i. as well as at 24-hours p.i. for tumor and other tis-
sues (Supplemental Table 1). For tumor targeting studies, differences
in mean %ID/g values between M21 (n = 7) and M21L tumor mice
(n = 5), as well as mice receiving 124I-PEG-dots (n = 5) or 124I-cRADY-PEG-
dot (n = 3) controls, were found to be maximal at 4-hours p.i. (P = 0.0015
for PEG-dot and M21L controls; P = 0.01 for cRADY-PEG-dot control),
remaining significantly elevated at 24 hours (P = 0.0015 and P = 0.004,
? The?Journal?of?Clinical?Investigation http://www.jci.org Volume 121 Number 7 July 2011
respectively), 48 hours (P = 0.001 and P = 0.003, respectively), 72 hours
(P = 0.015 and P = 0.005, respectively), and 96 hours (P = 0.005 for M21-
M21L). Tumor-to-muscle ratios for 124I-cRGDY-PEG-dots (n = 7) versus
those for 124I-PEG-dots (n = 5) were found to be statistically signifi-
cant at 24-hours p.i. (P = 0.001) and 72-hours p.i. (P = 0.006) but not at
4-hours p.i. (P =.35). Goodness-of-fit values (R2), along with their associ-
ated P values, were determined for the urine calibration curve (R2 = 0.973,
P = 0.01) as well as for the urine (R2 > 0.95, P = 0.047) and fecal
(R2 > 0.995, P < 0.002) cumulative %ID excretion curves using nonlinear
regression analyses (SigmaPlot, Systat, v. 11.0).
We acknowledge M. Gönen for providing assistance with biostatisti-
cal analyses and R. Toledo-Crow, S. Patel, and J. Lewis for technical
assistance. This work was supported by an NIH-American Recovery
and Reinvestment Act/Clinical and Translational Science Center
grant to M. Bradbury and U. Wiesner. The work was further sup-
ported by the Cornell Nanobiotechnology Center, a Science and
Technology Center program of the National Science Foundation
under agreement no. ECS-9876771, and by the In vivo Cellular and
Molecular Imaging Center P50 CA86438 grant. Technical services
provided by the Memorial Sloan-Kettering Cancer Center Small-
Animal Imaging Core Facility, supported in part by NIH Small-Ani-
mal Imaging Research Program (SAIRP) grant no. R24 CA83084 and
NIH center grant no. P30 CA08748, are gratefully acknowledged.
Received for publication March 23, 2011, and accepted in revised
form May 4, 2011.
Address correspondence to: Michelle S. Bradbury, Department of
Radiology, Sloan-Kettering Institute for Cancer Research, 1275
York Ave., New York, New York 10065, USA. Phone: 212.639.8938;
Fax: 212.794.4010; E-mail: firstname.lastname@example.org.
Erik Herz’s present address is: Exxon-Mobil Research and Engi-
neering, Paulsboro, New Jersey, USA.
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