MRI detection of VEGFR2 in vivo using a low molecular weight peptoid-(Gd)8-dendron for targeting

Article abstract of DOI:10.1021/ja105563a

The synthesis of a polylysine dendron containing eight GdDOTA units conjugated to a peptoid dimer known to have a high affinity for the vascular endothelial growth factor receptor 2 (VEGFR2) is described. This simple low molecular weight system with a molecular r₁ relaxivity of ～48 mM^-1 s^-1 is shown to enhance MR images of tumors grown in mice in vivo.

Full text of DOI:10.1021/ja105563a

Published on Web 08/26/2010
MRI Detection of VEGFR2 in Vivo Using a Low Molecular Weight
Peptoid-(Gd)8-Dendron for Targeting
Luis M. De Leo´n-Rodr´ıguez,† Angelo Lubag,† D. Gomika Udugamasooriya,‡ Bettina Proneth,‡
Rolf A. Brekken,‡ Xiankai Sun,† Thomas Kodadek,‡ and A. Dean Sherry*,†
AdVanced Imaging Research Center, Departments of Internal Medicine, Radiology, Biochemistry and Surgery,
UniVersity of Texas Southwestern Medical Center, 5323 Harry Hines BouleVard, Dallas, Texas 75390-9185
Received June 24, 2010; E-mail: Dean.Sherry@UTSouthwestern.edu
clearance rates can be tuned by molecular weight to allow sufficient
Abstract: The synthesis of a polylysine dendron containing eight
time to reach their target.11 Antibodies and peptides have been most
GdDOTA units conjugated to a peptoid dimer known to have a
widely used for targeting specific biomarkers, but such systems often
high affinity for the vascular endothelial growth factor receptor 2
suffer from poor in ViVo stability or costly production. More recently,
(VEGFR2) is described. This simple low molecular weight system
ꢀ-peptides and peptoids have gained interest as targeting moieties
with a molecular r1 relaxivity of ∼48 mM-1 s-1 is shown to
because they are easy to prepare, cost-effective, and stable toward
enhance MR images of tumors grown in mice in vivo.
peptidase and protease activity. Furthermore, screening methods to
identify highly specific peptoid targeting agents for specific biomarkers
on living cells have been reported.12 A strategy we have adopted for
Magnetic resonance imaging (MRI) is widely used for anatomical
imaging of soft body tissues and for measuring dynamic processes
creating targeted agents for molecular imaging by MRI consists of
such as perfusion, diffusion, and chemical exchange. Paramagnetic
(1) selection of a target specific moiety from a peptoid library, (2)
complexes (largely Gd3+, Fe2+, Mn2+) are commonly used to enhance
affinity optimization, (3) attachment of a small poly(GdDOTA)lysine
contrast differences by altering the inherent relaxation properties (T1,
dendrimer scaffold to the targeting peptoid, (4) affinity optimization
T2, T2*) of tissue water. MR contrast agents are typically given in
of the final conjugate, and (5) in ViVo testing. As an initial demonstra-
tion, we utilized a dimeric form of a 9-residue peptoid sequence
high doses (0.1 mmol/kg) and consequently are generally considered
too insensitive for molecular imaging applications. Consequently, MR
(GU40C4) known to bind with high affinity to the vascular endothelial
growth factor receptor 2 (VEGFR-2),12 an important target for tumor
contrast agents designed to target specific biostructures are often based
on nanoparticle or dendrimer platforms that allow significant amplifica-
metastasis. The fluorescein-tagged GU40C4 peptoid displayed a KD
) 91 ( 20 nM for VEGFR-2 (see Supporting Information). A third
generation poly(DOTA-lysine) dendron with a lysine linker (Figure
tion by an additive effect of multiple paramagnetic centers over a single
center.1 Although this approach does improve the sensitivity of MR
agents, changing from a simple low molecular weight complex to a
1) was prepared by standard solid phase and solution synthesis followed
large particle can have a substantial effect on tissue biodistribution
by addition of a maleimide group to the free R-amino of the linker
and clearance of the agent. Other factors that can be optimized to
(Scheme S1, Supporting Information). DOTA was selected as the Gd3+
decrease the amount of agent needed for detection include increasing
chelate since it forms complexes with high thermodynamic and kinetic
stability thus eliminating problems associated with metal release,
its affinity for a target (lowest KD).2 We recently demonstrated that a
single Gd3+-peptide conjugate targeted to a specific protein attached
to agarose beads could be detected by MRI at a local concentration of
∼4 µM and, based on those results, predicted that a Gd3+-based agent
with a molecular r1 ≈ 100 mM-1 s-1 should be able to detect biological
targets present at ∼690 nM.3 However, creating a single low MW
agent with an r1 ≈ 100 mM-1 s-1 has proven difficult even with highly
motionally restricted systems.4 A simpler approach would be to attach
a few Gd3+ chelates each having a more typical r1 to a targeting moiety
such that the molecular r1 sums to ∼100 mM-1 s-1. This has been
achieved by attaching several Gd3+ complexes to a variety of
functionalized scaffolds (e.g., polymers, hyperbranched polymers,
dendrimers)5-7 or to nanoparticles,8 but such large structures can add
new complexity by slowing renal filtration rates9 (glomerular filtration
threshold MW e 45 kDa)5 and even altering biodistribution of the
agent. For example, a PAMAM G4 dendrimer with ∼21 GdDTPA
plus four biotins on its surface for targeting (∼29 kD) is retained in
the vasculature of a tumor at 24 h simply due to the inherent enhanced
permeability and retention (EPR) property characteristic of macro-
molecules.10 This feature could make it difficult if not impossible to
differentiate a targeted versus a nontargeted macromolecule in tumors.
Polylysine-based dendrimers also show promise in that they can be
synthesized in specific sizes, they are biocompatible, and their blood
especially for applications in ViVo.13 Dendron conjugation to the
GU40C4 peptoid was accomplished by reaction of the thiol group of
a Cys residue of the peptoid and the maleimide moiety of the dendron.
The r1 relaxivity of the peptoid-Gd8-dendron conjugate (1 in Figure
1) was 13.8 ( 0.2 mM-1 s-1 (37 °C, pH 7, 23 MHz) per Gd3+ ion,
slightly higher than the r1 of the unconjugated Gd8-dendron (12.3 (
0.5 mM-1 s-1). These r1 relaxivities are similar to that reported for
Gadomer 17 (16.5 mM-1 s-1 at 25 °C, 20 MHz), a dendritic contrast
agent composed of three third generation lysine dendrons attached to
a trimesoyl triamide central core.14 The affinity between conjugate 1
and VEGFR-2, as measured by displacement of the fluorescein-peptoid,
was about 7-fold weaker (KD ) 703 ( 172 nM) than unmodified
GU40C4, presumably reflecting the steric bulk introduced by the Gd8-
dendron. To test this, a longer linker was introduced between the
peptoid and the Gd8-dendron (2 in Figure 1). This modification yielded
a conjugate with an increased binding affinity (KD ) 215 ( 67 nM)
and somewhat higher r1 (15.1 ( 0.2 mM-1 s-1, 37 °C, pH 7, 23 MHz).
At 9.4T, the measured r1 relaxivity of 2 was 6.1 mM-1 s-1 per Gd at
37 °C so this translates to a molecular relaxivity of ∼48 mM-1 s-1
under the conditions used for in ViVo imaging. The r1 value of 2 bound
to VEGFR2 on agarose beads did not change significantly so one can
reasonably assume that this will be its relaxivity in ViVo as well.
Porcine aortic endothelial (PAE) and PAE cells expressing human
† Advanced Imaging Research Center.
‡ Departments of Internal Medicine, Radiology, Biochemistry, and Surgery.
VEGFR2 (PAE/KDR) (2.5 × 105 receptors per cell) were chosen for initial
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C O M M U N I C A T I O N S
Figure 1. Gd8-dendron GU40C4 conjugates with different linker chains.
MR imaging. The receptor concentration per cell volume (∼5 µm cell
radius) was ∼790 nM for PAE/KDR while the control PAE cells had no
human VEGFR2. Thus, the local concentration of VEGFR2 in PAE/KDR
cells was predicted to be within the detection limit for a Gd3+ agent having
a molecular r1 of 100 mM-1 s-1.3 T1 weighted images of both cell lines
exposed to 2 or the Gd8-dendron lacking the peptoid (Figure S4) showed
that the image intensity of the PAE cell sample did not change when
exposed to either 1.5 µM 2 or the nonconjugated Gd8-dendron while the
PAE/KDR cells showed a significant increase in image contrast only when
exposed to 2, reflecting specific binding of 2 to VEGFR2 on these cells.
ICP-MS analysis of the cells postimaging showed there was negligible
Gd3+ associated with either cell line exposed to the nonconjugated Gd8-
dendron while the PAE/KDR cells exposed to 2 had ∼900 nM Gd3+.
These results show that cell receptors expressed at this level can be detected
by MRI using these simple targeted agents.
Next, we moved to an animal model to evaluate whether VEGFR2
expression could be detected in ViVo using this low MW agent (∼8.6
kD). Mice with MDA-MB-231 tumor xenografts known to express
VEGFR215 were imaged before and after administration of 2 (0.008
mmol/kg). Those results were compared with images of other mice
after injection of a scrambled 9-residue peptoid-Gd8-dendron conju-
gate (∼ 8.4 kD) (0.008 mmol/kg) that does not bind VEGFR2 (Figure
S3). As shown in Figure 2 (left panel), tumors in mice treated with 2
were maximally enhanced at ∼4 h postinjection (Figure 2A) while
the image intensity in tumors after injection of the control peptoid
Gd8-dendron conjugate (Figure 2B) returned to baseline levels by 4 h
(Figure 2 right panel). The kidneys of these animals were also
monitored during the study. After injection of the control peptoid Gd8-
dendron, the kidneys quickly darkened as a result of a reduction in T2
as expected during clearance of a Gd3+ agent at high imaging fields
(Figure 2B).16 The kidney intensity in animals postinjection of an
equivalent dose of 2 did not darken significantly until much later in
the study (Figure 2A). This indicates that 2 is largely sequestered
elsewhere during these early time points. It has been reported that
VEGFR2 is also expressed in other tissues (e.g., kidney, liver) in mice17
which might explain some of the time dependent contrast differences.
The kidney signal intensity in mice treated with 2 and the control
peptoid returned to pretreatment levels at 48-72 h postinjection.
In conclusion, we have presented a low molecular weight general
targeting platform by combining a peptoid sequence derived from
a chemical library with a Gd8-dendron to generate a high T1
relaxivity probe for molecular imaging of VEGFR2 in ViVo by MRI.
We anticipate that this platform technology will be generally useful
for other targets that exist in tissues at these levels as well.
Figure 2. (Left) 9.4 T MR T1-weighted coronal images of nude mice with
subcutaneous cell tumor MDA-MB-231 xenografts at various time points
following the intravenous (i.v.) tail injection of Gd8-dendron peptoid
conjugates. (A) images of a tumor-bearing mouse before and after i.v. tail
injection of 0.008 mmol/kg of conjugate 2. (B) Images of another tumor-
bearing mouse before and after tail i.v. injection with 0.008 mmol/kg of an
indentical Gd8-dendron conjugated to a control scrambled peptoid. The green
and yellow arrows point to a kidney and tumor, respectively. (Right) Tumor
signal intensities (s.i.’s) in mice injected with 2 (gray bars) or the control
peptoid (white bars) (s.i. is the average of 3 mice) normalized to the tumor
intensity prior injection. A t-test (two-tailed, unequal variance) was used
to compare s.i. before injection and at 0.5 or 4 h postinjection of the animals
injected with the control peptoid (+) or 2 (*) respectively. A p < 0.05 was
considered statistically significant.
Acknowledgment. Financial support from the National Institutes
of Health (RR02584, CA115531, and CA126608) is gratefully
acknowledged.
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Supporting Information Available: Calculations, detailed experi-
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discussed in this work. This material is available free of charge via the
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