Molecular Platform for Design and Synthesis of Targeted Dual-Modality Imaging Probes

Article abstract of DOI:10.1021/acs.bioconjchem.5b00028

We report a versatile dendritic structure based platform for construction of targeted dual-modality imaging probes. The platform contains multiple copies of 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) branching out from a 1,4,7-triazacyclononane-N,N′,N″-triacetic acid (NOTA) core. The specific coordination chemistries of the NOTA and DOTA moieties offer specific loading of ^68/67Ga³⁺ and Gd³⁺, respectively, into a common molecular scaffold. The platform also contains three amino groups which can potentiate targeted dual-modality imaging of PET/MRI or SPECT/MRI (PET: positron emission tomography; SPECT: single photon emission computed tomography; MRI: magnetic resonance imaging) when further functionalized by targeting vectors of interest. To validate this design concept, a bimetallic complex was synthesized with six peripheral Gd-DOTA units and one Ga-NOTA core at the center, whose ion T₁ relaxivity per gadolinium atom was measured to be 15.99 mM^-1 s^-1 at 20 MHz. Further, the bimetallic agent demonstrated its anticipated in vivo stability, tissue distribution, and pharmacokinetic profile when labeled with ⁶⁷Ga. When conjugated with a model targeting peptide sequence, the trivalent construct was able to visualize tumors in a mouse xenograft model by both PET and MRI via a single dose injection.

Full text of DOI:10.1021/acs.bioconjchem.5b00028

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Article
A Molecular Platform for Design and Synthesis
of Targeted Dual-modality Imaging Probes
Amit Kumar, Shanrong Zhang, Guiyang Hao, Gedaa Hassan, Saleh Ramezani, Su-tang Lo, Koji
Sagiyama, Masaya Takahashi, A. Dean Sherry, Orhan K Oz, Zoltán Kovács, and Xiankai Sun
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.5b00028 • Publication Date (Web): 23 Jan 2015
Downloaded from http://pubs.acs.org on February 18, 2015
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Bioconjugate Chemistry
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A Molecular Platform for Design and Synthesis of Targeted Dualꢀ
modality Imaging Probes
Amit Kumar1, Shanrong Zhang2, Guiyang Hao1, Gedaa Hassan1, Saleh Ramezani1, Koji Sagiyaꢀ
ma2, SuꢀTang Lo1, Masaya Takahashi2, A. Dean Sherry2, Orhan, K. Öz1, Zoltan Kovacs2, Xiankai
Sun1,2*
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1Department of Radiology, 2Advanced Imaging Research Center, University of Texas Southwestern Medical Center, Dallas, Texas 75390, USA
ABSTRACT: We report a versatile dendritic structure based platform for construction of targeted dualꢀmodality imaging probes.
The platform contains multiple copies of 1,4,7,ꢀ10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic acid (DOTA) branching out from a
1,4,7ꢀtriazacyclononaneꢀN,N',N''ꢀtriacetic acid (NOTA) core. The specific coordination chemistries of the NOTA and DOTA moieꢀ
ties offer specific loading of 68/67Ga3+ and Gd3+, respectively, into a common molecular scaffold. The platform also contains three
amino groups which can potentiate targeted dualꢀmodality imaging of PET/MRI or SPECT/MRI (PET: Positron Emission Tomogꢀ
raphy; SPECT: Single Photon Emission Computed Tomography; MRI: Magnetic Resonance Imaging) when further functionalized
by targeting vectors of interest. To validate this design concept, a bimetallic complex was synthesized with six peripheral Gdꢀ
DOTA units and one GaꢀNOTA core at the center, whose ion T1 relaxivity per gadolinium atom was measured to be 15.99 mMꢀ1sꢀ1
at 20 MHz. Further, the bimetallic agent demonstrated its anticipated in vivo stability, tissue distribution, and pharmacokinetic proꢀ
file when labeled with 67Ga. When conjugated with a model targeting peptide sequence, the trivalent construct was able to visualize
tumors in a mouse xenograft model by both PET and MRI via a single dose injection.
INTRODUCTION
amount a few orders of magnitude higher than PET or
SPECT.12ꢀ14
Molecular imaging is gaining importance for noninvasive asꢀ
sessment of biological events in animal and human subjects.
Through in vivo realꢀtime visualization and imaging quantifiꢀ
cation of biological targets of interest, a better understanding
of the biological processes underlining the initiation and proꢀ
gression of diseases can be obtained for more efficacious diꢀ
agnosis and treatment.1ꢀ2 However, a single imaging technique
is often unable to deliver all the necessary information reꢀ
quired for a definitive clinical decision. To date, successful
attempts have been seen to combine two or more imaging moꢀ
dalities in order to overcome the shortcomings of single moꢀ
dality systems for optimal visualization and better quantitative
delineation of targets of interest. Consequently, multimodal
imaging techniques have become a norm in both clinical pracꢀ
tice and preclinical research as evidenced by the implementaꢀ
tion of PET/CT (PET: Positron Emission Tomography; CT:
Computed Tomography) and PET/MRI (MRI: Magnetic Resꢀ
onance Imaging) procedures to patient care and the increasing
use of a variety of combinations of the currently available
imaging techniques in translational or basic biomedical reꢀ
search, such as MRI/optical and PET/NIRF (NIRF: Nearꢀ
infrared Fluorescence).3ꢀ8 The fusion of PET and MRI is espeꢀ
cially desirable because technically their physical features are
complementary. While radionuclideꢀbased imaging techꢀ
niques, either PET or SPECT (Single Photon Emission Comꢀ
puted Tomography), are extremely sensitive and quantitative,
which allows the studies of biological events or processes at
the molecular and cellular level, their spatial resolution is limꢀ
ited (> 1 cm for current clinical scanners).9ꢀ11 On the other
hand, while MRI is able to provide high spatial resolution (<
0.1 cm) with exquisite soft tissue contrast even without exogeꢀ
nous contrast agents, contrast agents are often required to
highlight specific biological or physiological processes in an
Given that more and more PET/MRI systems will likely be
used in the practice of diagnostic radiology, it becomes imꢀ
portant to develop dual modality imaging probes that can realꢀ
ize the full potential of both modalities.
To take advantage of the strengths of PET and MRI, our apꢀ
proach is to design and synthesize a dualꢀmodality agent scafꢀ
fold offering a “single pharmacological behavior” for both
imaging acquisitions. The merging of PET and MRI probe
moieties to a single molecular platform would facilitate coꢀ
location and crossꢀvalidation of each modality in targeted reꢀ
gions of interest (two measures of one event). While MRI can
provide the exact location of the probe, motion artifact correcꢀ
tion, and PET partial volume correction, PET can afford better
imaging quantification for higher detection sensitivity. Furꢀ
ther, given that the proton MRI contrast actually reflects the
map of the proton density, a coꢀlocalized PET signal distinct
from the proton background could make the MRI contrast
more identifiable, which further improves the MRI sensitivity.
Indeed several nanoparticleꢀbased PET/MRI agents have been
reported with enhanced magnetic relaxivities and considerable
promise as dualꢀmodality imaging agents.15ꢀ17 However, a naꢀ
noparticle platform for PET/MRI probe development presents
challenges. First, the nanoparticleꢀbased PET/MRI probes are
not single molecular entities and often have questionable in
vivo stability or molecular integrity. Second, to serve the purꢀ
pose of diagnostic imaging, contrast agents are desired to have
a reasonable blood circulation halfꢀlife for efficient accumulaꢀ
tion in targets and minimal nonspecific deposition in the monꢀ
onuclear phagocyte system (MPS) organs after systemic adꢀ
ministration. However, it is well known that most nanopartiꢀ
cles if not all possess suboptimal in vivo distribution as shown
by rapid sequestration into MPS organs and thereafter slow
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modality imaging probe design include the apparent in vivo
1
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3
stability (due to the inertness of the GdꢀDOTA and GaꢀNOTA
complexes) and the defined molecular formula with selective
loading of two metal ions.
4
5
6
7
8
Synthesis. The desired ligand H21L was synthesized in 7 steps
with an overall yield of approximately 15%. The structure of
1
each intermediate was verified by H NMR and 13C NMR as
well as mass spectroscopy. The synthesis route to compound
H21L is outlined in Scheme 1 and its functionalization is deꢀ
picted in Scheme 2. Compound 2 was obtained from bis(3ꢀ
aminopropyl)amine and carbobenzyloxy (Cbz) protected lyꢀ
sine in three steps (see Supporting Information). The free priꢀ
mary amine of 2 was coupled to the carboxylic acid bearing
side arms of NOTAꢀ(OH)3 (Scheme 1) via carbodiimide
chemistry to form 3. The Nꢀphthaloyl protected amino groups
of 3 were freed by deprotection via hydrazine to afford 4,
which contains six amino groups serving as points of attachꢀ
ment for the DOTA units.
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Figure 1. The dualꢀmodality molecular probe design
clearance. These properties present potential health hazards18ꢀ
20 which could impede their translation toward clinical applicaꢀ
tions. Third, the majority of nanoparticleꢀbased PET/MRI
agents provide T2 contrast, only a handful are T1ꢀbased agents,
which are preferred in the cases of low proton density in the
target tissues. To date, a wide range of macromolecules, such
Scheme 1. Synthetic route to ligand H21L
as perfluorocarbon emulsions,15 silica nanoparticles,16,
21ꢀ22
23ꢀ25
liposomal vesicles,17,
dendrimers,26ꢀ32 and polymers,33
have been explored for the development of gadoliniumꢀbased
T1 contrast agents. Of the macromolecular systems, denꢀ
drimers, which possess definitive molecular structures and
formula, have shown a promising role. For instance, the denꢀ
drimer systems of poly(amido amine) (PAMAM) have been
used as blood pool,34 liver,35 renal,36 lymphatic,37 and tumorꢀ
specific38 contrast agents. However, the GdꢀPAMAM comꢀ
plexes rarely afford an ionic relaxivity greater than 11 mM-1s-1
per gadolinium ion.39ꢀ40 When such dendrimer platforms are
used to develop PET/MRI or SPECT/MRI agents, it is chalꢀ
lenging to achieve precise control of radioisotope loading into
specific chelating moieties. Herein, we present a dualꢀ
modality molecular probe design (Figure 1) carrying six
1,4,7,ꢀ10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀtetraacetic
acid
The Nꢀhydroxy succinamide activated DOTA ester, 5, was
reacted to the six amino groups forming 6. Attempts to deproꢀ
tect the Boc groups of compound 6 led to partial deprotection
of the Cbz groups. To overcome this problem, the ligand was
fully deprotected by 30% HBr in an acetic acid solution. The
synthesized ligand, H21L, was fully characterized. The ligand
carries six DOTA units intended for chelating Gd3+ and one
NOTA unit intended for chelating Ga3+. Since Gd3+ and Ga3+
can be specifically loaded into the DOTA and NOTA moieꢀ
ties, respectively, based on their ionꢀsize preference, the bimeꢀ
tallic molecular complex is expected to carry the two metal
ions in a specified ratio so as to facilitate PET imaging based
quantification.
(DOTA) moieties branched out from single 1,4,7ꢀ
a
triazacyclononaneꢀN,N',N''ꢀtriacetic acid (NOTA) core strucꢀ
ture. In this work, we demonstrate an exclusive loading of
Gd3+ into the DOTA units affording a T1 ion relaxivity of
15.99 mMꢀ1sꢀ1 at 20 MHz, while the NOTA core remains
available to form a chelate with 67/68Ga3+ for nuclear imaging.
If the targeted accumulation event is rapid, 68Ga (t1/2 = 68 min;
β+ = 89%, Eβ+
= 1.92 MeV; EC = 11%) will be used for
max
PET imaging; otherwise, 67Ga (t1/2 = 3.26 d; γ = 184 keV) will
be used for SPECT imaging.
RESULTS AND DISCUSSION
The dualꢀmodality molecular probe design also possesses
three amino groups for potential conjugation with multiple
copies of targeting molecules, thereby providing multivalent
effect to the construct for better specific binding to the biologꢀ
ical target of interest. Of note, the strong interaction between
the targeting molecule tethered probe and the corresponding
target, triggered by multivalent effect, is expected to further
enhance the relaxivity of the probe. For proof of concept we
conjugated each amine with a model integrin αvβ3 targeting
peptide, c(RGDyK). The main features of this molecular dualꢀ
Functionalization of ligand H21L was performed by utilizing
the previously mentioned amino groups. Maleimido groups
were tethered to amines using the commercially available reaꢀ
gent of Nꢀsuccinimidyl 3ꢀmaleimidopropionate (Scheme2). The
model targeting peptide, c(RGDyK), was tethered to the maleiꢀ
mido carrying ligand H21L via thiolꢀmaleimide coupling. Givꢀ
en that targeting molecules attached to the bimetallic complex
may not be able to stretch out for specific binding because of
the steric hindrance from the construct, additionally we introꢀ
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Bioconjugate Chemistry
duced a PEG12 linker between the complex and targeting vecꢀ
tor.
or 68Ga3+ into the NOTA core of complex of Gd6H3L was then
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2
3
4
5
6
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8
carried out at pH 3ꢀ5 in an HEPES buffer. The cold Gd6LGa
complex was purified and characterized by HPLC and mass
spectrometry. Various concentration of Gd6LGa were injected
to inductively coupled plasma mass spectrometry (ICPꢀmass)
and the ratio of Gd:Ga was calculated to be 6:1 (see Supportꢀ
ing Information). The Gd6L68Ga was purified and characterꢀ
ized by radioꢀHPLC using the cold complex as reference.
Scheme 2. Synthesis of integrin-αvβ3 targeted ligand scaffolds
NH
NH
N
HN
H2
H2
N
O
N
H
1) SPDP
2) Dithiothreitol
N
H
O
O
NH
NH
O
O
O
H2N
HN
NH
HN
HS
O
N
N
H
H
N
H
NH
OH
Coordination chemistry of the NOTA core of H21L. To
prove the efficient removal of Gd3+ from the NOTA core durꢀ
ing the complexation procedure, the same complexation proꢀ
cedure was followed using Eu3+ instead of Gd3+. Chemically,
Eu3+ and Gd3+ have similar reactivities towards NOTA and
12 O
O
O
H
N
HN
O
O
O
O
9
HO
HO
c(RGDyK)-PEG12SH
O
OH
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c(RGDyK)
OH
O
N
HO
O
O
N
1
DOTA. Since Eu3+ is only weakly paramagnetic, the HꢀNMR
N
O
N
OH
O
N
O
OH
signals of its complexes are relatively sharp and also paraꢀ
magnetically shifted. This facilitates relatively straightꢀ
forward structural characterization by NMR spectroscopy. To
accomplish this, the Eu3+ complex of H21L, were synthesized
via two procedures, i.e., with and without a challenge by exꢀ
cess DTPA, followed by HPLC purification. Both complexes
HN
N
HO
NH
N
O
N
HO
N
O
O
O
R
OH
O
NH
N H
HO
OH
N
O
N
O
N
O
HN
R
N
N
N
O
H
N
N
O
HO
N
O
N
O
OH
H
O
H
OH
N
OH
O
N
N
1
H
O
O
were characterized by H NMR spectroscopy (400 MHz).
N
O
O
Figures 2a and 2b show the 1H NMR spectra of Eu3+ complexꢀ
es of H21L with and without the DTPA challenge, respectively.
The DTPA treated Eu3+ complex (Figure 2a) showed sharp
peaks in the NOTA region (around 3 – 4 ppm) suggesting a
metal free NOTA core as opposed to the untreated complex
(Figure 2b), which showed broad peaks indicating the interacꢀ
tion of metal with the NOTA core. This characterization
along with the mass spectroscopy results of Gd7L and Gd6H3L
indicates the exclusive Gd3+ coordination with the six DOTA
moieties of H21L. Further, the formation of Gd6LGa was conꢀ
firmed by the molar ratio of Gd:Ga determined by ICPꢀmass
(supporting information. These results demonstrate that Gd3+
and Ga3+ have been exclusively loaded to the DOTA and
NOTA moieties as designed, respectively, for the preparation
of the desired dualꢀmodality molecular imaging probe.
O
N
NH
N
HO
N
H
N
HO
O
O
HN
O
N
N
N
R
OH
O
N
N
N
O
O
OH
HO
N
N
OH
OH
O
O
OH
H21L; R = H
O
N
O
O
O
O
N
OH
O
H21L[c(RGDyK)]3; R = HO
O
O
O
O
O
c(RGDyK)-SH
N
H
O
HN
HN
N
H21L(Mal)3; R =
NH
O
O
H
N
O
O
N
S
O
O
N
H
O
NH
HN
NH2
c(RGDyK)-PEG12SH
OH
O
H21L[PEG12c(RGDyK)]3; R = HO
O
N
H
O
HN
HN
O
NH
H
N
O
O
O
H
O
O
O
N
N
S
N
H
12
O
O
NH
HN
NH2
The thiol group without a spacer (zero) or with a PEG12 linker
was introduced to c(RGDyK) using the commercially availaꢀ
ble
reagent:
2ꢀpyridyldithiolꢀtetraoxaoctatriacontaneꢀNꢀ
hydroxysuccinimide (SPDP). The thiol terminated c(RGDyK)
peptides with zero and PEG12 linker were subsequently added
to H21L(Mal)3 to afford Gd6H3L[c(RGDyK)]3
Gd6H3L[PEG12c(RGDyK)]3, respectively.
and
Complexation with Gd3+ and Ga3+ to form Gd6LGa. The
thermodynamic stability constants of GdꢀDOTA (logKGdꢀDOTA
= 24.7), GdꢀNOTA (logKGdꢀNOTA = 14.3), and GaꢀNOTA
(logKGaꢀNOTA = 31.0)41ꢀ42 suggest that when ligand H21L is
treated with six equivalents of Gd3+, its complex can be
formed exclusively with the DOTA units. The bimetallic comꢀ
plex of H21L, Gd6LGa, can then be formed by incorporation of
Ga3+ into the NOTA core. However, considering that the reacꢀ
tion of L with the stoichiometric amount of Gd3+ may be slow
and could lead to partial complexation of the DOTA units, the
first complexation was performed with excess of Gd3+ to give
complete chelation of both the DOTA and NOTA units formꢀ
ing Gd7L. This was confirmed with mass spectroscopy (see
Figure 2. 400 MHz 1H NMR spectra of a) Eu3+ complex of H21L
treated with excess DTPA b) untreated Eu3+ complex of H21L, and
c) EuꢀDOTA complex.
Relaxometric measurements. The T1 relaxivity of Gd6LGa
was measured at 20 MHz, 25 °C, to be 95.98 mMꢀ1sꢀ1 with R2
value greater than 0.99 (Each Gd3+ in the complex accounted
for 15.99 mMꢀ1sꢀ1 (ion relaxivity). Under the same conditions,
the relaxivity of Magnavist was found to be 4.1 mMꢀ1sꢀ1. Imꢀ
pressively, the T1 relaxivity of Gd6H3L was approximately 4
times higher than that of GdꢀDOTA complex and comparable
1
Supporting Information) as well as H NMR spectra using
Eu3+ instead of Gd3+. Later, Gd3+ was efficiently removed from
the NOTA core by treating the complex with excess diethyꢀ
lene triamine pentaacetic acid (DTPA) (the log K of Gdꢀ
DTPA42 is approximately eight orders of magnitude higher
than that of GdꢀNOTA). The incorporation of either cold Ga3+
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to the GdꢀPAMAM complexes, which have been reported with
that kidney clearance would be even faster once the positively
the ion relaxivity up to 11 mMꢀ1sꢀ1.39ꢀ40 It is noteworthy that a
similar relaxivity was also reported for a polylysineꢀbased
contrast agent (MW 17,453) with 24 GdꢀDOTA units.43ꢀ44 At
the physiological temperature of 37 °C, the relaxivity of
Gd6LGa was unchanged while its relaxivity in rat serum at 25
°C increased to 153.1 mMꢀ1sꢀ1. These observations suggest
two things; first, the lack of temperature dependence between
charged amino groups are removed from the agent through
conjugation with a targeting vector. Indeed when the amine
groups of the agent were capped with c(RGDyK), the kidney
uptake was impressively reduced to ca. 3 %ID/g at 2 h p.i.
1
2
3
4
5
6
7
8
The pharmacokinetics of Gd6L67Ga showed
a
twoꢀ
compartment profile with t1/2α = 11.3 min and t1/2β = 7.3 h,
quite different compared to nanoparticleꢀbased imaging agents
which can be described by one compartment models.46ꢀ47 The
t1/2α reveals that the tissue distribution of Gd6L67Ga was relaꢀ
tively rapid while the reasonably long t1/2β enables the probe to
accumulate in its targeted tissues without incurring high backꢀ
ground uptake. This indicates that the probe behaves similarly
to other clinically available small molecule contrast agents.48
The presence of amine groups on the ligand provides the ease
of incorporation of polyethylene glycol (PEG) chains into the
probe for optimization of the in vivo kinetics if necessary.
The in vitro and in vivo stabilities of Gd6L67Ga were evaluated
through incubation in rat serum and by mouse urine metabolite
analysis, respectively. The bimetallic complex remained intact
during the entire course of study, which further validates this
molecular probe platform design.
25
appended GdꢀDO3Aꢀmonoamide complexes limit the relaxivꢀ
ity somewhat at 25 C. Given that water exchange is faster at
37 C, the expected decline in relaxivity with increasing temꢀ
peratures is offset by faster water exchange and a more favorꢀ
able relaxivity at 37 C. This was an unanticipated yet welꢀ
̊C and 37 ̊C suggests that the water exchange rate in the
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̊
̊
comed added advantage for this probe platform design. The
higher relaxivity in serum indicates that Gd6LGa does interact
with one or more serum proteins.
Table 1. Tissue distribution data and pharmacokinetic parameꢀ
ters of Gd6L67Ga in normal Balb/c mice (n = 4). Data are preꢀ
sented as mean ± standard deviation.
Organ
%ID/g
4h
24h
1h
Figure 3. T1ꢀweighted MR images of samples recorded at 1.0 T at
25 ̊C. Samples of Magnevist (a → e) and Gd6H3L (v → z) conꢀ
taining a series of decreasing concentrations from 0.4 mM, 0.2
mM, 0.1 mM, 0.04 mM, to 0.02 mM.
Blood
Heart
Lung
3.51 ± 0.22
2.63 ± 0.84
0.94 ± 0.40
1.78 ± 0.87
2.13 ± 0.81
0.21 ± 0.06
0.21 ± 0.02
0.38 ± 0.09
1.39 ± 0.24
1.21 ± 0.13
2.59 ± 0.5
1.39 ± 0.11
MR imaging and relaxivity measurements of Gd6LGa at
1.0 T. The MR imaging potential of Gd6LGa was evaluated at
magnetic field of 1.0 T. Figure 3 shows the T1 weighted imagꢀ
ing of Gd6LGa in comparison with Magnevist at a series of
decreasing concentrations (0.4 mM – 0.02 mM determined by
ICPꢀmass). The images of Gd6LGa were obviously brighter
than those of Magnevist (4.1 mMꢀ1 sꢀ1) at each equivalent conꢀ
centration. The T1 relaxivity of Gd6LGa was measured at
113.36 mMꢀ1sꢀ1 (T1 ion relaxivity: 18.89 mMꢀ1sꢀ1) at 1.0 T
Liver
Kidney
Spleen
Muscle
Fat
101.32 ± 4.32 57.42 ± 17.29 43.90 ± 5.12
0.73 ± 0.19
0.27 ± 0.09
0.27 ± 0.10
0.08 ± 0.01
0.69 ± 0.07
0.38 ± 0.13
0.27 ± 0.10
0.07 ± 0.02
0.42 ± 0.12
0.08 ± 0.02
0.06 ± 0.02
0.02 ± 0.00
Brain
(25 ̊C). Since the agent is intended for conjugation to a targetꢀ
ing moiety for receptorꢀbased imaging, such interactions could
further enhance the relaxivity of the agent in vivo.
Twoꢀcompartment clearance profile (min)
t1/2α 11.33 ± 1.31 t1/2β
438.37 ± 15.11
In vivo tissue distribution, pharmacokinetics, and stability
of Gd6LGa. As compared to other nanoparticleꢀbased dual
modality imaging platforms, our molecular probe design is
expected to have a reasonable blood circulation halfꢀlife and
an efficient clearance profile from the organs. To evaluate the
in vivo behavior of this probe design, Gd6LH3 was radioꢀ
labeled with 67Ga (t1/2 = 3.26 d) so that the tissue biodistribuꢀ
tion could be evaluated over an extended period. As shown in
Table 1, the bimetallic complex, Gd6L67Ga, indeed displayed a
rapid and efficient clearance profile from all major organs
except kidneys in normal mice. The high renal uptake can be
attributed to the net positive charge on the probe due to the
presence of amine groups.45 However, an efficient renal clearꢀ
ance is obvious after the initial high uptake as seen from the
decrease of the kidney uptake level from 101.0 ± 4.3 %ID/g at
1 h p.i. to 43.0 ± 5.1 %ID/g at 24 h p.i. One would anticipate
Receptorꢀbinding assay of targeted Gd6LGa complexes.
Two integrin αvβ3 targeted Gd6LGa complexes
Gd6H3L[c(RGDyK)]3 and Gd6H3L[PEG12c(RGDyK)]3) were
prepared to validate that our molecular construct design can be
utilized for specific imaging of cancer biomarkers. The αvβ3ꢀ
binding affinities were determined by a competitive cellꢀ
binding assay using 125Iꢀechistatin (PerkinElmer) as the αvβ3ꢀ
specific radioligand. The bestꢀfit IC50 values (inhibitory conꢀ
centration where 50% of the 125Iꢀechistatin bound on U87MG
cells are displaced) of c(RGDyK), Gd6H3L[c(RGDyK)]3 and
Gd6H3L[PEG12c(RGDyK)]3) were measured to be 199 nM,
489 nM, and 37 nM (Supporting Information), respectively.
The significantly improved αvβ binding affinity of
Gd6H3L[PEG12c(RGDyK)]3) clearly indicates the critical role
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Page 5 of 11
Bioconjugate Chemistry
of the PEG12 linker in the desired targeting property of our design concept, we believe that it can be readily modified or
1
molecular scaffold design.
adapted to reach the desired MRI sensitivity.
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3
4
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7
8
9
In conclusion, we have successfully demonstrated a molecular
platform intended for targeted dualꢀmodality imaging of
PET/MRI or SPECT/MRI, which carries quantifiable numbers
of 67/68Ga3+ and Gd3+ ions in their specific chelation moieties.
This probe design displays a very favorable high T1 relaxivity
for MRI contrast enhancement. With the current rapid growth
of hybrid PET/MRI systems in diagnostic radiology, our moꢀ
lecular design of dualꢀmodality agents possessing a “single
pharmacological behavior” offers a versatile platform for the
development of multimodality imaging agents with potential
applications for noninvasive molecular profiling of various
diseases.
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EXPERIMENTAL PROCEDURES
General Materials and Methods All reactions were carried
out under N2 atmosphere in degassed dried solvents. Commerꢀ
cially available starting materials were purchased from comꢀ
mercial vendors and used directly without further purification
unless otherwise stated. All aqueous solutions were prepared
with MilliꢀQ water. Bulk solvents were removed using rotary
evaporator under reduced pressure at 40 °C. Trace solvents
were removed under high vacuum. Matrixꢀassisted laser deꢀ
sorption/ionization (MALDI) mass spectra were acquired on
an Applied Biosystems Voyagerꢀ6115 mass spectrometer.
Radiolabeled conjugates were purified by Light Cꢀ18 SepꢀPak
cartridges (Waters, Milford, MA). High performance liquid
chromatography (HPLC) was performed on a Waters Xterra
Shield RP Cꢀ18 semiprep column (250 × 10 mm, 10 ꢁm) and
read by a Waters 2996 photodiode array detector and an inꢀ
line Shell Jr. 2000 radioꢀdetector. The mobile phase was H2O
with 0.1% trifluoroacetic acid (TFA) (solvent A) and acetoniꢀ
trile with 0.1% TFA (solvent B). The gradient consisted of 0%
B to 80% B in 0−40 min at 4.0 mL/min flow rate. The radioacꢀ
tivity of excised tissue samples and radioactive standards were
counted by a Wizard2 300 automatic γꢀcounter (PerkinElmer).
Figure 4. Representative MRI (upper panel), PET (lower panel –
left), and fused PET/MR (lower panel – right) images of U87MG
tumor xenograft in SCID mice at 1 h post injection of
Gd6L[PEG12c(RGDyK)]368Ga. The white arrow indicates the
tumor site.
PET/MR Imaging using Gd6L[PEG12c(RGDyK)]
68Ga. To
3
overcome the sensitivity difference between PET and MR, the
radioactivity of 68Ga was controlled in the labeling of
Gd6H3L[PEG12c(RGDyK)]3. The final dose injected into each
animal carried about 6.0 mg of the final compound with an
activity of 100ꢀ150 µCi (Gd: ca. 0.027 mmol/kg; c(RGDyK):
2.0 µmol). Figure 4 shows in vivo dual PET/MR images of
integrin ανβ3 positive U87MG tumor enabled by a single dose
Synthesis Procedures
Compound 3. To a solution of the protected acid 1 (0.10 g,
0.15 mmol) in CH3CN (1.0 mL) were added the deprotected
amine 2 (0.40 g, 0.60 mmol), dicyclohexylcarbodiimide (0.15
g, 0.83 mmol) and triethylamine (0.30 g, 0.27 mmol). The
resultant solution was stirred for 12 h, filtered and the solvent
evaporated. The crude product was purified by flash chromaꢀ
tography (ethyl acetate) to give 3, a NOTA derivative, as a
injection of Gd6L[PEG12c(RGDyK)]
68Ga. The average tumor
3
uptake measured by PET was 0.88 %ID/g. It should be noted
that the tumor uptake value was reduced by roughly 3 times
when the injected amount of the agent increased from 0.1 mg
to 6.0 mg. This decrease also indicates the integrin ανβ3 imagꢀ
ing specificity. The observed MR contrast enhancement in the
tumor was 3.06 (n = 3) when compared to the contrast before
injection.
1
white solid (0.26 g, 0.10 mmol, 67%). H NMR (400 MHz,
CDCl3): δ 8.73 (bs, 6H), 7.71 (m, 12H), 7.61 (m, 12H), 7.23
(m, 15H), 5.56 (bs, 2H), 4.99 (m, 5H), 4.74 (m, 3H), 3.79ꢀ3.27
(m, 24H), 3.25ꢀ2.77 (m, 13H), 2.60ꢀ2.17 (m, 4H), 2.15ꢀ1.71
(m, 12H), 160 (m, 4H), 1.38 (m, 43H). 13C NMR (100 MHz,
CDCl3): δ 172.5, 168.3, 156.8, 136.5, 134.1, 133.9, 131.8,
128.4, 127.9, 123.3, 123.2, 79.0, 69.8, 66.5, 64.8, 49.3, 45.9,
44.3, 40.5, 35.5, 35.3, 325, 32.2, 29.3, 28.3, 27.9, 26.8, 22.6.
MS (MALDI) m/z calcd for C141H168N18O30: 2594.2; found:
2595.9 ([M + H]+).
To date, few PET/MRI or SPECT/MRI agents with a defined
structure have been reported. In vivo pH monitoring PET/MRI
agents carrying one GdꢀDOTA unit along with 18F or 68Ga as
PET contrast have been reported.49 Bimetallic complexes carꢀ
rying one GdꢀDOTA conjugated to 68GaꢀNOTA and one Gdꢀ
DOTA conjugated to cold Cu or InꢀDOTA do exist.50ꢀ51 Howꢀ
ever, they were not designed to serve the goal of providing
targeted contrast to both imaging techniques. To provide proof
of concept, the probe design in this work consists of only six
gadolinium chelatable DOTA units. While the anticipated T1
relaxivity enhancement was achieved, we recognize that furꢀ
ther amplification of MRI sensitivity is necessary in order to
realize the practical application of the molecular probe design
as dualꢀmodality imaging agents. Given the versatility of the
Compound 4. To a solution of 3 (0.26 g, 0.11 mmol) in ethaꢀ
nol (1.0 mL) was added hydrazine monohydrate (0.1 mL, 2.0
mM) and the mixture was stirred for 12 h at room temperature.
After the reaction, the precipitate was removed by filtration.
The filtrate was evaporated and extracted with CH2Cl2 (3 × 10
mL). The combined organic layers were evaporated to give 4
as light yellow oil (0.15 g, 0.08 mmol, 80%). This compound
1
was used for the next step without further purification. H
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Bioconjugate Chemistry
Page 6 of 11
NMR (400 MHz, CD3OD): δ 7.31 (s, 15H), 4.99 (m, 6H), 4.66 (1 x) was added the thiol carrying c(RGDyK)ꢀSH (0.014 g,
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2
3
4
5
6
7
8
(m, 3H), 3.68ꢀ3.39 (m, 10H), 3.38ꢀ3.31 (m, 6H), 3.20ꢀ2.94 (m,
17H), 2.93ꢀ2.72 (m, 9H), 2.65ꢀ2.30 (m, 7H), 1.95ꢀ1.81 (m,
6H), 1.78ꢀ1.57 (m, 6H), 1.55ꢀ1.20 (m, 44H). 13C NMR (100
MHz, CD3OD): δ 173.7, 173.1, 157.6, 136.9, 128.9, 127.3,
126.8, 126.5, 82.6, 77.3, 65.9, 64.5, 50.2, 48.8, 45.9, 44.5,
42.3, 38.5, 38.1, 36.7, 30.3, 29.1, 27.8, 26.6, 25.2 MS
(MALDI) m/z calcd for C93H156N18O18: 1814.1; found: 1816.3
([M + H]+).
0.029 mmol) and the solution was allowed to stir for 18 h. The
solution was purified by reverse phase HPLC using water and
acetonitrile solvent mixture to give H21L[c(RGDyK)]3 as a
white solid. (0.010 g, 0.002 mmol, 33.3%). MS (MALDI) m/z
calcd for C264H420N72O90S3 : 6136.9; found: 6139.1 ([M + H]+).
Compound c(RGDyK)PEG12SH. To the DMF solution (1
mL) of the αvβ3 integrin targeting peptide c(RGDyK) (0.040 g,
0.064 mmol) (Peptides International Inc, Kentucky) dissolved
in DMF (1.0 mL) was added Nꢀ 2ꢀpyridyldithiolꢀ
tetraoxaoctatriacontaneꢀNꢀhydroxysuccinimide (0.060 g, 0.065
mmol) (Thermo Scientific, IL) and the solution was allowed to
stir for 6 h. The solvent was evaporated and the product neuꢀ
tralized and purified by reverse phase HPLC using water and
acetonitrile solvent mixture and lyophilized. The resultant
white solid was dissolved in DMF (1 mL) and dithiothreitol
(0.010 g, 0.065 mmol) was then added and the solution was
allowed to stir for 3 h. The solvent was evaporated and the
product neutralized and purified by reverse phase HPLC using
water and acetonitrile solvent mixture to give
c(RGDyK)PEG12SH as a colorless viscous liquid. (0.025 g,
0.019 mmol, 30.2%). MS (MALDI) m/z calcd for
C57H98N10O22S: 1306.6; found: 1307.5 ([M + H]+).
Compound 6. To a solution of 4 (0.15 g, 0.08 mmol) in DMF
9
(1 mL) were added
5 (0.53 g, 0.80 mM), an Nꢀ
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hydroxysuccinimide ester of DOTA, and the mixture was
stirred for 24 h at room temperature. The solvent was evapoꢀ
rated and the product purified by reverse phase HPLC using
water and acetonitrile solvent mixture to give 6 as a white
solid (0.29 g, 0.06 mmol, 70%). 1H NMR (400 MHz, CDCl3):
δ 7.40ꢀ7.18 (s, 15H), 5.15ꢀ4.99 (m, 6H), 4.71 (m, 6H), 4.38ꢀ
4.83 (m, 26H), 3.82ꢀ3.36 (m, 61H), 3.30ꢀ2.62 (m, 17H), 2.60ꢀ
2.30 (m, 12H), 2.23ꢀ1.98 (m, 10H), 1.99ꢀ1.80 (m, 10H), 1.79ꢀ
0.96 (m, 174H). 13C NMR (100 MHz, CD3OD): δ 172.8,
172.6, 170.8, 170.6, 157.3, 137.2, 128.2, 127.6, 127.2, 84.5,
81.5, 65.8, 64.7, 53.6, 51.6, 49.2, 48.5, 45.3, 43.6, 42.0, 40.1,
36.5, 31.9, 27.2, 28.5, 27.2, 27.1, 22.7. MS (MALDI) m/z
calcd for C261H456N42O60: 5139.4; found: 5138.7 ([M + H]+).
Compound H21L[PEG12c(RGDyK)]3. To the malemide carꢀ
rying ligand, H21L(Mal)3 (0.030 g, 0.007 mmol), dissolved in
PBS (1 ×) was added the thiol carrying peptide c(RGDyK)ꢀSH
(0.038 g, 0.028 mmol) and the solution was allowed to stir for
18 h. The solution was purified by reverse phase HPLC using
water and acetonitrile solvent mixture to give
H21L[PEG12c(RGDyK)]3 as a white solid. (0.019 g, 0.002
mmol, 35.3%). MS (MALDI) m/z calcd for C345H579N75O129S3
: 7936.0; found: 7936.7 ([M + H]+).
Ligand H21L. To a solution of 6 (0.10 g, 0.02 mmol) was addꢀ
ed 30% HBr in AcOH (2 mL) and the solution was allowed to
stir for 4 h. The solvent was evaporated, the product neutralꢀ
ized and purified by reverse phase HPLC using water and aceꢀ
tonitrile solvent mixture to give H21L as a white solid (0.06 g,
1
0.02 mmol, 75%). H NMR (400 MHz, CD3OD): δ 4.42ꢀ3.62
(m, 89H), 3.59ꢀ3.32 (m, 67H), 3.24ꢀ2.85 (m, 48H), 2.80ꢀ2.30
(m, 22H), 2.34ꢀ2.03 (m, 17H), 2.06ꢀ1.85 (m, 14H), 1.88ꢀ1.65
(m, 20H), 1.65ꢀ1.39 (m, 11H), 1.31 (m, 6H) δ MS (MALDI)
m/z calcd for C153H270N42O54 : 3561.9; found: 3563.0 ([M +
H]+).
Compound Gd7L. The free ligand H21L (0.060 g, 0.013
mmol) was dissolved in water (1 mL) and the pH was adjusted
to 7.0 with NaOH (0.1 M). To this solution was added an exꢀ
cess amount of GdCl3.6H2O and the pH was readjusted to 6.5
and allowed to stir at room temperature overnight. The pH of
the resultant solution was raised above 8 using 1 M aqueous
NaOH, causing the excess Gd3+ to precipitate as Gd(OH)3. The
solution was filtered and the pH was readjusted to 7.0 using 1
N HCl. the solution was purified using HPLC and the fractions
pooled together and lyophilized to give a white solid. (0.051 g,
0.011 mmol, 84.6%). MS (MALDI) m/z calcd for
C153H261Gd7N42O60 : 4644.2; found: 4645.1 ([M + H]+).
Compound H21L(Mal)3. To the free ligand H21L (0.200 g,
0.056 mmol) dissolved in DMF (1 mL) was added triethyl
amine
(0.022
g,
0.224
mmol)
and
Nꢀ(γꢀ
maleimidobutyryloxy)succinimide and the solution was alꢀ
lowed to stir for 24 h. The solvent was evaporated; the product
neutralized and purified by reverse phase HPLC using water
and acetonitrile solvent mixture to give H21L(Mal)3 as a white
solid. (0.102 g, 0.052 mmol, 45.1%). MS (MALDI) m/z calcd
for C174H285N45O63 : 4015.0; found: 4016.3 ([M + H]+).
Compound c(RGDyK)ꢀSH. To the DMF solution (1 mL) of
the commercially available αvβ3 integrin targeting peptide
c(RGDyK) (0.030 g, 0.048 mmol) (Peptides International Inc,
Kentucky) was added Nꢀsuccinimidyl 3ꢀ(2ꢀpyridyldithio)ꢀ
propionate (0.020 g, 0.064 mmol) (Thermo Scientific, IL) and
the solution was allowed to stir for 6 h. The solvent was evapꢀ
orated; the product neutralized and purified by reverse phase
HPLC using water and acetonitrile solvent mixture and lyophiꢀ
lized. The resultant white solid was dissolved in DMF (1.0
mL) and dithiothreitol (0.010 g, 0.065 mmol) was then added.
The solution was allowed to stir for 3 h. The solvent was
evaporated and the product neutralized and purified by reverse
phase HPLC using water and acetonitrile solvent mixture to
give c(RGDyK)ꢀSH as a white solid. (0.103 g, 0.014 mmol,
29.5%). MS (MALDI) m/z calcd for C30H45N9O9S : 707.3;
found: 708.3 ([M + H]+).
Compound Gd6H3L. To the solution of Gd7L (0.05 g, 0.011
mmol), DTPA (0.1 mM, 1mL) was added. The solution was
stirred at room temperature for 2 h and then purified using
HPLC. The desired fractions were pooled together and lyophiꢀ
lized to give Gd6H3L as a white solid (0.04 g, 0.01 mmol,
81%). MS (MALDI) m/z calcd for C153H264Gd6N42O60: 4489.3;
found: 4634.9 ([M + K+ 6H2O]+).
Compound Eu6H3L. The free ligand H21L (0.030 g, 0.008
mmol) was dissolved in water (1 mL) and the pH was adjusted
to 7.0 with NaOH (0.1 M). To this solution was added an exꢀ
cess amount of EuCl3.6H2O and the pH was readjusted to 6.5
and allowed to stir at room temperature overnight. The pH was
raised above 8.0 using 1.0 N aqueous NaOH, which caused the
excess Eu3+ to precipitate as Eu(OH)3. The solution was filꢀ
tered and the pH was readjusted to 7.0 using 1.0 N HCl. To the
resulting solution, DTPA (0.1 mM, 1mL), was added and the
solution was purified using HPLC to give the desired complex.
The desired fractions were pooled together and lyophilized to
Compound H21L[c(RGDyK)]3. To the malemide carrying
ligand, H21L(Mal)3, (0.020 g, 0.005 mmol) dissolved in PBS
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Bioconjugate Chemistry
give Eu6LH3 as a white solid. (0.042 g, 0.009 mmol, 54%). MS sulting solution, DTPA (0.1 mM, 1mL), was added and the
1
2
3
(MALDI) m/z calcd for C153H252Eu6N42O54 : 4453.4; found:
solution was purified using HPLC to give the desired complex.
The desired fractions were pooled together and lyophilized to
give Gd6H3L[c(RGDyK)]3 as a white solid. (0.005 g, 0.001
4454.5 ([M + H]+).
Compound Cold Gd6LGa. The Gadolinium complex,
Gd6H3L (0.001 g, 0.225 µmol) was dissolved in a solution of
4ꢀ(2ꢀhydroxyethyl)ꢀ1ꢀpiperazineethanesulfonic acid (HEPES,
pH = 6.5, 1.0 M, 1.0 mL). To the resulting solution was added
a solution of GaCl3 (0.0001 g, 0.567 µmol) in 0.6 N HCl (0.3
mL) and the resulting solution was stirred for 1 h. To the mixꢀ
ture was added 500 ꢁL of 5.0 mM ethylendiaminetetraacetic
acid (EDTA) and the mixture was stirred for another 5 min at
room temperature (EDTA was used to remove nonspecifically
bound or free GaCl3 from the complex). The purification of
the bimetallic complex was carried out by passing the mixture
through a preconditioned SepꢀPak Cꢀ18 heavy cartridge. After
thorough rinsing (3 × 5 mL, water) of the cartridge, the bimeꢀ
tallic complex, Gd6LGa, was eluted by an ethanol water mixꢀ
ture (70:30). The product was purified with HPLC and characꢀ
terized by mass spec. MS (MALDI) m/z calcd for
mmol,
48%).
MS
(MALDI)
m/z
calcd
for
4
5
6
7
8
9
C264H402Gd6N72O90S3 : 7064.4; found: 7085.0 ([M + Na]+).
Compound Gd6L[c(RGDyK)]368Ga. In a 1.5 mL eppendorf
tube containing 80 ꢁg Gd6H3L[c(RGDyK)]3 complex in 1mL
of HEPES (pH = 6.5) solution, was added a solution of 15.0
mCi of 68GaCl3 in 0.6 N HCl. The reaction mixture was incuꢀ
bated at 75 °C for 0.5 h on a shaker. To this solution was addꢀ
ed DTPA (5.0 mM, 5.0 ꢁL) and the reaction mixture was inꢀ
cubated for 5 min at room temperature. The 68Gaꢀlabeled conꢀ
jugate, Gd6L[c(RGDyK)]368Ga, was purified by passing the
mixture through a preconditioned SepꢀPak Cꢀ18 light carꢀ
tridge. After thorough rinsing (3 × 5 mL, water) of the carꢀ
tridge, Gd6L[c(RGDyK)]368Ga was eluted by an ethanolꢀwater
mixture (70:30) to give 9.0 mCi of the labeled compound. The
product was analyzed by radioꢀ HPLC to determine the radioꢀ
chemical purity of the product. The radiochemical purity of
the compound was determined to be higher than 95%.
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C
153H264GaGd6N42O60 : 4555.28; found: 4556.9 ([M + H]+).
Compound Gd6L68Ga. In a 1.5 mL eppendorf tube containꢀ
ing 30 ꢁg Gd6H3L complex in 1.0 mL of HEPES (pH = 6.5)
solution, was added a solution of 8.0 mCi of 68GaCl3 in 0.6 N
HCl. The reaction mixture was incubated at 75 °C for 0.5 h on
a shaker. To this solution was added DTPA (5.0 mM, 5.o ꢁL)
and the reaction mixture was stirred for 5 min at room temperꢀ
ature (DTPA was used to remove nonspecifically bound or
free 68Ga from Gd6L68Ga). The Gd6L68Ga complex was puriꢀ
fied by passing the mixture through a preconditioned SepꢀPak
Cꢀ18 light cartridge. After thorough rinsing (3 × 3 mL, water)
of the cartridge, Gd6L68Ga was eluted by an ethanolꢀwater
mixture (70:30) to give 3.6 mCi of labeled compound. The
product was first analyzed by a Rita Star Radioisotope TLC
Analyzer (Straubenhardt, Germany) on instant thinꢀlayer
chromatography (ITLCꢀSG) plates (Pall Life Sciences, East
Hills, NY) and then by radioꢀ HPLC to determine the radioꢀ
chemical purity of the product. The compound was determined
to have more than 95% purity.
Compound Gd6H3L[PEG12c(RGDyK)]3 The c(RGDyK)
modified ligand H21L[PEG12c(RGDyK)]3 (0.019 g, 0.002
mmol) was dissolved in water (1.0 mL) and the pH was adꢀ
justed to 7.0 with NaOH (0.1 M). To this solution was added
an excess of GdCl3.6H2O and the pH was again adjusted to 6.5
and allowed to stir at room temperature overnight. The pH was
raised above 8.0 using 1.0 N aqueous NaOH, which caused the
excess Gd3+ to precipitate as Gd(OH)3. The solution was filꢀ
tered and the pH was readjusted to 7.0 using 1.0 N HCl. To the
resulting solution, DTPA (0.1 mM, 1.0 mL), was added and
the solution was purified using HPLC to give the desired comꢀ
plex. The desired fractions were pooled together and lyophiꢀ
lized to give Gd6H3L[PEG12c(RGDyK)]3 as a white solid.
(0.006 g, 0.001 mmol, 51%). MS (MALDI) m/z calcd for
C345H561Gd6N75O129S3 : 8864.3; found: 8864.7 ([M + H]+).
Compound Gd6L[PEG12c(RGDyK)]3Ga. The gadolinium
complex, Gd6H3L[PEG12c(RGDyK)]3 (0.001 g, 0.112 µmol)
was dissolved in an HEPES solution (pH = 6.5, 1.0 M, 1.0
mL). To the resulting solution was added a solution of GaCl3
(0.001 g, 0.567 µmol) in 0.6 N HCl (0.3 mL) and the resulting
solution was stirred for 1 h. To the mixture was added 500 ꢁL
of 5.0 mM of EDTA and the mixture, was allowed to incubate
for another 5 min at room temperature (EDTA was used to
remove nonspecifically bound or free GaCl3). The purification
of Gd6L[PEG12c(RGDyK)]3Ga was carried out by passing the
mixture through a preconditioned SepꢀPak Cꢀ18 heavy carꢀ
tridge. After thorough rinsing (3 × 5 mL, water) of the carꢀ
tridge, Gd6L[PEG12c(RGDyK)]3Ga was eluted by an ethanol
water mixture (70:30). The product was characterized by mass
spec. MS (MALDI) m/z calcd for C345H558GaGd6N75O129S3 :
8930.9; found: 8954.8 ([M + Na]+).
Compound Gd6L[PEG12c(RGDyK)]368Ga. For PET/MR
imaging, a slightly different labeling procedure was followed.
To a 1.5 mL eppendorf tube containing 6.0 mg of
Gd6H3L[PEG12c(RGDyK)]3 complex in 1.0 mL of HEPES
(pH = 6.5) solution was added a solution of 300 ꢁCi of
68GaCl3 in 0.6 N HCl. The reaction mixture was incubated at
75 °C for 10 min on a shaker. To this solution was added
DTPA (5.0 mM, 5.0 ꢁL) and the reaction mixture was incuꢀ
bated for 1 min at room temperature. The 68Gaꢀlabeled conjuꢀ
gate was purified by passing the mixture through a precondiꢀ
Compound Gd6L67Ga. In a 1.5 mL eppendorf tube containing
30 ꢁg Gd6H3L complex in 1.0 mL of HEPES (pH = 6.5) soluꢀ
tion, was added a solution of 4.0 mci of 67GaCl3 in 0.6 N HCl.
The reaction mixture was shaken and incubated at 75 °C for
0.5 h. To this solution, was added DTPA (5.0 mM, 5.0 ꢁL)
and the reaction mixture was incubated for 5 min at room
temperature. The Gd6L67Ga complex was purified by passing
the mixture through a preconditioned SepꢀPak Cꢀ18 light carꢀ
tridge. After thorough rinsing (3 × 3 mL, water) of the carꢀ
tridge, Gd6L67Ga was eluted by an ethanolꢀwater mixture
(70:30) to give 2.1 mCi of the labeled compound. The product
was first analyzed by a TLC Analyzer on ITLCꢀSG plates and
then by radioꢀHPLC to determine the radiochemical purity of
the product. The compound was determined to have more than
95% purity.
Compound Gd6H3L[c(RGDyK)]3 The c(RGDyK) modified
ligand H21L[c(RGDyK)]3 (0.010 g, 0.002 mmol) was disꢀ
solved in water (1 mL) and the pH was adjusted to 7 with
NaOH (0.1 M). To this solution was added an excess of
GdCl3.6H2O and the pH was again adjusted to 6.5 and allowed
to stir at room temperature overnight. The pH was raised
above 8.0 using 1.0 N aqueous NaOH, which caused the exꢀ
cess Gd3+ to precipitate as Gd(OH)3. The solution was filtered
and the pH was readjusted to 7.0 using 1.0 N HCl. To the reꢀ
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Bioconjugate Chemistry
Page 8 of 11
tioned SepꢀPak Cꢀ18 plus cartridge. After thorough rinsing (3
Ultra relaxometer (Oxford Instruments, UK). Longitudinal
× 5 mL, water) of the cartridge, Gd6L[PEG12c(RGDyK)]368Ga
was eluted by an ethanolꢀwater mixture (70:30) to give 180ꢀ
200 ꢁCi of the labeled compound. The product was analyzed
by radioꢀ HPLC to determine the radiochemical purity of the
product. The radiochemical purity of the compound was deꢀ
termined to be higher than 95%.
relaxation times were measured by using the inversionꢀ
recovery pulse sequence (180°ꢀtꢀ90°). The T1 relaxivities were
determined by the linear regression analysis of the water proꢀ
ton relaxation rates in solutions ranging in concentration from
0.005 to 12.0 mM, in Millipore water in triplicates.
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MRI imaging and relaxivity measurements at 1.0 T. The
T1ꢀweighted MR images of samples in 0.5 mL microfuge
tubes were collected using Aspect Imaging M2TM 1.0 T
(43.5ꢀ45 MHz, 60 mm diameter volume coil) Gradient Echo
Spoiled External Averaging (GREꢀEXT) sequence. For imagꢀ
ing, the following parameters were used: TR = 14.2 ms; effecꢀ
tive echo time (TE) = 2.8 ms; FOV 64 × 100 mm2, data matrix
= 256 × 256, averaging = 4, slice = 1 mm. The T1ꢀmaps of the
samples at 1.0 T were determined from a series of multiꢀT1
(0.01 ms to 5 s) Inversion Recovery Spin Echo sequence (IRꢀ
SE with minimum TE = 7.0 ms); TR= 10 s; FOV= 64 × 100
mm2; matrix = 128 × 128; averaging = 1, steady state scans =
10), fitted and calculated using Matlab code. All the fits for T1
values used to calculate the longitudinal relaxivity, r1, had
fitting coefficients, R2 ≥ 0.99. Three trials were performed.
The Gd and Ga metal concentrations of samples were deterꢀ
mined by inductively coupled plasma mass spectrometry (ICPꢀ
MS)
PET/MR Imaging using Gd6L[PEG12c(RGDyK)]368Ga. The
MR and PET imaging studies were performed on a 1.0 T MR
scanner (Aspect Imaging, Shoham, Israel) using a 35 mm
mouse body coil, and a Siemens Inveon Multimodality
PET/CT system (Siemens Medical Solutions Inc., Knoxville,
TN, USA), respectively. Ten minutes prior to imaging, the
animals were anesthetized using 3% isofluorane at room temꢀ
perature until stable vitals were established. Once the animals
were sedated, they were placed onto a custom made interꢀ
changeable bed between the MR and PET scanners under 2%
isofluorane anesthesia for the duration of the imaging. A T1ꢀ
weighted gradient echo, spoiled sequence (echo
time/repetition time [TE/TR] 3/15 msec; field of view [FOV]
80 × 100 mm; slice thickness 0.8 mm; matrix 256 × 256;
number of excitations [NEX]ꢂ=ꢂ3; flip angleꢂ=ꢂ25°) was perꢀ
formed for each mouse. Each mouse was injected with 100 –
150 µCi (Gd: ca. 0.027 mmol/kg) via the tailꢀvein. MR scans
were obtained preꢀcontrast and 1 h after contrast medium inꢀ
jection.
Biodistribution and pharmacokinetics studies of Gd6L67Ga.
Male BALB/C mice were injected with 300 µCi of Gd6L67Ga
complex to evaluate the tissue distribution of the tracer in
mice. Mice were sacrificed 1 h, 4 h, and 24 h post injection
(p.i.). The organs of interest (blood, heart, lung, liver, spleen,
kidney, stomach, muscle, fat, small intestine, large intestine,
and brain) were harvested, weighed, and radioactivity was
quantified using a γꢀcounter. Standards were prepared and
counted along with the tissue samples to calculate the percentꢀ
ageꢀinjected dose per gram (%ID/g). To determine the pharꢀ
macokinetic parameters, mice injected with the tracer were
blood sampled from the retroꢀorbital sinus at 2 min, 5 min, 10
min, 30 min, 1 h, 2 h, 24 h, and 48 h p.i. and quantified using a
γꢀcounter. The pharmacokinetic parameters were calculated
based on a twoꢀcompartment open model.
In vitro and ex vivo stability of Gd6L67Ga. The in vitro staꢀ
bility test was performed in rat serum. Briefly, Gd6L67Ga
complex (100 ꢁCi, 5 ꢁL) was added into 100 ꢁL of rat serum
(n = 3). The solution was incubated for 1 h, 4 h 24 h and 48 h
at 37 °C, respectively. The solution was vortexed and centriꢀ
fuged for 5 min at the speed of 21,000 g. The supernatant was
then analyzed by HPLC. For the in vivo stability evaluation,
each male mouse was injected with 600 ꢁCi of Gd6L67Ga
complex in 100 ꢁL of saline via the tail vein. The urine samꢀ
ples were collected at 1h, 4 h 24 h and 48 h p.i. and then anaꢀ
lyzed by HPLC.
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Integrin αvβ3 receptorꢀbinding assay. The αvβ3 integrinꢀ
binding affinities of c(RGDyK), Gd6H3L[c(RGDyK)]3 and
Gd6H3L[PEG12c(RGDyK)]3 were determined by a competitive
cellꢀbinding assay using 125Iꢀechistatin (PerkinElmer) as the
αvβ3ꢀspecific radioligand. The experiments were performed on
U87MG human glioblastoma cells following a previously
reported method.52 Briefly, U87MG cells were grown in RPMI
1640 medium supplemented with penicillin, streptomycin, and
10% (v/v) fetal bovine serum (FBS) at 37 °C under 5% CO2.
Suspended U87MG cells in the binding buffer (20 mM Tris,
pH 7.4, 150 mM NaCl, 2.0 mM CaCl2, 1.0 mM MgCl2, 1.0
mM MnCl2, 0.1% bovine serum albumin) were seeded on mulꢀ
tiwell DV plates (Millipore) with 5 × 104 cells per well and
then incubated with 125Iꢀechistatin (10,000 cpm/well) in the
presence of increasing concentrations (0–5000 nM) of each
c(RGDyK) peptide conjugate for 2 h. The final volume in each
well was maintained at 200 ꢁL. At the end of incubation, unꢀ
bound 125Iꢀechistatin was removed by filtration followed by
three rinses with cold binding buffer. The retentate was colꢀ
lected and the radioactivity was measured using a γꢀcounter.
The bestꢀfit IC50 values (inhibitory concentration where 50%
of the 125Iꢀechistatin bound on U87MG cells are displaced) of
PET imaging was acquired with the mouse mounted on the
interchangeable bed directly following the MR acquisition. A
15 min PET static scan was performed and reconstructed using
Fourier Rebinning and Ordered Subsets Expectation Maximiꢀ
zation 3D (OSEM3D) algorithm. Reconstructed MR and PET
images were fused and analyzed using the VivoQuant software
(Invicro LLC, Boston, USA). For quantification, regions of
interest were placed on the tumor and muscle. The latter was
used for normalization. The resulting quantitative data were
expressed in intensity and %ID/g for MR and PET images.
c(RGDyK),
Gd6H3L[c(RGDyK)]3
and
ASSOCIATED CONTENT
Gd6H3L[PEG12c(RGDyK)]3 were calculated by fitting the data
with nonlinear regression using GraphPad Prism (GraphPadꢀ
Software, Inc.). Experiments were repeated with quintuplicate
samples.
Supporting Information. Supporting schemes, synthesis of comꢀ
pounds, ICP quantification are available free of charge via the
Internet at http://pubs.acs.org.”
Relaxivity measurements at 0.5 Tesla (T). The T1 values
were recorded at 23 MHz (0.5 T) at 25 °C by using a Maran
AUTHOR INFORMATION
ACS Paragon Plus Environment

Page 9 of 11
Bioconjugate Chemistry
Corresponding Author
*Correspondence to Xiankai Sun, Ph.D., Department of Radioloꢀ
gy and Advanced Imaging Research Center, University of Texas
Southwestern Medical Center at Dallas, 2201 Inwood Road, Texꢀ
as 75390ꢀ8542, USA;
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Eꢀmail: Xiankai.Sun@UTSouthwestern.edu.
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Author Contributions
The manuscript was written through contributions of all authors.
All authors have given approval to the final version of the manuꢀ
script.
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Funding Sources
This work was partially supported by the Prostate Cancer Reꢀ
search Program of the United States Army Medical Research and
Materiel Command (W81XWHꢀ12ꢀ1ꢀ0336) and the Dr. Jack
Krohmer Professorship Funds.
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Bioconjugate Chemistry
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