Synthesis of 64CuII- bis(dithiocarbamatebisphosphonate) and its conjugation with superparamagnetic iron oxide nanoparticles: In vivo evaluation as dual-modality PET-MRI agent

Article abstract of DOI:10.1002/anie.201007894

A novel bifunctional chelator combines a dithiocarbamate group for binding the positron-emitter ⁶⁴Cu (red spheres) for PET imaging and a bisphosphonate group (green ellipsoids) for strong binding to several inorganic materials, such as MRI cont

Full text of DOI:10.1002/anie.201007894

DOI: 10.1002/anie.201007894
Imaging Technology
6
4
II
Synthesis of Cu –Bis(dithiocarbamatebisphosphonate) and Its
Conjugation with Superparamagnetic Iron Oxide Nanoparticles:
In Vivo Evaluation as Dual-Modality PET–MRI Agent**
Rafael Torres Martin de Rosales,* Richard Tavarꢀ, Rowena L. Paul, Maite Jauregui-Osoro,
Andrea Protti, Arnaud Glaria, Gopal Varma, Istvan Szanda, and Philip J. Blower*
The synergistic combination of positron emission tomography
PET) and magnetic resonance imaging (MRI) is likely to
them with colloidal stability. The polymeric coatings are
typically bound relatively weakly to the surface of the SPIOs,
(
[
4]
become the next generation of dual-modality scanners in
medical imaging. These instruments will provide us with
accurate diagnoses thanks to the sensitive and quantifiable
signal of PET and the high soft-tissue resolution of MRI.
Furthermore, patients will receive less radiation dose and
spend less time in the procedure relative to current dual-
modality scanners (e.g. PET–computed tomography (CT)).
As a consequence, there has been increasing interest recently
which results in a lack of stability over time. One solution to
this problem is to cross-link the polymer units at the surface of
[
5]
the nanoparticles. However, there are concerns for the
translatability of these compounds due to toxic chemicals
[
4b]
used in the synthesis.
An alternative to radiolabeling the coatings of SPIO
particles is to label their inorganic surface directly with a
molecule that binds to both a PET isotope and the nano-
particle, leaving the polymeric coating unaffected. In this
regard, we have recently reported that bisphosphonates (BPs;
[
1]
in the development of dual-modality PET–MRI agents.
The majority of the PET–MRI agents reported to date are
based on the combination of PET isotopes with superpar-
9
9m
Figure 1) radiolabeled with a suitable isotope ( Tc) for
[
2]
amagnetic iron oxide (SPIO) nanoparticles. These magnetic
nanoparticles are ideal for the purpose, having a proven
record of biocompatibility and a track record of extensive use
in the clinic as MRI contrast agents for imaging the
[
3]
reticuloendothelial and lymphatic systems. The radiolabel-
ing of SPIOs has been done to date by often complicated
chemical conjugation with their coatings, which are com-
monly biocompatible polymers such as dextran that provide
[
*] Dr. R. Torres Martin de Rosales, Dr. R. Tavarꢀ, Dr. R. L. Paul,
Dr. M. Jauregui-Osoro, Dr. A. Protti, Dr. A. Glaria, Dr. G. Varma,
I. Szanda, Prof. P. J. Blower
Division of Imaging Sciences & Biomedical Engineering
King’s College London
4th Floor, Lambeth Wing, St. Thomas’ Hospital, London SE1 7EH
(
UK)
Figure 1. Schematic representations of a bisphosphonate (BP; top)
and the conjugation reaction between the BP-based PET tracer [ Cu-
E-mail: rafael.torres@kcl.ac.uk
philip.blower@kcl.ac.uk
Homepage: http://www.kcl.ac.uk/schools/medicine/research/
imaging/
64
(
dtcbp) ] and the dextran-coated iron oxide nanoparticle MRI probe
2
Endorem/Feridex (bottom).
[
**] This work was funded by the KCL Centre of Excellence in Medical
Engineering funded by the Wellcome Trust and the EPSRC under
grant WT 088641/Z/09/Z. The authors are also grateful for support
from the KCL-UCL Comprehensive Cancer Imaging Centre, funded
by CRUK and APSRC, in association with the MRC and DoH
single photon emission computed tomography (SPECT) bind
strongly to SPIO nanoparticles such as the dextran-coated
MRI contrast agent Endorem/Feridex, and that the binding is
[
6]
(
England), Guy’s and St. Thomas’ Charity, the Department of
stable in vitro and in vivo. BPs hence have remarkable
potential in the development of radionuclide-based SPIO
imaging agents for dual-modality studies. Herein, we report
the synthesis and characterization of a novel bifunctional BP
conjugate that has been designed to bind to both SPIO
Health’s NIHR Biomedical Research Centres funding scheme, an
EPSRC Fellowship (to M.J.O.), and the Wellcome Trust for an
equipment grant supporting the purchase of the PET-CT scanner.
We thank D. Thakor and K. Sunassee for technical support and K.
Shaw for Cu production. PET=positron emission tomography,
MRI=magnetic resonance imaging.
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64
nanoparticles for MR imaging and Cu for PET imaging. Cu
is an isotope that is gaining attention for its favorable
properties (half-life 12.7 h, 18% b , 39% b , 43% electron
capture) for PETand radionuclide therapy. To bind Cu, we
introduce the use of dithiocarbamate (dtc) as chelating group.
The dtc group is a well-known ligand in coordination
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201007894.
+
À
[
7]
64
Re-use of this article is permitted in accordance with the Terms and
Conditions set out at http://onlinelibrary.wiley.com/journal/
1
0.1002/(ISSN) 1433–7851/homepage/2002_onlineopen.html
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[
8]
chemistry that binds to all transition metals, including
BP (4) was then treated with CS2 to form the desired
bifunctional chelator (dtcbp; Scheme 1).
6
4
copper, but its use as a Cu chelator for PET imaging has
[9]
64
been neglected. The compound formed, [ Cu(dtcbp) ]
The ligand dtcbp has been designed to bind Cu ions
through the dtc group to leave two BP groups free for binding
to the surface of an iron oxide nanoparticle. A major concern
in the design of dtcbp and other bifunctional metal chelators
2
(
Scheme 1, Figure 1), has great affinity for iron oxide nano-
particles and other inorganic materials such as hydroxyapatite
II
was whether Cu ions could coordinate to both the dtc and BP
groups. Indeed, BPs have been reported to be good ligands for
[
10]
Cu.
Spectroscopic studies demonstrate, however, that
dtcbp preferentially binds copper ions through its dtc group,
and not the BP group. First, ESIMS studies of a solution of
[
Cu(dtcbp) ] demonstrate the presence and stoichiometry of
2
2
À
the desired complex (ions observed: [MÀ2H] ,
2
À
2À
[
MÀ3H+Na] and [MÀ4H+2Na] ) (see the Supporting
II
Information). Second, titration of Cu ions into a solution of
dtcbp results in the appearance of an absorption band in the
UV/Vis spectrum with l = 440 nm, characteristic of square-
max
II
[8]
planar Cu –bis(dithiocarbamate) complexes (Figure 2). The
II
Scheme 1. Synthesis of dtcbp and [Cu(dtcbp) ]. Reagents and condi-
Figure 2. A) UV/Vis titration of dtcbp upon the addition of Cu ions
2
tions: a) Na CO (10 equiv), CH CN, 708C for 70 h; b) H , 10% Pd/C,
(0–0.5 equiv) showing the increase in absorbance of the band at
2
3
3
2
EtOH, 48 h (R=Me, Et); c) 5m HCl, reflux for 16 h; d) 1. Phosphorous
acid (1.5 equiv), PCl (3.4 equiv), sulfolane, 678C for 3 h; 2. H O,
l=440 nm due to the formation of [Cu(dtcbp) ]. B) Plot of absorbance
2
II
3
2
at l=440 nm against [Cu ]/[dtcbp] ratio. The absorbance increases
II
1
008C for 1 h; e) CS (19 equiv), NaOH (7 equiv), THF, 24 h;
until the [Cu ]/[dtcbp] ratio is 0.5, confirming the expected stoichiom-
2
f) 0.5 equiv Cu(OAc) , H O.
etry.
2
2
II
(
(
HA) and rare-earth metal oxides such as gadolinium oxide
Gd O ). Furthermore, we demonstrate that conjugation with
intensity of this band increases until 0.5 equivalents of Cu
ions are present, which is consistent with the formation of the
2
3
clinically approved SPIOs gives nanoparticles that can be
used for in vivo PET–MR lymphatic imaging (Figure 1).
Initial attempts to insert a dtc group into a BP were made
by reaction of carbon disulfide with the amino group of
alendronate, a primary amino-BP. The dtc–BP conjugate
compound was formed and isolated in low yield, but lacked
stability, readily decomposing at pH ꢀ 7 to release the starting
materials. Dithiocarbamates derived from primary amines are
known to be unstable under acid conditions. Therefore, we
adopted the strategy of using a secondary amine instead.
Monomethylation of amino-BPs was unfeasible because of
desired complex [Cu(dtcbp) ]. Furthermore, the data fit well
to a 2:1 ligand/metal binding isotherm, which gives a value for
2
2
log K of ꢁ 10.1 (K = [ML ]/[M][L] ). The lack of involvement
2
of the BP in copper binding is demonstrated by IR spectros-
copy. Characteristic BP bands are observed in dtcbp at 954
À1
and 999 cm attributable to symmetrical and asymmetrical
À1
[11]
PÀO vibrations, and at 1078 cm for n(P=O) vibrations.
The frequency of these vibrations remains unchanged after
copper binding. n(CS) vibrations usually found at around
À1
1000 cm , which often provide information about the den-
ticity of the DTC ligand, were not observed because of
overlap with the intense BP bands. Copper binding, however,
the insolubility in organic solvents and high pK (ca. 12) of the
a
À1
amino groups of amino-BPs. A different synthetic strategy
elicits a strong band at 1335 cm owing to n(N-CSS),
(
Scheme 1) was chosen by combining a carboxylic acid for the
suggesting a high degree of single bond character after
metal complexation, as previously seen for other transition-
metal–bis(DTC) complexes.
formation of a BP and a methylated secondary amine
separated by an ethylene spacer (3). The N-methylamino-
[
12]
5
510
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Radiolabeling of dtcbp with Cu to form [ Cu(dtcbp) ]
spot becomes light green after staining the TLC plate with
Dittmer–Lesterꢀs reagent, indicating the presence of phos-
phorus (Figure 3B (3)). Free, nonradioactive Cu, on the other
2
6
4
was achieved by mixing an aliquot of a solution of Cu(OAc)
2
in water with an aqueous solution of dtcbp in carbonate buffer
at pH 9. As with the cold complex, the complexation proceeds
instantaneously and quantitatively, and no heating is
required. However, the high affinity and stability of the BP
group to several inorganic materials (see below) made
6
4
hand, migrates with R = 0.66, as found for Cu (Figure 3B
F
(4)).
6
4
The stability of [ Cu(dtcbp) ] was confirmed in phos-
2
phate-buffered saline (PBS) and human serum for at least
48 h. Incubation at 378C in these media showed no decom-
position during this time using the TLC method described
above. Furthermore, the complex does not decompose under
the TLC conditions used (up to 50 mm EDTA), thus
demonstrating high inertness towards ligand substitution.
Subjecting the complex to more challenging conditions such
as incubation in 3 mm EDTA solution at pH 4 results in
6
4
characterization particularly troublesome. Indeed, [ Cu-
dtcbp) ] irreversibly binds to most chromatographic materi-
(
2
als such as silica, silica-based reverse-phase (RP) (C18 and
C8), polymer-based RP, and Al O stationary phases when
2
3
using common HPLC and TLC solvents, including ion-pairing
conditions. Ion-exchange stationary phases also resulted in
irreversible binding of the compound. Finally, radiolabeling
yields were calculated using silica gel TLC with 15–50 mm
6
4
partial decomposition only after 5 h. To determine if [ Cu-
ethylenediaminetetraacetic acid (EDTA) in 10% NH OAc/
(dtcbp) ] binds to serum proteins, these were precipitated by
4
2
6
4
MeOH (50/50) as the mobile phase. Using this system, “free”
addition of ethanol. Thus, in serum, [ Cu(dtcbp) ] appears to
2
6
4
64
Cu moves with a R = 0.66 (15 mm EDTA), whereas [ Cu-
bind completely to proteins. However, the binding was
reversed if an insoluble material with known affinity towards
F
(
dtcbp) ] has a value of R = 0.04 (Figure 3A). Very efficient
2
F
6
4
BPs, such as HA, was added to the serum–[ Cu(dtcbp) ]
2
mixture at various time points within 48 h. This resulted in
6
4
complete binding of [ Cu(dtcbp) ] to HA, suggesting that the
2
binding to serum proteins is weak and that the complex is
inert to transchelation by copper-binding biomolecules pres-
ent in human serum.
BPs are well-known strong binders of several inorganic
materials, including calcium salts such as HA, and metal
[
13]
oxides such as TiO , ZrO , SiO , and Fe O . Indeed, we
2
2
2
3
4
6
4
tested the binding of [ Cu(dtcbp) ] to several of these salts
2
showing high binding (ꢁ 97%) to HA, Fe O , and calcium
3
4
4
6
carbonate (CC; Figure 4). Interestingly, [ Cu(dtcbp) ] also
2
6
4
Figure 3. A) Radio-TLC chromatograms of free Cu (R =0.66, top)
F
6
4
and [ Cu(dtcbp) ] (R =0.04, bottom). Vertical lines represent R
F
2
F
values 0 (left) and 1 (right); B) Pictures of TLC plates showing:
) [Cu(dtcbp) ] at R =0.04 under white light; 2) the same TLC plate
1
2
F
under UV light (l=254 nm); 3) the same plate under white light after
being stained with Dittmer–Lester’s reagent; 4) free Cu under white
light, after being stained with a concentrated solution of diethyl
dithiocarbamate. All TLC plates were made of silica gel and developed
with 15 mm EDTA in 10% NH OAc/MeOH (50:50).
4
6
4
Figure 4. In vitro binding study of [ Cu(dtcbp) ] in 50 mm tris(hydroxy-
2
methyl)aminomethane pH 7 at room temperature to various inor-
À1
labeling (10 GBq/mg, radiochemical yield = 100%) was
found when dtcbp concentrations of ꢁ 0.15 mm were used.
ganic materials (1 mgmL ) after 1 h incubation. Abbreviations:
hydroxyapatite (HA); calcium carbonate (CC); calcium phosphate
(CP); b-tricalcium phosphate (b-CP); calcium pyrophosphate (CPy),
and calcium oxalate (CO).
6
4
To prove the chemical identity of [ Cu(dtcbp) ], the non-
2
radioactive compound was analyzed using the same TLC
method. Thus, [Cu(dtcbp) ] stays at the baseline of the TLC
2
plate, which is in agreement with its radioactive analogue
(
Figure 3B, images 1, 2, and 3). [Cu(dtcbp) ] can be seen by
binds to rare-earth metal oxides of the type M O (M = Gd,
2
2
3
visible light owing to its absorbance at l = 440 nm (Figure 3B
Er, Eu, Yb). It is also worth noting that the presence of two
II
64
(
1)), which is characteristic of Cu –bis(dithiocarbamate)
BP moieties in [ Cu(dtcbp) ] increases the binding capabil-
2
ligand to metal charge transfer (LMCT) transitions, and it is
UV-active at l = 254 nm (Figure 3B (2)). Furthermore, the
ities to these materials when compared to mono-BP com-
pounds, which seem to be selective for HA among the calcium
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Communications
[
14]
64
salts.
[ Cu(dtcbp) ], however, binds well to HA, CC,
2
calcium phosphate (CP), and b-tricalcium phosphate (b-CP).
6
4
The affinity of [ Cu(dtcbp) ] towards Fe O allows us to
2
3
4
demonstrate the potential of this compound for the synthesis
of dual-modality PET–MR imaging agents. Labeling of
clinically available SPIO nanoparticles (Endorem/Feridex)
6
4
with [ Cu(dtcbp) ] was performed as follows: Endorem
2
6
4
solution (15 mL) was added to a solution of [ Cu(dtcbp) ],
2
and the mixture was heated at 1008C for 15 min. This step is
necessary to achieve maximum radiochemical yields (pre-
sumably by assisting the BP groups to permeate the loosely
bound dextran coating and bind to the iron oxide surface of
the nanoparticles). Limiting the heating period to 15 min
64
Figure 5. In vivo PET–MR imaging studies with [ Cu(dtcbp) ]–
2
[
6]
maintains the colloidal stability of the solution. The
Endorem in a mouse. A,B) Coronal (top) and short axis (bottom) MR
images of the lower abdominal area and upper hind legs showing the
popliteal lymph nodes (solid arrows) before (A) and after (B) footpad
injection of [ Cu(dtcbp) ]–Endorem. C) Coronal (top) and short-axis
(bottom) NanoPET–CT images of the same mouse as in (B) showing
the uptake of [ Cu(dtcbp) ]–Endorem in the popliteal (solid arrow)
and iliac lymph nodes (hollow arrow). D) Whole-body NanoPET–CT
images showing sole uptake of [ Cu(dtcbp) ]–Endorem in the popliteal
and iliac lymph nodes. No translocation of radioactivity to other
tissues was detected.
radiolabeled nanoparticles (hydrodynamic size = (108 Æ
6
0) nm) were then purified by centrifugal filtration of the
6
4
colloidal solution with a 10 kDa molecular weight cut-off
2
6
4
(
(
(
MWCO) membrane, which removes unbound [ Cu-
6
4
2
dtcbp) ]. Radiolabeling yields of 95% were obtained
100% radiochemical purity). The stability of the nano-
particle–BP interaction was studied for [ Cu(dtcbp) ]–
2
64
2
6
4
2
Endorem in PBS and human serum by separating the
nanoparticles from the media using centrifugation and
6
4
1
00 kDa MWCO filters at several time points. [ Cu(dtcbp) ]
2
remained bound quantitatively to the magnetic nanoparticles
in both media at 378C for at least 48 h. We also studied the
other inorganic materials, such as HA and rare-earth oxides.
6
4
64
II
stability of [ Cu(dtcbp) ]–Endorem in high concentrations of
The ligand dtcbp binds Cu efficiently to form Cu –bis(di-
thiocarbamatebisphosphonate), and the complex is stable
2
6
4
EDTA (10 mm) at pH 4, showing that Cu remains associated
6
4
with Endorem for at least 24 h, which is in contrast to
in vitro for at least 2 days. [ Cu(dtcbp) ] is not as thermody-
2
6
4
[
Cu(dtcbp) ], for which extensive decomposition is evident
namically stable or kinetically inert under highly acidic
conditions or in the presence of high concentrations of
2
within 5 h. Thus, it seems that conjugation to the nano-
particles or the protective effect of the dextran polymer
coating prevents transchelation in vitro.
EDTA as complexes derived from macrocyclic ligands (t <
=
2
1
5 min in 5m HCl at 908C compared to 154 h for Cu-CB-
6
4
[7,16]
In vivo PET–MR imaging studies with [ Cu(dtcbp) ]–
TE2A),
but is sufficiently inert to metal transchelation
2
Endorem were carried out sequentially in a 9.4 T NMR
magnet and a NanoPET–CT scanner (Figure 5). The lym-
phatic system was chosen as in vivo model because of the
clinical need for accurate quantification of lymph node
uptake using imaging, especially in oncologic studies in
while retaining the advantage of fast metal-binding kinetics at
room temperature. This is an important factor when radio-
6
4
labeling temperature-sensitive compounds. [ Cu(dtcbp) ]
2
binds several inorganic materials with high affinity, including
Endorem/Feridex and several rare-earth metal oxides that
have promising MR contrast properties, such as Gd O , or
9
9m
which the uptake of SPIO nanoparticles and
Tc colloids
2
3
in sentinel lymph nodes have been shown to provide a
luminescent properties, such as Eu O . The PET–MR dual-
2
3
64
[15]
measure of cancer spread. First, T *-weighted MR images
modality imaging capabilities of [ Cu(dtcbp) ]–Endorem
2
2
of the lower abdominal area and legs of an anaesthetized
C57BL/6 mouse were obtained, and the popliteal lymph
nodes were located (Figure 5A, solid arrows). The mouse was
then injected in the footpads with 2 MBq (20 mL, 44 mg Fe)
were demonstrated in vivo by showing that it accumulates
6
4
in draining lymph nodes. [ Cu(dtcbp) ]–Endorem should
2
allow easy and accurate quantification of its uptake in vivo
using the PET–MR instrumentation currently in develop-
6
4
[17]
[
Cu(dtcbp) ]–Endorem. After 3 h, the animal was imaged
ment. Radiolabeling of iron or rare-earth oxide materials
2
6
4
again using the same parameters and showed significant
decrease in signal and hence accumulation of Endorem in the
popliteal lymph nodes (Figure 5B). The mouse was then
transferred to the NanoPET–CT scanner and an image
acquired. Uptake in the popliteal lymph nodes and, to a
lesser extent, in the iliac lymph nodes was observed (Fig-
with [ Cu(dtcbp) ] and other BP-based radiotracers in
2
combination with BP-targeting/stability molecules could be
used as a clean and simple method to synthesize targeted
PET–MR or PET–optical-imaging agents.
Received: December 14, 2010
Revised: February 17, 2011
Published online: May 4, 2011
6
4
ure 5C,D), confirming co-location of Cu and Endorem in
draining lymph nodes.
In summary, we have described the design, synthesis, and
characterization of dtcbp, a novel bifunctional chelator
containing a dithiocarbamate group for binding the PET
Keywords: chelates · copper · imaging agents · nanoparticles ·
rare earths
.
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isotope Cu, and a BP group for strong binding to Fe O and
3
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