A Cyanine-Bridged Somatostatin Hybrid Probe for Multimodal SSTR2 Imaging in Vitro and in Vivo: Synthesis and Evaluation

Article abstract of DOI:10.1002/cbic.202000791

Multimodal imaging probes have attracted the interest of ongoing research, for example, for the surgical removal of tumors. Modular synthesis approaches allow the construction of hybrid probes consisting of a radiotracer, a fluorophore and a targeting unit. We present the synthesis of a new asymmetric bifunctional cyanine dye that can be used as a structural and functional linker for the construction of such hybrid probes. ⁶⁸Ga-DOTATATE, a well-characterized radiopeptide targeting the overexpressed somatostatin receptor subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our cyanine dye, thus providing additional information along with the data obtained from the radiotracer. We tested the SSTR2-targeting and imaging properties of the resulting probe ⁶⁸Ga-DOTA-ICC-TATE in vitro and in a tumor xenograft mouse model. Despite the close proximity between dye and pharmacophore, we observed a high binding affinity towards SSTR2 as well as elevated uptake in SSTR2-overexpressing tumors in the positron emission tomography (PET) scan and histological examination.

Full text of DOI:10.1002/cbic.202000791

Combining Chemistry and Biology
Accepted Article
Title: Synthesis and Evaluation of a Cyanine-Bridged Somatostatin
Hybrid Probe for Multimodal SSTR2-Imaging In Vitro and In Vivo
Authors: Isabelle Heing-Becker, Carsten Grötzinger, Nicola Beindorff,
Sonal Prasad, Sarah Erdmann, Samantha Exner, Rainer
Haag, and Kai Licha
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.202000791
Link to VoR: https://doi.org/10.1002/cbic.202000791
01/2020

10.1002/cbic.202000791
ChemBioChem
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Synthesis and Evaluation of a Cyanine-Bridged Somatostatin
Hybrid Probe for Multimodal SSTR2-Imaging In Vitro and In Vivo
Isabelle Heing-Becker,[a] Carsten Grötzinger,[b] Nicola Beindorff,[c] Sonal Prasad,[c] Sarah Erdmann,[b]
Samantha Exner,[b] Rainer Haag[a] and Kai Licha*[a]
Abstract: Multimodal imaging probes have attracted the interest of
ongoing research, e.g. for surgical removal of tumors. Modular
synthesis approaches allow for the construction of hybrid probes
consisting of a radiotracer, fluorophore and targeting unit. We present
the synthesis of a new asymmetric bifunctional cyanine dye, which
can be used as a structural and functional linker for the construction
imaging, which enables the resection of affected areas.[4] Beyond
intraoperative imaging, hybrid probes are also interesting for the
evaluation of new drug candidates because the same label can
be used for subsequent in vitro, in vivo and ex vivo studies. In this
way, difficulties in data interpretation resulting from different
labels with different biological behavior are circumvented.
of such hybrid probes. 68Ga-DOTATATE,
a
well-characterized
For these purposes, labels consisting of a fluorophore being either
attached to a
chelator for radionuclide complexation[5] or
radiopeptide targeting the overexpressed somatostatin receptor
subtype 2 (SSTR2) in neuroendocrine tumors, was labeled with our
cyanine dye, thus providing additional information along with the data
obtained from the radiotracer. We tested the SSTR2-targeting and
imaging properties of the resulting probe 68Ga-DOTA-ICC-TATE in
vitro and in a tumor xenograft mouse model. Despite the close
proximity between dye and pharmacophore, we observed a high
binding affinity towards SSTR2 as well as elevated uptake in SSTR2-
overexpressing tumors in the positron emission tomography (PET)
scan and histological examination.
subjected to 18F-fluorination[6] have been developed and
conjugated to specific targeting molecules such as small
molecules,[7] peptides,[8] antibodies[9] or polymers.[10] When
building up hybrid probes, the question arises of how to position
the three components radiotracer, fluorophore and targeting unit.
This aspect is mainly dictated by the availability of free
conjugatable sites of the components. The vast majority of hybrid
probes either contain an extra linking platform (based e.g. on
lysine[11] or cyclooctyne[12]) or use the radionuclide chelator as the
linker between fluorophore and targeting unit.[3]
In our study, we present a new design approach for the
construction of multimodal imaging probes and employ the
fluorophore as the linking unit between chelator and targeting
moiety. Among the different classes of fluorescent dyes, cyanine
dyes possess a high structural modifiability that can be used to
introduce various functional groups.[13] We exploited this aspect
Introduction
Multimodal imaging using PET or SPECT (single photon emission
computed tomography) together with fluorescence imaging has
emerged as a promising tool for the development of new
diagnostics and treatment options.[1] Instead of using different
imaging agents for different imaging modalities in various
experimental setups, only one probe is used for all imaging
procedures. Obvious advantages for preclinical and clinical
applications include the complementary data obtained from both
modalities having different sensitivities, resolutions and
penetration depths along with a reduction of time, costs and
resources needed.[2]
to design and synthesize
a new asymmetric bifunctional
indocarbocyanine (ICC) dye which can serve as a linker between
two heterofunctional groups.
To exemplify the usage of this new ICC dye, we decided to focus
on the radiopeptide 68Ga-DOTATATE that has attracted particular
interest for the construction of multimodal imaging probes.[3,14] As
somatostatin analogs, the peptides Phe1-Tyr3-octreotate (TATE)
and the closely related Phe1-Tyr3-octreotide (TOC) target the
somatostatin receptor (SSTR) subtype 2, which can be
overexpressed in neuroendocrine tumors (NETs).[15] Their
radiolabeled forms 68Ga-DOTATATE and 68Ga-DOTATOC are
the workhorses in the diagnosis of NETs and represent well-
characterized, clinically used radiodiagnostics for PET imaging of
SSTR2.[16] In these compounds, the DOTA chelator is connected
via the N-terminal amine of the peptide, leaving only the remaining
functional groups of the peptide for the attachment of a
fluorophore. However, Edwards et al. found that further
functionalization of the Lys-residue in TATE with a cyanine dye
A major application of hybrid probes lies in the translation into
image-guided oncologic surgery.[3] The different modalities help
the surgeon to first localize the tumor via the PET or SPECT scan,
followed by the identification of tumor margins via fluorescence
[a]
I. Heing-Becker, Prof. Dr. R. Haag, Dr. Kai Licha
Institut für Chemie und Biochemie
Freie Universität Berlin
Takustr. 3
led to
a poor in vivo tumor uptake and indicates that
14195 Berlin (Germany)
E-mail: kai.licha@fu-berlin.de
Dr. C. Grötzinger, Dr. S. Erdmann, Dr. S. Exner
Department of Hepatology and Gastroenterology
Charité – Universitätsmedizin Berlin
Augustenburger Platz 1
functionalization of certain residues in TATE can significantly
diminish the peptide functionality towards SSTR2.[17] In other
approaches for multimodal SSTR2-imaging, Ghosh et al. used a
chelator with two binding sites as the linker for a cyanine dye and
the peptide TOC as well as a linking platform to connect all three
components.[18] In this case, the dye was intentionally positioned
far away from the peptide to minimize interactions.
[b]
[c]
13353 Berlin
Dr. N. Beindorff‡, Dr. S. Prasad‡°
‡BERIC - Berlin Experimental Radionuclide Imaging Center
°Department of Nuclear Medicine
Charité - Universitätsmedizin Berlin
Augustenburger Platz 1
13353 Berlin
1
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Scheme 1. Different design approaches (a-c) for SSTR2-targeting hybrid probes using either the peptide, the chelator or a platform as the linking structure. d) In
this study, the dye is used as the linker. e) Synthesis of the new asymmetric bifunctional indocarbocyanine dye 2 and DOTA-ICC label 3 as well as the chemical
structure of the 68Ga-DOTA-ICC-TATE probe 4 with the cyanine dye bridging the DOTA-chelator and the peptide TATE.
Results and Discussion
Our study was conceived to investigate whether SSTR2-targeted
imaging is possible when using our bifunctional cyanine dye as
the linker for the DOTA chelator and the targeting peptide TATE,
with the dye being placed directly next to the pharmacophore. The
resulting Ga-DOTA-ICC-TATE probe was examined in cell
experiments and in an SSTR2-overexpressing tumor mouse
model to evaluate its targeting and imaging properties on the in
vitro, in vivo and ex vivo level.
Synthesis
Different strategies for the molecular design of somatostatin-
based hybrid probes have been developed (Scheme 1A-C) using
the targeting peptide, the chelator or
a
dedicated
2
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Figure 1. In vitro assessment of the hybrid probe. (A) Cellular uptake studies with RIN1038 insulinoma cells overexpressing GFP-fused SSTR2 at their surface
(green). The cells were incubated in medium alone (left) or medium plus 1 µM DOTA-ICC-TATE (red, right). (B) Concentration-dependent displacement of the
radioligand Tyr11-SST14 by DOTA-ICC-TATE and natGa-DOTA-ICC-TATE.
component as the linker. To the best of our knowledge, employing
the fluorophore as the linker for SSTR2-targeting hybrid probes
has not been done so far. We herein present a synthetic approach
that opens up new possibilities for the construction of multimodal
imaging probes. We used this strategy to create a dye-linked
hybrid probe for multimodal SSTR2-imaging (Scheme 1D). To this
end, we selected the clinically used radiotracer 68Ga-DOTATATE
and inserted a cyanine dye between the chelator and peptide.
This molecular design was achieved by synthesizing an
indocarbocyanine dye carrying two different conjugatable
functional groups at the aromatic moieties. Whereas the
functionalization of the indolic N-substituents is widely known and
applied,[19] our design approach leaves these N-substituents in
principle open to modifications so that physicochemical properties
like solubility or aggregation behavior can be adjusted.
We synthesized the asymmetric ICC dye 1 from a phthalimide
functionalized Fischer’s base, N,N-diphenylformamidine and a
carboxylic acid containing indolenine precursor, leading to a good
yield of 35% in terms of asymmetric dye synthesis (Scheme 1E).
Hydrazinolysis according to the Ing-Manske procedure in
Gabriel’s synthesis using hydrazine monohydrate in methanol
yielded the new bifunctional ICC dye 2. For the upscaling of this
step, it was crucial to use a microwave setup for the heating, short
heating times and repeated addition of hydrazine monohydrate in
order to obtain good yields of up to 80%. Performing the
hydrazinolysis with the indodicarbocyanine (IDCC) analog led to
conditions were required being incompatible with acid-labile
protecting groups.
Afterwards, conventional coupling of the N-terminus of TATE to
the carboxyl group of the fluorophore was performed, followed by
the removal of the remaining protective groups. In comparison to
other design approaches, the resulting hybrid probe becomes
relatively compact with the dye in direct proximity to the
pharmacophore.
Spectroscopic properties
The new bifunctional ICC dye 2 has a high absorption coefficient
of 130 000 L/(mol cm) in water with an absorption maximum at
551 nm and an emission maximum at 570 nm (Figure S1). For the
natGa-DOTA-ICC label, the absorption coefficient amounts to a
good value of 80 000 L/(mol cm) with an absorption maximum at
552 nm and an emission maximum at 570 nm. No significant
changes in the fluorescence behavior compared to the free dye 2
were observed.
In vitro studies
Toxicity assays demonstrated that the conjugate DOTA-ICC-
TATE does not inhibit either metabolism or proliferation of human
cells, up to a concentration of 10 µM (Figure S2).
The cellular uptake of the DOTA-ICC-TATE conjugate and the
internalization of SSTR2 were analyzed. Therefore, RIN1038
cells overexpressing an SSTR2-GFP fusion protein were used,
allowing direct detection via GFP fluorescence. After incubation
of the cells with the conjugate (1 µM) for 30 minutes, the strong
fluorescent signal arising from the ICC dye could be clearly
colocalized with the internalized vesicle-associated GFP-fused
SSTR2 (Figure 1A), thus indicating the retention of the receptor-
binding and endocytosis-stimulating properties of the peptide in
vitro. According to the quantitative receptor internalization assay,
DOTA-ICC-TATE stimulates the internalization of the SSTR2-
GFP fusion protein in a concentration-dependent manner at a
subnanomolar EC50 value of 0.42 nM (Figure S2). The binding
affinity towards SSTR2 was further supported by a competitive
radioligand binding assay using 125I-labeled Tyr11-somatostatin-
a
decrease of the yield, unfortunately, because longer
polymethine chains are more prone towards decomposition by
nucleophilic attacks. The water-soluble bifunctional ICC dye 2
represents a unique linker platform for various applications, with
a carboxylic acid on the one hand and an aliphatic amine that
allows reactions with electrophiles under mild conditions on the
other hand.
The amine functionality of this dye was used to attach a tert-butyl
protected, HSTU-activated DOTA chelator to obtain the DOTA-
ICC label 3 in a yield of 55% (Scheme 1E). For PET or SPECT
studies, the DOTA chelator can in general form stable complexes
with 64Cu, 68Ga and 111In.[20] The methylene unit between the
aromatic moiety and the amine of the dye was essential for the
amide coupling. Without this additional methylene unit, harsh
3
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Figure 2. (A) Illustrative PET/MRI image of an SSTR2-overexpressing BON tumor bearing living mouse, acquired 41 minutes after tail vein injection of 68Ga-DOTA-
ICC-TATE. Pink arrows indicate the SSTR2-overexpressing BON tumor, white arrows indicate the wildtype BON tumor. The scale bar is representative for all
images. (1) T1 coronal MRI image. (2) PET coronal image. (3) T2 FSE 2D transverse MRI and fusion with PET image. (B) Ex vivo biodistribution data in xenograft-
bearing nude mice (n=3) 1 h after injection of 68Ga-DOTA-ICC-TATE.
14 as a tracer in BON-SSTR2 cells (Figure 1B), yielding similar
and low IC50 values of 0.77 ± 0.3 nM for DOTA-ICC-TATE and
0.93 ± 0.3 nM for natGa-DOTA-ICC-TATE. These values are in the
range of published values for DOTATATE and Ga-DOTATATE,[21]
suggesting that the close vicinity of the cyanine dye to the peptide
TATE does not substantially influence the peptide functionality in
vitro.
the uptake of our probe in the BON tumor. According to the
PET/MRI and biodistribution data, the clearance route is
predominantly renal with a high uptake in the kidneys (~30% IA/g).
Attaching a fluorophore to 68Ga-DOTATATE usually causes
higher renal uptake compared to the dye-free 68Ga-
DOTATATE,[21,23] leading to values of up to 70% IA/g.[14b,18a] The
same applies, usually to a smaller extent, for the uptake in the
liver, which is also influenced by non-polar structural moieties as
typically given by fluorophores. In our study, the predominance of
the renal clearance route compared to the hepatobiliary route may
be attributed to the relatively hydrophilic indocarbocyanine dye
with a short polymethine chain length, no additional benzene
moieties and the presence of one sulfonate group. The tumor-to-
background ratios 1 h after injection were moderate for the
muscles and low for blood. Other studies with SSTR2-targeting
hybrid probes observed similar values 3-4 h after injection,[14b,18a]
which could be improved when imaging at later time points, e.g.
24 h or 48 h after injection using 67Ga.[18c]
In vivo imaging and biodistribution
Radiolabeling of the DOTA-ICC-TATE conjugate with 68Ga was
performed at 95 °C within 500 s. Purification yielded the 68Ga-
DOTA-ICC-TATE probe 4 (Scheme 1E) in high radiochemical
purity (> 95%) with a molar activity of 40 GBq/µmol.
In vivo PET imaging was performed 41-48 minutes after injection
of 8.8 – 14.3 MBq (0.2 – 0.3 nmol) of the 68Ga-DOTA-ICC-TATE
probe into nude mice bearing an SSTR2-overexpressing tumor
on the right and a wildtype tumor on the left shoulder (Figures S3-
5). As shown in Figure 2A, the tumor could be well visualized by
the PET/MRI scan. The uptake of the probe in the SSTR2-
overexpressing tumor was higher than in the wildtype tumor with
average standard uptake values of 6.6 ± 1.3% IA/mL in the
SSTR2-overexpressing tumor and uptake values of 4.6 ±
0.3% IA/mL in the wildtype tumors. Substantial uptake of the
probe was also seen in the kidneys and the bladder, but generally
low in other organs.
Ex vivo biodistribution data were obtained 1 h after injection in the
same tumor mouse model (Figure 1B). The data is in good
agreement with the PET/MRI scans with uptake values of
6.7 ± 1.4% IA/g in the SSTR2-overexpressing tumor, being higher
than in the wildtype tumor (4.1 ± 0.2% IA/g). In other organs, a
high accumulation was found in the kidneys (~30% IA/g) and a
moderate uptake in the liver (~5% IA/g). Uptake in the wildtype
BON tumors may be explained by the expression of other relevant
SSTR subtypes, such as SSTR3 and SSTR5.[22] It is known from
the literature that somatostatin analogs display the second
highest affinity for SSTR5 and a moderate affinity for SSTR3.[21a]
These receptor subtypes are expressed at a comparatively high
level in the wildtype BON cell line, which most likely contributes to
Histological examination
When tumor sections of mice sacrificed 1 h p.i. were examined
using a confocal laser microscope, no fluorescence above
background level was detected. In particular, no differences
between sections from SSTR2-overexpressing and wildtype
tumor were found. We assumed that the tracer dose chosen for
the PET/MRI scan was not high enough for fluorescence imaging
on the histological level due to the inferior sensitivity of this
method. We therefore repeated the experiment with the same
tumor mouse model and injected 6 nmol of natGa-DOTA-ICC-
TATE. Mice were sacrificed 5 h after injection and the tumors
excised. Upon confocal laser scanning microscopy of tumor
sections from these mice (Figure 3A), we detected clear
differences in the signals arising from the ICC dye in the probe
between the SSTR2-overexpressing (mean grey value 32.8 ± 5.4)
and the wildtype BON tumor (mean grey value 12.4 ± 3.3), found
to be statistically significant (P = 0.005). The necessity of using a
higher dose for fluorescence microscopy due to its sensitivity is
4
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Figure 3. Histological ex vivo examination of BON wildtype and SSTR2-overexpressing BON tumors from xenograft-bearing nude mice (n=3) injected with natGa-
DOTA-ICC-TATE. (A) Images from the confocal laser scanning microscopy. (B) Quantitative analysis of the images. ***P = 0.005.
not unexpected as other studies have also used picomolar doses
for PET or SPECT and nanomolar doses for fluorescence imaging
or microscopy.[7,18b,24] Further studies are required to determine
Experimental Section
General information
the optimal dose that can be used both for PET and for ex vivo
examinations afterwards, e.g. for cancer staging or intraoperative
frozen section analysis. However, increasing the amount of
peptide for fluorescence imaging might weaken the image
contrast for PET or SPECT due to a lower specific activity of the
tracer.[18b,25]
Chemicals were purchased from commercial sources (like Sigma Aldrich,
TCI, AlfaAesar, ABCR and Fluka). Indolenine precursors and the protected
DOTA chelator were synthesized according to published procedures. H,
1
13C and 71Ga NMR spectra in solution were measured with the
spectrometers ECX 400 (400 MHz) and ECP 500 (500 MHz) from JEOL
and Avance 500 (500 MHz) and Avance 700 (700 MHz) from Bruker. Mass
spectra were measured on a 6210 ESI-TOF and 6230 ESI-TOF from
Agilent. UV/VIS spectra were recorded using a PerkinElmer LAMBDA 950
UV/VIS/NIR spectrometer and fluorescence spectra recorded using a
JASCO FP-6500 spectrometer. NP automated column chromatography
was done on a CombiFlash Rf (Teledyne ISCO) using prepacked silica
columns (30 µm). Purification of compounds using size exclusion
chromatography was done on a Sephadex column (NAP-25, Sephadex G-
25 DNA) with water as eluent.
Conclusion
We present a new modular design approach for the synthesis of
multimodal imaging probes based on a bifunctional cyanine dye
serving as a structural and functional linking unit. Our study
demonstrates that a cyanine dye can serve as the linker in the
comparatively compact SSTR2-targeting hybrid probe 68Ga-
DOTA-ICC-TATE with direct proximity between dye and
pharmacophore. Using radionuclide and fluorescence imaging
modalities, we performed SSTR2-imaging in vitro, in vivo and ex
vivo in SSTR2-expressing cell and tumor mouse models. Our dye-
bridged hybrid probe showed excellent in vitro SSTR2-affinity and
peptide functionality, whereas the in vivo PET imaging using 68Ga
at an early imaging time point needs further improvements for
higher tumor-to-background ratios. Histological ex vivo
examination using fluorescence microscopy showed high
specificity of the hybrid probe for the SSTR2-overexpressing
tumor in comparison to the wildtype tumor.
Synthesis
N-(Hydroxymethyl)-phthalimide. To a solution of phthalimide (10.0 g,
68.0 mmol, 1.0 eq.) in H2O (35 mL) was added a formaldehyde solution
(37 % in H2O, 5.1 mL). After heating the suspension to 105 °C for 1 h under
heavy stirring, a clear and colourless solution was obtained. Storing the
solution in the freezer overnight led to the precipitation of a colourless solid,
which was filtered off and washed with ice-cold water. The filter cake was
dried in vacuo at 30 °C and afforded the product as a colourless solid (10.9
1
g, 61.5 mmol, 90%). H NMR (d6-DMSO, 400 MHz, ppm) δ = 7.93 - 7.88
(m, 4H; -CH), 6.39 (t, J = 7.0 Hz, 1H; -OH), 4.96 (d, J = 6.9 Hz, 2H; -CH2);
13C NMR (d6-DMSO, 101 MHz, ppm) δ = 167.4, 134.8, 131.6, 123.4, 60.1;
MS (ESI+): m/z C9H7NO3 [M+Na]+ calculated: 200.0324, found: 200.0318.
While previous reports mainly focused on the use of Cy5- or Cy7-
based dyes for image-guided surgery, we employed a Cy3-based
fluorophore due to chemical considerations during the
establishment of the synthesis. In comparison to Cy7-based
probes, a Cy3-based probe has its own eligibility because in vitro
and histological ex vivo examinations after PET or SPECT studies
can be easily performed using conventional fluorescence
microscopes for detection. Future studies aim to expand our
synthetic approach to a Cy5-based construct with additional
benefits for the field of image-guided surgery.
1,3,3-Trimethyl-2-methylene-5-(phthal-imidemethyl)-indolenine.
Fischer’s base (10.9 mL, 10.7 g, 51.6 mmol, 1.0 eq.) was dissolved in
concentrated H2SO4 (55 mL). To the red-brown solution, N-
(hydroxymethyl)-phthalimide (10.9 g, 61.5 mmol, 1 eq.) was added
portionwise within 45 min. The brown solution was stirred at room
temperature for 75 h and poured onto ice-cooled water (60 mL). After
addition of ammonia solution (12.5% in water, 400 mL) and filtration, the
filter cake was dissolved in dichloromethane (350 mL) and washed with
demineralized water (2 x 300 mL). The aqueous phase was extracted with
dichloromethane (2 x 400 mL), dried over Na2SO4, filtered and the solvent
removed under reduced pressure. After automated column
5
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10.1002/cbic.202000791
ChemBioChem
FULL PAPER
chromatography (silica gel, eluent dichloromethane/methanol with 0–5%
and 0–0.5% methanol), recrystallization with dichloromethane and ethanol
(1:1) and washing with methanol was done and afforded the product as a
yellow to red solid with minor impurities (8.9 g, ca. 27 mmol, ca. 52%). 1H
NMR (CDCl3, 400 MHz, ppm) δ = 7.81 (dd, J = 5.4 Hz, J = 3.0 Hz, 2H; -
CH), 7.67 (dd, J = 5.5 Hz, J = 3.1 Hz, 2H; -CH), 7.24 (d, J = 1.8 Hz, 1H; -
CH), 7.18 (d, J = 1.8 Hz, 1H; -CH), 6.44 (d, J = 8.0 Hz, 1H; -CH), 4.74 (s,
2H; -CH2), 3.81 (s, 2H; -CH2), 2.98 (s, 3H; -CH3), 1.30 (s, 3H; -CH3); 13C
NMR (CDCl3, 126 MHz, ppm) δ = 168.3, 163.0, 146.3, 138.1, 134.0, 132.4,
128.9, 126.6, 123.4, 123.0, 104.7, 73.5, 44.2, 41.8, 30.0, 28.9; MS (ESI+):
m/z C21H20N2O2 [M+H]+ calculated: 333.1604, found: 333.1649, [M+Na]+
calculated: 355.1423, found: 355.1468.
Dye 1. 5-Carboxy-1,3,3-trimethyl-2-methylene-1-(4-sulfobutyl)indolenine
(1.12 g, 4.28 mmol, 1.0 eq.) and N,N-diphenylformamidine (420 mg, 4.28
mmol, 1.0 eq.) were dissolved in acetic anhydride (34.3 mL) and heated to
120 °C for 20 min. To the red-brown solution was then added a suspension
of 1,3,3-trimethyl-2-methylene-5-(phthalimidomethyl)indoline (1.02 g, 4.28
mmol, 1.0 eq.) and sodium acetate (525 mg, 12.8 mmol, 3.0 eq.) in acetic
anhydride (17.1 mL). Afterwards, acetic acid (1.55 mL) was added
dropwise, which led to a colour change to pink-violet. The reaction mixture
was stirred at 120 °C for 30 min. The solvent was removed under reduced
pressure and the residue taken up in DMF for precipitation of the dye by
adding diethyl ether (3x). The precipitate was purified by automated
column chromatography (silica gel, eluent dichloro-methane/methanol with
0–50% methanol) and afforded product 1 as a pink-red solid (1.01 g, 1.48
mmol, 35%). Rf = 0.5 (DCM/MeOH 1:1); 1H NMR (d4-MeOD, 500 MHz,
ppm) δ = 8.57 (t, J = 13.5 Hz, 1H; -CH), 8.16 - 8.12 (m, 2H; -CH), 7.94 –
7.89 (m, 2H; -CH), 7.89 - 7.83 (m, 2H; -CH), 7.64 (d, J = 1.7 Hz, 1H; -CH),
7.53 (dd, J = 8.2 Hz, J = 1.7 Hz, 1H; -CH), 7.44 (d, J = 8.7 Hz, 1H; -CH),
7.40 (d, J = 8.3 Hz, 1H; -CH), 6.59 (d, J = 13.6 Hz, 1H; -CH), 6.55 (d, J =
13.3 Hz, 1H; -CH), 4.95 (s, 2H; -CH2), 4.21 (t, J = 7.4 Hz, 2H; -CH2), 3.75
(s, 3H; -CH3), 2.95 (t, J = 6.8 Hz, 2H; -CH2), 2.08 - 1.98 (m, 2H; -CH2), 1.82
(s, 6H; -CH3), 1.79 (s, 6H; -CH3); 13C NMR (d6-DMSO, 176 MHz, ppm) δ =
175.5, 173.3, 167.7, 167.0, 149.8, 145.4, 141.9, 141.2, 140.4, 134.6, 131.6,
130.6, 127.8, 123.3, 121.7, 112.1, 110.9, 104.7, 102.7, 50.8, 49.2, 48.3,
43.7, 40.8, 31.8, 27.5, 27.1, 26.0, 22.5; MS (ESI+): m/z C38H39N3O7S
[M+H]+ calculated: 682.2588, found: 682.2581, [M+Na]+ calculated:
704.2406, found: 704.2408.
3 as a pink-red solid (59.5 mg, 51.1 µmol, 56%). Rf = 0.3 (DCM/MeOH 1:1);
1H NMR (d4-MeOD, 400 MHz, ppm) δ = 8.55 (t, J = 13.4 Hz, 1H; -CH), 8.13
- 8.09 (m, 2H; -CH), 7.56 (d, J = 1.5 Hz, 1H; -CH), 7.44 - 7.37 (m, 3H; -
CH), 6.61 (d, J = 13.7 Hz, 1H; -CH), 6.55 (d, J = 13.2 Hz, 1H; -CH), 4.50
(b, 2H; -CH2), 4.19 (t, J = 6.7 Hz, 2H; -CH2), 3.75 (s, 3H; -CH3), 3.64 - 3.37
(m, 4H; -CH2), 3.21 - 3.00 (b, 4H), 2.95 - 2.89 (m, 4H; -CH2), 2.83 - 2.37
(m, 8H; -CH2), 2.36 - 2.14 (b, 4H; -CH2), 2.07 - 1.92 (m, 6H; -CH2), 1.79 (s,
6H; -CH3), 1.78 (s, 6H; -CH3), 1.58 - 1.55 (m, 2H; -CH2), 1.50 (s, 9H; -CH3),
1.48 (s, 18H;- CH3); 13C-NMR (d4-MeOD, 176 MHz, ppm) δ = 177.8, 175.5,
174.5, 174.4, 174.2, 171.8, 152.4, 147.2, 143.1, 142.8, 141.9, 138.7, 132.4,
129.3, 124.6, 123.0, 112.8, 111.8, 105.8, 104.2, 82.8, 57.3, 56.8, 51.6,
51.0, 50.0, 49.9, 45.0, 43.9, 43.4, 32.3, 28.5, 28.1, 27.0, 23.5; MS (ESI+):
C60H90N8O13S [M+Na]+ calculated: 1185.6246, found: 1185.6234,
[M+2Na-3C4H8]2+ calculated: 520.2127, found: 520.2144.
natGa-DOTA-ICC label. A mixture of TFA/demineralized water/phenol/
thioanisol/1,2-ethandithiol (82.5:5:5:5:2.5, 1.5 mL) was added to the
DOTA-ICC label 3 (61.6 mg, 52.9 µmol, 1.0 eq.) and stirred for 4 h at room
temperature. The solvent was removed under reduced pressure, the
residue taken up with water and washed with DCM. The aqueous phase
was lyophilized. Preparative HPLC was performed in an isocratic mode on
a high-pressure gradient system (stainless steel), equipped with a
Shimadzu LC-8A pump, Shimadzu CBM-20A controller, variable
wavelength UV detector from Knauer and a Rheodyne injector with 10 mL
sample loop. The stationary phase was a prepacked LUNA HILIC diol
column (200 Å, 5 µM, 250 x 21.2 mm) from Phenomenex with precolumn.
The eluent was a degassed MeCN/water mixture (75 % MeCN + 50 mM
ammonium formate) and a flow rate of 20 mL/min was applied. Detection
was performed at 480 nm. The appropriate fractions were lyophilized to
afford the deprotected DOTA-ICC label (14.9 mg), which was then
dissolved in water (800 µL). GaCl3 was added and the pH value adjusted
to 4 using NaOH. The reaction mixture was heated at 80 °C for 30 minutes.
The mixture was diluted with water, dialyzed (regenerated cellulose (Carl
Roth), MWCO = 500 g/mol, water, 3 d) and lyophilized to afford the
product as a pink-red solid (14.0 mg, 13.2 µmol, 25% over 2 steps). 1H
NMR (D2O/d3-MeCN 5:2, 700 MHz, ppm) δ = 8.80 (t, J = 13.5 Hz, 1H),
8.35 (d, J = 1.6 Hz, 1H), 8.35 – 8.33 (m, 1H), 7.77 (d, J = 1.7 Hz, 1H), 7.68
(dd, J = 8.1, 1.6 Hz, 1H), 7.65 (dd, J = 11.9, 8.2 Hz, 2H), 6.73 (d, J = 13.7
Hz, 1H), 6.66 (d, J = 13.2 Hz, 1H), 4.76 (d, J = 3.3 Hz, 2H), 4.40 (t, J = 7.4
Hz, 2H), 4.27 (dd, J = 15.2, 3.8 Hz, 3H), 4.24 (s, 2H), 4.15 (d, J = 11.2 Hz,
2H), 4.12 (s, 1H), 4.10 (s, 1H), 4.06 (s, 3H), 4.03 (s, 3H), 3.95 (s, 3H), 3.76
(s, 3H), 3.74 – 3.72 (m, 3H), 3.59 (ddt, J = 14.3, 8.3, 4.4 Hz, 6H), 3.50 (td,
J = 14.3, 4.4 Hz, 2H), 3.22 (t, J = 7.4 Hz, 2H), 2.21 (dq, J = 21.9, 7.6 Hz,
4H), 2.06 (s, 6H), 2.04 (s, 6H); 13C NMR (D2O/d3-MeCN 5:2, 176 MHz,
ppm) δ = 176.6, 174.6, 173.5, 173.2, 171.5, 171.0, 169.1, 151.1, 145.5,
142.1, 141.8, 140.9, 136.6, 131.1, 130.0, 128.1, 123.6, 121.6, 111.9, 110.8,
104.1, 102.5, 63.4, 61.5, 59.7, 57.4, 57.3, 54.7, 54.5, 50.7, 49.8, 49.0, 44.0,
42.8, 42.5, 39.2, 31.7, 27.6, 27.2, 26.0, 22.1; 71Ga NMR (D2O, 153 MHz,
ppm) δ = -25.0; UV/Vis (H2O): λmax (ε) = 552 nm (80.000 L/(mol cm));
fluorescence (H2O): λex = 530 nm,, λem = 570 nm; MS (ESI+):
C48H63GaN8O13S [M+Na]+ calculated: 1082.3305, found: 1082.3322; MS
(ESI-): C48H63GaN8O13S; [M-H]+ calculated: 1059.3413, found: 1059.3402.
Dye 2. Dye 1 (50.0 mg, 73.3 µmol, 1.0 eq.) was dissolved in methanol
(3.75 mL, purged with Argon) and hydrazine monohydrate (10.7 µL,
11.0 mg, 220 µmol, 6.0 eq.) was added in two portions within 15 minutes.
The reaction mixture was heated to 90 °C in the microwave for 30 minutes.
The solvent was removed under reduced pressure. Purification of the
residue
by
column
chromatography
(silica
gel,
eluent
dichloromethane/methanol with 0 – 60% methanol) afforded product 2 as
a pink-red solid (32.5 mg, 58.9 µmol, 80%). Rf = 0.05 (DCM/MeOH 1:1); 1H
NMR (d4-MeOD, 500 MHz, ppm) δ = 8.57 (t, J = 13.5 Hz, 1H; -CH), 8.11 -
8.07 (m, 2H; -CH), 7.60 (d, J = 1.7 Hz, 1H; -CH), 7.47 (d, J = 7.9 Hz, 1H; -
CH), 7.38 (d, J = 3.6 Hz, 1H; -CH), 7.36 (d, J = 3.1 Hz, 1H; -CH), 6.55 (d,
J = 10.3 Hz, 1H; -CH), 6.52 (d, J = 10.6 Hz, 1H; -CH), 4.21 (t, J = 7.3 Hz,
2H; -CH2), 3.95 (s, 2H; -CH2), 3.73 (s, 3H; -CH3), 2.96 (t, J = 6.7 Hz, 2H; -
CH2), 2.07 - 2.01 (m, 2H; -CH2), 1.82 (s, 6H; -CH3), 1.81 (s, 6H; -CH3); 13
C
NMR (d4-MeOD, 176 MHz, ppm) δ = 176.9, 176.3, 152.2, 144.9, 143.5,
142.6, 141.5, 139.3, 136.7, 131.7, 129.7, 124.4, 123.2, 112.4, 111.4, 104.6,
104.3, 51.6, 50.7, 50.3, 45.8, 45.1, 31.9, 30.8, 28.4, 28.2, 27.1, 23.5;
UV/Vis (H2O): λmax (ε)= 551 nm (130.000 L/(mol cm)); fluorescence (H2O):
λex = 530 nm,, λem = 570 nm; MS (ESI+): m/z C30H37N3O5S [M+H]+
calculated: 552.2454, found: 552.2515, [M+Na]+ calculated: 574.2346,
found: 574.2332.
DOTA-ICC label 3. The tert-butyl protected DOTA chelator (68.5 mg, 109
µmol, 1.2 eq.) and HSTU (45.5 mg, 127 µmol, 1.4 eq.) were dissolved in
DMF (750 µL) and DIPEA (23.1 µL, 17.6 mg, 136 µmol, 1.5 eq.) was added.
The reaction mixture was stirred for 1 h at room temperature. Compound
2 (50.0 mg, 90.6 µmol, 1.0 eq.) was dissolved in demineralized water (1.2
mL) and added with DIPEA (46.2 µL, 35.1 mg, 272 µmol, 3.0 eq.) to the
reaction mixture. After stirring for 15 h at room temperature, the solvent
was removed under reduced pressure. The residue was purified twice by
DOTA-ICC-TATE conjugate. The peptide conjugate was synthesized in a
0.05 mmol scale on a Thr-preloaded Wang Resin. The synthesis was
carried out on a PTI synthesizer (Protein Technologies, USA) with double
couplings of each amino acid (5 eq. amino acid for 40 min) in DMF. Both
cysteines were introduced as MMT protected building blocks. The DOTA-
ICC label 3 (100 µmol, 2.0 eq.) was coupled manually using HATU (100
µmol, 2.0 eq.) and DIPEA (200 µmol, 4.0 eq.) in DMF (2 mL) for 4 h.
Afterwards, MMT protecting groups were cleaved by treating the resin 5x
with a mixture of DCM:TFA:TIS (94:1:5) (3 mL) for 2 min. Followed by a
DCM and DMF wash, the cyclization using N-chlorosuccinimide (2 eq.) in
DMF for 15 min was carried out. The final cleavage from the resin was
done in TFA:TIS:H2O (95:2.5:2.5) for 3 h. The crude DOTA-ICC-TATE
conjugate was purified by preparative HPLC (RP-C18, 0-5 min 95/5, water
(0.1% TFA)/MeCN (0.1% TFA); 5-60 min 10/90, water (0.1%TFA)/MeCN
(0.1% TFA)) using a Gilson PLC 2020 personal Purification System (Gilson
Inc., Middleton, WI, USA) including a Nucleodur column (VP250/32 C18
HTec, 5μm) from Macherey-Nagel with a flow rate of 30 mL/min. The
automated
column
chromatography
(silica
gel,
eluent
dichloromethane/methanol with 0–100% methanol) and afforded product
6
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000791
ChemBioChem
FULL PAPER
product was gained in two peaks as a pink powder (9.5 mg, 4.7 μmol, 5 %)
and analyzed by UPLC-UV using an Aquity UPLC H-Class with a
quaternary solvent manager, a Waters autosampler and an Aquity UPLC-
BEH RP-C18 column (1.7 μm, 2.1 x 50 mm) from Waters with a flow rate
of 0.6 mL/min connected to a Waters UV detector and a QDa detector and
the following gradient used with solvents A and B (A = H2O + 0.1%TFA; B
= MeCN + 0.1%TFA): 0 – 1.5 min (with 5% B in A); from 1.5 – 13 min (5%
B to 95% B in A); 13 – 13.9 min (with 95% B in A); 13.91 – 15 min (with
5% B in A). Detection was performed at 220 nm. UV/Vis (H2O): λmax (ε) =
553 nm (74.000 L/(mol cm)); fluorescence (H2O): λex = 530 nm,,
λem = 570 nm; m/z C97H128N18O24S3 [M+2H]2+ calculated: 1013.93, found:
1013.81 and 1013.62, [M+H+Na]2+ calculated: 1024.93, found: 1024.75
and 1025.01, [M+3H]3+ calculated: 676.29, found: 676.25 and 676.35.
natGa-DOTA-ICC-TATE conjugate. GaCl3 (217 µg, 1.24 nmol, 5.0 eq.) in
205.6 µL HEPES buffer and 25 µL MeCN was added to DOTA-ICC-TATE
(500 µg, 247 nmol, 1.0 eq.). The pH of the solution was adjusted to pH =
4 using HCl (1 M) and the reaction mixture heated to 90 °C for 1 h. After
cooling down, the purification was done by using a Sephadex column and
water as eluent. The fractions were lyophilized, dissolved in 1.0 mL water
and the concentration was determined via absorption spectroscopy using
an absorption coefficient of 80.000 L/(mol cm) to be 64 nmol. Analytical
RP-HPLC of the natGa-labeled peptide conjugate in comparison to the Ga-
free peptide conjugate was performed on an Agilent 1200 system (Agilent,
Waldbronn, Germany) equipped with an Eclipse XDB-C18 bonded silica
(5 µm, 50 x 4.6 mm) column (Agilent, Waldbronn, Germany) with a flow
rate of 1 mL/min and the column at 55 °C. Elution was performed using a
linear gradient with solvents A and B (A = H2O + 0.1%TFA; B = MeCN +
0.1%TFA) with a gradient of 20 – 60% B in A over 20 min. Detection was
performed at 543 nm.
AlamarBlue™ redox indicator (Thermo Fisher, Waltham, MA, USA) were
added on top of each well, incubated for 3 – 4 h and the resulting
fluorescence was measured using an EnVision Multilabel Plate Reader
(Perkin Elmer, Waltham, MA, USA). Afterwards, the supernatant was
removed; cells were fixated with 4% v/v formaldehyde for 10 min and
stained with 1 µg/mL DAPI in PBS/0.1% v/v Triton for another 10 min. Four
fields per well were imaged using an IN Cell Analyzer 1000 (GE Healthcare,
Buckinghamshire, UK) with a 4x objective and nuclei were counted by
Investigator software (GE Healthcare, Buckinghamshire, UK). All values
were normalized to the control treated with vehicle and analyzed using
GraphPad Prism 5.04.
Endocytosis assay. Rat insulinoma RIN1038 cells stably expressing a
ratSSTR2-GFP fusion protein almost exclusively show green cell
membrane fluorescence in the resting state while in the presence of a
functional agonist, the receptor-GFP fusion protein is translocated to a
perinuclear endocytic vesicle compartment. This shift is readily recognized
by inspection of the fluorescent image and can be detected by software-
based quantitation algorithms. After an incubation time of 30 min with 1 µM
DOTA-ICC-TATE in RPMI1640 medium at 37 °C, cells were fixed for
10 min using 4% formaldehyde in PBS, air-dried and mounted on glass
slides with ImmuMount (Thermo Fisher Scientific, Waltham, MA, USA).
Mounted cells were imaged using a confocal laser-scanning microscope
(LSM510, Carl Zeiss, Jena, Germany) with a helium-neon laser at 488 and
543 nm, BP505-525 and LP560 emission filters and a 63x NeoFluar oil
immersion objective. For quantitative analysis and determination of the
concentration of half-maximal internalization effect (EC50), eleven
concentrations of DOTA-ICC-TATE (3 pM – 300 nM) were used in the
same cell model in quadruplicates in a 96 well plate. After an incubation
time of 30 min with 1 µM DOTA-ICC-TATE in RPMI1640 medium at 37 °C,
cells were fixed for 10 min using 4% formaldehyde plus 0.1 µg/mL DAPI in
PBS before adding 100 µL of PBS per well. For automated microscopy,
microscopic fluorescence images were recorded by an IN Cell Analyzer
1000 (GE Healthcare) with 20x magnification at 5 frames per well in a 96
well format. Image processing was performed with In Cell Investigator
software (GE Healthcare) applying the provided granularity algorithm. The
output of the analysis was indicated as vesicle area/cell and was utilized
for calculation of concentration-response curves resulting in the
determination of half-maximal activity (EC50).
In vitro binding affinity assay. 10 nmol Tyr11-somatostatin-14
(Tyr11-SST14, Bachem, Bubendorf, Switzerland) were iodinated by the
chloramine T method[26] with 1 mCi carrier-free Na125I (NEZ033L010MC,
Perkin Elmer, Waltham, MA, USA) and purified by HPLC (Analytic HPLC
1200 Series, Agilent, Santa Clara, CA, USA). For competitive radioligand
binding assays, 40,000 BON cells or BON-SSTR2 cells (overexpressing
human SSTR2)[22] were seeded in 96-well plates and grown overnight. The
next day, cells were incubated in binding buffer (50 mM Hepes, pH 7.4,
5 mM MgCl2, 1 mM CaCl2, 0.5 % w/v BSA, complete protease inhibitors
(Roche, Mannheim, Germany)) containing 100,000 cpm (counts per
minute) [125I]-Tyr11-SST14 and increasing concentrations of unlabeled
peptide. After 30 min at 37 °C, cells were washed with ice-cold washing
buffer (50 mM Tris-HCl, pH 7.4, 125 mM NaCl, 0.05 % w/v BSA), lysed
with 1 N NaOH, transferred to vials and measured in a gamma counter
(Wallac 1470 Wizard, Perkin Elmer, Waltham, MA, USA). The obtained
cpm values were analyzed with GraphPad Prism 5.04 and IC50 values
were calculated by nonlinear regression (one site - fit logIC50, least squares
fit).
Radiolabeling with 68Ga
Radiolabeling experiments were performed on a synthesis module
(Modular Lab PharmTracer) which allows fully automated cassette-based
labelling of Gallium tracers utilizing a Pharmaceutical grade 68Ge/68Ga
Generator (GalliaPharm, 1.85 GBq, GMP) both purchased from Eckert &
Ziegler GmbH. Cassettes were GMP-Conform, sterile and used without
pre-conditioning of the cartridges. Gallium-generator at 1-month post
calibration was eluted with aqueous HCl (0.1 M, 7 mL) and the eluate was
purified on an ion-exchange cartridge followed by elution directly into the
reactor pre-heated at 40 °C using 1 mL of 0.1 M HCl in acetone. An aliquot
of DOTA-ICC-TATE, 20 µg (stock solution, 1 µg/µL in water) was mixed
with 500 µL HEPES buffer (0.1 M in WFI, pH 7) and added to the reactor
before starting the synthesis. The reaction mixture (pH 3-4) was then
heated for 500 s at 95 °C. After the reaction, the reactor was cooled with
0.5 mL of saline following the transfer of the contents of the reactor on a
C18-cartridge (SEPPAK, Waters GmbH) for post-purification. After
washing the C18-cartridge with saline, the product was eluted with 1 mL
of ethanol/water (1:1) and diluted with 1.5 mL saline for animal
experiments. RP-HPLC (Knauer) with a Eurospher II column (C18, 250 ×
4 mm) was used to quantify the radiochemical purity of 68Ga-DOTA-ICC-
TATE. The HPLC was equipped with an Azura P.6.1L pump coupled with
ultraviolet
(Azura
UVD
2.1L)
and
radiometric
(γ-Raytest-
Isotopenmessgeräte GmbH) detectors. The gradient elution system used
mobile phase A (100% acetonitrile) and mobile phase B (deionized H2O
containing 0.1% trifluoroacetic acid) and a flow rate of 1.0 mL/min. Starting
with 0% A and 100% B, the gradient was increased to 100% A over 25 min
and finally returned to initial gradient conditions within 5 minutes.
Radiochemical purity of the tracer was found to be ≥ 95%, radiochemical
yield was about 40% and specific activity was calculated as 40 GBq/µmol
of peptide.
In vivo imaging and biodistribution
Tumor Model. Neuroendocrine BON and BON-SSTR2 cells (3x106) were
suspended in
a volume of 150 µL 0.9% NaCl and inoculated
subcutaneously on the right and left shoulder of female nude NMRI-
Foxn1nu /Foxn1nu mice (Janvier Labs, Saint-Berthevin, France). After 2-3
weeks of tumor growth, the tumor size was sufficient for imaging. Animal
care followed institutional guidelines and all experiments were approved
by local animal research authorities (approval number G0192/08).
PET/MRI Imaging. Tomographic imaging was performed using the
dedicated small animal 1 Tesla nanoScan PET/MRI (Mediso, Hungary).
Tumor bearing mice (n=3) were anaesthetized with isoflurane and were
In vitro tests
Toxicity tests. BON-SSTR2 cells were seeded in quadruplicates in 96
well plates at a density of 5,000 cells per well and grown overnight. Cells
were treated with the indicated concentrations of DOTA-ICC-TATE in
100 µL medium per well. Metabolic activity and cell number were
determined after another 96 h. For this, 100 µL medium containing
7
This article is protected by copyright. All rights reserved.

10.1002/cbic.202000791
ChemBioChem
FULL PAPER
given an injection of 8.8 – 14.3 MBq 68Ga-DOTA-ICC-TATE in a volume of
150 µL into the tail vein for PET. PET scans were performed for 20 min
starting 41-48 min after injection of the probe. The uptake of 68Ga-DOTA-
ICC-TATE in the BON and BON-SSTR2 tumors was determined by
manual contouring of a volume-of-interest (VOI) of the PET image using
PMOD 3.5 (PMOD Technologies Ltd., Switzerland). An average standard
uptake value was computed from the 10 hottest voxels within the 68Ga-
DOTA-ICC-TATE positive lesions. MRI-based attenuation correction of the
PET was conducted with the T1-weighted material map (matrix
144x144x164, with dimensions 0.5x0.5x0.6 mm3, TR:15 ms, TE 2.1 ms
and a flip angle of 25°). Further anatomic MRI scans were acquired using
a T2-weighted 2D fast-spin echo sequence (T2-FSE 2D) with the following
parameters: transverse sequentially, 224x224x26, 0.3x0.3x1.0 mm³, TR:
5409 ms, TE: 84 ms and a flip angle of 180°.
Biodistribution study. Tumor bearing mice (n=3) were injected with
approximately 5 MBq of 68Ga-DOTA-ICC-TATE in a volume of 150 µL
0.9% NaCl into a lateral tail vein via a catheter. Mice were sacrificed by
cervical dislocation and dissected 1 h after injection. Tumors, blood,
stomach, pancreas, small intestine, colon, liver, spleen, kidney, heart, lung,
muscle and femur samples were weighed and uptake of radioactivity was
measured by a gamma counter (Wallac 1470 Wizard, Perkin Elmer,
Waltham, MA, USA).
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Histological ex vivo examination
Tumor bearing mice (n=3) were injected with approximately 6 nmol of
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immersion objective.
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Acknowledgements
We thank Maria Glanz for her work on solid-phase peptide
synthesis. This study was financially supported by the Dahlem
Research School and the Studienstiftung des Deutschen Volkes
for PhD Scholarships and the Deutsche Forschungsgemein-
schaft for PET/MRI use.
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Conflict of Interest
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The authors declare no conflict of interest.
Keywords: cyanines • fluorescence • multimodal imaging • octreotate
• SSTR2
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Entry for the Table of Contents
Multifunctional cyanine dyes: Multimodal imaging agents can be built up based on an asymmetric cyanine dye connecting the
radionuclide and targeting unit. Insertion of a cyanine dye into the clinically approved radiopharmaceutical 68Ga-DOTATATE allowed
identification of cancer tissue in vivo and ex vivo using radionuclear and fluorescence imaging.
Isabelle Heing-Becker, Carsten Grötzinger, Nicola Beindorff, Sonal Prasad, Sarah Erdmann, Samantha Exner, Rainer Haag,
Kai Licha*
Keywords: cyanines • fluorescence • multimodal imaging • octreotate • SSTR2
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