Comparison of 68Ga-labeled RGD mono- and multimers based on a clickable siderophore-based scaffold

Article abstract of DOI:10.1016/j.nucmedbio.2019.09.002

Cyclic pentapeptides containing the amino acid sequence arginine-glycine-aspartic (RGD) have been widely applied to target α_vβ₃ integrin, which is upregulated in various tumors during tumor-induced angiogenesis. Multimeric cyclic RGD peptides have been reported to be advantageous over monomeric counterparts for angiogenesis imaging. Here, we prepared mono-, di-, and trimeric cyclic arginine-glycine-aspartic-D-phenylalanine-lysine (c (RGDfK)) derivatives by conjugation with the natural chelator fusarinine C (FSC) using click chemistry based on copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC). The α_vβ₃ binding properties of ⁶⁸Ga-labeled mono-, di-, and trimeric c(RGDfK) peptides were evaluated in vitro as well as in vivo and compared with the references monomeric [⁶⁸Ga]GaNODAGA-c(RGDfK) and trimeric [⁶⁸Ga]GaFSC(suc-c(RGDfK))₃. All ⁶⁸Ga-labeled c(RGDfK) peptides displayed hydrophilicity (logD = ?2.96 to ?3.80), low protein binding and were stable in phosphate buffered-saline (PBS) and serum up to 2 h. In vitro internalization assays with human melanoma M21 (α_vβ₃-positive) and M21-L (α_vβ₃-negative) cell lines showed specific uptake of all derivatives and increased in the series: mono- < di- < trimeric peptide. The highest tumor uptake, tumor-to-background ratios, and image contrast were found for the dimeric [⁶⁸Ga]GaMAFC(c(RGDfK)aza)₂. In conclusion, we developed a novel strategy for direct, straight forward preparation of mono-, di-, and trimeric c(RGDfK) conjugates based on the FSC scaffold. Interestingly, the best α_vβ₃ imaging properties were found for the dimeric [⁶⁸Ga]GaMAFC(c(RGDfK)aza)₂.

Full text of DOI:10.1016/j.nucmedbio.2019.09.002

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Comparison of 68Ga-labeled RGD mono- and multimers based on
a clickable siderophore-based scaffold
Piriya Kaeopookum, Milos Petrik, Dominik Summer, Maximilian
Klinger, Chuangyan Zhai, Christine Rangger, Roland Haubner,
Hubertus Haas, Marian Hajduch, Clemens Decristoforo
PII:
S0969-8051(19)30181-7
DOI:
https://doi.org/10.1016/j.nucmedbio.2019.09.002
NMB 8090
Reference:
To appear in:
Nuclear Medicine and Biology
Received date:
Revised date:
Accepted date:
12 July 2019
23 September 2019
30 September 2019
Please cite this article as: P. Kaeopookum, M. Petrik, D. Summer, et al., Comparison of
68Ga-labeled RGD mono- and multimers based on a clickable siderophore-based scaffold,
Nuclear Medicine and Biology(2019), https://doi.org/10.1016/j.nucmedbio.2019.09.002
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68
Comparison of Ga-labeled RGD mono- and multimers based on a
clickable siderophore-based scaffold
b
a
a
Piriya Kaeopookum a,d, Milos Petrik , Dominik Summer , Maximilian Klinger ,
a,e
a
a
c
Chuangyan Zhai , Christine Rangger , Roland Haubner , Hubertus Haas , Marian
Hajduch b, Clemens Decristoforo a,*
a Department of Nuclear Medicine, Medical University Innsbruck, Innsbruck, Austria
b Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacky
University, Olomouc, Czech Republic
c Division of Molecular Biology, Biocenter, Medical University Innsbruck, Innsbruck, Austria
d Research and Development Division, Thailand Institute of Nuclear Technology, Nakhonnayok,
Thailand
e School of Forensic Medicine, Southern Medical University, Guangzhou, China
* Corresponding author at: Universitätsklinik für Nuklearmedizin, Medizinische Universität
Innsbruck, Anichstr. 35, A-6020 Innsbruck, Austria. Tel.: +43 512 504 80951; fax: +43 512 504
6780951.
E-mail address: clemens.decristoforo@i-med.ac.at (C. Decristoforo).

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ABSTRACT
Cyclic pentapeptides containing the amino acid sequence arginine-glycine-aspartic (RGD) have
been widely applied to target αvβ3 integrin, which is upregulated in various tumors during tumor-
induced angiogenesis. Multimeric cyclic RGD peptides have been reported to be advantageous
over monomeric counterparts for angiogenesis imaging. Here, we prepared mono-, di-, and
trimeric cyclic arginine-glycine-aspartic-D-phenylalanine-lysine (c(RGDfK)) derivatives by
conjugation with the natural chelator fusarinine C (FSC) using click chemistry based on copper
68
(I)-catalyzed azide-alkyne cycloaddition (CuAAC). The αvβ3 binding properties of Ga-labeled
mono-, di-, and trimeric c(RGDfK) peptides were evaluated in vitro as well as in vivo and
compared with the references monomeric [68Ga]GaNODAGA-c(RGDfK) and trimeric
[68Ga]GaFSC(suc-c(RGDfK))3.
All 68Ga-labeled c(RGDfK) peptides displayed hydrophilicity (logD = -2.96 to -3.80), low
protein binding and were stable in phosphate buffered-saline (PBS) and serum up to 2 h. In vitro
internalization assays with human melanoma M21 (αvβ3-positive) and M21-L (αvβ3-negative)
cell lines showed specific uptake of all derivatives and increased in the series: mono- < di- <
trimeric peptide. The highest tumor uptake, tumor-to-background ratios, and image contrast were
found for the dimeric [68Ga]GaMAFC(c(RGDfK)aza)2.
In conclusion, we developed a novel strategy for direct, straight forward preparation of mono-,
di-, and trimeric c(RGDfK) conjugates based on the FSC scaffold. Interestingly, the best αvβ3
imaging properties were found for the dimeric [68Ga]GaMAFC(c(RGDfK)aza)2.
Keywords: gallium-68, RGD, angiogenesis, αvβ3 integrin, PET

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1. Introduction
Tumor angiogenesis is induced and exploited by tumor for providing oxygen and nutrients
through newborn blood vessels. This process is well recognized as an essential mechanism for
tumor growth and metastasis [1]. During angiogenesis, αvβ3 integrins are overexpressed on
angiogenic endothelial cells in pathological tissues but absent in normal endothelial cells and
most healthy organs [2-4]. Moreover, αvβ3 integrin shows recognition and high binding affinity
with the arginine-glycine-aspartic (RGD) motif; hence, αvβ3 is a target for tumor angiogenesis
imaging.
Fluorine-18 labeled galacto-RGD ([18F]galacto-RGD) was the first tracer for noninvasive
imaging of αvβ3 expression and was already evaluated in clinical trials [5]. This compound
revealed good pharmacokinetics and receptor specific uptake. Due to the low yields and complex
synthesis of [18F]galacto-RGD, gallium-68 (68Ga) analogues were developed, initially based on
1,4,7,10-tetraazacyclododecane-N,Nʹ,Nʹʹ,Nʹʹʹ-tetraacetic acid (DOTA), followed by other
bifunctional chelators (BFCs) like 1,4,7-triazacyclononane-N,Nʹ,Nʹʹ-triacetic acid (NOTA) and
its derivative NODAGA exhibiting improved targeting properties for tumor angiogenesis [6-8].
To enhance the binding affinity toward αvβ3, multimerization of cyclic RGD peptides with
various BFCs, linkers as well as different numbers of cyclic RGD motifs have been widely
explored. New BFCs possessing three conjugation sites, 1,4,7-triazacyclononane-N,Nʹ,Nʹʹ-tris[(2-
carboxyethyl)methylenephosphinic acid] (TRAP) and fusarinine C (FSC), were used for
synthesis of trimeric cyclic RGD conjugates [9-11]. The hexadecimeric cyclic RGD is the
multimer with the largest number of RGD motifs and showed a 125-fold increase of in vitro
binding affinity relative to the monomer [12]. In most cases the increasing number of RGD

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moieties revealed an enhanced binding affinity and improved tumour uptake as expected but also
higher kidney uptake was observed [13,14].
Multimeric cyclic RGD peptides have been developed by two major routes. For the first strategy,
cyclic RGD multimers (di-, tetra-, octa-, hexadecamer) were initially synthesized and
subsequently linked to the conjugation site of BFCs such as DOTA, NOTA, and NODAGA [12-
14]. It requires multi-step synthesis and purification as well as large amounts of c(RGD). For the
second approach, chelating systems possessing multiple conjugation sites, including TRAP, FSC,
and modified NOTA, were used as scaffold where one RGD moiety per each conjugation site
was coupled [9,11,15,16]. The advantage of this approach is the straightforward synthesis route
using the chelator not only for the complex formation but also as scaffold for the multimeric
radiopharmaceutical. However, due to the broad availability of BFCs like DOTA or NOTA, the
first approach is more commonly used.
In previous works, we reported that FSC, a cyclic hydroxamate siderophore, is a promising BFC
68
for Ga and can be used as scaffold for multimerization [10,17,18]. FSC-based trimeric cyclic
RGD with different linkers were synthesized. [68Ga]GaFSC(suc-c(RGDfK))3, conjugate with
succinic linker, exhibited better angiogenesis targeting properties than the monomeric
[68Ga]GaNODAGA-c(RGDfK)
and
the
trimeric
tracer
with
another
BFC
[68Ga]GaTRAP(c(RGDfK))3 [15]. We also showed the possibility to selectively introduce mono-
, di-, and trisubstituents to FSC by acylation of the free amines [19,20].
In this study, FSC-based mono-, di-, and trimeric c(RGDfK) conjugates were synthesized using
copper (I)-catalyzed azide-alkyne cycloaddition (CuAAC), a mild and high regioselective click
reaction, and labeled with 68Ga (Scheme 1). Binding properties toward αvβ3 integrin were studied
in vitro and in vivo. MicroPET/CT images in tumor model of all conjugates were compared with

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the references monomeric [68Ga]GaNODAGA-c(RGDfK) and trimeric [68Ga]GaFSC(suc-
c(RGDfK))3.
2. Materials and methods
2.1. General
All commercially available chemicals were of analytical grade and used without further
purification. Human melanoma M21 and M21-L cells were a kind gift from D.A. Cheresh,
Departments of Immunology and Vascular Biology, The Scripps Research Institute, La Jolla,
CA, USA. Human glioblastoma U87MG cells were obtained from American Type Culture
Collection (ATCC, Manassas, VA, USA). The 68Ge/68Ga generator (IGG100) was purchased
from Eckert & Ziegler Strahlen- und Medizintechnik AG (Berlin, Germany) with a nominal
activity of 1850 MBq and was eluted with 0.1 M HCl solution (Rotem Industries Ltd., Beer-
Sheva, Israel) using the fractionated elution approach.
Reversed-phase high performance liquid chromatography (RP-HPLC) analysis was performed
with an UltiMate 3000 UHPLC pump, an UltiMate 3000 autosampler, an UltiMate 3000 column
compartment, an UltiMate 3000 variable wavelength detector (Thermo Fisher Scientific, Vienna,
Austria) and a GabiStar radiometric detector (Raytest GmbH, Straubenhardt, Germany). An ACE
3 C18, 3 µm 100 Å, 150 x 3.0 mm column (ACE, Aberdeen, UK) and UV detection at 220 nm or
450 nm were employed. Acetonitrile (ACN)/H2O/0.1% trifluoroacetic acid (TFA) was used as
mobile phase at a flow rate of 0.6 mL/min with the following multistep gradients: 0.0–1.0 min
10% ACN, 1.0–10.0 min 10–30% ACN, 10.0–11.0 min 30–60% ACN, 11.0–13.0 min 60% ACN
(gradient A); and 0.0–1.0 min 10% ACN, 1.0–12.0 min 10–60% ACN, 12.0–14.0 min 60% ACN
(gradient B).

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Preparative RP-HPLC was carried out on a Gilson 322 HPLC pump with a Gilson UV/VIS-155
detector and a PrepFC automatic collector (Gilson International B.V., Limburg, Germany). A
Eurosil Bioselect Vertex Plus, C18A 5μm 300 Å, 30 × 8 mm precolumn and a Eurosil Bioselect
Vertex Plus, C18A 5μm 300 Å, 300 × 8 mm column (Knauer, Berlin, Germany) were employed
with a flow rate of 2 mL/min and UV detection at 220 nm. The mobile phase ACN/H2O/0.1%
TFA was used with several multistep gradients.
Radio-instant thin layer chromatography (radio-ITLC) was analyzed on a TLC scanner (Scan-
RAM, LabLogistic, Sheffield, UK) using TLC-SG strips (Varian, Lake Forest, CA, USA) with
two different mobile phases: 0.1 M sodium citrate (pH 5) and 1:1 (v/v) mixture of 1 M
ammonium acetate (NH4OAc) and methanol (MeOH) (pH 7).
The radioactivities of in vitro and in vivo samples were measured in a 2480 Automatic Gamma
Counter Wizard2 3” (PerkinElmer, Waltham, MA, USA).
2.2. [Fe]Fusarinine C ([Fe]FSC)
[Fe]FSC was extracted from Aspergillus fumigatus mutant strain ΔsidG. Fungi were cultured
under iron starvation condition as described by Schrettl et al. [21] with slightly changed
incubation time and extraction method as previously published [16]. [Fe]FSC: tR = 6.3 min
(gradient A). MALDI TOF-MS: m/z [M + H]+ = 780.55 [C33H51FeN6O12; exact mass: 779.63
(calculated)].
2.3. [Fe]MAFC and [Fe]DAFC
To limit the reaction site from three to two or one positions, one or two amino groups of
[Fe]FSC were acetylated to give acetyl[Fe]FSC ([Fe]MAFC) or diacetyl[Fe]FSC ([Fe]DAFC),
respectively, as previously described [19]. [Fe]MAFC: 1.9 mg, 8% yield, tR = 7.80 min (gradient

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A), MALDI TOF-MS: m/z [M + Na]+ = 844.65 [C35H53FeN6O13; exact mass: 821.67
(calculated)]. [Fe]DAFC: 7.9 mg, 32% yield, tR = 9.36 min (gradient A), MALDI TOF-MS: m/z
[M + Na]+ = 886.68 [C37H55FeN6O14; exact mass: 863.71 (calculated)].
2.4. [Fe]DAFC(alkyn), [Fe]MAFC(alkyn)2, and [Fe]FSC(alkyn)3
The coupling reagents 1-hydroxy-7-azabenzotriazole (HOAt) and O-(7-azabenzotriazol-1-
yl)-N,N,N´,N´-tetramethyl uroniumhexafluorophosphate (HATU) were mixed with 4-pentynoic
acid in dimethylformamide (DMF) in a molar ratio of 4:4:3. The mixture with an excess of 1.5,
3, or 4.5 equiv of 4-pentynoic acid was transferred to 1 equiv of [Fe]DAFC (5.9 mg, 6.9 µmol),
[Fe]MAFC (14.5 mg, 17.7 µmol), or [Fe]FSC (9.9 mg, 12.7 µmol), respectively. N,N-
diisopropylethylamine (DIPEA) was added to adjust pH to 10–11, and the reaction mixture was
stirred at RT for 1 h. Hereafter solvent was reduced in vacuo, and crude product was then
purified by preparative RP-HPLC followed by lyophilization to obtain pentynoyl[Fe]DAFC
([Fe]DAFC(alkyn)), dipentynoyl[Fe]MAFC ([Fe]MAFC(alkyn)2), or tripentynoyl)[Fe]FSC
([Fe]FSC(alkyn)3). [Fe]DAFC(alkyn): 4.2 mg, 64% yield, tR = 8.63 min (gradient B), MALDI
TOF-MS: m/z [M + H]+ = 944.44 [C42H59FeN6O15; exact mass: 943.79 (calculated)].
[Fe]MAFC(alkyn)2: 10.13 mg, 58% yield, tR = 9.57 min (gradient B), MALDI TOF-MS: m/z [M
+ H]+ = 982.53 [C45H61FeN6O15; exact mass: 981.84 (calculated)]. [Fe]FSC(alkyn)3: 7.3 mg,
57% yield, tR = 10.78 min (gradient B), MALDI TOF-MS: m/z [M + Na]+ = 1042.65
[C48H63FeN6O15; exact mass: 1019.89 (calculated)].
2.5. Cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-)
Synthesis of cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-) was performed as described by
Haubner et al [22,23]. Briefly, cyclic pentapeptides with side chain protecting groups, cyclo(-
Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(Dde)-), was synthesized by cyclization of linear

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pentapeptides from Fmoc-based solid-phase peptide synthesis (SPPS). To obtain free amino
group at lysine for further conjugation, selective deprotection of Dde was carried out using 2%
hydrazine in DMF. Purifications were performed by trituration with H2O followed by preparative
RP-HPLC. Cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-): 146.7 mg, 74% yield (based on linear
pentapeptide), tR = 12.38 min (gradient B). MALDI TOF-MS: m/z [M + H]+ = 912.64
[C44H65N9O10S; exact mass: 912.10 (calculated)].
2.6. Cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(N3)-)
To cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys-) (30.6 mg, 33.6 µmol), the mixture of
HOAt (9.1 mg, 67.1 µmol), HATU (25.6 mg, 67.1 µmol), and 5-azidopentanoic acid (7.2 mg,
50.4 µmol) in DMF was added. The reaction mixture was alkalized to pH 10–11 using DIPEA
and stirred at RT for 30 min. Solvent was evaporated, and residual was separated by preparative
RP-HPLC. Cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(N3)-): 22.7 mg, 65% yield, tR = 14.86
min (gradient B). MALDI TOF-MS: m/z [M + H]+ = 1038.51 [C49H72N12O11S; exact mass:
1037.23 (calculated)].
2.7. Cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-)
Cyclo(-Arg(Pbf)-Gly-Asp(OtBu)-D-Phe-Lys(N3)-) (22.7 mg, 21.9 µmol) was dissolved in
the solution of TFA/H2O/isopropylsilane (38:1:1 v/v) and stirred at RT. After 4 h, the reaction
mixture was dried in vacuo, and crude product was obtained by trituration with diethyl ether.
Hereon purification was performed on preparative RP-HPLC. Cyclo(-Arg-Gly-Asp-D-Phe-
Lys(N3)-) (c(RGDfK)N3): 11.0 mg, 69% yield, tR = 8.79 min (gradient B). MALDI TOF-MS:
m/z [M + H]+ = 729.08 [C32H48N12O8; exact mass: 728.80 (calculated)].
2.8. DAFC(c(RGDfK)aza), MAFC(c(RGDfK)aza)2, and FSC(c(RGDfK)aza)3

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DMF solution of 1 equiv [Fe]DAFC(alkyn) (1.83 mg, 1.94 µmol), [Fe]MAFC(alkyn)2 (1.78
mg, 1.81 µmol), or [Fe]FSC(alkyn)3 (1.66 mg, 1.63 µmol) were mixed with 2, 4, or 6 equiv of
c(RGDfK)N3,
respectively.
A
freshly
prepared
mixture
of
tris(3-
.
hydroxypropyltriazolylmethyl)amine (THPTA) (10 equiv) and CuSO4 5H2O (4 equiv) in H2O
was added to the DMF mixture followed by sodium ascorbate (200 equiv) solution. After stirring
at RT for 2 h, 100 equiv of disodium ethylenediaminetetraacetic acid (Na2EDTA) solution (50
mM, pH 4) was added to remove iron from molecules, and the reaction mixture was stirred for a
further 18 h. After solvent removal, the crude product was purified by preparative RP-HPLC.
DAFC(c(RGDfK)aza): 1.2 mg, 37% yield, tR = 12.68 min (gradient A). MALDI TOF-MS: m/z
[M + H]+ = 1620.24 [C74H110N18O23; exact mass: 1619.77 (calculated)]. MAFC(c(RGDfK)aza)2:
1.3 mg, 30% yield, tR = 12.82 min (gradient A). MALDI TOF-MS: m/z [M + H]+ = 2386.83
[C109H160N30O31; exact mass: 2386.61 (calculated)]. FSC(c(RGDfK)aza)3: 1.59 mg, 31% yield, tR
= 9.15 min (gradient B). MALDI TOF-MS: m/z [M + Na]+ = 3177.13 [C144H210N42O39; exact
mass: 3153.46 (calculated)].
2.9. NODAGA-c(RGDfK) and FSC(suc-c(RGDfK))3
Compounds were synthesized as described by Knetsch et al [7,10].
2.10. 68Ga-Labeling
To 5 μg of peptide, 200 μL [68Ga]GaCl3 (approx. 35–40 MBq) in 0.1 M HCl (fractionated
elution) was added. The pH of solution was adjusted to 4.5 by adding 40 μL of 1.1 M sodium
acetate (NaOAc). The labeling mixture was allowed at RT for 10 min. Radiochemical yield (%
RCY)
of
[68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2,
and
[68Ga]GaFSC(c(RGDfK)aza)3 were determined using RP-HPLC and/or radio-ITLC.
2.11. natGa-Complexation

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Gallium-complexes of all conjugates were synthesized using the same protocol as described
68
for Ga-labeling whereby [68Ga]GaCl3 was replaced by 100-fold molar excess of GaBr3 in 200
µL of 0.1 N HCl.
2.12. Distribution coefficient (logD)
[68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2,
or
[68Ga]GaFSC(c(RGDfK)aza)3 (1 µM) in 500 µL phosphate buffered-saline (PBS) were mixed
with 500 µL octanol. The mixture was vortexed at 1400 rpm for 15 min and subsequently
centrifuged at 2000 rcf for 2 min. Aliquots of aqueous and octanol phases were separately
collected and measured in a gamma counter. The logD values were calculated from the obtained
data (n = 6).
2.13. Stability assay
Stabilities of all three conjugates were tested in PBS (pH 7.4) and fresh human serum after
0,
30,
60,
and
120
min
incubation.
[68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2, or [68Ga]GaFSC(c(RGDfK)aza)3 (50 µL, 2 nmol) were
incubated in 450 µL PBS or human serum at 37 °C. Hereon, aliquots of PBS were injected
directly to RP-HPLC while serum aliquots were mixed with MeOH, vortexed, and centrifuged at
20000 rcf for 2 min. Supernatant was diluted with H2O and subsequently analyzed by RP-HPLC.
The stability was presented as radiochemical purity (% RCP) of radiotracer (n = 3).
2.14. Protein binding assay
Protein binding abilities were carried out by incubating [68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2, and [68Ga]GaFSC(c(RGDfK)aza)3 in human serum at 37 °C for
30, 60, and 120 min. After incubation, 25 µL of serum aliquot was passed through a size
exclusion spin columns MicroSpinTM G-50 (GE Healthcare, Vienna, Austria) by centrifugation at

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2000 rcf for 2 min. Protein binding ability was determined by measuring activities of eluate
(protein bound) and column (non-protein bound) in a gamma counter. The results were displayed
as percentage of protein bound to total activity (n = 3).
2.15. Receptor binding affinity assay
The half maximal inhibitory concentration (IC50) of [natGa]DAFC(c(RGDfK)aza),
[natGa]MAFC(c(RGDfK)aza)2, [natGa]FSC(c(RGDfK)aza)3and a control cyclo(-Arg-Gly-Asp-D-
Tyr-Val-) (c(RGDyV)) for binding αvβ3 integrin were evaluated in human melanoma M21 (αvβ3-
positive) cells using [125I]c(RGDyV) as radioligand. [125I]c(RGDyV) was produced as described
previously [22]. M21 cells were washed twice with PBS (pH 7.4) and then suspended in binding
buffer (20 mM tris(hydroxymethyl)aminomethane (TRIS), 0.1% bovine serum albumin (BSA), 1
mM CaCl2, 1 mM MgCl2, 10 µM MnCl2, 150 mM NaCl, pH 7.3) at a concentration of 1 × 107
cells/mL. In a 96-well MultiScreen Filter Plates HTS (1 μm glass fiber filter, Merck Millipore,
Darmstadt, Germany), aliquots of 106 cells were added and incubated in increasing concentration
[0.001–1000 nM] of [natGa]-bound conjugates or c(RGDyV) in triplicate. After 5 min,
[125I]c(RGDyV) (approx. 50000 cpm) was added and cells were incubated at 37 °C for 2 h under
shaking condition (200 rpm). The incubation was interrupted by suction of the medium and the
plate was washed thrice with ice-cold binding buffer. The filters were collected and the
remaining activities were measured in a gamma counter. IC50 values were calculated using a
nonlinear curve fitting in OriginPro 6.1 software (Northampton, MA, USA).
2.16. Internalization assay
Cell suspensions were prepared at a concentration of 2 × 106 cells/mL for human melanoma
M21 (αvβ3-positive) as well as M21-L (αvβ3-negative) and 6 × 106 cells/0.2 mL for U87MG
cells, in RPMI 1640 (Gibco, Invitrogen Corporation, Paisley, UK) containing 1% glutamine, 1%

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BSA, 1 mM CaCl2, 1 mM MgCl2, and 10 µM MnCl2. Aliquots of 1 mL of M21 and M21-L cells
or 0.2 mL of U87MG were transferred to Eppendorf tubes in triplicate for each cell line with
either PBS/0.5% BSA (total series) or c(RGDyV) in PBS/0.5% BSA with a final concentration
of 10 µM (nonspecific series) and incubated at 37 °C for 1 h. After adding 50 µL
[68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2,
or
[68Ga]GaFSC(c(RGDfK)aza)3 to a final concentration of 10 nM, cells were incubated at 37 °C
for 90 min. Incubation was interrupted by centrifugation at 5000 rpm for 2 min, medium
removal, and rapid rinsing twice with ice-cold TRIS buffer (25 mM TRIS, 0.1% BSA, 1 mM
CaCl2, 1 mM MgCl2, 10 µM MnCl2, 150 mM NaCl, pH 7.3). Thereafter, cells were incubated
twice with acid wash buffer (20 mM sodium acetate buffer, pH 4.5) at 37 °C for 5 min to wash
out surface-bound molecules, and supernatant was collected. Subsequently, cells were lysed by
treatment twice with 2 M NaOH, and fractions were combined (internalized fraction). All
collected samples and standard solutions were counted by a gamma counter. Protein amount in
NaOH fraction was determined using spectrophotometric analysis with Bradford reagent (Sigma-
Aldrich). Internalized activity was expressed as percentage of total activity per milligram protein
(% cpm/mg protein).
2.17. Animal experiment
All animal experiments were conducted in accordance with regulations and guidelines of the
Czech Animal Protection Act (No. 246/1992), and with the approval of the Czech Ministry of
Education, Youth, and Sports (MSMT-16402/2012-30), and the institutional Animal Welfare
Committee of the Faculty of Medicine and Dentistry of Palacky University in Olomouc.
Female 8–10-week-old SCID mice (Envigo, Horst, The Netherlands) were used for in vivo
experiments. The number of animals was reduced as much as possible (n = 3 per group and time

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point) in all experiments. Tumor xenografts were established near the front shoulder of SCID
mice by subcutaneous injection of human glioblastoma U87MG (αvβ3-positive), 5 × 106 cells in
200 μL appropriate medium and Matrigel (1:1). The tumors were allowed to grow until 0.5 cm3.
The tracer injection and small animal imaging were carried out under 2% isoflurane anaesthesia
(FORANE, Abbott Laboratories, Abbott Park, IL, USA) to minimize animal suffering and to
prevent animal motion.
2.17.1. Biodistribution studies
To evaluate ex vivo biodistribution, a group of 3 tumor-bearing mice were retro-orbitally
(r.o.) injected with [68Ga]GaDAFC(c(RGDfK)aza), [68Ga]GaMAFC(c(RGDfK)aza)2,
[68Ga]GaFSC(c(RGDfK)aza)3,
[68Ga]GaFSC(suc-c(RGDfK))3,
or
[68Ga]GaNODAGA-
c(RGDfK) (1–2 MBq/mouse, 1 µg peptide). Animals were sacrificed by cervical dislocation at
30 or 90 min post-injection (p.i.) for all tracers except [68Ga]GaFSC(suc-c(RGDfK))3 which was
studied only for 90 min p.i. Organs and tissues (blood, spleen, pancreas, stomach, intestines,
kidneys, liver, heart, lung, muscle, femur, tumor) were collected, weighed, and measured in a
gamma counter. Results were expressed as percentage of injected dose per gram tissue (%ID/g).
2.17.2. MicroPET/CT imaging
MicroPET and CT images were acquired with an Albira PET/SPECT/CT small animal
imaging system (Bruker Biospin Corporation, Woodbridge, CT, USA). Xenograft-bearing mice
were injected r.o. with [68Ga]GaDAFC(c(RGDfK)aza), [68Ga]GaMAFC(c(RGDfK)aza)2, or
[68Ga]GaFSC(c(RGDfK)aza)3 in a dose of 5–8 MBq corresponding to 1–2 μg of peptide per
animal. Anaesthetized animals were placed in prone position in the Albira system before the start
of imaging. Static PET/CT images were acquired over 40 min starting 90 min after injection. A
10-min PET scan (axial FOV 148 mm) was performed, followed by a double CT scan (axial

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FOV 110 mm, 45 kVp, 400 μA, at 400 projections). Scans were reconstructed with the Albira
software (Bruker Biospin Corporation, Woodbridge, CT, USA) using the maximum likelihood
expectation maximization (MLEM) and filtered backprojection (FBP) algorithms. After
reconstruction, acquired data was viewed and analyzed with PMOD software (PMOD
Technologies Ltd., Zurich, Switzerland).
2.18. Statistical analysis
Differences in tumor uptake among the 5 c(RGDfK) peptides were analyzed using GraphPad
Prism 8.1.1 (GraphPad Software Inc.; La Jolla, USA) with a one-way ANOVA followed by a
Holm-Sidak post hoc test for multiple comparison. The level of significant was set to a P value
of less than 0.05.
3. Results
3.1. Peptide synthesisand coupling to the chelators
Synthesis of deprotected cyclo(-Arg-Gly-Asp-D-Phe-Lys(N3)-) (c(RGDfK)N3) using Fmoc
protocols, cyclization, deprotection of lysine side chain, azido introduction, and deprotection of
all protecting groups was carried out in good yield. Mono-, di-, and trimeric c(RGDfK) were
achieved from CuAAC of c(RGDfK)N3 and alkynic siderophores in moderate yields.
Straightforward synthesis of all conjugates is presented in Scheme 1.
3.2. 68Ga-Labeling
At the stated labeling conditions (pH 4.5; RT; 10 min incubation),
[68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2,
and
[68Ga]GaFSC(c(RGDfK)aza)3 were obtained in almost quantitative % RCP (> 99%) and used
without further purification.

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3.3. In vitro characterization
LogD values of all three conjugates revealed high hydrophilicity. Conjugates with less
c(RGDfK) motifs showed higher hydrophilicity as follows: [68Ga]GaDAFC(c(RGDfK)aza) (-
3.80 ± 0.06) > [68Ga]GaMAFC(c(RGDfK)aza)2 (-3.54 ± 0.11) > [68Ga]GaFSC(c(RGDfK)aza)3 (-
2.96 ± 0.21).
Stability studies in PBS (pH 7.4) and human serum at 37 °C revealed high stability for all
compounds in both media for up to 2 h (see Table 1).
Incubation of all [68Ga]-labeled c(RGDfK)aza in human serum exhibited low protein binding
between 3–9% of total activity. Compounds with less number of c(RGDfK) peptide showed
lower values ([68Ga]GaDAFC(c(RGDfK)aza)
[68Ga]GaFSC(c(RGDfK)aza)3) at any time point.
<
[68Ga]GaMAFC(c(RGDfK)aza)2
<
Binding affinities to αvβ3 integrin of metal bound mono-, di-, and trimeric c(RGDfK) conjugates
are shown in Fig. 1. IC50 values from displacement assays using M21 cells with increasing
concentration of the inhibitory peptides were 17.0 ± 0.8 nM for c(RGDyV) (control), 80.3 ± 2.0
nM for [natGa]DAFC(c(RGDfK)aza), 3.9 ± 1.2 nM for [natGa]MAFC(c(RGDfK)aza)2, and 1.7 ±
0.2 nM for [natGa]FSC(c(RGDfK)aza)3. These results revealed the enhancement of binding
affinity with increasing number of RGD binding motifs.
Results from In vitro internalization studies using M21 (αvβ3-positive) and M21-L (αvβ3-
negative) cells are presented in Fig. 2. The internalized activities (% cpm/mg protein) in M21
cells without blockade were extended with increasing number of c(RGDfK) moiety, 2.2 ± 0.3 for
[68Ga]GaDAFC(c(RGDfK)aza), 4.5 ± 0.8 for [68Ga]GaMAFC(c(RGDfK)aza)2, and 9.8 ± 1.0 for
[68Ga]GaFSC(c(RGDfK)aza)3. The corresponding activities in M21 cells with blockade and
M21-L cells with or without blockade were reduced to 0.2 to 0.7% confirming the receptor-

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specific internalization of all compounds. In U87MG cells, corresponding values were
considerably lower with 1.2 ± 0.1, 2.9 ± 0.3, and 2.6 ± 0.1, for [68Ga]GaDAFC(c(RGDfK)aza),
[68Ga]GaMAFC(c(RGDfK)aza)2, and [68Ga]GaFSC(c(RGDfK)aza)3, respectively, blocking
reduced the values to 0.9–1.4%. (see Supporting Information Fig. S1).
3.4. Biodistribution studies
Biodistribution data and tumor-to-tissue ratios of all three studied and two reference 68Ga-
labeled c(RGDfK) compounds in tumor-bearing SCID mice at 90 min p.i. are presented in Fig. 3
and Fig. 4, respectively. The results found in this study differed from in vitro evaluations. The
tumor uptake was as follows: [68Ga]GaMAFC(c(RGDfK)aza)2 > [68Ga]GaFSC(c(RGDfK)aza)3
>
[68Ga]GaDAFC(c(RGDfK)aza).
Among
the studied
compounds,
monomeric
[68Ga]GaDAFC(c(RGDfK)aza) showed the lowest tumor uptake (2.73 ± 0.28 %ID/g) as
expected. This value was 2-fold, but not significantly (P = 0.2), higher than that of the reference
[68Ga]GaNODAGA-c(RGDfK) (1.35 ± 0.39 %ID/g) while uptakes in all the rest organs were
comparable. Trimeric [68Ga]GaFSC(c(RGDfK)aza)3 revealed similar activity accumulations in
tumor (3.98 ± 0.64 %ID/g) and all other tissues, except spleen, relative to standard trimeric
[68Ga]GaFSC(suc-c(RGDfK))3
(4.95
±
1.10
%ID/g).
Surprisingly,
dimeric
[68Ga]GaMAFC(c(RGDfK)aza)2 exhibited unexpected high tumor uptake (8.19 ± 0.41 %ID/g),
which was significantly higher than that of the trimeric [68Ga]GaFSC(c(RGDfK)aza)3 (P =
0.003) and [68Ga]GaFSC(suc-c(RGDfK))3 (P = 0.02). All radiolabeled compounds showed fast
blood clearance (< 0.3 %ID/g) but high accumulation in kidneys. Highest uptake in each organ
was mostly found for [68Ga]GaFSC(c(RGDfK)aza)3. Statistical comparison of tumor uptakes
among the 5 conjugates is presented in Supporting Information, Fig. S2.

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Tumor-to-background ratios (Fig. 4), neglecting tumor-to-spleen ratios which were
comparable in all compounds and tumor-to-femur which was lowest in [68Ga]GaNODAGA-
c(RGDfK),
were
ranked
as
follows:
[68Ga]GaMAFC(c(RGDfK)aza)2
>
[68Ga]GaDAFC(c(RGDfK)aza)
>
[68Ga]GaFSC(suc-c(RGDfK))3
~
[68Ga]GaNODAGA-
c(RGDfK) ~ [68Ga]GaFSC(c(RGDfK)aza)3. The same trend was also observed at 30 min p.i.
(See. Supporting Information, Fig. S3). Moreover, [68Ga]GaMAFC(c(RGDfK)aza)2 revealed
excellent tumor-to-blood ratio (44.38
±
5.58) which was 2.7-fold higher than
[68Ga]GaFSC(c(RGDfK)aza)3 (16.56 ± 1.35). [68Ga]GaFSC(c(RGDfK)aza)3 showed the lowest
ratios in all organs due to its high retention especially in kidneys.
3.5. MicroPET/CT imaging
MicroPET/CT imaging was performed in SCID mice bearing U87MG tumor to compare the
image properties and in vivo pharmacokinetic of mono-, di-, and trimeric counterparts (Fig. 5).
The visualization intensities of all studied conjugates in tumor followed the order of:
[68Ga]GaMAFC(c(RGDfK)aza)2
[68Ga]GaDAFC(c(RGDfK)aza) while intensities in kidneys were as follows:
[68Ga]GaFSC(c(RGDfK)aza)3 [68Ga]GaMAFC(c(RGDfK)aza)2
>
[68Ga]GaFSC(c(RGDfK)aza)3
>
>
>
[68Ga]GaDAFC(c(RGDfK)aza). These results confirmed the tumor-targeting abilities and kidney
retentions found in ex vivo biodistribution studies (Fig. 3). Besides the tumor and kidneys,
urinary bladder showed intense visualization, confirming the main elimination route via renal
excretion.
4. Discussion

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The first 68Ga-labeled cyclic RGD compounds for PET/CT imaging of αvβ3 integrin
expression were [68Ga]GaDOTA-c(RGDfK) and [68Ga]GaNOTA-c(RGDyK) [6,24] showing the
potential to image angiogenesis. To improve image contrast by enhancing the binding affinity to
αvβ3 integrin, multivalent concepts have been applied by several research groups and showed an
enhanced localization of multimeric cyclic RGD peptides in αvβ3 integrin-expressing tumors
[13,14,25]. Most multivalent peptides were di- and tetrameric cyclic RGD peptides employing
DOTA and NOTA as BFCs. Comparison to the monomeric RGD counterpart, both di- and
tetrameric conjugates displayed improved tumor targeting due to their avidity effect, enhanced
statistical rebinding, and prolonged target retention from increased tracer concentration.
Nonetheless, dimeric c(RGD) was superior to tetrameric c(RGD) analogues in term of tumor-to-
tissue ratios due to the slower elimination from non-target tissues of the larger tetrameric
molecules [14,26].Recently, several groups have described the use of azide or alkyne derivatized
scaffolds based on the TRAP [27], DOTA [28], and HBED-CC [29], for multiple conjugation of
targeting moieties through CuAAC click chemistry, a method for highly selective reaction under
mild condition.
In this work, we successfully developed novel mono-, di-, and trimeric c(RGDfK) probes
based on the FSC scaffold applying CuAAC click chemistry as an alternative conjugation
strategy. We compared them with previously published conjugates, [68Ga]GaFSC(suc-
c(RGDfK))3 and [68Ga]GaNODAGA-c(RGDfK), and studied the effect of multivalency on
targeting characteristics.
Cyclic RDGfK was prepared and modified at the ɛ-amino function with 5-azidopentanoic
acid to obtain c(RGDfK)N3 containing an azide group for CuAAC. Terminal alkynyl moieties,
which are compatible with the azide, were introduced to [Fe]FSC, [Fe]MAFC, and [Fe]DAFC.

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The yields from conjugation of c(RGDfK)N3 with alkynyl[Fe]FSC or its derivatives via CuAAC
were almost quantitative and subsequent demetalation were about 85% as confirmed by HPLC
monitoring (see also Supporting Information Fig. S4). Finally, products FSC-based
c(RGDfK)aza were successfully obtained in 30-37% overall yields after purification and could
further be radiolabeled with 68Ga in quantitative yields. Losses were mainly due to the
demetalation and the final HPLC purification step, this could potentially be refined using
alternative strategies. Removing iron before conjugation of the peptides, as used with other
BFCs, Cu2+ complexation by the click product could a significant disadvantage. Using chelate
cages such as sulfur and cyanide for copper removal may result the cleavage of disulfide bonds
in biomolecules and toxicity issues, respectively. Recently, Notni et al. established a method for
removal of Cu2+ from TRAP-conjugates after click reaction by using NOTA as a transchelator
[30]. Using FSC and derivatives as iron-complex form, whereby the chelating moiety was fully
occupied by Fe3+, Cu2+ was unable to bind to the conjugation product. Additionally, we used an
excess amount of chelating ligand THPTA, which tightly bound to Cu2+/Cu+, to protect Cu+ from
oxygen. This ensured removal of excess of Cu2+ in one step together with removal of iron after
the CuAAC click reaction by means of EDTA, thereby simplifying this approach.
The logD values (-2.96 to -3.80) revealed a high hydrophilicity of all compounds that was
diminished
with
increasing
number
of
c(RGDfK)
moieties.
Monomeric
[68Ga]GaDAFC(c(RGDfK)aza) displayed a slightly higher hydrophilicity than standard
[68Ga]GaNODAGA-c(RGDfK)
[68Ga]GaFSC(c(RGDfK)aza)3
(-3.80
vs
-3.6) [7]
whereas
than the
trimeric
revealed
lower
hydrophilicity
reference
[68Ga]GaFSC(suc-c(RGDfK))3 (-2.96 vs -3.6) [15]. These deviations can be explained by the
hydrophilicity of the FSC scaffold and lipophilicity of the carbon chain and the 1H-1,2,3-triazole

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linker, respectively. Protein binding that increased slightly with larger number of c(RGDfK)
moieties was generally low with values between 3.3 to 8.9%. Stability tests in serum and PBS
demonstrated the stability of all conjugates up to 2 h, thus, indicating their suitability for imaging
over periods matching the half-live of gallium-68.
The determined IC50 values from displacement assays demonstrated the expected
enhancement of the binding affinity to αvβ3 integrin in the series mono-, di-, and trimeric
c(RGDfK), which is in line with findings using a NOTA-based scaffold [26]. This was
confirmed by internalization assays in αvβ3-positive M21 cells where the trimeric RGD
derivatives showed higher cell uptake than di- and monomeric analogues, respectively (Fig. 2).
Receptor-specificities of all conjugates were evidenced by low uptake values (0.2 to 0.7%) in
M21 cells with blockade and M21-L (αvβ3-negative) cells. However, in U87MG cells lower
values for the trimer were found with only 2.6% vs 2.9% for the dimer and 1.2% for the
monomer. The overall lower values, despite using higher numbers of U87MG cells as compared
to M21 cells, also confirms the reported lower receptor expression in this cell line [31].
In vivo behavior in SCID mice bearing αvβ3-positive U87MG glioblastoma xenograft
provided
expected
enhancement
of
tumor
accumulation
of
the
trimer
[68Ga]GaFSC(c(RGDfK)aza)3 compared to monomeric [68Ga]GaDAFC(c(RGDfK)aza) and
[68Ga]GaNODAGA-c(RGDfK), which was in line with our original findings with
[68Ga]GaFSC(suc-c(RGDfK))3. Nonetheless, for dimeric [68Ga]GaMAFC(c(RGDfK)aza)2 by far
the highest uptake in U87MG tumors was found with 8.19 ± 0.41 %ID/g. This trend was
confirmed by microPET/CT imaging. It is in contrast to the higher affinity to αvβ3-Integrin and
the more efficient binding affinity to M21 cells of the trimer. This observation could be
explained by the lower density of αvβ3-integrins on U87MG cells and was partly confirmed by in

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vitro binding results. It also should be noted that the increase in binding affinity between di- and
trimeric FSC-RGD conjugates was less pronounced than between monomer and dimer.
Likewise, Liu has proved that one of the most important factors for the increase of αvβ3 affinity
of multimeric cyclic RGD is bivalency [26,32]. Another reason for the better in vivo tumour
targeting
of
dimeric
[68Ga]GaMAFC(suc-c(RGDfK))2
over
trimeric
[68Ga]GaFSC(c(RGDfK)aza)3 could be related to differences in pharmacokinetics, i.e. better and
more rapid diffusion into tissue due to lower steric hindrance of a smaller molecule, lower
protein binding values, and lower lipophilicity, having a larger effect than the enhancement of
αvβ3 integrins binding affinity by additional RGD motifs.
In contrast, kidney uptake, blood and other major organ values increased as expected with
increasing multimerization and were very comparable for the respective monomers and trimers.
Exceptions were observed in spleen, which showed the highest accumulation for the dimer
[68Ga]GaMAFC(c(RGDfK)aza)2, as well as liver, intestine, and stomach, which exhibited
comparable uptakes for [68Ga]GaMAFC(c(RGDfK)aza)2 and [68Ga]GaFSC(c(RGDfK)aza)3.
These four organs are known to express αvβ3 integrin [9], supporting the higher targeting
efficiency of [68Ga]GaMAFC(c(RGDfK)aza)2. These improved targeting properties resulted in
the highest tumor-to-tissue ratios among all investigated compounds.
Also some other studies showed that multivalency does not always improve the in vivo tumor
uptake. Notni et al. [33] developed 68Ga-labeled prostate-specific membrane antigen (PSMA)
inhibitor conjugates, using TRAP as a chelator and scaffold. Gallium-68 labeled mono-, di-, and
trimeric lysine-urea-glutamic motifs (DBCO1, DBCO2, DBCO3) exhibited comparable tumor
uptake in imaging studies, even slightly higher for the dimer. The reduced tumor accumulation of
the trimer [68Ga]GaDBCO3 was explained by the influence of molar total administrated amount

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of biologically active compound. The authors concluded in their paper that molar activity had a
more pronounced influence than structural and in vitro parameters. The applied molar activity in
our study may therefore not have been optimal for all compounds.
Trimeric compounds showing lower tumor uptakes than dimeric counterpart were also
observed in our previous work with the FSC scaffold [20,34]. Multimeric minigastrin analogues
were prepared and tested in vitro and in vivo. At the studied time point, 1 h, dimeric conjugates
exhibited higher tumor accumulations than the trimeric conjugates in biodistribution studies. At
longer observation periods (2 h and 4 h) trimers revealed improved tumor uptake values. These
results may support our data on [68Ga]GaFSC(c(RGDfK)aza)3 at 90 min p.i., possibly not long
enough for optimal target accumulation.
In a recent comparison of 4 different di- and trimeric RGD-conjugates including
[68Ga]GaFSC(suc-c(RGDfK))3, the highest tumor targeting was found for trimeric compounds,
however a dimeric [68Ga]GaDOTA-E-[c(RGDfK)]2 revealed similar tumor-to-tissue ratios as
compared to its trimeric counterparts and the difference was more pronounced in a tumor model
with higher expression of αvβ3 integrins [11]. The potential effect of simultaneous binding of
more than one RGD unit to αvβ3 integrins by multimeric RGD tracers seemed to be unlikely due
to limited spacer length between the RGD units [26].
5. Conclusion
Mono- and multimeric RGD bioconjugates based on the FSC scaffold were directly accessible
applying click chemistry. The dimeric [68Ga]GaMAFC(c(RGDfK)aza)2 was found to be superior
to the trimeric [68Ga]GaFSC(c(RGDfK)aza)3 due to its higher tumor uptake and tumor-to-tissue

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ratios in vivo together with highest visualization intensity in PET/CT imaging. Although trimeric
[68Ga]GaFSC(c(RGDfK)aza)3 demonstrated the highest αvβ3-integrin affinity in vitro, its in vivo
tumor-to-tissue ratios were even poorer than the monomeric [68Ga]GaDAFC(c(RGDfK)aza.
Multivalency improved binding affinity from mono- to di- and mono- to tri-, but not from di- to
trimeric counterparts. Bivalency was dominant over the multivalency in this study. However,
different trends may be observed in other cases depending on the molecular structure, type of
BFC, and cyclic RGD arrangement. [68Ga]GaMAFC(c(RGDfK)aza)2 shows promising
characteristics for angiogenesis imaging, and it could be further applied to dual-targeting by
introducing an additional targeting moiety to the FSC scaffold.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors gratefully acknowledge the financial support of the Royal Thai Government
Scholarships (PhD scholarship to P. Kaeopookum), the Austrian Science Foundation (FWF;
grant P 25899-B23 to C. Decristoforo and Euregio SupErA IPN95-B30 to H. Haas), Ministry of
Education, Youth and Sports of the Czech Republic (LO1304; to M. Petrik) and European
Regional Development Fund - Project ENOCH (No. CZ.02.1.01/0.0/0.0/16_019/0000868; to M.
Petrik). We would like to thank Heinz Zoller and Nadja Baumgartner, Department of Internal
Medicine II, Medical University Innsbruck, for making MALDI-TOF analysis available and Leo
Gruber, Department of Radiology, Medical University Innsbruck for support in statistical
evaluation.

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Appendix A. Supplementary data
Fig. S1 showing the internalization studies in U87MG cell line at 90 min incubation
Fig. S2 comparing relative tracer uptake in U87MG tumor of all RGD peptides at 90 min p.i.
Fig. S3 showing the tumor-to-tissue ratios of [68Ga]Ga-c(RGDfK)aza in SCID mice bearing
U87MG tumor at 30 min p.i.
Fig. S4 showing HPLC monitoring of click reaction and iron removal of
[Fe]DAFC(c(RGDfK)aza)
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Table 1
Distribution coefficient (logD), protein binding, and stability in PBS (pH 7.4) and human serum
of mono-, di-, and trimeric [68Ga]Ga-c(RGDfK)aza.
Stability (% RCP)
Incubation
time (n = 3)
logD
(n = 6)
Protein binding (%)
[68Ga]Ga-c(RGDfK)aza
(n = 3)
PBS (n = 3)
serum (n = 3)
[68Ga]GaDAFC(c(RGDfK)aza)
-3.80 ± 0.06
30
60
4.6 ± 0.7
99.9 ± 0.0
99.6 ± 0.2
3.4 ± 0.0
99.8 ± 0.1
99.7 ± 0.1

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120
30
3.3 ± 0.8
99.6 ± 0.3
99.7 ± 0.1
99.9 ± 0.0
99.8 ± 0.1
99.7 ± 0.3
99.4 ± 0.4
99.5 ± 0.6
99.3 ± 0.6
99.8 ± 0.2
99.8 ± 0.2
99.5 ± 0.3
99.8 ± 0.2
99.7 ± 0.2
99.3 ± 0.7
[68Ga]GaMAFC(c(RGDfK)aza)2
[68Ga]GaFSC(c(RGDfK)aza)3
-3.54 ± 0.11
-2.96 ± 0.21
5.8 ± 0.7
6.2 ± 0.7
7.2 ± 0.9
8.3 ± 0.6
8.7 ± 0.8
8.9 ± 1.1
60
120
30
60
120