18F-FP-PEG2-β-Glu-RGD2: A symmetric integrin αvβ3-targeting radiotracer for tumor PET imaging

Article abstract of DOI:10.1371/journal.pone.0138675

Radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive determination of integrin α_vβ₃ expression in tumors. In this study, we performed radiosynthesis and biological evaluation of a new ¹⁸

Full text of DOI:10.1371/journal.pone.0138675

RESEARCH ARTICLE
18
F-FP-PEG -β-Glu-RGD : A Symmetric
2
2
Integrin α β -Targeting Radiotracer for
v 3
Tumor PET Imaging
1☯¤
2☯
1
1
1
Kongzhen Hu , Xiaolan Tang , Ganghua Tang *, Shaobo Yao , Baoguo Yao ,
1
1
1
1
1
Hongliang Wang , Dahong Nie *, Xiang Liang , Caihua Tang , Shanzhen He
1 Department of Nuclear Medicine, the First Affiliated Hospital, Sun Yat-Sen University, Guangzhou,
510080, China, 2 College of Materials and Energy, Southern China Agricultural University, Guangzhou,
510642, China
☯
These authors contributed equally to this work.
a11111
¤ Current address: Department of Chemistry, Stony Brook University, New York, 11794, United Stated of
America
*
gtang0224@126.com (GT); niedahong@126.com (DN)
Abstract
OPEN ACCESS
Radiolabeled cyclic arginine-glycine-aspartic (RGD) peptides can be used for noninvasive
determination of integrin α β expression in tumors. In this study, we performed radiosynth-
Citation: Hu K, Tang X, Tang G, Yao S, Yao B, Wang
v 3
18
18
2 2
H, et al. (2015) F-FP-PEG -β-Glu-RGD : A
esis and biological evaluation of a new F-labeled RGD homodimeric peptide with one 8-
Symmetric Integrin α -Targeting Radiotracer for
v
β
3
18
amino-3,6-dioxaoctanoic acid (PEG ) linker on the glutamate β-amino group ( F-FP-
2
Tumor PET Imaging. PLoS ONE 10(9): e0138675.
doi:10.1371/journal.pone.0138675
PEG -β-Glu-RGD ) as a symmetric PET tracer for tumor imaging. Biodistribution studies
2
2
1
8
showed that radioactivity of F-FP-PEG -β-Glu-RGD was rapidly cleared from blood by
2
2
Editor: Effie C Tsilibary, National Center for Scientific
Research Demokritos, GREECE
1
8
predominately renal excretion. MicroPET-CT imaging with F-FP-PEG -β-Glu-RGD2
2
revealed high tumor contrast and low background in A549 human lung adenocarcinoma-
bearing mouse models, PC-3 prostate cancer-bearing mouse models, and orthotopic trans-
Received: July 5, 2015
Accepted: September 2, 2015
Published: September 23, 2015
18
planted C6 brain glioma models. F-FP-PEG -β-Glu-RGD exhibited good stability in vitro
2
2
and in vivo. The results suggest that this tracer is a potential PET tracer for tumor imaging.
Copyright: © 2015 Hu et al. This is an open access
article distributed under the terms of the Creative
Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Introduction
Data Availability Statement: All relevant data are
within the paper.
Angiogenesis is the process of formation of new vessels in avascular tissue. It plays a key role in a
variety of processes such as rheumatoid arthritis [1], psoriasis [2], cardiovascular diseases [3],
diabetic retinopathy [4], and tumor growth, as well as tumor metastasis [5]. The angiogenic pro-
cess depends on vascular endothelial cells (ECs) migration and invasion [6]. Integrin αvβ3
involves in the migration of ECs during formation of new blood vessels and is highly expressed
only in activated ECs of tumor neovasculature, not in normal cells or quiescent ECs [7]. Mono-
meric cyclic arginine-glycine-aspartic (RGD) peptides and analogs showed high affinity and
Funding: This work was funded by the National
Natural Science Foundation (No. 81571704, No.
8
1371584, No.81201116), the Science Technology
Foundation of Guangdong Province (No.
014A020210008, No.2013B021800264), Science
and Technology Planning Project of Guangzhou (No.
011J5200025, No. 201510010145), the Wu Jieping
2
selectivity for the integrin α β [8]. Thus, several radiolabeled monomeric RGD peptides and
v 3
2
analogs have been developed for monitoring and quantifying integrin α β expression noninva-
sively in tumors with positron emission tomography (PET) [9–16]. For example, F-galacto-
Medical Foundation (320675013203), and Sun Yat-
Sen University (No. 80000-3126132).
v 3
1
8
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
1
8
Competing Interests: The authors have declared
that no competing interests exist.
RGD [11] and F-AH111585 [10] have been developed in clinical studies for PET imaging of
integrin α β . To further improve the binding affinity of peptide-based probes, multimeric RGD
v 3
peptides have been reported and shown that the dimeric RGD peptides as imaging agents, as
18
exemplified by F-FP-PRGD (Fig 1) for clinical PET imaging of integrin α β [9], are better
2
v 3
than monomeric, tetrameric and octameric RGD peptides [12–14]. Thus, many radiolabled
dimeric RGD peptides with asymmetric α-glutamate (Glu) linker group have been prepared and
evaluated as integrin α β -targeted radiotracers [14–16]. Symmetric linkers provided a conve-
v 3
18
nient method to synthesize dual-receptor-targeting tracers, such as F-FB-AEADP-BBN-RGD
AEADP = 3,3’-(2-aminoethy- lazanediyl)-dipropanoic acid] (Fig 1) [17]. However, as far as we
[
know, the radiolabeled symmetric cyclic RGD dimeric peptide as integrin α β -targeting radio-
v 3
tracers have not been reported yet.
We previously reported a symmetric β-glutamate linker Fmoc-β-Glu(OSu)-OSu for prepa-
1
8
ration of F-FP-PEG -β-Glu-RGD (Fig 1), showing Fmoc-β-Glu(OSu)-OSu as a good sym-
3
2
metric linker for coupling homodimers and heterodimers [18–20]. In this study, we designed a
1
8
new symmetric RGD homodimer peptide PEG -β-Glu-RGD , which was labeled with
F
2
2
+
+
(
97% β decay; maximum β energy = 0.64 MeV, t = 109.8 min) by conjugation with 4-nitro-
1/2
phenyl 2- F-fluoropropionate ( F-NFP). The symmetrical F-labeled PEG -β-Glu-RGD
1
8
18
18
2
2
1
8
(
F-FP-PEG -β-Glu-RGD ) (Fig 1) was then used for noninvasive imaging of integrin α β
2 2 v 3
expression in tumor-bearing animals to evaluate its usefulness.
Materials and Methods
Chemicals and Equipment
All chemicals purchased were of analytical grade and used without further purification unless
otherwise indicated. RGD was purchased from APeptide Co., Ltd (Shanghai, China). Mass
spectrometry (MS) was obtained on a Quattro/LC mass spectrometer by electro-spray ioniza-
tion. SEP-PAK light QMA and Oasis HLB cartridges were obtained from Waters Corporation
(
Milford, MA, USA). SEP-PAK light QMA cartridges were preconditioned with 5 mL
NaHCO aqueous (8.4%) and 10 mL water before use. Oasis HLB cartridges were precondi-
3
tioned with 10 mL ethanol and water before use. Analytical HPLC was performed using an
Agilent 1200 Series HPLC system equipped with a Easeatech AQ-C18 (4.6 × 150 mm, 5 μm;
Welch Materials, Inc) at the flow rate of 1 mL/min. The gradient program started from 98%
solvent A (0.1% trifluoroacetic acid in water): 2% solvent B (0.1% trifluoroacetic acid in
MeCN) ramped to 90% solvent A: 10% solvent B at 8 min, and ramped to 20% solvent A: 80%
solvent B at 20 min. The elution profile was detected with an ultraviolet detector (Agilent inter-
face 35900E, Agilent Technologies, USA) at 254 nm and a B-FC-3200 high energy PMT Detec-
tor (Bioscan. Washington DC, USA). Radioactivity was measured by a calibrated ion chamber
(
Capintec CRC-15R) or a gamma counter (γ-counter) (GC-1200, USTC Chuangxin Co. Ltd.
Zonkia Branch, China).
Synthesis of FP-PEG -β-Glu-RGD
2
2
The Fmoc-protected β-glutamic acid activated ester linker Fmoc-β-Glu(OSu)-OSu was pre-
pared according to Fig 2 as previously reported [19]. Fmoc-beta-Glu(OtBu)-OH (100 mg,
0
.235 mmol) was hydrolyzed with trifluoroacetic acid (TFA) and isolated with the HPLC sys-
tem to give Fmoc-beta-Glu(OH)-OH. After the collected fractions were combined and lyophi-
lized to give a powder, to a solution of Fmoc-beta-Glu(OH)-OH in 2 mL of N, N-
dimethylforamide (DMF) were added N-hydroxysuccinimide (HOSu) (0.126 mg, 1.10 mmol)
and dicyclohexylcarbodiimide (DCC) (0.226 mg, 1.10 mmol). The resulting mixture was stir-
red at room temperature for 10 h. The dicyclohexylurea by-product was filtered off. The filtrate
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
1
8
18
18
Fig 1. Chemical structures of F-PP-PRGD
2
,
2 2
F-FB-AEADP-BBN-RGD, F-FP-PEG -β-Glu-RGD , and Fmoc-β-Glu(OSu)-OSu.
doi:10.1371/journal.pone.0138675.g001
was evaporated to dryness under vacuum to give a crude product, which was then taken up in
mL of methylene chloride. The insoluble solid was filtered off. The filtrate was concentrated
3
to about 1 mL. The solution was added dropwise into 30 mL of ether. The desired product was
precipitated as white solid, which was dried in vacuo. The yield was 60% (79.4 mg, 0.141
+
1
mmol). MS (ESI, m/z): 564.2 ([M+H] ). H-NMR (CDCl ) δ (ppm): 7.22–7.82 (m, 8H, 9-fluor-
3
enyl aromatic hydrogen), 5.34 (s, 1H, NH), 4.86 (m, 1H, CH), 4.68 (d, 2H, COOCH ), 4.40 (m,
2
1
H, COOCH CH), 2.64 (m, 8H, succinimide group), 2.42 (d, 4H, CH CO).
2 2
Fig 2. Synthetic route of Fmoc-β-Glu(OSu)-OSu.
doi:10.1371/journal.pone.0138675.g002
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
The PEG -β-Glu-RGD homodimer peptide was synthesized by APeptide Co., Ltd (Shang-
2
2
hai, China) according to solid-phase peptide synthesis (SPPS) strategy as described in Fig 3.
+
MS (ESI, m/z): 1495.8 ([M+H] ) [19].
Synthesis of FP-PEG -β-Glu-RGD was performed by reaction of PEG -β-Glu-RGD with
2
2
2
2
4
4
6
-nitrophenyl 2-fluoropropionate (NFP). In brief, to a solution of anhydrous NFP (1 mg,
.7 μmol) in anhydrous DMSO (200 μL) was added PEG -β-Glu-RGD (1 mg, 0.67 μmol in
2
2
00 μL of DMSO) and DIPEA (40 μL). The reaction mixture was stirred for 2 h at 50°C. After
quenching with 0.5 mL 5% acetic acid, the final product was purified by preparative HPLC and
+
lyophilized to afford FP-PEG -β-Glu-RGD as a white powder. MS (ESI, m/z): 1570.4 ([M+H] ).
2
2
1
8
Radiosynthesis of F-FP-PEG -β-Glu-RGD
2
2
18
18
18
No-carrier-added F-fluoride was obtained through the nuclear reaction O (p, n) F by irra-
diation of a more than 95% O-enriched water target with a 10-MeV proton beam on the PET
trace cyclotron (IBA Technologies, Belgium). F-NFP was synthesized according to the
reported procedure [20,21]. To the dried F-NFP residue was added a solution of PEG -β-
18
18
18
2
Glu-RGD (100 μg) in 20 μL of DIPEA and 0.2 mL of anhydrous DMSO (Fig 4). The reaction
2
mixture was heated to 40°C for 5 min, and then was quenched with 0.5% acetic acid (1 mL)
and water (10 mL). The mixture solution was passed through an Oasis HLB cartridge and the
cartridge was washed with water (10 mL). The desired product was eluted from the cartridge
1
8
with 2 mL of ethanol. The solvent was removed by a stream of nitrogen at 50°C. The F-
labeled peptide was formulated in normal saline and passed through a 0.22 μm Millipore filter
into a sterile vial for experiments.
Cells and Animals
This study was carried out in strict accordance with the recommendations in the Guide for the
Care and Use of Laboratory Animals of the Ministry of Science and Technology of the People's
Republic of China. The experimental protocols were approved by the Committee on the Ethics
Fig 3. The route of PEG
2
-β-Glu-RGD
2
via solid-phase peptide synthesis (SPPS).
doi:10.1371/journal.pone.0138675.g003
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
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8
2 2
Fig 4. Radiosynthesis of F-FP-PEG -β-Glu-RGD .
doi:10.1371/journal.pone.0138675.g004
of Animal Experiments of the First Affiliated Hospital, Sun Yat-Sen University (Permit Num-
ber: [2012]001). All surgeries were performed under chloral hydrate anesthesia, and all efforts
were made to minimize suffering, to reduce the number of animals used, and to use alternatives
to in vivo techniques, if available.
Cell lines and mice were obtained from the Laboratory Animal Center of Sun Yat-Sen Uni-
versity. The cells were cultivated in RPMI 1640 medium with a physiologic glucose concentra-
tion (1.0 g/L) containing 5% fetal calf serum at 37°C in a humidified atmosphere of 5% CO2
and 95% air. In the study, 20 normal mice, 20 nude mice and 5 rats were used. Among them,
sixteen normal mice were used for biodistribution analysis, four normal mice were used for in
vivo metabolism, and twenty nude mice and five rats were used for making tumor-bearing
models. Mice or rats were housed 5 animals per cage under standard laboratory conditions at
2
5°C and 50% humidity. Every day mice and rats were observed for signs of ill health and no
animal death was found. Eight PC-3 tumor-bearing models were generated by subcutaneous
6
injection of 5 × 10 tumor cells into the right shoulder of male athymic nude mice. Twelve
A549 human lung adenocarcinoma-bearing models were generated by subcutaneous injection
6
of 2 × 10 tumor cells into the left shoulder of male athymic nude mice. Five orthotopic trans-
6
planted C6 brain glioma models were made by injection of 2 × 10 tumor cells into the brain of
rat. MicroPET-CT studies were performed on the mice 1–4 weeks after inoculation when the
tumor diameter reached 0.6–1.0 cm (3–4 weeks after inoculation for PC-3 models and C6
brain glioma models, and 1–2 weeks for A549 models).
Biodistribution Studies
18
For single-isotope ( F) biodistribution studies, sixteen normal Kunming mice or eight A549
lung adenocarcinoma-bearing nude mice were injected with 1.48–2.96 MBq (40–80 μCi) of
1
8
F-FP-PEG -β-Glu-RGD in 100–200 μL of saline through the tail vein. The mice were kept
2
2
anesthetized with 5% chloral hydrate solution after tracer administration. Radioactivity in the
syringe before and after administration was measured in a calibrated ion chamber. The animals
were sacrificed by cervical dislocation at various times after injection, blood was obtained
through the eyeball vein, the organs of interest (blood, brain, heart, lung, liver, kidney, pan-
18
creas, spleen, stomach, and intestine) were rapidly dissected and weighed, and F radioactivity
was counted with a γ-counter. All measurements were background-subtracted and decay-cor-
rected to the time of injection, then averaged together. Data were expressed as a percentage of
the injected dose per gram of tissue (%ID/g) (n = 4 per group).
In Vitro Stability and in Vivo Metabolism
18
For the in vitro experiment, a sample of F-FP-PEG -β-Glu-RGD (1.48 MBq, 10 μL) dis-
2
2
solved in normal saline was added to 200 μL of mouse serum and incubated at 37°C. An aliquot
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
of the serum sample was passed through a 0.22 μm Millipore filter and injected into a radio-
18
HPLC column to analyze the stability of F-FP-PEG -β-Glu-RGD in mouse serum within 2
2
2
h. The experiment was performed using 3 separate samples. The metabolic stability of
1
8
F-FP-PEG -β-Glu-RGD was evaluated in normal Kunming mice (n = 3). Each mouse was
2
2
18
injected with F-FP-PEG -β-Glu-RGD at dose of 3.7–14.8 MBq (100–400 μCi) in saline via a
2
2
tail vein. After 30 min post-injection, the urine was carefully collected and analyzed by radio-
HPLC.
MicroPET-CT Imaging
PET imaging of tumor-bearing mice was carried out using the Inveon small animal PET/com-
18
puted tomography (CT) scanner (Siemens). 3.7 MBq (100 μCi) of F-FP-PEG -β-Glu-RGD
2
2
was injected intravenously in conscious animals via the tail vein. A few minutes later the mice
were anesthetized with 5% chloral hydrate solution (6 mL/kg). Ten-minute static PET images
were acquired at four time points (30, 60, 90, and 120 min) postinjection. The images were
reconstructed by two-dimensional ordered-subset expectation maximum (OSEM). For the
integrin receptor-blocking experiment, RGD (4 mg/kg) was injected with 3.7 MBq of
1
8
F-FP-PEG -β-Glu-RGD into PC-3 tumor-bearing mice (n = 4). At 1 h after injection, the
2
2
1
0-min static microPET scans were acquired. For each microPET scan, regions of interest
(
ROIs) were drawn over the tumor, normal tissue, and major organs on decay-corrected
whole-body coronal images using Inevon Research Workplace 4.1 software. The maximum
radioactivity concentration (accumulation) within a tumor or an organ was obtained from
mean pixel values within the multiple ROI volume, which was converted to MBq/mL/min by
using a conversion factor. Assuming a tissue density of 1 g/mL, the ROIs were converted to
MBq/g/min and then divided by the administered activity to obtain an imaging ROI-derived %
ID/g. The mice were sacrificed at the end of the study for PET imaging.
Statistical Analysis
Data were expressed as mean ±SD. Statistical analysis was performed with SPSS software, ver-
sion 13.0 (SPSS Inc.), for Windows (Microsoft). A P value of less than 0.05 was considered to
indicate statistical significance.
Results
Chemistry and Radiochemistry
The symmetric RGD homodimer peptide PEG -β-Glu-RGD was synthesized by using Fmoc/
2
2
t-Bu-protected solid-phase peptide synthesis (Fig 3). Resin-cyclic(RGD) was coupled with
symmetric Fmoc-β-Glu(OSu)-OSu linker (Fig 2) and further reacted with cyclic(RGD) peptide
to afford resin-cyclic(RGD)-β-Glu(Fmoc)- cyclic(RGD). After removal of the Fmoc-group,
Boc-PEG -OH was attached to the free primary amine of the β-Glu group in resin-cyclic
2
(
RGD)-β-Glu-cyclic(RGD). The resin-peptide was released from the solid support and protect-
ing groups were removed by TFA treatment to afford homodimer peptide PEG -β-Glu-RGD .
2
2
PEG -β-Glu-RGD was isolated by semi-preparative HPLC and lyophilized. The purity and
2
2
identity of the final product was confirmed by HPLC and ESI-MS spectrometry [19].
1
8
18
F-Radiolabeling of PEG -β-Glu-RGD homodimer with F-NFP was performed in
2
2
DMSO for 5 min (Fig 4). The desired product was purified by an Oasis HLB cartridge, which
greatly reduced the total synthesis time. The decay-corrected radiochemical yield was 60 ± 10%
1
8
(
n = 8) from F-NFP with high radiochemical purity (> 95%). The total radiochemical yield
1
8
18
of F-FP-PEG -β-Glu-RGD was 18 ± 3% (n = 10, decay-uncorrected) based on F-fluoride
2
2
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
1
8
within 110 min and the specific radioactivity of F-FP-PEG -β-Glu-RGD was more than
1
2
2
1
8
14.0 GBq/μmol. The radiochemical purity of F-FP-PEG -β-Glu-RGD was over 95%.
2 2
In vivo Biodistribution
The biodistribution data of F-FP-PEG -β-Glu-RGD in normal Kunming mice are summa-
18
2
2
18
rized in Table 1. The radioactivity had fast clearance from blood. F-FP-PEG -β-Glu-RGD
2
2
cleared predominantly through the renal pathway as evidenced by high kidneys uptake and
rapid washout, with 14.0 ± 3.7%ID/g, 4.66 ± 1.23%ID/g, 2.23 ± 0.20%ID/g, and 0.73 ± 0.07%
ID/g at 5, 30, 60, and 120 min postinjection, respectively. The liver showed moderate uptake of
radioactivity and a relatively slow washout rate. Other tissues including brain, spleen, bone,
stomach, and muscle, showed relatively low uptake and rapid clearance of radioactivity.
1
8
F-FP-PEG -β-Glu-RGD showed the similar biodistribution and pharmacokinetics charac-
2
2
teristics to the previous reported RGD radiotracers [14,16].
2
18
Biodistribution of F-FP-PEG -β-Glu-RGD in A549 lung adenocarcinomamice- bearing
2
2
18
mice is shown in Table 2. F-FP-PEG -β-Glu-RGD had avid uptake (3.38 and 2.68% ID/g) in
2
2
A549 tumor at 30 and 60 min postinjection, respectively. The A549 tumor-to-muscle ratio (tar-
get-to-background ratio) decreased slightly from 3.60 at 30 min to 3.12 at 60 min postinjection.
Biodistribution studies also demonstrated high kidneys uptake and rapid washout. Bone uptake
was relatively low, suggesting no in vivo defluorination of the tracer.
Stability and Metabolism
18
Radio-HPLC analysis demonstrated that F-FP-PEG -β-Glu-RGD in serum was proven to be
2
2
1
8
stable. F-FP-PEG -β-Glu-RGD was kept more than 95% intact in mouse serum at 37°C for 2
2
2
1
8
h. The stability in vivo and fate of F-FP-PEG -β-Glu-RGD over 30 min was analyzed in
2
2
18
urine. There were almost no metabolites of F-FP-PEG -β-Glu-RGD detectable in urine, sug-
2
2
1
8
gesting that F-FP-PEG -β-Glu-RGD is metabolically stable during its excretion via the renal
2
2
route.
MicroPET-CT Imaging Study
18
MicroPET-CT imaging was carried out with F-FP-PEG -β-Glu-RGD in PC-3 prostate can-
2
2
cer-bearing mouse models (Fig 5A) and A549 human lung adenocarcinoma-bearing mouse
models (Fig 5B). The tumors were clearly visible with high contrast to the contralateral back-
ground at all time points measured from 30 to 120 min and had the highest uptake at 30 min
18
postinjection of F-FP-PEG -β-Glu-RGD . High renal uptake at early time points was found
2
2
and bladder accumulation was also observed, suggesting that the tracer is mainly excreted via
the renal-bladder route. Radioactivity accumulations in tumors and muscles were quantified
by measuring the ROIs encompassing the entire organ on the coronal PET images (Fig 5C).
18
The results demonstrated that F-FP-PEG -β-Glu-RGD had significantly higher uptake in
2
2
tumors than that in muscles and also had high tumor/muscle uptake ratios. In addition, the
18
tumor uptake of F-FP-PEG -β-Glu-RGD in PC-3 prostate tumor-bearing mice was higher
2
2
than that in A549 human lung adenocarcinoma-bearing mice (3.38 ± 0.44% ID/g vs.
.85 ± 0.35%ID/g, n = 4, P < 0.05) at 60 min postinjection (Fig 5C), possibly due to higher
2
level expression of integrin α β in PC-3 prostate cancer than that in A549 human lung
v 3
adenocarcinoma.
18
The integrin α β binding specificity of F-FP-PEG -β-Glu-RGD in PC-3 prostate tumor
v 3
2
2
was confirmed by blocking studies (Fig 6). As shown in Fig 6A and 6B, the tumor uptake of
1
8
F-FP-PEG -β-Glu-RGD at 1 h postinjection was significantly inhibited by co-injection of an
2
2
excess dose of RGD (3.38 ± 0.44%ID/g for no blocking vs. 1.82 ± 0.23%ID/g for blocking, n = 4
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
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8
a
Table 1. Biodistribution of F-FP-PEG
2
-β-Glu-RGD
2
in Normal Mice .
Organ
5min
30min
60min
120min
Blood
4.66 ± 0.57
1.71 ± 0.28
2.40 ± 0.45
2.80 ± 0.24
3.55 ± 1.12
2.20 ± 0.25
14.0 ± 3.7
1.65 ± 0.52
0.86 ± 0.23
1.07 ± 0.39
1.32 ± 0.58
2.01 ± 0.67
0.97 ± 0.39
4.66 ± 1.23
0.98 ± 0.19
1.18 ± 0.17
0.85 ± 0.25
0.92 ± 0.13
0.96 ± 0.37
1.25 ± 0.18
0.85 ± 0.25
0.66 ± 0.13
1.13 ± 0.45
1.79 ± 0.33
0.54 ± 0.28
2.23 ± 0.20
0.64 ± 0.07
1.17 ± 0.33
0.71 ± 0.24
0.86 ± 0.23
0.73 ± 0.07
0.48 ± 0.15
0.30 ± 0.08
0.34 ± 0.09
0.39 ± 0.15
0.81 ± 0.16
0.23 ± 0.06
0.73 ± 0.07
0.42 ± 0.15
0.91 ± 0.31
0.38 ± 0.18
0.71 ± 0.05
0.40 ± 0.06
Brain
Heart
Lung
Liver
Pancreas
Kidney
Spleen
Intestine
Muscle
Stomach
Bone
1.88 ± 0.68
2.03 ± 0.95
1.65 ± 0.75
1.37 ± 0.41
1.68 ± 0.28
a
Means ± SD (n = 4). Data are average % ID/g.
doi:10.1371/journal.pone.0138675.t001
per group, P < 0.05), which is obvious evidence that there is high expression of integrin αvβ3
receptor in the tumor.
PET images of orthotopic-transplanted brain glioma rat models are shown in Fig 7. A low
1
8
uptake of F-FP-PEG -β-Glu-RGD was detected in normal brain tissue, but a high uptake in
2
2
18
brain glioma tissue was observed. At 60 min post-injection of F-FP-PEG -β-Glu-RGD ,
2
2
uptake ratio of glioma tissue to white matter and glioma tissue to gray matter was 2.63 ± 0.53%
1
8
ID/g and 1.61 ± 0.32%ID/g respectively, which were a little higher than those of F-FDG
2.24 ± 0.42%ID/g and 1.45 ± 0.21%ID/g, n = 5, P> 0.05, respectively). The results demonstrate
(
1
8
that F-FP-PEG -β-Glu-RGD is a useful PET tracer for brain cancer imaging.
2
2
a
Table 2. Biodistribution of 18F-FP-PEG
2 2
-β-Glu-RGD in Nude Mice Bearing A549 Xenografts .
Organ
30min
60min
Blood
2.12 ± 0.55
1.62 ± 0.08
1.99 ± 0.19
2.04 ± 0.12
3.14 ± 0.04
1.48 ± 0.14
4.63 ± 1.04
2.55 ± 0.15
2.49 ± 0.34
0.94 ± 0.18
1.61 ± 0.04
1.17 ± 0.21
3.38 ± 0.23
3.60
1.59 ± 0.57
1.41 ± 0.14
1.47 ± 0.03
1.65 ± 0.10
2.35 ± 0.26
1.15 ± 0.08
2.66 ± 0.25
1.72 ± 0.13
1.64 ± 0.13
0.86 ± 0.06
1.22 ± 0.21
0.88 ± 0.10
2.68 ± 0.25
3.12
Brain
Heart
Lung
Liver
Pancreas
Kidney
Spleen
Intestine
Muscle
Stomach
Bone
Tumor
Tumor to Muscle ratio
a
Means ± SD (n = 4). Data are average % ID/g.
doi:10.1371/journal.pone.0138675.t002
PLOS ONE | DOI:10.1371/journal.pone.0138675 September 23, 2015
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
Fig 5. Small animal PET imaging and quantification. (A) MicroPET-CT imaging of PC-3 prostate tumor-
1
8
bearing mice at 30, 60, 90, and 120 min after injection of F-FP-PEG
2 2
-β-Glu-RGD . (The red arrows
indicated PC-3 prostate tumor). (B) MicroPET-CT imaging of A549 human lung adenocarcinoma-bearing
1
8
mice at 30, 60, 90, and 120 min after injection of F-FP-PEG
2 2
-β-Glu-RGD . (The red arrows indicated A549
lung adenocarcinoma). (C) Time-activity curves of PC-3 prostate tumor, A549 lung adenocarcinoma and
1
8
2 2
muscle after intravenous injection of F-FP-PEG -β-Glu-RGD .
doi:10.1371/journal.pone.0138675.g005
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
1
8
Fig 6. Tumor uptake of F-FP-PEG
2
-β-Glu-RGD
2
was inhibited by co-injection of an excess dose of RGD. (A) MicroPET-CT images of PC-3 prostate
1
8
tumor-bearing mice without/with a blocking dose of RGD (4 mg/kg body weight) at 60 min after injection of F-FP-PEG
indicated PC-3 prostate tumor). (B) The tumor uptake of F-FP-PEG -β-Glu-RGD in PC-3 prostate tumor and PC-3 prostate tumor treated with cold RGD
2 2
-β-Glu-RGD . (The red arrows
1
8
2 2
blocking at 60 min after injection (mean ±SD, n = 4 per group).
doi:10.1371/journal.pone.0138675.g006
Discussion
Based on our previous works [18–20], we used a symmetric β-glutamate linker Fmoc-β-Glu
18
(
OSu)-OSu to prepare the symmetric F-FP-PEG -β-Glu-RGD peptide as an integrin α β -
2 2 v 3
targeting PET radiotracer. β-Glutamic acid was a substrate for glutamine synthetase in biosyn-
thesis [22,23]. The symmetric PEG -β-Glu linker was used to improve in vivo pharmacokinetic
n
properties of drugs [24]. In our work, the Fmoc-β-Glu(OSu)-OSu linker was comprised of one
Fmoc-protecting amino group and two activated carboxylic acid ester groups. The protruding
1
8
free PEG-amino group in PEG -β-Glu-RGD encountered less steric hindrance for F-labeling
2
2
1
8
with F-NFP than that in PEG-α-Glu-RGD [9,16]. Compared with the asymmetric α-glu-
2
tamic acid activated ester group (α-glutamate linker), the symmetric β-glutamic acid activated
ester group (β-glutamate linker) could be used to prepare symmetric homodimer peptides by
conjugating with the free amino groups of the peptides via forming amide bonds. Also, the
symmetric β-glutamate linker was easy to prepare only one heterodimer product with definite
pure compound structure due to the symmetric nature of the linker. In comparison with the
1
8
existing tracer F-FP-PRGD , having the overall uncorrected radiochemical yield of 10–15%
2
18 −
(
n = 5) starting from F and the measured specific activity of 114 ± 72 GBq/μmol within a
total synthesis time of around 3 h [25], F-FP-PEG -β-Glu-RGD gave a little high radiochem-
18
2
2
18
ical yield (18 ± 3%, decay-uncorrected) based on F-fluoride and specific radioactivity of more
than 114.0 GBq/μmol within a short total synthesis time of 110 min. In addition, the symmetric
Fmoc-β-Glu(OSu)-OSu linker could exhibit definite non-toxicity and could be easily prepared
without tedious synthesis, compared with the symmetric AEADP linker, which was recently
used for coupling peptide heterodimer [17]. Therefore, the Fmoc-β-Glu(OSu)-OSu linker pro-
vides a general method for fast assembly of various peptide homodimers and heterodimers.
1
8
F-FP-PRGD is a potential asymmetric RGD homodimer PET tracer, recently approved
2
by the FDA (IND 104150) for a first-in-human test [9,14]. In the RGD dimer the close two
cyclic RGD motifs may lead to the enhanced integrin α β binding rate or the reduced dissocia-
v 3
tion rate of the cyclic RGD peptide from the integrin α β [8]. Similar structure to asymmetric
v 3
PLOS ONE | DOI:10.1371/journal.pone.0138675 September 23, 2015
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
Fig 7. MicroPET-CT fused images of orthotopic transplanted C6 brain glioma model rats at 60 min
1
8
18
2 2
post-injection of F-FDG and F-FP-PEG -β-Glu-RGD . (The red arrows indicate the tumor).
doi:10.1371/journal.pone.0138675.g007
1
8
18
F-FP-PRGD [14,15], F-FP-PEG -β-Glu-RGD with symmetric β-glutamate linker instead
2
2
1
2
8
of asymmetric α-glutamate linker of F-FP-PRGD does not affect the binding affinity of
2
RGD homodimer to integrin α β . Especially, the two RGD motifs of the symmetric RGD
2
v 3
18
dimer peptide ( F-FP-PEG -β-Glu-RGD ) are equally binding to the integrin α β receptor
2
2
v 3
and may significantly increase “local RGD concentration” of the receptor-binding site. Thus,
1
8
symmetric F-FP-PEG -β-Glu-RGD should possess better binding affinity to integrin α β
v 3
2
2
18
and higher accumulation in tumors than F-FP-PRGD ; but this needs to be further investi-
2
gated. The in vivo blocking experiment demonstrated that a coinjection of excess amounts of
18
RGD significantly inhibited uptake of F-FP-PEG -β-Glu-RGD in the PC-3 tumor, confirm-
2
2
ing the integrin α β -binding specificity in vivo. In addition, comparison of the PET imaging
v 3
1
8
18
results of F-FP-PRGD and F-FP-PEG -β-Glu-RGD revealed that they had comparable
2
2
2
tumor uptake and non-specific tissue uptake at the different tumor-bearing models [14].
18
The biodistribution results showed high uptake of F-FP-PEG -β-Glu-RGD in the kidneys
2
2
and in tumors (PC-3 prostate tumor and A549 human lung adenocarcinoma), low radioactiv-
ity accumulation in other organs, and rapid clearance from the body, which were similar to the
1
8
observed biodistribution results of F-FP-PRGD in U87MG tumor [14]. The in vivo biodistri-
2
18
bution results of F-FP-PEG -β-Glu-RGD were also correlated well with the microPET data
of model mice. Also, F-FP-PEG -β-Glu-RGD exhibited excellent stability in vitro and in
2
2
18
2
2
PLOS ONE | DOI:10.1371/journal.pone.0138675 September 23, 2015
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A Symmetric Integrin αvβ3-Targeting Radiotracer for Tumor PET Imaging
18
vivo. No defluorination of F-FP-PEG -β-Glu-RGD was observed, as no visible bone uptake
2
2
was found in mice biodistribution and in microPET imaging of model mice. These observa-
tions were consistent with other RGD homodimer radiotracers reported previously [14–16]. In
addition, PET images of orthotopic transplanted glioma models showed low uptake of
1
8
F-FP-PEG -β-Glu-RGD in normal brain tissue and high uptake in glioma tissue. The results
2
2
18
demonstrate that F-FP-PEG -β-Glu-RGD can be a potential tracer for PET imaging of cere-
2
2
bral and peripheral tumors.
Conclusion
In this study, we successfully designed and synthesized a novel integrin α β receptor-targeting
v 3
symmetric homodimeric peptide, PEG -β-Glu-RGD , using a symmetric linker Fmoc-β-Glu
2
2
1
8
(
OSu)-OSu. F-FP-PEG -β-Glu-RGD showed high tumor uptake and good tumor-to-back-
2 2
ground contrast in PC-3, A549 and C6 brain glioma tumor models. The symmetric homodi-
mer tracer is easy to prepare and has high specificity in tumor models, which makes it a
promising PET tracer for tumor angiogenesis imaging.
Acknowledgments
We thank Kan Du, Liangjun Rao, Xinchong Shi and Qingqiang Tu for assistance with this
work.
Author Contributions
Conceived and designed the experiments: KH GT XT. Performed the experiments: KH XT GT
SY HW XL BY SH. Analyzed the data: KH GT XT. Contributed reagents/materials/analysis
tools: KH DN GT CT. Wrote the paper: KH GT XT.
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