In vivo MR/optical imaging for gastrin releasing peptide receptor of prostate cancer tumor using Gd-TTDA-NP-BN-Cy5.5

Article abstract of DOI:10.1016/j.bmc.2010.04.040

Magnetic resonance imaging (MRI) has become the leading imaging tool for providing fine anatomical and physiology details. Optical imaging is offering a sensitive and specific method for in vivo molecular imaging of targeting molecules. The goal of this s

Full text of DOI:10.1016/j.bmc.2010.04.040

Bioorganic & Medicinal Chemistry 19 (2011) 1085–1096
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
In vivo MR/optical imaging for gastrin releasing peptide receptor
of prostate cancer tumor using Gd-TTDA-NP-BN-Cy5.5
Ying-Hsiu Lin a, , Kasala Dayananda a, , Chiao-Yun Chen b,c, Gin-Chung Liu b,c, Tsai-Yueh Luo d,
Hui-Sheng Hsu e, Yun-Ming Wang a,
*
a Department of Biological Science and Technology, National Chiao Tung University, 75 Bo-Ai Street, Hsinchu 300, Taiwan
b Department of Medical Imaging, Kaohsiung Medical University Hospital, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan
c Department of Radiology, Kaohsiung Medical University, 100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan
d Isotope Application Division, Institute of Nuclear Energy Research, 1000 Wenhua Road, longtan, Taoyuan County 325, Taiwan
e Graduate Institute of Medicine College of Medicine and Department of Medical Imaging, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University,
100 Shih-Chuan 1st Road, Kaohsiung 807, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Magnetic resonance imaging (MRI) has become the leading imaging tool for providing fine anatomical and
physiology details. Optical imaging is offering a sensitive and specific method for in vivo molecular imag-
ing of targeting molecules. The goal of this study is to design, synthesize, and characterize a new target-
specific dual contrast agent for MR and optical imaging. Hence, [Gd(TTDA-NP)(H2O)]2ꢀ was prepared and
characterized. In addition, an 8-amino acid Bombesin analogue (BN) peptide substrate, which can target
prostate, breast, and colon cancer, was synthesized by solid-phase peptide synthesis and subsequently
Received 25 February 2010
Revised 13 April 2010
Accepted 15 April 2010
Available online 21 April 2010
Keywords:
conjugated with [Gd(TTDA-NP)(H2O)]2ꢀ to form BN conjugated Gd-TTDA-NP-BN. The water-exchange
Magnetic resonance imaging
Gastrin releasing peptide (GRP) receptor
Bombesin
298
rate ðk Þ for [Gd(TTDA-NP)(H2O)]2ꢀ (110 ꢁ 106 sꢀ1) is significantly higher than that of [Gd(DTPA)
ex
(H2O)]2ꢀ complex and the rotational correlation time (
s
R) for [Gd(TTDA-NP)(H2O)]2ꢀ (145 ps) is also
higher than those of [Gd(TTDA)(H2O)]2ꢀ (104 ps) and [Gd(DTPA)(H2O)]2ꢀ (103 ps). The Gd-TTDA-NP-BN
shows remarkable high relaxivity (7.12 mMꢀ1 sꢀ1) comparing to that of [Gd(TTDA-NP)(H2O)]2-. The fluo-
rescence studies showed that the Gd-TTDA-NP-BN could efficiently enter PC-3 cells. Additionally, the
human cancer cells xenografts using Gd-TTDA-NP-BN-Cy5.5 as an optical imaging probe clearly visualized
subcutaneous PC-3 tumor and demonstrated its targeting ability to the gastrin releasing peptide (GRP)
receptor overexpression. Furthermore, the biodistribution studies demonstrated significantly high tumor
uptake (25.97 1.07% ID/g) and high tumor-to-normal tissue ratios at one hour post-injection of Gd-
TTDA-NP-BN-Cy5.5 in the animal model. These results suggest that the Gd-TTDA-NP-BN-Cy5.5 is a supe-
rior probe for in vivo optical imaging. Importantly, the MR imaging studies showed notable signal
enhancement (44.9 4.2%) on the tumor, indicating a high level accumulation of the contrast agent within
the PC-3 tumor sites. Hence, targeting of prostate cancer cells was observed under in vitro and in vivo MR
imaging studies using Gd-TTDA-NP-BN contrast agent. We conclude that Gd-TTDA-NP-BN and Gd-TTDA-
NP-BN-Cy5.5 can be potentially used as the contrast agents for targeting GRP receptor overexpressing cells
and tumors.
Peptide
Fluorescence
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
requiring long scan times as well as from a finite half-life and
radiation dosage preventing longitudinal imaging in patients.9 Mag-
Molecular imaging is providing a sensitive and specific method
for the detection and localization of the biochemical appearance
in vivo.1 Recently, some progress in tumor-targeted imaging by
positron emission tomography (PET)2–4 has been made with the
assist of Bombesin complexes labeled with radioactive isotopes.5–8
However, radionuclide imaging may suffer from low photon counts
netic resonance imaging (MRI) offers several advantages over other
clinical diagnostic techniques in molecular imaging, including high
spatial resolution, noninvasiveness, high anatomical contrast, and
lack of harmful radiation.10–12 On the other hand, sensitivity of
MRI in depicting a small molecule is constrained by the ubiquitous
protons in the body, resulting in a high background and low signal
to noise ratio (SNR). Hence, the alternative amplification strategies
using smart contrast agents are required. In addition, noninvasive
imaging protease activity is very important for personalized diagno-
sis and therapy in treating diseases.9,13
* Corresponding author. Tel.: +886 3 5721212x56972; fax: +886 3 5729288.
E-mail address: ymwang@mail.nctu.edu.tw (Y.-M. Wang).
These two authors contributed equally.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.04.040

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Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
Over the past few years, optical imaging techniques have been
2. Results and discussion
established as powerful tools for in vivo molecular imaging of
specific target structures. Although poor light penetration through
tissues makes their utilization in human difficult, these techniques
have been effectively used in preclinical small animal research.14,15
Near-infrared (NIR) fluorescence imaging, which uses neither ion-
izing radiation nor radioactive materials, is emerging as a comple-
ment to nuclear imaging methods. The cyanine dye 5.5 (Cy5.5), a
NIR fluorescent dye has proven to be a promising in vivo probe.
It has an absorbance maximum at 675 nm and emission maximum
at 694 nm and can be detected in vivo at subnanomolar quantities
and at depths sufficient for experimental and clinical imaging.16,17
Bombesin (BN) is a small peptide containing 14 amino acids (Glu-
Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2). It
was originally isolated from the skin of amphibian Bombina oriental-
is.18,19 The BN-like peptides have very high binding affinity for the
gastrin releasing peptide (GRP) receptor and produce a wide range
of biological responses in peripheral tissues as well as in the central
nervous system, including stimulation of gastrointestinal hormone
release, exocrine secretion, and maintenance of circadian
rhythms.20,21 In addition, it has been documented that the BN pep-
tides promote proliferation in cancer cells.22 The overexpression of
GRP receptor has been demonstrated in a large number of tumors,
including prostate, breast, colon, and small cell lung cancer.23–25 In
the past few years, the highly GRP receptor overexpressing tumor
is emerging as an attractive target for both cancer therapy and early
detection and Bombesin or its analogues labeled with different
radionuclides had been widely used for tumor imaging.26,27
2.1. Synthesis of Gd-TTDA-NP-BN and Gd-TTDA-NP-BN-Cy5.5
In this study, we reported the Gd-TTDA-NP-BN and Gd-TTDA-
NP-BN-Cy5.5 complexes for molecular MR imaging and optical
imaging. Scheme 1 shows the schematic representation of the syn-
thesis of Gd-TTDA-NP-BN, Gd-TTDA-NP-BN-Cy5.5, and Eu-TTDA-
NP-BN. The ligand TTDA-NP-BN was synthesized in four steps. In
the first step, 3,6,10-triazadodecane-3,10-tetraacetic acid (tetra)-
tert-butyl ester (TTDA-4est) was treated with 2-hydroxy-5-nitro-
benzyl bromide in the presence of anhydrous K2CO3 in CH3CN at
80 °C for 20 h under nitrogen atmosphere. The resulting product
(TTDA-NP) was purified by silica gel column chromatography. Sub-
sequently, the hydrogenation was performed in the presence of Pd/
C catalyst in methanol at room temperature. The product (TTDA-
NP-NH2) was purified by column chromatography. In the next step,
it was required to convert the aromatic amine to a more reactive
isothiocyanate functional group for peptide conjugation. TTDA-
NP-NH2 was treated with thiophosgene in THF at 60 °C for over-
night. The crude product (TTDA-NP-NCS) was purified by column
chromatography. Finally, the Bombesin analogue (BN) (Lys-Gly-
Gly-Gly-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) peptide was con-
jugated with TTDA-NP-NCS ligand, to obtain TTDA-NP-BN after
preparative HPLC purification. The ESI mass spectrum shown in
Figure S1 (see Supplementary data) confirms its identity. The Gd-
TTDA-NP-BN complex was obtained by complexation of Gd(III)
ion with TTDA-NP-BN ligand in water at room temperature for
18 h. The resulting complex was identified by MS (ESI), as shown
in Figure S2. The Gd-TTDA-NP-BN-Cy5.5 was achieved through
conjugation of NHS-Cy5.5 ester with amino group of lysine residue
of the Gd-TTDA-NP-BN, purified by preparative HPLC, and identi-
fied by MS (ESI).
Thermodynamically stable, water-soluble, and highly paramag-
netic Gd(III) complexes, each bearing a multidentate ligand and at
least one coordinated water molecule, have demonstrated high
relaxivity and have therefore been served as versatile MR imaging
contrast agents. Recently, our group and other research groups
have developed asymmetry poly(aminocarboxylates) derivatives
of
3,6,10-tri(carboxymethyl)-3,6,10-triazadodecanedioic
acid
2.2. 17O NMR study
(TTDA) and their Gd(III) complexes have shown to possess fast
water-exchange rates.28–32 Although, the water-residence lifetime
has a minor influence on relaxivity of small molecular contrast
agents but it has a significant influence on relaxivity of macromo-
lecular contrast agents.33,34 The ligand TTDA was chosen because
the water-residence lifetimes for [Gd(TTDA)(H2O)]2ꢀ and its deriv-
atives are significantly lower than that of [Gd(DTPA)(H2O)]2ꢀ
(DTPA = 3,6,9-tri(carboxymethyl)-3,6,9-triazaundecanedioic acid)
and has reached the optimal value. Recently, Bombesin conjugated
[Gd(DOTA)(H2O)]ꢀ derivative as a MRI contrast agent.35 However,
the detailed MR imaging study has not yet reported. In this regard,
we synthesized and characterized a new contrast agent for optical
and MR imaging studies. The (N-(1-methylene(p-isohiocyanatoph-
enol)di(carboxymethyl) triazadodecanedioic acid (TTDA-NP) was
conjugated with Bombesin analogue (Lys-Gly-Gly-Gly-Gln-Trp-
Ala-Val-Gly-His-Leu-Met-NH2) peptide (TTDA-NP-BN), BN conju-
gated to target GRP receptor expressing tumor cells. The synthetic
strategy of Gd-TTDA-NP-BN was depicted in Scheme 1. Subse-
quently, cyanine dye was conjugated to Gd-TTDA-NP-BN complex
for optical imaging studies. We also evaluated the cell cytotoxicity
of Gd-TTDA-NP-BN to PC-3 cells which is a human prostate cancer
cell line with overexpressing gastrin releasing peptide (GRP) recep-
tors and KB cells, human nasopharyngeal epidermal carcinoma cell
line, lacking of the expression of GRP receptors. In addition, in vitro
assays were tested using PC-3 cells to evaluate the cellular uptake
and cellular retention. In vitro and in vivo optical imagings were
studied to demonstrate the targeting ability toward GRP receptor
overexpression cells and tumors. Furthermore, in vitro and
in vivo T1-weighted MR imaging studies of cells and tumor-bearing
mice were also investigated.
Kinetic parameters were determined for [Gd(TTDA-NP)(H2O)]2ꢀ
by variable-temperature 17O NMR studies. The chemical shifts
(Dxr), the longitudinal (1/T1r), and transverse (1/T2r) relaxation
rates were analyzed simultaneously (using Eqs. 1S–9S in the Supple-
mentary data). The data for [Gd(TTDA-NP)(H2O)]2ꢀ are plotted in
Figure S3 and the corresponding curves represent the results of
the best fitting of the data according to Eqs. (1S–9S). As shown in
the Table S1, the large number of parameters is influencing the data
obtained by the different techniques. However, the 17O NMR tech-
nique has an advantage that the outer-sphere contributions to the
relaxation rates are negligibly small, which is a result of the oxygen
nucleus being closer to the paramagnetic center when it is bound in
the inner sphere. The scalar coupling constant (A/⁄) of [Gd(TTDA-
NP)(H2O)]2ꢀ is very similar to those which are obtained for other
Gd(III) complexes (ꢀ3.2 0.3 and ꢀ3.8 0.2 ꢁ 106 rad sꢀ1 for [Gd
(TTDA)(H2O)]2ꢀ and [Gd(DTPA)(H2O)]2ꢀ, respectively) with one in-
ner-sphere water molecule. In the overall temperature range, the
transverse 17O relaxation rates (1/T2r) decrease with an increasing
temperature, indicating that this system is in the fast exchange re-
gime.36 Hence, 1/T2r is determined by the relaxation rate of the coor-
dinated water molecule (1/T2m), which is influenced by the water-
residence lifetime (sM = 1/kex), the longitudinal electronic relaxation
rate (1/T1e), and the scalar coupling constant (A/⁄). The k298
ex
(110 13 ꢁ 106 sꢀ1) value of [Gd(TTDA-NP)(H2O)]2ꢀ is similar to
those of other [Gd(TTDA)(H2O)]2ꢀ derivatives, which is significantly
higher than that of [Gd(DTPA)(H2O)]2ꢀ ðke2x98 ¼ 4:1 ꢂ 0:3 ꢁ 106 sꢀ1Þ.
Moreover, the value of correlation time (sR) for Gd(III) complex with
TTDA-NP was obtained at 298 K from the fitting of 17O NMR. The sR

Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
1087
ButOOC
ButOOC
COOtBu
COOtBu
ButOOC
COOtBu
COOtBu
1) 2-Hydroxy-5-nitrobenzyl bromide,
N
N
H
N
N
N
N
º
K2CO3/CH3CN, 80
C
ButOOC
2) H2/Pd, CH3OH
HO
NH2
1
2
ButOOC
ButOOC
HO
COOtBu
COOtBu
BN(P)
R
N
N
N
ButOOC
ButOOC
COOtBu
COOtBu
Thiophosgene,
THF
N
N
N
DMSO
NH
BN(P)
N
R
HO
NCS
H
S
4
3
HOOC
HOOC
COOH
COOH
1.GdCl3
O
O
1. 94.5%TFA,
2.5%EDT
2.5%H2O,
1%TIS
N
N
N
O
O
O
O
N
N
N
Gd
HO
NH
O
O
O
NH
S
S
N BNH
H
NH
BNH
5
6
EuCl3
Cy5.5-NHS
O
O
O
O
O
O
O
O
O
O
O
O
N
N
Eu
N
N
N
Gd
N
O
O
O
O
O
NH
S
O
NH
S
NH
BN
NH
BN
Cy5.5
7
8
Scheme 1. Synthetic scheme of Gd-TTDA-NP-BN, Gd-TTDA-NP-BN-Cy5.5, and Eu-TTDA-NP-BN.
Table 1
value of [Gd(TTDA-NP)(H2O)]2ꢀ (145 ps) is higher than those of
[Gd(TTDA)(H2O)]2ꢀ (104 ps)37 and [Gd(DTPA)(H2O)]2ꢀ (103 ps).38
Relaxivity (r1) of [Gd(TTDA-NP)(H2O)]2ꢀ, [Gd-TTDA-NP-BN] [Gd(TTDA)(H2O)]2ꢀ, and
[Gd(DTPA)(H2O)]2ꢀ, at 37.0 0.1 °C and 20 MHz
Complex
pH
Relaxivity r1/mMꢀ1sꢀ1
2.3. Luminescence study
[Gd(TTDA-NP)(H2O)]2ꢀ
[Gd-TTDA-NP-BN]
7.4 0.1
7.4 0.1
7.5 0.1
7.6 0.1
4.16 0.08
7.12 0.05
3.85 0.03
3.89 0.03
[Gd(TTDA)(H2O)]2ꢀa
[Gd(DTPA)(H2O)]2ꢀb
To determine the number of inner sphere water by luminescence
method,39–41 the [Eu(TTDA-NP)(H2O)]2ꢀ complex was synthesized.
The luminescence lifetime (s) has been measured in both water
a
Data was obtained from Ref. 37.
Ref. 42.
and deuterium oxide upon excitation at 395 nm. The [Eu(TTDA-
NP)(H2O)]2ꢀ complex contains 0.92 and 0.72 inner-sphere water
molecules calculated by using (Eqs. 1 and 2) as follows
b
NP)(H2O)]2ꢀ conjugated with BN peptide reduces the tumbling rate
and enhances its relaxivity. It is known that [Gd(TTDA-NP)(H2O)]2ꢀ
has one inner-sphere water molecule and exhibits higher water-
exchange rate than those of [Gd(DTPA)(H2O)]2ꢀ derivatives.42
Significantly, the relaxivity of [Gd(TTDA-NP)(H2O)]2ꢀ complex is
slightly higher than those of [Gd(TTDA)(H2O)]2ꢀ and [Gd(DTPA)-
q ¼ 1:05½1=sH O ꢀ 1=sD Oꢃ
ð1Þ
ð2Þ
2
2
q ¼ ð½1=sH O ꢀ 1=sD Oꢃ ꢀ 0:25Þ ꢁ 1:2
2
2
where q is the number of water molecules bound to metal ions, sH2O
luminescence half-life in water solution and sD2O is the lumines-
cence half-life in deuterium oxide solution, respectively. The q value
is obtained from the luminescence study.
(H2O)]2ꢀ
.
2.4. Relaxivity
2.5. Cell cytotoxicity
In order to obtain the relaxivity of Gd(III) complex, we mea-
sured the proton spin-lattice relaxation time for [Gd(TTDA-
NP)(H2O)]2ꢀ and Gd-TTDA-NP-BN complexes at 37.0 0.1 °C and
20 MHz. The relaxivity (r1) values were shown in Table 1. The
Gd-TTDA-NP-BN exhibits substantially higher longitudinal relaxiv-
In vitro cell cytotoxicity is an important parameter for Gd-TTDA-
NP-BN complex to apply to molecular imaging. In this study, the
MTT assay was performed to evaluate cytotoxicity of Gd(III)
complex against PC-3 and KB cells. The various concentrations of
Gd(III) complex were incubated with PC-3 and KB cell lines, after
24 h the cell viability was examined. Figure 1 showed that more than
ity than that of [Gd(TTDA-NP)(H2O)]2ꢀ
, because [Gd(TTDA-

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90% cell viability was observed after 24 h of incubation under high
concentration of Gd-TTDA-NP-BN complex. These results indicated
that the Gd-TTDA-NP-BN exhibited low cytotoxicity.
2.6. In vitro optical imaging
In this study, Eu-TTDA-NP-BN was used to evaluate the specific-
ity of Gd-TTDA-NP-BN to PC-3 cells using fluorescence microscopy.
The Eu-TTDA-NP-BN complex was incubated with PC-3 cells at 4.0
and 37.0 °C. The specific tumor cell uptake of the Bombesin ana-
logue conjugated complex (Eu-TTDA-NP-BN) was visualized under
fluorescence microscopy. Figure 2A–C showed that the fluores-
cence image of PC-3 cells incubated with or without of Eu-TTDA-
NP-BN, at 4.0 and 37.0 °C, respectively. These results indicated that
the PC-3 cells had produced strong fluorescence intensity at
37.0 °C. In contrast, the PC-3 cells have shown weak fluorescence
at 4.0 °C, because the complex only binds to cell membrane and
cannot enter into cells at low temperature. Hence, the cellular up-
take of Eu-TTDA-NP-BN was high at the physiological temperature,
comparing to at low temperature.
Additionally, we synthesized the near-infrared dye conjugated
with Gd(III) complex (Gd-TTDA-NP-BN-Cy5.5) and carried out an
in vitro study to demonstrate the targeting of probe to GRP recep-
tor. In order to assess the binding specificity of Gd-TTDA-NP-BN-
Cy5.5 to GRP receptor, the complex was incubated with PC-3 and
KB cells. The fluorescence images were studied, as shown in Figure
3A and B. Figure 3A shows the red fluorescence originated from
targeting of probe to PC-3 cells and the cell image with nucleus
stained with blue DAPI dye was overlay with Cy5.5 fluorescence
image. The resulted fluorescence images clearly demonstrate that
the Gd(III) complex interacts only with cells, but not with nucleus.
In addition, the PC-3 cells had displayed strong fluorescence. These
data confirmed that the Gd-TTDA-NP-BN-Cy5.5 complex could be
specifically target to PC-3 cells. In contrast, KB cells did not exhibit
red fluorescence as shown in Figure 3B because of no GRP receptor
overexpression. These results imply that Gd-TTDA-NP-BN-Cy5.5
could efficiently bind to GRP receptor.
2.7. In vivo optical imaging
The in vivo optical imaging studies were performed in PC-3
tumor, which is GRP receptor overexpressed. The solution of
Gd-TTDA-NP-BN-Cy5.5 complex (10 nmol) was injected to nude
mice bearing PC-3 and KB tumors through tail vein. Figure 4A–C
illustrated in vivo optical images of mice at 0.5 h after injection
of Gd-TTDA-NP-BN-Cy5.5 by white light, NIR fluorescence, and
color mapping, respectively. The NIR imaging demonstrates the
Figure 2. Fluorescence microscope imaging (A) PC-3 Cells incubated without Eu-
TTDA-NP-BN, (B) PC-3 cells incubated with Eu-TTDA-NP-BN at 4.0 °C, and (C) PC-3
cells incubated with Eu-TTDA-NP-BN at 37.0 °C.
uptake of Gd-TTDA-NP-BN-Cy5.5 in the region of the tumor. As
shown in Figure 4B, a strong fluorescence enhancement was ob-
served for PC-3 tumor cells. In contrast, no obvious fluorescence
enhancement was found for negative KB tumor owing to lack of
GRP receptor expression. These results indicate that the Gd-
TTDA-NP-BN-Cy5.5 has the ability to target PC-3 tumor in vivo.
Furthermore, optical imaging was conducted at predetermined
time intervals from 0.5 to 24.0 h after injection, as shown in Figure
5A and B. The subcutaneous PC-3 tumor could be clearly visualized
Figure 1. Cytotoxicity of Gd-TTDA-NP-BN incubated with KB cells (black) and PC-3
cells (gray) for 24 h. Concentration dependence (100–1000
lM) of MTT assay graph
observed in vitro cell viability after incubating of Gd-TTDA-NP-BN.

Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
1089
Figure 3. Fluorescence microscopy assays of Gd-TTDA-NP-BN-Cy5.5 binding to GRP receptor. The signals of Gd-TTDA-NP-BN-Cy5.5 (10
lM) and DAPI are displayed in red
and blue, respectively.
Figure 4. Mice bearing human prostate cancer xenograft (PC-3 cells) and human nasopharyngeal cancer xenograft (KB cells) were injected Gd-TTDA-NP-BN-Cy5.5 (10 nmol).
Optical images showed results 0.5 h after injection. (A) White light, (B) NIR fluorescence, (C) Color mapping of B.
from the surrounding background tissue from 0.5 to 24.0 h post-
injection, whereas the negative tumor could not be visualized at
all time points. In addition, the quantification analysis of these
images was performed. The fluorescence intensities of the tumors
and the tissues as a function of time were represented in Figure 5C.
The tumor uptake reached to a maximum in 0.5 h and slowly ex-
hausted over time. Thus, we proved that the Gd-TTDA-NP-BN
had high degree of specificity for GRP receptor overexpressing
tumor.
2.8. Biodistribution study
To investigate the biodistribution of Gd-TTDA-NP-BN-Cy5.5
complex, the mice bearing PC-3 and KB tumors were sacrificed
after optical imaging at one hour post-injection of Gd-TTDA-NP-
BN-Cy5.5. Tumors and other organs were separated and subjected
to optical imaging. The fluorescence images of the whole organ and
tumors were obtained, as shown in Figure 6A, and % ID/g values
were calculated for each organ, as illustrated in Figure 6B. The

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Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
Figure 5. (A) In vivo fluorescence images of subcutaneous PC-3 and KB tumors bearing nude mice after intravenous injection of Gd-TTDA-NP-BN (10 nmol). Images were
acquired at 0.5, 1.0, 4.0, and 24 h post-injection. (B) Color mapping of A, (C) Fluorescence intensity in the region of the tumor as a function of time with Gd-TTDA-NP-BN-
Cy5.5.
major uptake of Gd-TTDA-NP-BN-Cy5.5 was observed in tumor,
kidney, and stomach. The biodistribution data obtained at one hour
post-injection indicated that the uptake of PC-3 tumor was signif-
icantly higher than that of KB tumor. On the other hand, the bone
showed minor uptake which may be the release of free Gd(III) ion
from the complex. The peak percentage per dose gram (% ID/g) val-
ues for PC-3 tumor, bladder, and stomach were 25.97 1.07,
16.94 2.15, and 16.26 1.87, respectively. Tumor-to-blood, tu-
mor-to-muscle, tumor-to-kidney, and tumor-to-liver ratios are
illustrated in Figure 6C. These results are consistent with the opti-
cal imaging. It clearly demonstrates that the Gd-TTDA-NP-BN-
Cy5.5 is capable of targeting GRP receptor on the PC-3 tumor.
2.9. In vitro MR imaging
To demonstrate the potential utility of the Bombesin analogue
conjugated [Gd(TTDA-NP)(H2O)]2ꢀ, the in vitro MR imaging studies
were conducted with PC-3 and KB cells using a 3.0-T MR imaging

Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
1091
Figure 6. (A) Representative images of dissected organs of mice bearing PC-3 and KB tumors sacrificed one hour after intravenous injection of Gd-TTDA-NP-BN-Cy5.5 at a
dose of 10 nmol equivalent Cy5.5/mice. (1) liver, (2) lung, (3) heart, (4) blood, (5) PC-3 tumor, (6) KB tumor, (7) muscle, (8) pancreas, (9) spleen, (10) bladder, (11) intestines,
(12) stomach, (13) bone, (14) skin, and (15) kidney, (B) Biodistribution of the Gd(III) complex at one hour post-injection, (C) Tumor-to-normal tissue ratios.
PC-3 cells were 2.8 2.4, 32.0 2.6, 68.7 4.1, and 21.6 2.8, respec-
tively. The PC-3 cells showed remarkable signal enhancement com-
scanner. Figure 7A–E showed the in vitro T1-weighted MR images
of cells without contrast agent or with [Gd(DTPA)(H2O)]2ꢀ
[Gd(TTDA-NP)(H2O)]2ꢀ
Gd-TTDA-NP-BN, and Gd-TTDA-NP-BN
,
paring to KB cells, among which Gd-TTDA-NP-BN showed
a
,
maxima of 68.7% enhancement. These in vitro MR imaging results
indicated that Gd-TTDA-NP-BN could efficiently target to GRP recep-
tor overexpressing PC-3 cells. The [Gd(DTPA)(H2O)]2ꢀ and [Gd(TTDA-
NP)(H2O)]2ꢀ did not show significant signal enhancement on the PC-
3 and KB cells, because these contrast agents were not conjugated
with BN peptide. In addition, the competitive study for Gd-TTDA-
NP-BN with fourfold excess of BN peptide substrate was performed.
Bombesin analogue peptide and Gd-TTDA-NP-BN competed on bind-
with fourfold excess of BN peptide, respectively. The enhancement
(%) was calculated by the following (Eq. 3):
SIpost ꢀ SIpre
Enhancementð%Þ ¼
ꢁ 100%
ð3Þ
SIpre
where SIpre is the value of signal intensity for cells untreated with the
contrast agent and SIpost is the value of signal intensity for cells trea-
ted with the contrast agent. The signal enhancement (%) values for

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Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
NP-BN, and the T1-weighted images were performed over
0.4–24 h. Figure 8 shows the comparison of the T1-weighted fast
spin-echo images of mice bearing tumor of pre- (Fig. 8 top) and
post- (Fig. 8 bottom) intravenous injection of Gd-TTDA-NP-BN,
[Gd(TTDA-NP)(H2O)]2ꢀ, and Gd-TTDA-NP-BN with fourfold excess
of BN peptide, respectively. The enhancement (%) was calculated
by the following (Eq. 4):
ðSIpost=SIp:postÞ ꢀ ðSIpre ꢀ SIp:pre
Þ
Enhancementð%Þ ¼
ꢁ 100%
ð4Þ
ðSIpre=SIp:pre
Þ
where SIpost is the signal intensity after injection, SIp.post is the signal
intensity of the phantom after injection, SIpre is the signal intensity
before injection, and SIp.pre is the signal intensity of the phantom be-
fore injection. The region-of-interest analysis over the PC-3 and KB
tumors at 24 min post-injection of Gd-TTDA-NP-BN indicated that
the signal enhancement percentages were 44.9 4.2 and
10.45 1.8, respectively. These results showed a significant signal
enhancement on the PC-3 tumors. However, no remarkable signal
enhancement changes on the KB tumors were observed. The
Gd-TTDA-NP-BN with fourfold excess of BN had shown significant
lower signal enhancement than that of Gd-TTDA-NP-BN. Conversely,
no notable signal enhancement change for [Gd(TTDA-NP)(H2O)]2ꢀ
was observed. It clearly demonstrates that Gd-TTDA-NP-BN complex
has the ability to target GRP receptor on the PC-3 tumors. Further-
more, the targeting ability of Gd-TTDA-NP-BN was also proven by
comparing the signal enhancement (%) of Gd-TTDA-NP-BN as a
function of time with those of [Gd(TTDA-NP)(H2O]2ꢀ and Gd-TTDA-
NP-BN with fourfold excess of BN peptide, as shown in Figure 9A–C.
These results imply that the signal enhancement of Gd-TTDA-NP-
BN is steady over an hour and then starts to decrease. On the other
hand, the Gd[(TTDA-NP)(H2O)]2ꢀ and Gd-TTDA-NP-BN with fourfold
excess of BN did not showed any changes on their signal enhance-
ment over 24 h. Moreover, the signal enhancements (%) of in vivo
MR imaging are in good agreement with in vitro MR imaging. Hence,
the Gd-TTDA-NP-BN is capable of targeting the GRP receptor of the
PC-3 tumor.
Figure 7. (1) The in vitro T1-weighted MR images of PC-3 and KB cell after the
treatment of (A) without contrast agent, (B) [Gd(DTPA)(H2O)]2ꢀ, (C) [Gd(TTDA-
NP)(H2O)]2ꢀ, (D) Gd-NP-TTDA-BN, (E) Gd-TTDA-NP-BN with fourfold excess of BN,
(2) Color mapping of (1).
ing to GRP receptor, and the signal enhancement was drastically re-
duced because of only small amount of Gd-TTDA-NP-BN uptake by
PC-3 cells. These results showed that Gd-TTDA-NP-BN had significant
high signal enhancement over other non-BN conjugated Gd(III)
complexes.
2.10. In vivo MR imaging
The in vivo targeting ability of Gd-TTDA-NP-BN complex was
examined with the mice bearing PC-3 and KB tumors. The mice
bearing tumors (n = 5) were prepared by subcutaneous injection
of the PC-3 and KB cells into the left and right lateral thighs,
respectively. The MR imaging on mice was studied at predeter-
mined time intervals after the intravenous injection of Gd-TTDA-
3. Conclusion
In summary, we successfully synthesized Bombesin analogue
conjugated Gd(III) complex for molecular optical and MR imaging.
A significant increase on relaxivity (r1) for Gd-TTDA-NP-BN was
Figure 8. The in vivo T1-weighted MR images of mice bearing PC-3 (right) and KB (left) xenografts pre- and post-injection of Gd-TTDA-NP-BN (0.1 mmol/kg), MR imaging was
performed with a clinical 3.0-T MR scanner and animal coil, scanned by a Fast gradient echo pulse sequence (TR/TE/flip angel = 800/12/10°).

Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
1093
thiazol-2-yl]-2,5-diphenyltetrazoplium bromide (MTT), and
phosphate buffered saline were purchased from Sigma–Aldrich.
3,6,10-Triazadodecane-3,10-tetraacetic acid (tetra)-tert-butyl ester
(TTDA-4est) (1) was prepared using a previously published meth-
od.28,43 tert-Butylbromoacetate was synthesized according to the
reported procedure.44 Fluorescence and luminescence properties
were measured using Varian Cary Eclipse fluorescence spectropho-
tometer and 1H (400 MHz), 13C (100 MHz), and 17O (54.2 MHz) NMR
spectra were recorded on a Varian Gemini-400 spectrometer. The
HPLC experiments were performed on an Amersham ÄKTAbasic 10
instrument equipped with an Amersham UV-900 detector and
Amersham Frac-920 fraction collector. Supelco RP-C18 column
(5
l
m, 4.6 ꢁ 250 mm and 5 m, 10 ꢁ 250 mm) was used. LC-mass
l
spectral analyses were performed with Waters Micromass-ZQ, mass
spectrometry. The concentration of Gd(III) complex was determined
by ICP-MS with a Perkin–Elmer OPTIMA 2000.
4.2. Synthesis
4.2.1. Synthesis of TTDA-NP (2)
TTDA-4-est (1) (11.19 g, 19.5 mmol) and K2CO3 (5.39 g,
39.0 mmol) were suspended in anhydrous CH3CN(100 mL) and stir-
red for 20 min. Then the reaction mixture was treated with 2-hydro-
xy-5-nitrobenzyl bromide (5.72 g, 23.4 mmol) and refluxed at 80 °C
for 24 h under nitrogen atmosphere. The reaction was cooled to
room temperature and then the salt was filtered. The filtrate was
concentrated under the reduced pressure. The resulting product
was purified by silica gel column chromatography (hexane/ethyl
acetate = 3:1), to give TTDA-NP-NO2 (11.78 g, 69.2%). MS(ESI): m/z
calad: 725.88, found: 725.65 [M+H]+. 1H NMR (400 MHz, CDCl3): d
1.43 (m, 36H, –C–(CH3)3), 1.80 (b, 2H, –CH2–), 2.72 (q, 4H, –CH2–),
3.00 (t, J = 6.0 Hz, 4H, –CH2–), 3.38 (s, 8H, –CH2–), 4.04 (b, 2H,
–CH2–), 8.00 (s, 1H, Ar-H), 8.10 (dd, 2H, Ar-H).
Subsequently, the Pd/C (240 mg) was added to the stirring
solution of TTDA-NP-4 ester (9.03 g, 12.4 mmol) in CH3CN (50 mL)
under 1.5 atm H2. This step was repeated until the pressure decreas-
ing was stopped. Then the solvent was removed under reduced pres-
sure. The product was purified by column chromatography (SiO2,
Figure 9. Plots of signal enhancements (%) of the tumor as a function of time with
dichloromethane/methanol = 19:1), to give
2 (TTDA-NP-NH2)
(A) Gd-TTDA-NP-BN, (B) Gd-TTDA-NP-BN with fourfold excess of BN, (C) [Gd(TTDA-
(7.67 g, 88.7%). 1H NMR (400 MHz, CDCl3): d 1.44 (m, 36H,–
C(CH3)3), 1.74 (m, 2H,–CH2–), 2.68 (q, 4H,–CH2–), 2.94 (t, J = 6.5 Hz,
4H,–CH2–), 3.37 (s, 8H,–CH2–), 3.86 (b, 2H,–CH2–), 6.40 (s, 1H, Ar-
H), 6.54 (d, 2H, Ar-H).13C NMR (100 MHz, CDCl3): d 170.6, 170.5,
168.9, 166.4, 150.4, 138.4, 116.5, 81.6, 81.2, 81.0, 56.0, 55.8, 55.7,
51.9, 51.5, 51.4, 28.1.
NP)(H2O)]2ꢀ
.
observed. The cell cytotoxicity results showed that the Gd-TTDA-
NP-BN complex had low cytotoxic. Furthermore, in vitro optical
imaging results reveal that this Gd(III) complex efficiently targets
to PC-3 tumor cells. The biodistribution studies further showed a
high level accumulation of the Gd-TTDA-NP-BN-Cy5.5 complex
on the GRP receptor overexpression tumor cells. In addition, Gd-
TTDA-NP-BN complex has the ability to target PC-3 tumor cells,
as proven by in vitro and in vivo MR imaging studies. Thus, the
Gd-TTDA-NP-BN-Cy5.5 can be potentially used as a dual contrast
agent for optical/MR imaging of prostate cancer.
4.2.2. Synthesis of TTDA-NP-NCS (3)
TTDA-NP-NH2 (2) (5.36 g, 7.87 mmol) was dissolved in THF
(40 mL) and the resulting reaction mixture was cooled in an ice
bath and then thiophosgene (1.9 mL, 25.97 mmol) was slowly
added under nitrogen atmosphere. The stirring was continued for
3 h and then the solvent was removed under the reduced pressure.
Purification by chromatography (silica, dichloromethane/metha-
nol = 19:1), produces a yellow oil substance 3 (2.24 g, 38.7%). 1H
NMR (400 MHz, CDCl3): d 1.45 (m, 36H,–C–(CH3)3), 1.92 (b, 2H,–
CH2–), 2.79 (b, 4H,–CH2–), 3.00 (b, 4H,–CH2–), 3.36 (s, 8H,–CH2–),
3.87 (b, 2H,–CH2–), 7.03 (s, 1H, Ar-H), 7.11 (d, 2H, Ar-H).13C NMR
(100 MHz, CDCl3): d 170.5, 170.4, 157.4, 126.5, 122.7, 121.6,
117.4, 81.3, 81.1, 56.0, 55.8, 55.7, 51.8, 51.2, 47.5, 28.1.
4. Experimental
4.1. General methods
All commercially available chemical reagents were used without
further purification. Silica gel, palladium/charcoal (Pd/C), 1,2-eth-
anedithiol (EDT), and ninhydrin were purchased from Merck. 2-Hy-
droxy-5-nitrobenzyl bromide and bromoacetylbromide were
purchased from Alfa aesar. Diisopropylethylamine (98%) (DIPEA),
N-methylmorpholin 99% (NMM), triethyl silane, and hydrazine
monohydrate (99%) were purchased from Acros. Trifluoroacetic acid
(99%), piperidine (99.5%), pyridine, DL dithiothretiol, europium
chloride, gadolinium chloride, cyanine dye (Cy5.5), 3-[4,5-dimethyl-
4.2.3. Synthesis of peptide
The solid-phase peptide synthesis was performed on a Rink
Amide resin (0.63 mmol/g loading) by using the Fmoc chemistry.
They were assembled on a PS-3 (Automated Peptide Synthesizer).
The side chain protecting groups of trifunctional amino acids were
trifluoroacetic acid labile. BN peptide (Bombesin analogue) was

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Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
synthesized on a 0.128 mmol scale using a fourfold molar excess of
Fmoc-protected amino acids (0.512 mmol), which were activated
by using fourfold excess of bromo-tris-pyrrolidino phosphonium
hexafluorophosphate (PyBoP) in the presence of N-methylmorpho-
made using a 20 MHz relaxometer operating at 37.0 0.1 °C (NMS-
120 Minispec, Bruker). Before each measurement, the relaxometer
was tuned and calibrated. The values of r1 were determined from
six data points generated by an inversion-recovery pulse sequence.
The proton spin-lattice relaxation (T1) was measured by using var-
ious concentrations of Gd(III) complexes at 37.0 0.1 °C in PBS buf-
fer (pH 7.4).
a
lin (NMM) (20% v/v) in dimethylformamide. N -Fmoc protecting
groups were removed by treating the resin-attached peptide with
piperidine (20% v/v) in dimethylformamide. The Bombesin ana-
logue functionalized resin (0.5 g, 0.63 mmol/g, 0.31 mmol) was
deprotected with the piperidine solution and was washed with
the dimethylformamide as described above.
4.4. Luminescence method for establishing solution hydration
state
4.2.4. Conjugation of ligand to peptide substrate (4)
Luminescence lifetime data has been obtained for the Eu(III)
complex to determine the number of inner-sphere water mole-
In a separate vial, TTDA-NP-NCS (0.914 g, 1.24 mmol) and diiso-
propylethylamine (0.16 g, 1.24 mmol) were dissolved in 10 mL of
dimethyl sulfoxide. The resulting solution was added to the depro-
tected resin, and nitrogen gas was bubbled through the mixture
for 24 h. The peptide solution was removed. The resin was washed
with dimethylformamide and methanol, and then was dried under
vacuum. A solution of 95% trifluoroacetic acid, 2.5% 1, 2-ethanedi-
thiol, 1.5% water, and 1.0% triethyl silane (10 mL) were added to
the resin, and the nitrogen was bubbled through the mixture for
2 h. Then the cleaved peptide was precipitated in ice-cold diethyl
ether and centrifuged at 1500 rpm for 5 min. Purification was per-
formed by preparative HPLC using a C18 column. Elution was per-
formed with a solvent system consisting of 0.1% CF3COOH/H2O
(solvent B) and CF3COOH/CH3CN (solvent A), by applying the follow-
ing gradient: from 95% solvent B to 40% solvent A over 100 min at
flow rate of 1 mL/min (retention time: 17.5 min by UV detector at
256, 280 nm). The collected fractions were combined and lyophi-
lized to yield the product 4 as a pink powder (229.6 mg, 42.3%).
MS (ESI): m/z calcd: 1751.98, found: 1751.20 [M+H]+.
cules in the aqueous solution.39–41 The luminescence lifetime (
s)
has been determined in both H2O and D2O. The complex containing
the number of inner-sphere water was calculated by (Eqs. 1 and 2).
The q value is obtained from the luminescence study.
4.5. Variable-temperature 17O NMR measurement
The measurement of the 17O transverse relaxation rates, longi-
tudinal relaxation rates, and chemical shifts was carried out with
a Varian Gemini-400 spectrometer, equipped with a 10 mm probe,
by using an external D2O lock. The Varian 600 temperature control
unit was used to stabilize the temperature in the range 278–338 K.
Solutions containing 5.5% of the 17O isotope were used. The inver-
sion-recovery of method was applied to measure longitudinal
relaxation rates, 1/T1, and the Carr-Purcell-Meiboom-Gill spin-echo
technique was used to obtain transverse relaxation rates, 1/T2. The
solution was introduced into spherical glass containers, fitting into
ordinary 10 mm NMR tubes, in order to eliminate magnetic sus-
ceptibility corrections to chemical shifts.
4.2.5. Synthesis of Gd-TTDA-NP-BN (6)
TTDA-NP-BN (100 mg, 0.057 mmol) was dissolved in deionized
water (8 mL) and pH 6.0–7.0 was adjusted by slowly adding so-
4.6. Cell culture and animal model
dium hydroxide. Then 340
l
L of GdCl3 (161.25 mM) was added
PC-3 cell is a human prostate cancer cell line overexpressing
gastrin releasing peptide (GRP) receptors.45 KB cell is a human
nasopharyngeal epidermal carcinoma cell line, lacking of the
expression of GRP receptors. All cells were obtained from American
Type Culture Collection (Manassas, VA). PC-3 and KB cells were
cultured in RPMI 1640 medium (GIBCO) and Ham’s F12 K medium
(GIBCO), respectively. All media were supplemented with 10% of
fetal bovine serum, sodium bicarbonate (1.5 g/L), sodium pyruvate
(1.0 mM), and non-essential amino acid (0.1 mM). Cells were cul-
and the mixture was stirred for 48 h at 37 °C. The completion of
complex formation was confirmed by xylenol orange test. The
solution was filtered and the filtrate was lypophilized. A white
powder product 6 was obtained. The purity of Gd-TTDA-NP-BN
was determined by HPLC and the identity was confirmed by mass.
MS (ESI): m/z calcd: 1904.19, found: 1908.60 [M+4H]+.
4.2.6. Synthesis of Eu-TTDA-NP-BN (7)
TTDA-NP-BN (20 mg, 0.014 mmol) was dissolved in deionized
water (3 mL) and pH 6.0–7.0 was adjusted by slowly adding so-
dium hydroxide. The resulting solution was stirred for 10 min, fol-
tured at 37 °C in a humidified 5% CO2 atmosphere. BALB/
cAnN.Cg-Foxn1nu/CrlNarl mice (5 weeks old, male) were purchased
from the National Laboratory Animal Center, Taipei, Taiwan. Ani-
mal experiments were performed in accordance with the institu-
lowed by the addition of 72.4 lL of EuCl3 (161.25 mM). The stirring
was continued for 48 h at 37 °C. Subsequently, the solution was fil-
tered and the filtrate was lyophilized to give 7. The purity of Gd-
TTDA-NP-BN was analyzed by HPLC and identified by mass. MS
(ESI): m/z calcd: 1998. 91, found: 1999.40 [M+H]+.
tional guidelines. PC-3 and KB cells in 100 l
L (106 cells) PBS
were subcutaneously injected into five nude mice. The cells were
mixed in equal volume with Matrigel and injected into the right
(PC-3) and left (KB) lateral thighs of the mice. An MR imaging
experiment was performed 2 weeks after the tumor implantation,
at which time the tumors were measured to be at least 100 mm3.
This method produces a high yield of tumor in the lateral thighs
of nude mice.
4.2.7. Synthesis of Gd-TTDA-NP-BN-Cy5.5 (8)
Gd-TTDA-NP-BN (2.42 mg, 1.47 lmol) was dissolved in 0.1 M
sodium borate (Na2B4O7) at pH 8.3 and then NHS-Cy5.5 (2 mg,
1.78 mL) was added. The resulting reaction mixture was stirred
in dark, at room temperature for 12 h and purified by preparative
HPLC. The collected fractions were combined and lyophilized to af-
ford 8, (3.5 mg, 75.1%). MS(ESI): m/z calcd: 2800.11, found:
2801.50 [M+H]+.
4.7. Cell cytotoxicity
PC-3 and KB cell lines were used to measure the in vitro cell
cytotoxicity of Gd-TTDA-NP-BN. An amount of 105 cells was plated
in each well of the 96-well plates for 24 h. Then, the Gd-TTDA-NP-
BN were added at the desired concentrations (from 0.125 to
2.0 mM Gd(III) complex per well). After 24 h of incubation, the
supernatant was removed and the cells were washed three times
with PBS. Cell viability was estimated using the 3-(4,5-dimethyl-
4.3. Relaxation time measurement
Relaxation times T1 of aqueous solutions of Gd(III) complex
were measured to determine relaxivity r1. All measurements were

Y.-H. Lin et al. / Bioorg. Med. Chem. 19 (2011) 1085–1096
1095
thiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) conversion
test. Briefly, MTT (50 L) solution was added to each well. After
2 h of incubation, each well was treated with dimethyl sulfoxide
(50 L) with pipetting. Absorption at 570 nm was measured on
ELISA reader. The data represent the average of sixteen wells.
The viability of untreated cells was assumed to be 100%.
[Gd(DTPA)(H2O)]2ꢀ
,
(diluted in 1 mL medium, 1 mM Gd) at
l
37.0 0.1 °C for 30 min, and then washed three times with PBS buf-
fer. Competitive targeting of free BN peptide to Gd-TTDA-NP-BN was
also studied with co-incubates of Gd-TTDA-NP-BN and fourfold ex-
cess of free BN peptide. All samples were scanned by a fast gradient
echo pulse sequence (TR/TE/flip angle = 100/5.8/10°).
l
4.8. In vitro specific targeting study of Eu-NP-TTDA-BN
4.13. In vivo MR imaging
The specific targeting study was investigated by using Eu-TTDA-
NP-BN complex. PC-3 cell lines were used to measure the in vitro
specific targeting study. About 105 cells were plated in each well
of the 6-well plates for 24 h. Then, Eu-TTDA-NP-BN was added at
the predetermined concentrations (1 mM, Eu-TTDA-NP-BN per
well) and incubated at 4.0 and 37.0 °C. After one hour the superna-
tant was removed and the cells were washed three times with PBS
buffer. Then, the cells were treated with paraformaldehyde (4%,
0.5 mL) solution for 10 min to fix the cells, and washed with PBS
buffer. Finally, the cells were inspected using a confocal fluores-
cence microscopy (exciter, 360/30 nm; emitter, 400/20 nm).
Nude mice bearing PC-3 and KB tumors were studied by MR
imaging when the subcutaneous tumor xenografts reached a vol-
ume of 100 mm3. A solution of Gd-TTDA-NP-BN (0.1 mmol/kg)
was injected via the tail vein. The MR imaging of pentobarbital-
anesthetized mice were performed at 0, 1, 6, and 24 h respectively,
using a 3.0-T magnetic resonance scanner and a high-resolution
animal coil (3.8 cm diameter, GE homemade). All animals were
scanned using a T1-weighted fast spin-echo sequence (TR/TE/flip
angle = 100/5.8/10°) for imaging.
Acknowledgment
4.9. In vitro optical imaging of Gd-TTDA-NP-BN-Cy5.5
Funding from National Science Council of Taiwan (Grant Nos.
NSC 98-2627-M-009-009, NSC 97-2113-M-009-016-MY3, and
NSC 97-2623-7-037-001-NU) is gratefully acknowledged.
PC-3 and KB cell lines were also used to measure the in vitro
specific targeting of Gd-TTDA-NP-BN-Cy5.5. About 105 cells were
plated in each well of the 24-well plates for 24 h. Then, Gd-
TTDA-NP-BN-Cy5.5 was added at the predetermined concentra-
Supplementary data
tions (10 lM per well). After one hour of incubation at 37 °C, the
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.04.040.
supernatant was removed and the cells were washed three times
with PBS. In addition, the nucleus was stained with 40,6-diamidi-
no-2-phenylindole (200 nM) solution for 3 min, followed by wash-
ing with PBS buffer three times, and the cells were inspected using
fluorescence microscopy, a ZEISS Axio imager M1 microscope
stand with TFT monitor equipped with DAPI filters (exciter, 360/
30 nm; emitter, 400/20 nm) and Cy5.5 filters (exciter, 650/20 nm;
emitter, 675/35 nm) to observe the cellular localization.
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station 2000 MM digital imaging system from molecular imaging
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ment, mice (n = 5) were injected via tail vein with 10 nmol of
Gd-TTDA-NP-BN-Cy5.5 and subjected to optical imaging at various
time points of post-injection. All NIR fluorescence images were ac-
quired using 10 s exposure times.
4.11. Biodistribution study
Gd-TTDA-NP-BN-Cy5.5 (10 nmol) was intravenously injected
into male nude mice. The mice (n = 5) were sacrificed one hour
after the intravenous injection of Gd-TTDA-NB-BN-Cy5.5. Blood,
lung, liver, spleen, heart, muscle, pancreas, bladder, intestines, kid-
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