Gadolinium complex of DO3A-benzothiazole aniline (BTA) conjugate as a theranostic agent

Article abstract of DOI:10.1021/jm401206t

A gadolinium complex of 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid (DO3A) and benzothiazole-aniline (BTA) of the type [Gd(DO3A-BTA)(H ₂O)] has been prepared for use as a single molecule theranostic agent. The kinetic inertness and r

Full text of DOI:10.1021/jm401206t

Article
pubs.acs.org/jmc
Gadolinium Complex of DO3A-benzothiazole Aniline (BTA)
Conjugate as a Theranostic Agent
Hee-Kyung Kim,†,‡ Min-Kyoung Kang,† Ki-Hye Jung,‡ Sun-Hee Kang,§ Yeoun-Hee Kim,#
Jae-Chang Jung,§ Gang Ho Lee,∥ Yongmin Chang,*,⊥,† and Tae-Jeong Kim*,‡
‡
§
∥
†Department of Medical & Biological Engineering, Department of Applied Chemistry, Department of Biology, Department of
⊥
Chemistry, Department of Diagnostic Radiology and Molecular Medicine, Kyungpook National University, 80 Daehakro, Bukgu,
Daegu, 702-701, Korea
#Cheil Eye Hospital, Cheil Eye Research Institute, 803-2 Sinam-1dong, Dong-gu, Daegu, 701-820, Korea
S
* Supporting Information
ABSTRACT: A gadolinium complex of 1,4,7,10-tetraazacyclo-
dodecane-1,4,7-trisacetic acid (DO3A) and benzothiazole-ani-
line (BTA) of the type [Gd(DO3A-BTA)(H2O)] has been
prepared for use as a single molecule theranostic agent. The
kinetic inertness and r1 relaxivity (= 3.84 mM−1 s−1) of the
complex compare well with those of structurally analogous Gd-
DOTA. The same complex is not only tumor-specific but also
intracellular, enhancing MR images of cytosols and nuclei of
tumor cells such as MCF-7, MDA-MB-231, and SK-HEP-1.
Both DO3A-BTA and Gd(DO3A-BTA) reveal antiproliferative
activities as demonstrated by GI50 and TGI values obtainable
from the cell counting kit-8 (CCK-8) assays performed on these
cell lines. Ex vivo and in vivo monitoring of tumor sizes provide
parallel and supportive observations for such antiproliferative
activities.
employed as multimodal imaging probes such as magnetic
resonance imaging (MRI)/optical and MRI/SPECT.7,8
Despite such widespread use of benzothiazole-based BFCs,
their uses have been limited solely to molecular imaging in
vitro, not to mention their application as therapeutic agent in
vivo. It is thus to be highly motivated to investigate the
possibility of expanding the scope of benzothiazole-based BFCs
to the area of theranostics (a coinage of diagnostic and
therapy). This notion can be justified as we observe the recent
thrust to put together imaging and therapeutic functions in a
single molecule.
In particular, our motivation comes from the observations
that the complexes of DO3A-benzothiazole conjugate (IV)
serve as multimodal MRI/optical probes. Thus, we would like
to pursue further the therapeutic propensity of the closely
related Gd complex of the type [Gd(DO3A-benzothiazole
aniline)(H2O)] (1) in the hope that it may serve as a single
molecule theranostic agent. In this connection, it has to be
noted that although numerous types of gadolinium nano-
particles (GdNPs) with multiple functionalities have been
developed,9 most of which carry either multimodal,10
therapeutic,11 or theranostic functionality,12 their application
INTRODUCTION
■
Benzothiazoles are fused bicyclic systems possessing diverse
biological properties such as anti-inflammatory, antimicrobial,
and anticancer effects. As such, a great deal of research activities
has been carried out in the past two decades in an effort to
develop various benzothiazole derivatives with high antitumor
activity.1 Some representative examples include compounds I
shown in Chart 1.2
Even further derivatization of benzothiazole(s) is feasible,
and their antitumor properties may be utilized in combination
with molecular imaging and therapy. One such endeavor may
include the formation of bifunctional chelates (BFCs)
incorporating benzothiazole. For instance, the fluorescent
rhenium complex conjugated to 2-(4-aminophenyl)-
benzothiazole (compounds II, Chart 1) has been reported to
enter MCF-7 breast cancer cells. On the basis of these
observations, the analogous radioactive complexes (M = 99mTc,
186Re, 188Re) have been proposed as potential radiopharma-
ceuticals for single photon emission computed tomography
(SPECT) and radioimmunotherapy (RIT).3,4 Benzothaizoles
labeled with radioisotopes such as 99mTc (compounds III,
Chart 1) and 11C have been proposed as potential probes for β-
amyloid plaques in the brain.5,6 More recently, conjugates of
benzothiazoles with 1,4,7,10-tetraazacyclododecane-1,4,7-tris-
tert-butyl acetate (DO3A) (compounds IV, Chart 1) have been
Received: August 6, 2013
Published: September 24, 2013
© 2013 American Chemical Society
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Chart 1. Various Benzothiazoles Used in Imaging and Therapy
(0.1%, v/v) and an acetonitrile solution of TFA (0.1%, v/v) with a 30
min isocratic elution (95 vs 5%) at a flow rate of 1.4 mL/min was used.
The purity of all products was determined with purity above 95% by
elemental analysis. The purity of DO3A-BTA and Gd(DO3A-BTA)
was double-checked by analytical and semipreprative reversed-phase
HPLC with a UV−vis detection probing at 240 nm (Supporting
Information Figure S5).
in nanomedicine is still in its infancy due to the toxicological
problems inherently connected with slow excretion rates.13
In contrast, however, small molecule Gd-chelates would
provide a promising substitute to eliminate or overcome the
problems inherent to GdNPs-based pharmaceuticals. Gd-
motexafin is a novel example for an antineoplastic drug capable
of disrupting cancer cell antioxidant systems, thus contributing
to cellular death. This same complex has demonstrated an
additional functionality of radiosensitizing the cancer.14 A more
recent example includes a heterobimetallic Gd−Pt complex
incorporating DTPA and terpyridine (terpy) as chelate. This
complex is known to intercalate DNA due to the presence of
the Pt(terpy) moiety, an analogue of cisplatin. The author has
proposed that the Gd/Pt complex might have potentiality in
gadolinium neutron-capture (GdNCT).15 The use of Gd
complexes (including GdNPs) in GdNCT has become the
topic of recent interest,16,17 as they are closely related to the
well-established boron neutron-capture therapy (BNCT).18,19
We now wish to report the design and synthesis of Gd(DO3A-
BTA) for use as a theranostic agent. To the best of our
knowledge, we believe this complex to be a rare example of
small molecule Gd-chelate based theranostic agent.
Synthesis and Characterization. tert-Butyl 2,2′,2″-(10-(2-(4-
(Benzo[d]thiazol-2-yl)phenylamino)-2-oxoethyl)-1,4,7,10-tetraaza-
cyclododecane-1,4,7-triyl)triacetate. To a mixture of DO3A(tBu)3
(1.1 g, 2.2 mmol) and NaHCO3 (0.5 g, 6.6 mmol) in DMF (10 mL)
was added dropwise a solution of N-(4-(benzo[d]thizol-2-yl)phenyl)-
2-chloroacetamide (1.0 g, 3.3 mmol) in DMF (10 mL). After stirring
for 18 h at RT, water (200 mL) was added dropwise and the mixture
was stirred for 2 h at RT. The precipitate was filtered and washed with
water. The crude product thus obtained was further purified by
column chromatography (silica, CH2Cl2/MeOH, 97:3) to give a white
1
solid. Yield: 0.5 g (30%). H NMR (CDCl3): δ = 8.09−8.07 (d, 2H,
phenyl), 8.02−8.00 (d, 1H, benzothiazole), 7.92−7.90 (d, 2H,
phenyl), 7.86−7.85 (d, 1H, benzothiazole), 7.47−7.43 (td, 1H,
benzothiazole), 7.36−7.32 (td, 1H, benzothiazole), 3.05−2.85 (m,
8H, −CH2CO2, −CH2CONH), 2.61−2.02 (m, 16H, CH2 in the
cyclen ring). Anal. Calcd for C41H64N6O7S·4HCl: C, 53.13; H, 6.96;
N, 9.07; S, 3.46. Found: C, 52.93; H, 6.62; N, 8.97; S, 3.17. HR-
FABMS (m/z): calcd for C41H60N6O7SNa, 803.4142 ([MNa]+);
found, 803.4142 ([MNa]+).
EXPERIMENTAL SECTION
DO3A-BTA. Deprotection of tert-butyl was performed as follows:
tert-Butyl 2,2′,2″-(10-(2-(4-(benzo[d]thiazol-2-yl)phenylamino)-2-ox-
oethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetate (1.0 g, 1.3
mmol) dissolved in a mixture of TFA/CH2Cl2 (1:1, 20 mL) was
stirred at RT for 24 h, after which the solution was evaporated under a
reduced pressure. The crude product was dissolved in MeOH (10 mL)
and precipitated with diethyl ether (100 mL) to yield a yellowish white
solid. Yield: 0.6 g (68%). 1H NMR (methanol-d4): δ = 8.00−7.95 (m,
4H, phenyl), 7.79−7.77 (m, 2H, benzothiazole), 7.53−7.50 (t, 1H,
benzothiazole), 7.43−7.40 (t, 1H, benzothiazole), 3.86−3.61 (m, 8H,
−CH2CO2, −CH2CO), 3.50−3.14 (m, 16H, CH2 in the cyclen
ring). Anal. Calcd for C29H46N6O12S·5H2O: C, 49.56; H, 6.60; N,
11.96; S, 4.56. Found: C, 49.57; H, 6.20; N, 12.33; S, 4.45. HR-
FABMS (m/z): calcd for C29H36N6O7SNa, 635.2264 ([MNa]+);
found, 635.2264 ([MNa]+). HPLC (analytical, 2.1 mm × 250 mm;
flow rate, 1.4 min/L; retention time, 24.65 min).
■
General Remarks. All reactions were carried out under an
atmosphere of dinitrogen using the standard Schlenk techniques.
Solvents were purified and dried using standard procedures. N-(4-
(Benzo[d]thiazol-2-yl)phenyl)-2-chloroacetamide4 and DO3A(tBu)3
(= 1,4,7,10-tetraazacyclododecane-1,4,7-tris-tert-butyl acetate)25 were
prepared according to the literature methods. All other commercial
reagents were purchased from Aldrich and used as received unless
otherwise stated. Deionized water was used for all experiments. The
1H NMR experiment was performed on a Bruker Advance 400
spectrometer by the Center for Instrumental Analysis, Kyungpook
National University (KNU). Chemical shifts were given as d values
with reference to TMS as an internal standard. Coupling constants are
in Hz. Elemental analyses were performed by Center for Instrumental
Analysis, KNU in Daegu. FAB-mass spectra were obtained by using a
JMS-700 model (Jeol, Japan) mass spectrophotometer by Korea Basic
Science Institute (KBSI). The HPLC system (high pressure liquid
chromatography, KNAUER, Berlin, Germany) equipped with a
Develosil ODS-HG-5 column (6.0 mm × 250 mm; Nomura Chemical
Co., Ltd., Aichi, Japan) was used for the measurement. Elution
conditions were as follows: a mixture of an aqueous solution of TFA
Gd(DO3A-BTA). DO3A-BTA (0.5 g, 0.8 mmol) was dissolved in
water (15 mL) to which was added gadolinium(III) chloride
hexahydrate (0.3 g, 0.8 mmol). The mixture was stirred at RT for
18 h, during which time pH of the solution was periodically adjusted
to 7.0−7.5 with NaOH (1.0 M). Water was removed by evaporation,
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and the remaining oily product was taken up in a minimum amount of
water (∼5 mL) to be added dropwise to acetone for precipitation. The
white precipitate thus formed was removed by filtration, washed with
acetone, and dried under vacuum. The precipitate (∼0.6 g) was
redissolved in a minimum amount of water to elute in Amberite XAD
16N (25 g). Any inorganic salts such as NaCl and the Gd3+ ion were
removed initially by eluting with water and the product by MeOH (25
mL). The yellowish white solid left after removal of MeOH was
washed several times with acetone and dried under vacuum to yield an
off-white, hygroscopic solid (0.5 g, 82%). The absence of the free Gd3+
ion was further confirmed by the xylenol orange test, and the purity
(above 95%) determined by HPLC (analytical, 2.1 mm × 250 mm;
flow rate, 1.4 min/L; retention time, 21.87 min). Anal. Calcd for
C29H59GdN6O20S·13H2O: C, 34.79; H, 5.94; N, 8.39; S, 3.26. Found:
C, 34.87; H, 4.09; N, 8.63; S, 2.93. HR-FABMS (m/z): calcd for
C29H34GdN6O7S, 768.1445 ([MH]+); found, 768.1456 ([MH]+).
Transmetalation Kinetics and Determination of Kinetic
Constants. This experiment was performed according to the
literature method.20 It is based on the evolution of the water proton
of view (FOV); 192 × 128 matrix size; 1.2 mm slice thickness; number
of acquisition (NEX) = 8. Images were obtained for 240 min after
injection. The anatomical locations with enhanced contrast were
identified with respect to heart, kidney, and liver on postcontrast MR
images. For quantitative measurement, signal intensities in specific
regions of interest (ROI) measured using Advantage Window software
(GE Medical, USA). The contrast-to-noise ratio (CNR) was calculated
using eq 1, where SNR is the signal-to-noise ratio.
CNR = (SNR post − SNR pre
)
(1)
In Vivo Therapy. The subcutaneous tumor model was established
in a nude mouse as described in tumor model session in order to
investigate the therapeutic effect of Gd(DO3A-BTA) in vivo. When
the average volume of MDA-MB-231 reached 50 mm3 six to seven
weeks after implantation of cells, two groups of mice with each bearing
five of those were assigned, respectively, to the tests for control and
Gd(DO3A-BTA). The volume of the solution in each test was 70 μL
(0.1 mmol/kg, 35−50 mM stock solution) and injected once a day for
3 weeks. Tumor sizes were measured at given time points either with a
caliper or with MR images. Tumor size measured by caliper is given as
V/V0 (V0 = the initial volume), while that measured by MRI is
calculated using eq 2.
P
relaxation rate (R1 ) of a buffered solution (phosphate buffer, pH 7.4)
containing 2.5 mmol/L gadolinium complex and 2.5 mmol/L ZnCl2.
Then 10 μL of a 250 mmol/L solution of ZnCl2 is added to 1 mL of a
buffered solution of the paramagnetic complex. The mixture is
vigorously stirred, and 300 μL is taken up for the relaxometric study. A
control study, run on Gd-DO3A-BT (Gadovist), Gd-EOB-DTPA
(Primovist), Gd-BOPTA (Multihance), Gd-DTPA-BMA (Omniscan),
and Gd-DOTA (Dotarem) with zinc acetate, has given results identical
to those obtained in the presence of ZnCl2. The R1P relaxation rate is
obtained after subtraction of the diamagnetic contribution of the
proton water relaxation from the observed relaxation rate R1 = (1/T1).
The measurements were performed on a 3 T whole body system
(Magnetom Tim Trio, Siemens, Germany) at room temperature.
Relaxivity. T1 measurements were carried out using an inversion
recovery method with a variable inversion time (TI) at 1.5 T (64
MHz). The magnetic resonance (MR) images were acquired at 35
different TI values ranging from 50 to 1750 ms. T1 relaxation times
were obtained from the nonlinear least-squares fit of the signal
intensity measured at each TI value. For T2 measurements, the CPMG
(Carr−Purcell−Meiboon−Gill) pulse sequence was adapted for
multiple spin−echo measurements. Then 34 images were acquired
with 34 different echo time (TE) values ranging from 10 to 1900 ms.
T2 relaxation times were obtained from the nonlinear least-squares fit
of the mean pixel values for the multiple spin−echo measurements at
each echo time. Relaxivities (R1 and R2) were then calculated as an
inverse of relaxation time per mM. The determined relaxation times
(T1 and T2) and relaxivities (R1 and R2) are finally image-processed to
give the relaxation time map and relaxivity map, respectively.
tumor size = number of pixels of tumor × pixel area
(2)
× slice thickness
Biodistribution. Gd(DO3A-BTA) was administered intravenously
as a bolus (0.1 mmol/kg) in a tail vein of nine female mice bearing
MDA-MB-231 tumor (balb/c nude mice; 18−25 g). The mice were
anesthetized and killed by means of exsanguinations from the vena
cava at each time point (after 1, 6, and 24 h injection time). The
gadolinium concentration was measured in tissues (liver, spleen, gall
bladder, bladder, kidneys, lung, tumor, blood, heart, brain, and
intestine). The gadolinium concentration was determined by digesting
the tissues with HNO3 (65%) at 180 °C for 120 min and measuring
the concentration in the clear diluted solution by inductively coupled
plasma atomic emission spectrometry (ICP-AES, Optima 7300DV,
PerkinElmer, USA). Detection limit of this method is 0.01 ppm.26
Cell Culture. The culture medium consisted of Dulbecco’s
Modified Eagle’s Medium (DMEM; Gibco Invitrogen, Carlsbad,
CA) or Roswell Park Memorial Institute (RPMI, Gibco Invitrogen,
Carlsbad, CA), 10% (v/v) fetal bovine serum (FBS), and 1% (v/v)
penicillin−streptomycin. Cells were plated at a density of 2 × 105
cells/35 mm dish, incubated during overnight for stabilization and
then treated with Gd for 24 h in serum depletion media.
Tumor Model. The MCF-7 and MDA-MB-231 tumor cells (106
cell mL−1) suspended in DMEM or RPMI-1640 medium without fetal
bovine serum and penicillin−streptomycin were inoculated into
subcutaneous tissue (sc) of female BALB/c nude mice (7 weeks,
18−25 g of body weight). The nude mice were anesthetized (1.5%
isoflurane in oxygen). The MR experiments were carried out 6 or 7
weeks after tumor cell implantation.27
Cell Fractions for MRI. MCF-7, MDA-MB-231, and SK-HEP-1
cells were plated 2 × 105 in 35 mm dishes. The DMEM or RPMI
growth medium was removed, and then Gd(DO3A-BTA) (100 μM)
in DMEM or RPMI serum depletion media for 24 h. The cells were
washed with phosphate buffered saline (PBS) and harvested. For cell
fractionation, cancer cells were lysed by three cycles of freezing and
thawing in PBS, and lysates centrifuged at 1000 rpm for 5 min at 4 °C.
The supernatant was cytosolic fraction, and the first pellet was
resuspended in radioimmunoprecipitation assay (RIPA) buffer for 1 h
at 4 °C and centrifuged at 12,000 rpm for 10 min at 4 °C. The
supernatant corresponded to RIPA buffer-soluble membrane fraction
and the final pellet contained nuclei and cell organelles. Collected cells
were performed MR imaging by using a 1.5T MRI scanner.
MR Imaging and Image Analysis. In vitro MR images were
taken with a 1.5 T (T) MR unit (GE Healthcare, Milwaukee, WI,
USA) equipped with a surface coil. The imaging parameters for SE
(Spin Echo) are as follows: repetition time (TR) = 300 ms; echo time
(TE) = 12 ms; 15 mm field of view (FOV); 256 × 256 matrix size; 1.2
mm slice thickness; number of acquisition (NEX) = 20.
All animal experiments were performed in accordance with the rules
of the animal research committee of Kyungpook National University.
Seven-week normal female balb/c nude mice with weights of 18−25 g
were used for the MRI. Fifteen-week-old female balb/c nude mice
bearing MCF-7 tumor were used for the MRI. The mice were
anesthetized by 1.5% isoflurane in oxygen. Measurements were made
before and after injection of Gd(DO3A-BTA) and Gd-DOTA
(Dotarem) via tail vein. The amount of CA per each injection is 0.1
mmol [Gd]/kg for MR images. After each measurement, the mouse
was revived from anesthesia and placed in the cage with free access to
food and water. During these measurements, the animals were
maintained at approximately 37 °C using a warm water blanket. MR
images were taken with a 1.5 T (T) MR unit (GE Healthcare,
Milwaukee, WI, USA) equipped with a homemade small animal RF
coil. The coil was of the receiver type with its inner diameter being 50
mm. The imaging parameters for spin−echo (SE) are as follows:
repetition time (TR) = 300 ms; echo time (TE) = 13 ms; 8 mm field
Cell Viability Assay (in Vitro Growth Inhibition). MCF-7,
MDA-MB-231, and SK-HEP-1 cells were plated 1 × 104 in 96-well
plate. The DMEM or RPMI growth medium was removed, and then
Gd(DOTA-BTA) in DMEM or RPMI serum depletion media for 24
h. Then the live cell count was assayed using CCK-8 (Dojindo,
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Sunnyvale, CA) according to the manufacturer’s protocol. In brief, 10
μL of CCK-8 solution was added to each well, and the samples were
incubated for 4 h before the absorbance was measured at 450 nm.
Fluorescence and UV Microscope Images of Cells. Gd-
(DOTA-BTA) injected tissue was harvested and fixed with 4%
paraformaldehyde (PFA). The tissue was then dehydrated through an
ethanol series, cleared by soaking in xylene, embedded in paraffin, and
sectioned (5 mm) using a microtome RM 2125RT (Leica, Wetzlar,
Germany). The specimen was incubated overnight with monoclonal
antibodies against β-tubulin. Images were captured using a Zeiss
fluorescence microscope (TRITC-546 nm, UV-385 nm).
BTA), were prepared according to the literature method with a
slight modification (Supporting Information Scheme S1).
DO3A-BTA can be readily prepared by acid hydrolysis with
TFA of the corresponding tert-butyl ester which is in turn
prepared from simple conjugation of DO3A(tBu)3 with
benzothiazole−chloroacetylaniline. The reaction of DO3A-
BTA with GdCl3 resulted in the formation of the corresponding
Gd(III) complex of the type [Gd(DO3A-BTA)(H2O)] as a
hygroscopic white solid.
The formation of both DO3A-BTA and its Gd-complex was
confirmed by microanalysis and spectroscopic techniques such
FACS Analysis. Cells (∼1 × 106) were suspended in 100 μL of
PBS, and 200 μL of 95% ethanol were added while vortexing. Then the
cells were incubated at 4 °C for 1 h, washed with PBS, and
resuspended in 250 μL of 1.12% sodium citrate buffer (pH 8.4)
together with 12.5 μg of RNase. Incubation was continued at 37 °C for
30 min. The cellular DNA was then stained by applying 250 μL of
propidium iodide (PI) (50 μg/mL) for 30 min at room temperature.
The PI stained cells were analyzed by fluorescent activated cell sorting
(FACS) on a FACScan flow cytometer to determine relative DNA
content based on red fluorescence.
Cell Morphology. Primary cultured normal conjunctival cell
(passage 4−5) and cancer cells were plated 2 × 105 in 35 mm dishes.
Cells were exposed with Gd(DOTA-BTA) for 24 h with serum
depletion media. The morphological features of cultured cells were
observed by phase-contrast microscopy.
1
as H NMR, HRFAB- and MALDI-TOF mass spectrometry.
The Gd(III) complex was identified as a hydrate of 13 water
molecules [Gd(DO3A-BTA)(H2O)]·13H2O as confirmed by
microanalytical results.
Kinetic Inertness. The kinetic inertness for Gd(DO3A-
BTA) is typically represented by the evolution of the
P
normalized paramagnetic longitudinal relaxation rates, R1 (t)/
P
R1 (0) as a function of time along with those for Gd-DOTA,
Gd-BOPTA, Gd-EOB-DTPA, Gd-DO3A-BT, and Gd-DTPA-
BMA for comparative purposes (Supporting Information
P
Figure S1). Here, the relative value of R1 at any time t is a
good estimator of the extent of transmetalation of gadolinium
by zinc. Of various endogenous ions, Zn2+ has been noted to
displace more gadolinium ions due to its higher concentration
in the blood than any other ions such as Cu(II), Ca(II), and
K(I).20 As might be expected, our new Gd complex exhibits
high kinetic stability comparable with those of Gd-DO3A-BT
and Gd-DOTA employing the same type of macrocyclic
chelate. Virtually no significant changes in relaxivity is observed
either with DOTA or DO3A for as long as 3 days. In contrast,
however, other MRI contrast agents (CAs) employing the
acyclic DTPA analogues show significant drops in R1 for the
same period of time.
RESULTS AND DISCUSSION
Synthesis. The bifunctional chelate and its Gd(III)
complex, each abbreviated as DO3A-BTA and Gd(DO3A-
■
Table 1. Relaxivity Data of Gd-DOTA and Gd(DO3A-BTA)
in PBS (64 MHz, 293K)
r1 (mM−1 s−1
)
r2 (mM−1 s−1
)
Gd(DO3A-BTA)
Gd-DOTA
3.84 0.19
3.69 0.12
4.07 0.19
3.98 0.19
Relaxivity. Gd(DO3A-BTA) reveals relaxivities in PBS
comparable well with those of Gd-DOTA (Table 1). The
Figure 1. (a) T1-weighted MR images of MCF-7, MDA-MB-231, and SK-HEP-1 cell fractions incubated with Gd(DO3A-BTA). (b) UV−vis images
of MCF-7 tumor tissue of mice (×400): tissue stained with β-tubulin (left), tissue obtained by tail vein injection with Gd(DO3A-BTA) (middle),
and merged image of two (right). (c) UV−vis images of MDA-MB-231 tumor tissue (×100): tissue stained with PI (left), tissue obtained by tail vein
injection with Gd(DO3A-BTA) (middle), and merged image of two (right). (d) CNR profiles and in vivo MR axial images of mice bearing MDA-
MB-231 tumor obtained with Gd(DO3A-BTA) and Gd-DOTA, measured 1 h after injection.
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Figure 2. (a) phase-contrast microscopic images of morphological changes in cultured MCF-7, MDA-MB-231, and SK-HEP-1 cells with Gd(DO3A-
BTA). (b) Proliferation of normal conjunctival cell, MCF-7, MDA-MB-231, and SK-HEP-1 after treatment with various concentrations of
Gd(DO3A-BTA). (c) Flow cytometric (left) and quantitative analysis of apoptosis for MCF-7, MDA-MB-231, and SK-HEP-1 cells after treatment
with Gd(DO3A-BTA). Apoptosis was analyzed as a sub-G1 fraction by FACS.
Interaction with HSA. Proton longitudinal paramagnetic
relaxation rate of Gd(DO3A-BTA) (Supporting Information
Figure S2) and the corresponding binding constant Ka
(Supporting Information Table S1) provide the evidence for
the noncovalent lipophilic interaction between the chelate
DO3A-BTA. One may expect a higher Ka value with DO3A-
BTA than with other DOTA derivatives devoid of any aromatic
moieties. We have found Ka for Gd(DO3A-BTA) to be greater
almost by an order of one than those for Gd-DOTA and Gd-
EOB-DTPA (Supporting Information Table S1). Gd-DOTA is
a well-known extracellular fluid (ECF) agent incorporating
DOTA as a chelate, and one may expect little of such
interaction. Gd-EOB-DTPA, a liver-specific MRI CA, also
exhibits unexpectedly low interaction in spite of the presence of
aromatic moiety in the DTPA ligand.21,22
Table 2. In Vitro Activities of DO3A-BTA and Gd(DO3A-
BTA) in Cancer Cell Lines
MCF-7
MDA-MB-231
SK-HEP-1
DO3A-BTA
GI50 [μM]
715.57
669.57
625.33
TGI [μM]
1923.28
1637.75
1656.83
Gd(DO3A-BTA)
GI50 [μM]
258.48
555.89
237.47
637.82
203.43
706.73
TGI [μM]
presence of the lipophilic BTA moiety in DO3A-BTA seems to
exert little effect on the thermodynamic stability of Gd(DO3A-
BTA) in PBS. In comparison, its presence seems to be a little
more significant in HSA because of the lipophilic interaction
with HSA. This point will be discussed in the next section.
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and Gd(DO3A-BTA) have revealed antitumor characteristics
against three cell lines such as MCF-7, MDA-MB-231, and SK-
HEP-1. All revealed apoptotic characteristics such as cell
shrinkage, apoptotic bodies, and detachment from the plate on
their treatment.
Morphological changes in cultured cell lines in the presence
of Gd(DO3A-BTA) become apparent at the gadolinium
concentration of 200 μM and above with all cell lines. With
DO3A-BTA, even higher dosage is required to observe such a
change (Figure 2a). The antiproliferative activities of DO3A-
BTA and Gd(DO3A-BTA) measured by CCK-8 assays render
the growth suppression of the cell lines in terms of cell viability
for MCF-7, MDA-MB-231, and SK-HEP-1 (Figure 2b).
According to corresponding growth-inhibition 50 (GI50) and
total-growth-inhibition (TGI) values (Table 2), Gd(DO3A-
BTA) is a little more potent than DO3A-BTA in that the
former shows lower GI50 and TGI values for all three cell lines.
When TGI values are compared among the three tumor cells,
MCF-7 is the most vulnerable toward the Gd complex, and yet
as far as GI50 is concerned, SK-HEP-1 shows the lowest value.
We further investigated the effects of Gd(DO3A-BTA) on the
progression of cancer cell lines through the cell cycle and on
the induction of apoptotic cell death. The proportion of cells in
sub-G1 phase gradually increases in all cell lines with the
increase in the gadolinium concentration (Figure 2c). Here, it is
to be noted that the DNA content in the sub-G1 phase
amounts to the quantitative indication of the progress of
apoptosis. Here the mechanism of action of DO3A-BTA or its
Gd complex is the subject of further investigation, and the one
suggested by others in connection with analogous benzothia-
zoles, i.e., compounds I (Chart 1), may be assumed to be
operative as follows: selective uptake into sensitive cells
followed by arylhydrocarbon receptor (AhR) binding and
translocation into the nucleus, induction of the cytrochrome
P450 isoform (CYP) 1A1, conversion of the drug into an
electrophilic reactive intermediate, and formation of extensive
DNA adducts resulting in cell death.1,16
Figure 3. (a) Ex vivo images of MDA-MB-231 tumors derived from
balb/c nude mice treated with Gd(DO3A-BTA) and saline after the
end of treatment. (b) Changes in tumor volumes (t test, p < 0.05) as
measured by a caliper.
Tumor Targeting. Tumor-targeting nature of Gd(DO3A-
BTA) can be visualized by adopting the literature method
(Figure 1a).23 Tumors employed for the examinations are two
breast cancer cells MCF-7 and MDA-MB-231 and hepatocarci-
noma SK-HEP-1, all of which exhibit strong enhancement
mostly at the cytosol and the nucleus, thus demonstrating
intracellular targeting nature of the present agent. It is observed
for the first time that Gd(DO3A-BTA) is present both in
cytosol and nucleus, although BTA itself is known to be present
in both of them (vide infra for the possible mechanism of
action by Gd(DO3A-BTA)). The exact amounts of gadolinium
in the cytosol and the nucleus of tumor cells are provided in
Table S4 (Supporting Information). Ex vivo targeting experi-
ments provide paralleled observations for cell permeability. An
almost complete overlap is made between UV−vis images of
MCF-7 and that stained with a fluorophore, β-tubulin (Figure
1b). The same observations are made with MDA-MB-231 as
well (Figure 1c). The degree of signal enhancement with
Gd(DO3A-BTA), as expressed by contrast-to-noise ratio
(CNR), is even higher and persists longer in vivo than Gd-
DOTA (Figure 1d).
The therapeutic effect of Gd(DO3A-BTA) in vivo was
examined on MDA-MB-23,1 revealing the lowest GI50 of the
three cell lines. Expectedly, tumor growth is suppressed on
treatment with Gd(DO3A-BTA), while a sharp increase is
observed on treatment with saline alone. The difference in the
tumor sizes is visualized in the corresponding ex vivo images
(Figure 3a), and changes in tumor volume can be measured by
a caliper as well (Figure 3b). MR monitoring of the change in
tumor sizes provides parallel and supportive observations for
such an antiproliferative activity (Supporting Information
Figure S4).
The amount of Gd accumulated in the tumor site for 24 h is
40−220 μg Gd/g tumor (Supporting Information Figure S5
and Table S3), which falls within the range of the optimal
amount (50−200 μg Gd/g tumor) for GdNCT.16 This
observation implies potentiality of Gd(DO3A-BTA) as a new
and practical radiopharmaceuticals as well.
All in all, good correlation between the in vitro, in vivo, and
ex vivo assays demonstrates intracellular tumor targeting nature
of the present agent. Most notably, the formation of neither the
DO3A-BTA conjugate nor Gd(DO3A-BTA) would change
such tumor specificity inherent in BTA. These observations will
be treated in more detail in connection with therapeutic
properties of Gd(DO3A-BTA) (vide infra).
Therapy. Various BTA derivatives anchored on water-
soluble moieties such as lysine and alanine are well-known for
their anticancer characteristics and thus have been widely
employed for imaging and therapeutic purposes.24 In this
regard, it would be interesting to know whether structural
modification of BTA with such a chelate as DOTA and its Gd
complex would render an additional entry to highly water-
soluble therapeutic agents. Indeed, both DO3A-BTA conjugate
CONCLUSIONS
■
In conclusion, we have synthesized a bifunctional chelate
DO3A-BTA and its gadolinium complex of the type [Gd-
(DO3A-BTA)(H2O)] to put into a new entry as a single
theranostic agent. The kinetic inertness of the complex
compares well with that of structurally related Gd-DOTA. Its
R1 relaxivity is 3.84 mM−1 s−1, slightly higher than that of Gd-
DOTA (R1 = 3.69 mM−1 s−1). The biodistribution pattern of in
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vivo MRI compares well with those of DTPA-based, liver-
specific MRI CAs such as Gd-EOB-DTPA and Gd-BOTPA in
that heart and abdominal aorta are enhanced specifically and
that enhancement endures as long as 1 h. The Gd complex is
intracellular as well as tumor-specific, as confirmed by MR
images of cytosols and nuclei of MCF-7, MDA-MB-231, and
SK-HEP-1 cells. The antiproliferative activities of DO3A-BTA
and its gadolinium complex Gd(DO3A-BTA) were demon-
strated by GI50 and TGI values obtainable from the CCK-8
assays performed on the above cell lines. The Gd complex is a
little more potent than the DO3A-BTA conjugate in that the
former shows lower GI50 and TGI values for all three cell lines.
When TGI values are compared among the three tumor cells,
MCF-7 is the most vulnerable toward the Gd complex, and yet
as far as GI50 is concerned, SK-HEP-1 shows the lowest value.
Apoptosis of cancer cells was confirmed by the observations of
the increase of the portion of cells in the sub-G1 phase in line
with an increase in the Gd concentration.
CAs, contrast agents; ECF, extracellular fluid; AhR, arylhy-
drocarbon receptor; CYP, cytrochrome P450 isoform
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ASSOCIATED CONTENT
■
S
* Supporting Information
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AUTHOR INFORMATION
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ACKNOWLEDGMENTS
■
This work was partially supported by NRF through the Basic
Science Research Program (2012-0006388 and 2011-0015353)
and the Ministry of Commerce, Industry, and Energy through
the Regional Technology Innovation Program (RTI01-01-01).
We are most grateful to Dr. Ji-Ae Park (Molecular Imaging
Research Center, Korea Institute of Radiological & Medical
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kinetic stability measurement.
ABBREVIATIONS USED
■
DO3A, 1,4,7,10-tetraazacyclododecane-1,4,7-trisacetic acid;
BTA, benzothiazole aniline; GI50, growth inhibition 50; TGI,
total growth inhibition; BFCs, bifunctional chelates; SPECT,
single photon emission computed tomography; RIT, radio-
immunotherapy; MRI, magnetic resonance imaging; GdNCT,
gadolinium neutron-capture; GdNPs, gadolinium nanoparticles;
BNCT, boron neutron-capture therapy; TI, inversion time;
CPMG, Carr−Purcell−Meiboon−Gill pulse sequence; TE,
echo time; TR, repetition time; SE, spin echo; FOV, field of
view; NEX, number of acquisition; ROI, regions of interest;
CNR, contrast to noise ratio; DMEM, Dulbecco’s Modified
Eagle’s Medium; RPMI, Roswell Park Memorial Institute; FBS,
fetal bovine serum; HSA, human serum albumin; PBS,
phosphate buffered saline; RIPA, radioimmunoprecipitation
assay; CCK-8, cell counting kit-8; PFA, paraformaldehyde; PI,
propidium iodide; FACS, fluorescent activated cell sorting;
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