Preclinical evaluation of DO3A-Act-AQ: A polyazamacrocyclic monomeric anthraquinone derivative as a theranostic agent

Article abstract of DOI:10.1021/mp4004089

An anthraquinone conjugated macrocyclic chelating agent, 2,2′,2″-(10-(2-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino) -2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid or DO3A-Act-AQ, was synthesized by reacting trisubstituted cyclen (DO3A) with 2-chloro-N-(9,10-dioxo-9,10-dihydro-anthracen-1-yl)-acetamide and radiolabeled with ⁶⁸GaCl₃ in 84% efficiency and a specific activity of 4.62 MBq/nmol. The IC₅₀ value for BMG-1 cells was 0.1 mM, while the same concentration of DO3A-Act-AQ rendered no significant toxicity in HEK cells. The exposure of BMG-1 cells with 0.1 mM DO3A-Act-AQ displayed a time-dependent increase in apoptosis (40.7% at 4 h and 53% at 24 h), and the effect was 2.8- and 3.6-fold % higher as seen in HEK cells. An increase in S-phase cell population suggested S-phase arrest concomitant with induction of apoptosis in BMG-1 cells reaching to 4.5 times after 24 h with respect to control cells. DNA binding studies on CT-DNA (calf thymus) revealed a quenching pattern in the presence of DO3A-Act-AQ (10-70 μM), and the Stern-Volmer quenching constant was 2.4157 × 10⁶ L mol^-1, indicative of strong binding with ds-DNA. A decrease in the positive and negative bands of CT-DNA was seen at 278 nm and 240 nm, respectively, on addition of 0.05 mM of DO3A-Act-AQ in CD studies. ⁶⁸Ga-DO3A-Act-AQ was stable in vitro in both PBS and human serum for at least 2 h. The in vivo blood kinetics study performed on normal rabbits indicated fast clearance with t_1/2(F) = 40 ± 0.3 min and t_1/2(S) = 3 h 30 min ± 0.1 min. Ex vivo biodistribution analysis displayed a favorable tumor-to-muscle ratio of 8.4 after 2 h in athymic nude mice xenografted with BMG-1 cells, suggesting the specificity of ⁶⁸Ga-DO3A-Act-AQ toward tumors.

Full text of DOI:10.1021/mp4004089

Article
pubs.acs.org/molecularpharmaceutics
Preclinical Evaluation of DO3A-Act-AQ: A Polyazamacrocyclic
Monomeric Anthraquinone Derivative as a Theranostic Agent
Anupriya Adhikari,†,‡ Anupama Datta,*,† Manish Adhikari,§ Kanchan Chauhan,† Krishna Chuttani,†
Sanjiv Saw,⊥ Abha Shukla,‡ and Anil K. Mishra*,†
†Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, Defence Research &
Development Organization, Brig S K Mazumdar Road, Delhi-110054, India
‡Department of Chemistry, Kanya Gurukul Campus, Gurukul Kangri Vishwavidyalaya, Haridwar-249404, India
§Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Defence Research & Development
Organization, Brig S K Mazumdar Road, Delhi-110054, India
⊥Division of Clinical PET, Institute of Nuclear Medicine and Allied Sciences, Defence Research & Development Organization, Brig S
K Mazumdar Road, Delhi-110054, India
S
* Supporting Information
ABSTRACT: An anthraquinone conjugated macrocyclic che-
lating agent, 2,2′,2″-(10-(2-(9,10-dioxo-9,10-dihydroanthracen-
1-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetic acid or DO3A-Act-AQ, was synthesized by
reacting trisubstituted cyclen (DO3A) with 2-chloro-N-(9,10-
dioxo-9,10-dihydro-anthracen-1-yl)-acetamide and radiolabeled
with 68GaCl3 in 84% efficiency and a specific activity of 4.62
MBq/nmol. The IC50 value for BMG-1 cells was 0.1 mM, while
the same concentration of DO3A-Act-AQ rendered no
significant toxicity in HEK cells. The exposure of BMG-1
cells with 0.1 mM DO3A-Act-AQ displayed a time-dependent increase in apoptosis (40.7% at 4 h and 53% at 24 h), and the
effect was 2.8- and 3.6-fold % higher as seen in HEK cells. An increase in S-phase cell population suggested S-phase arrest
concomitant with induction of apoptosis in BMG-1 cells reaching to 4.5 times after 24 h with respect to control cells. DNA
binding studies on CT-DNA (calf thymus) revealed a quenching pattern in the presence of DO3A-Act-AQ (10−70 μM), and the
Stern−Volmer quenching constant was 2.4157 × 106 L mol−1, indicative of strong binding with ds-DNA. A decrease in the
positive and negative bands of CT-DNA was seen at 278 nm and 240 nm, respectively, on addition of 0.05 mM of DO3A-Act-
AQ in CD studies. 68Ga-DO3A-Act-AQ was stable in vitro in both PBS and human serum for at least 2 h. The in vivo blood
kinetics study performed on normal rabbits indicated fast clearance with t1/2(F) = 40 0.3 min and t1/2(S) = 3 h 30 min 0.1
min. Ex vivo biodistribution analysis displayed a favorable tumor-to-muscle ratio of 8.4 after 2 h in athymic nude mice
xenografted with BMG-1 cells, suggesting the specificity of 68Ga-DO3A-Act-AQ toward tumors.
KEYWORDS: anthraquinone, CT-DNA, BMG-1, HEK, gallium-68, PET, cancer
Anthraquinone, having a planar tricyclic structure, is the
backbone of many known antitumor drugs like doxorubicin
and mitoxantrone capable of targeting at the molecular/DNA
level.2 They are known for their foremost property of
intercalation between the DNA base pairs, causing an
unwinding of the helix leading to miscoding and possible cell
apoptosis.3 Gadolinium complexes of 1,4- and 1,5-diaminoan-
thraquinone conjugated 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA) based compounds are
reported to bind with the specific DNA sequence open-
d(ATCGAGACGTCTCGAT)2.4 Recently, mono- and dime-
INTRODUCTION
■
Despite phenomenal advances in the understanding of the
diseases, cancer still remains the leading cause of death
worldwide. Therefore, new strategies for combined diagnostic
and therapeutic are imperative for the early diagnosis of cancer
and appropriate therapeutic strategy.1 In addition to the
surgical removal of tumor tissue, other modes of pretreatment
include chemotherapy, radiotherapy, and immunotherapy.
Currently, a wide range of chemotherapeutic agents in the
form of alkylating agents, DNA intercalator, and antimetabo-
lites are known wherein they affect cell division or DNA
synthesis, leading to inhibition of cell growth and cell death.
There is a continuing interest in the development of new agents
in which the anthraquinone base structure represents an
attractive target for the design of new anticancer agent due to
its strong role in the control of cell proliferation.
Received: July 16, 2013
Revised: December 17, 2013
Accepted: December 22, 2013
Published: December 23, 2013
© 2013 American Chemical Society
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tallic Au(I) triphenylphosphine complexes derived from 1,2-,
1,4-, and 1,8-dialkynyloxyanthraquinone have been synthesized,
and their cytotoxicities on the cancer cell line (MCF-7) and
fluorescence cell imaging have been reported.5 1,4- and 1,8-Bis-
substituted anthracenedione derivatives inhibit the activity of
telomerase.6 Aminoacylhydroxyanthraquinone bearing glycyl,
valyl, lysyl, and tryptophanyl residues, 1,4-bis(2-
aminoethylamino)anthraquinone−amino acid conjugates, and
1,4-diamidoanthraquinone derivatives were reported to exhibit
significant DNA binding (intercalation) or cytostatic or
cytotoxic activities in cancer cells.7−15
In nuclear imaging, the use of stable complexes with
radiometals is a crucial factor for the development of metal-
based imaging and therapeutic agents. The lanthanide
(trivalent) complexes of polyazapolycarboxylic macrocycle
with seven to eight donor atoms such as with DOTA are
known for their strong complexation stability, unusually rigid,
highly symmetrical structure, and adopting the same geometry
in solution as well as the solid state in comparison with
noncyclic ligands such as DTPA, even though both are
octadentate chelate. Positron emission tomography (PET) is
a renowned imaging modality, and recently worldwide interest
in 68Ga as a radioisotope for PET which gives high-resolution
images of the functional processes in the body has enhanced
rapidly because of its appropriate properties for high-quality
PET imaging.16−18
The anthraquinone moiety has been much explored for
cellular/in vitro imaging, but in vivo studies using 68Ga have not
yet been exploited in detail. This prompted us to work with
anthraquinone and to determine its potential as a therapeutic
and PET imaging probe. The current work has been directed
toward the development of an imaging as well as therapeutic
agent. The potential of the ligand as a tumor-specific agent was
evaluated after its synthesis and radiolabeling with 68Ga. Here,
we report the synthesis of a macrocyclic ligand conjugated with
DNA binding ligand anthraquinone (DO3A-Act-AQ) showing
specificity to the tumor using PET imaging and biodistribution
performed on athymic nude mice xenografted with BMG-1
cells. DNA (calf thymus) binding studies were also performed
using fluorescence and circular dichroism (CD) spectroscopic
techniques. The therapeutic potential of the synthesized
macrocyclic derivative was assessed by determining the
protonation constant and complexation behavior of DO3A-
Act-AQ with therapeutic metals like Sm(III) and Lu(III). The
cytotoxicity, induction of apoptosis, and effect on cell cycle in
both cancer and normal cells were assessed by MTT assay and
flow cytometric methods, respectively, to further evaluate its
therapeutic behavior.
purchased from Aldrich (99.9%). Stock solutions of the
individual metal cations were prepared by dissolving respective
metal salts in water, and their concentration was determined by
complexometric titration with standardized Na2H2-EDTA
(ethylenediaminetetraacetic acid) and xylenol orange as an
indicator. Radiocomplexation and the radiochemical purity was
checked by instant thin layer chromatography (ITLC-SG)
strips and radio-TLC. High glucose Dulbecco’s modified
Eagle’s medium (HG-DMEM), trypsin-EDTA, bovine serum
albumin (BSA), Hank’s balanced salt solution (HBSS), crystal
violet, propidium iodide (PI), annexin V−PI kit, RNase A and
low melting agarose (LMA), and ethidium bromide (EB) were
obtained from Sigma-Aldrich, St. Louis, MO, USA. Phosphate-
buffered saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT), and citric acid were procured
from Himedia Laboratory Pvt. Ltd., Mumbai, India.
1
Instrumentation. H and 13C NMR spectra were recorded
on a Bruker Avance II 400 MHz (Switzerland). High-resolution
electrospray-ionization mass spectrometry (ESI-MS) was
carried out on Bruker micrOTOF-Q II and Agilent 6540
UHD accurate mass Q-TOF LC/MS. HPLC analyses were
performed on Agilent 1200 LC coupled to a UV detector (λ =
214 and 254 nm). The C-18 RP Agilent columns (5 μm, 4.6
mm × 250 mm, 5 μm, 9.4 mm × 250 mm) and mobile phase
(System A: 20% methanol and 80% water; System B: 10%
acetonitrile and 90% water) were used. The flow rate for
analytical HPLC was 0.5 mL/min, while that for preparative
HPLC was 10 mL/min. Flow cytometric measurements were
carried out using a FACS calibur flow cytometer (Becton
Dickinson and Co., Franklin Lakes, NJ, USA). Radiolabeling
studies and potentiometric measurements were carried out
using a γ-scintillation counter GRS230, ECIL (India) and an
automatic titration system consisting of Metrohm 713 pH
meter equipped with a Metrohm A.60262.100 electrode and
800 Dosino autoburet, respectively. CD spectra were measured
on a JASCO 815 spectrophotometer, and the MTT assay
absorbance was read at 570 nm using 630 nm as a reference
wavelength on an ELISA reader (BioTek instruments, Vinooski,
VT, USA). PET imaging was performed using a Discovery STE
16 (GE) system. UV−vis and fluorescence studies were
performed in a Biotek synergy H4 hybrid multiplate reader.
Synthetic Approach. tert-Butyl-2,2′,2″-(1,4,7,10-tetraa-
zacyclododecane-1,4,7-triyl)triacetate (1). Synthesis was
performed as reported in the literature (see also Scheme
1).19 Briefly, to a stirring solution of 1,4,7,10-tetraazacyclodo-
decane (1.5 g, 8.72 mmol) in dry acetonitrile (100 mL) was
added NaHCO3 (2.19 g, 26.0 mmol) at 0 °C. This was
followed by the dropwise addition of tert-butyl bromoacetate
(3.86 mL, 26.0 mmol). The reaction was further allowed to
reach 25 °C and stirred for 48 h. After 48 h the reaction
mixture was filtered, and the filtrate was evaporated under
pressure to obtain the crude product. It was purified by open
column chromatography on silica gel, Rf = 0.7 (eluent: CHCl3/
CH3OH: 9.5/0.5) to give the desired product as a white solid in
80% yield (3.5 g). The final product was well-characterized by
MATERIALS AND METHODS
■
Chemicals. 1,4,7,10-Tetraazacyclododecane was purchased
from Strem, France. 1-Aminoanthraquinone, tert-butylbromoa-
cetate, 2-chloroacetylchloride, potassium carbonate, potassium
iodide, N,N-diisopropylethylamine (DIPEA), pyridine, dry
acetonitrile, methanol, dichloromethane, toluene, trifluoroacetic
acid, water, chloroform, and ethanol were purchased from
Sigma-Aldrich, USA. Column chromatography was carried out
using silica MN60 (60−200 μm). Thin layer chromatography
(TLC) was checked on aluminum plates coated with silica gel
1160, F254 (Merck, Germany), and visualized under UV light
254 nm. For the potentiometric studies, the chemicals used
were of the highest analytical grade. The metal salts, SmCl3·
6H2O, LuCl3·6H2O, CuSO4·5H2O, and Ga(NO3)2·xH2O were
1
1H, 13C NMR, and mass spectrometry. H NMR (CDCl3, 400
MHz): δH = 3.18−3.28 (s, 6H, CH2), 2.89−2.94 (m, 16H,
CH2), 1.437 (s, 27H, CH3) ppm; 13C NMR (CDCl3, 100
MHz): δC = 170.52, 169.63, 81.94, 81.69, 58.22, 51.36, 51.24,
49.21, 48.79, 47.52, 28.24, 28.20 ppm; HR-ESI-MS calculated
for [C26H50N4O6 + Na]+: 537.3628; found [M + Na]+:
537.3631.
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(400 mg, 0.778 mmol) dissolved in toluene (100 mL)
compound 2 (279 mg, 0.93 mmol) was added after the
addition of KI (129 mg, 0.778 mmol) and DIPEA (0.2 mL)
under an inert atmosphere. The reaction was heated at 90 °C
for 18 h and monitored by TLC (CHCl3:CH3OH, 9:1) until
completion of the reaction. The solvent was removed under
vacuum, and chloroform was added to the oily residue which
was further given wash with water (2 × 100 mL). The organic
layer was collected separately and dried over Na2SO4, filtered,
and evaporated, yielding an orange oil which was further
purified using open column chromatography using eluent
(CHCl3/CH3OH, 9:1) to give product 3. Yield: 0.480 g, 80%.
1H NMR (400 MHz, CDCl3): δH = 12.38 (1H, NH), 8.95 (1H,
s, ArH), 8.2 (2H, d, J = 5.2 Hz, ArH), 8.0 (1H, t, ArH), 7.8
(1H, t, ArH), 2.1−4.2 (24H, br, CH2), 1.42 (27H, s, CH3)
ppm, 13C NMR (100 MHz, CDCl3): δC = 187.36, 182.47,
173.02, 172.95, 172.14, 141.42, 135.27, 134.72, 134.64, 134.11,
133.78, 132.71, 127.47, 127.14, 126.08, 122.83, 117.89, 81.85,
81.83, 57.98, 55.82, 55.71, 27.92, 27.87 ppm. HR-ESI-MS
calculated for [C42H59N5O9 + Na]+: 800.4210; found [M +
Na]+: 800.4181. HPLC, System B, λ = 254 nm; tR = 4.21 min.
2,2′,2″-(10-(2-(9,10-Dioxo-9,10-dihydroanthracen-1-yla-
mino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-
triyl)triacetic acid, DO3A-Act-AQ. Compound 3 (100 mg) was
added in a mixture of dichloromethane and trifluoroacetic acid
(1:1), 10 mL at 0 °C. The reaction mixture was brought to 25
°C and further stirred for additional 48 h. The solvent was
evaporated under vacuum, and the residue was dissolved in 2
mL of MeOH, followed by addition of 30 mL of diethyl ether at
0 °C and stirred for 1 h at room temperature producing a red
precipitate of compound 4 which was further purified by
Scheme 1. Representation for the Synthesis of DO3A-Act-
AQ
a
1
preparative HPLC. Yield: 60 mg, 76%. H NMR (400 MHz,
MeOD): δH = 9.0 (1H, br, NH), 7.28−8.1 (7H, bm, ArH),
2.1−4.2 (24H, m, CH2) ppm, 13C NMR (100 MHz, DMSO-
d6): δC = 186.05, 182.02, 174.98, 170.00, 140.26, 136.85,
135.19, 123.08, 121.59, 118.51, 115.90, 113.15, 54.78, 51.16,
49.20 ppm. HR-ESI-MS calculated for [C30H35N5O9 + Na]+:
632.2332; found [M + Na]+: 632.2347. HPLC, System A, λ =
254 nm; tR = 10.97 min.
a
Reagents and conditions: (a) 2-chloroacetyl chloride, pyridine,
acetonitrile, 40 °C, 16 h; (b) tert-butylbromoacetate, acetonitrile,
NaHCO3, 0 °C, 48 h; (c) DIPEA, KI, toluene, 90 °C, 18 h; (d)
DCM−TFA (1:1), 0 °C, 48 h.
Radiochemical Synthesis. Fresh 68Ga3+ [t1/2 68 min] was
eluted from 68Ge/68Ga generator system (IGG100, Eckert
Ziegler, Germany) using 1 mL of 0.1 M HCl (740 MBq) and
was trapped on a cation exchange Strata X-C cartridge column.
The column was washed with 1 mL solution of 80% acetone/
0.15 N HCl. 68Ga desorbed from the column with 400 μL of
98% acetone/0.05 N HCl mixture. The pH of purified 68Ga-
(III) was adjusted to 4.0 using 1 M HEPES (2-[4-(2-
hydroxyethyl)piperazin-1-yl]ethanesulfonic acid) buffer, and a
stoichiometric amount 100 μg of DO3A-Act-AQ was added to
the labeling vial. The mixture was heated at 90 °C for 10 min. It
was cooled and passed through C-18 cartridge (preconditioned
with C2H5OH and H2O) to remove excess free 68Ga by
washing the cartridge with water (1 mL), and finally the
compound was eluted from the cartridge with 200 μL of
H2O:C2H5OH (7:3). The labeling efficiency was checked by
ITLC-SG in CH3OH/NaOAc (1:1) and ACN/H2O (1:1)
solvent system.
2-Chloro-N-(9,10-dioxo-9,10-dihydroanthracen-1-yl)-
acetamide (2). 1-Amino-9,10-anthraquinone (0.5 g, 2.24
mmol) and pyridine (300 μL) were taken in toluene (50
mL) and heated at 40 °C under nitrogen. A solution of 2-
chloroacetylchloride (0.357 mL, 4.48 mmol) in toluene was
added dropwise to it. The reaction mixture was allowed to
reach 25 °C and left for overnight stirring. The solvent was
evaporated under pressure, and the residue obtained was
dissolved in 200 mL of CHCl3. The organic layer was washed
with water (3 × 50 mL) and dried over anhydrous Na2SO4.
The solvent was removed under vacuum yielding compound 2
as a yellow powder. Yield: 0.460 g, 68.6%. 1H NMR (400 MHz,
CDCl3): δH = 9.15 (dd, 1H, ArH), 8.30 (m, 1H, ArH), 8.20 (m,
1H, ArH), 8.10 (dd, 1H, J = 1.2 Hz, ArH), 7.75 (m, 3H, ArH),
4.31 (s, 2H, CH2), 13.1 (1H, Br, NH) ppm, 13C NMR (100
MHz, CDCl3): δC = 187.09, 182.53, 166.39, 140.84, 135.75,
134.54, 134.41, 134.12, 133.87, 132.75, 127.59, 127.09, 126.03,
123.42, 118.58, 43.38 ppm. HR-ESI-MS calculated for
[C16H10ClNO3 + H]+: 300.0427; found [M + H]+: 300.0424.
HPLC, System B, λ = 254 nm; tR = 2.57 min.
Octanol−Water Partition Coefficient (log Poctanol/water).
Log P was determined at physiological condition, pH 7.4 by the
“shake-flask” method.20 To a solution containing octanol (500
μL) and PBS (500 μL, pH 7.4) (obtained from saturated
octanol-PBS solution), 68Ga-DO3A-Act-AQ (50 μL, 1.85 MBq)
was added. The resulting solution was vortexed at room
tert-Butyl-2,2′,2″-(10-(2-(9,10-dioxo-9,10-dihydroanthra-
cen-1-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclodode-
cane-1,4,7-triyl)triacetate (3). To a stirring solution of 1,4,7-
tris(tert-butoxycarbonylmethyl)-1,4,7,10-tetraazacyclododecane
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temperature and centrifuged at 4000 γ for 15 min. Aliquots of
10 μL were taken from both octanol and saline phases, and the
activity was measured on a γ counter. The experiment was
performed in triplicate. The partition coefficient (P) was
calculated as log P.
In Vitro Studies. Cell Culture. Human brain malignant
glioma (BMG-1) and human embryonic kidney (HEK) cells
were procured from the National Centre for Cell Science, Pune
and were maintained at 37 °C in a humidified CO2 incubator
(5% CO2, 95% air) in RPMI 1640 and high glucose−DMEM,
respectively. Cells were supplemented with 5% fetal bovine
serum, 2 mM L-glutamine, 100 U/mL penicillin, and 100 μg/
mL streptomycin. Cells (1 × 106) were regularly subcultured
twice a week using 0.05% trypsin in 0.02% EDTA to maintain a
log phase growth of cells.
Cell Viability Assessment. The cytotoxicity of DO3A-Act-
AQ was assessed using the MTT colorimetric assay.21 HEK and
BMG-1 cells were seeded in 96-well plates (2 × 104 cells/200
μL per well) for 24 h. These cells were treated with DO3A-Act-
AQ at different concentrations (0.0−0.5 mM) for different time
periods (4 h, 24 and 48 h) at 37 °C. Untreated cells were used
as a control. After treatment, cells were incubated with MTT
(0.5 mg/mL) for 4 h at 37 °C at desired time intervals. The
medium was then carefully removed by aspiration, and the cells
were lysed in 200 μL of DMSO to dissolve the formazan
crystals. The enzymatic reduction of MTT to formazan crystals
was quantified by the optical density checked at 570 nm with
630 nm as a reference filter. The experiment was repeated three
times, and results have been expressed as the mean SD.
Determination of Phosphatidylserine Externalization.
Apoptosis induction in HEK and BMG-1 cells after treatment
with DO3A-Act-AQ was studied by flow cytometry using Sigma
(APOAF) annexin V-FITC apoptosis detection kit. Cells were
seeded in 96-well plates (1 × 103) cells per well and incubated
at 37 °C for 24 h. The stock solution (10 mM) of DO3A-Act-
AQ was diluted with culture medium to a total volume of 300
μL. Untreated cells were used as a control. Cells were treated
with 0.1 mM DO3A-Act-AQ. Cells were collected by
centrifugation (10 min, 1000 γ) after harvesting, washed with
PBS again, and resuspended in annexin-binding buffer (10 μg/
mL HEPES, 140 μg/mL NaCl, and 2.5 μg/mL CaCl2, pH 7.4).
Afterward, 5 μL of the annexin V conjugate was added to each
sample and 5 μL of PI staining solution (1 μg/mL). Cells were
incubated at room temperature for 15 min in dark. Flow
cytometric analysis was made within one hour of addition of
annexin-binding buffer to each tube. Annexin V-FITC
fluorescence was collected on the FL-1 channel, and PI
fluorescence was collected on the FL-2 channel on the log scale.
A minimum of 10 000 cells per sample were acquired and
analyzed using Cell Quest software.
Becton, Dickinson, and Co., Franklin Lakes, NJ) and ModFit
LT (version 2.0: Verity Software, Inc., Topsham, ME, USA)
software for acquisition and analysis. The analysis was done in
triplicate.
In Vitro Stability. The in vitro stability of 68Ga-DO3A-Act-
AQ was checked at the physiological pH. The complex was
incubated with PBS, and the ITLC was checked at different
time intervals up to 4 h to determine the stability of conjugate
in PBS. Further, serum stability was estimated using blood
samples collected from healthy volunteers by venipuncture
which was allowed to clot for almost 45 min at 37 °C in a
humidified incubator at 95% air/5% CO2. The blood samples
were centrifuged, and the human serum was collected by
filtration through 0.22 μm syringe filter. Human serum (1 mL)
was incubated with 50 μL of 68Ga-DO3A-Act-AQ (3.7 MBq),
and dissociation of the complexes was determined after 30, 60,
120, 180, and 240 min by running an ITLC using methanol−
ammonium acetate (1:1) as the developing solvent.
CD Spectroscopic Measurements. All CD spectroscopic
measurements were carried out with a continuous flow of
nitrogen purging the polarimeter, and the measurements were
performed at room temperature with 1 cm pathway quartz cells.
The CD spectra were run from 320 to 220 nm at a speed of 20
nm/min, and the buffer (5 μg/mL Tris−HCl, pH 7.4)
background was automatically subtracted. Data were recorded
at an interval of 0.1 nm. The CD spectrum of CT-DNA alone
(0.05 mM) was recorded as the control. The results were taken
as ellipticity in mdeg, and scans were accumulated and
automatically averaged. The concentration of CT-DNA was
kept constant at 0.05 mM, while 0.1 mM of compound in buffer
solution was added to CT-DNA (1:1).
Photophysical Properties. UV−vis absorbance and fluo-
rescence emission spectrum for DO3A-Act-AQ was measured
to check the λabsorbance and λexcitation and λemission range. A stock
solution of concentration 1 mM was prepared in PBS buffer,
and dilutions were done as per the requirement. A spectrum
was recorded from 200 to 800 nm range. The dilution prepared
was in the range of 10−70 μM.
Fluorescence Spectroscopic Studies. The fluorescence
emission spectra were measured at 298 K in the wavelength
range of 300−800 nm. Blanks corresponding to the Tris-HCl
buffer solution (pH 7.4) were subtracted to correct background
fluorescence. Increasing concentrations of compound (0.1−0.4
mM) was micropipetted directly into a 1.0 cm cell containing
0.005 mM ethidium bromide and 0.5 mM CT-DNA (total
volume 1 mL), and the reaction was performed at room
temperature. The synchronous fluorescence spectra was
recorded by scanning both excitation and emission wavelengths
simultaneously. The spectrofluorometric measurements of all
samples were carried out over the excitation wavelength range
of 200−700 at 1 nm intervals, and Δλ (a constant wavelength
interval between emission and excitation wavelength) was
changed in the range of 10−200 at 10 nm intervals (total of 20
Δλ increments). The EB-DNA system was excited at 530 nm,
and the emission spectrum was taken at 604 nm. The relative
binding of the compound with CT-DNA was determined by
using classical Stern−Volmer equation
Cell Cycle Analysis. Exponentially growing cells (HEK and
BMG-1) were treated with 0.1 mM of DO3A-Act-AQ for 4 and
24 h. Cells were collected by trypsinization and washed twice in
ice-cold PBS at pH 7.4. Cell suspensions were fixed by vigorous
addition of ice-cold 70% (v/v) ethanol and stored at 4 °C for a
minimum of 24 h prior to analysis. Cells at a density of
approximately 1 × 106 were treated with 200 μg/mL RNase A
for 30 min at 37 °C followed by 200 μL of propidium iodide
staining solution (20 μg/mL propidium iodide in PBS at pH
7.4) and incubated in the dark at room temperature for 10−15
min. The percentage of cells in sub-G1, G0/G1, S, and G2/M
phases of cell cycle was determined and analyzed from at least
three independent experiments using Cell Quest (version 3.0.1:
Io/I = 1 + Ksv[compound]
where Io and I are the fluorescence intensity in absence and
presence of the quencher, Ksv is the Stern−Volmer quenching
constant, and [compound] is the concentration of the
compound.
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The apparent binding constant (Kapp) was also calculated
from the equation,
surface blood and debris from the organs. Organs were
weighed, and the radioactivity was measured. The uptake of
68Ga-DO3A-Act-AQ in each tissue was calculated and ex-
pressed as percentage injected dose per gram of the tissue.
Scintigraphic Studies. PET imaging was performed on
athymic nude mice bearing tumor from BMG-1 cells xenografts
grown for 14 days on right forelimb (100−200 mm3). 68Ga-
DO3A-Act-AQ (3.7 MBq, 100 μL) was injected through a tail
vein of the animal to visualize the tumor uptake. PET images
were taken at 120 min post injection.
KEB[EB] = Kapp[compound]
where the complex concentration was the value at a 50%
reduction of the fluorescence intensity of EB and KEB = 1.0 ×
107 M−1 [EB] = 5.0 μM.
The stock solution of CT-DNA was prepared freshly in 5 μg/
mL Tris-HCl buffer, pH 7.4. The purity of the DNA was
checked by observing the ratio of the absorbance at 260/280
nm. The solution gave a ratio of >1.8 at A260/A280, which
indicates that DNA was sufficiently free from protein. The
concentration of DNA in stock solution was determined by UV
absorption at 260 nm using a molar absorption coefficient ε260
= 6600 L mol−1 cm−1.
Potentiometric Titration. A pH meter−electrode system
was calibrated for pH 4, 7, and 9 using Metroholm ion analysis
buffers before the titrations. The protonation constant of
DO3A-Act-AQ were determined potentiometrically by titrating
1 mM of DO3A-Act-AQ with 0.1 mM tetramethylammonium
hydroxide (TMAOH). Titrations were performed in the pH
range of 2−11.5 for protonation constants. The complexation
constants of DO3A-Act-AQ with Sm(III), Lu(II), Cu(II), and
Ga(III) were determined by direct pH potentiometric titration
(2−4 mM Mn+ and 2 mM ligand solutions), I = 0.1 g/L using
TMACl. Metal solutions were prepared by dissolving the
quantitable amounts of their salts in Milli-Q water. For
titrations of complexes, a 1:1 ligand to metal ratio was
prepared, and the experiment was performed in duplicate. The
protonation and stability constants were evaluated from
titration data using the program Tiamo 2.0.
Animal Model. All animal experiments were performed in
accordance with guidelines of Institute’s Animal Ethics
Committee (Reg. No. 8/GO/a/99/CPCSEA). For blood
clearance study, male New Zealand rabbits were used, and
athymic nude mice were used for PET imaging and ex vivo
biodistribution studies. Animals were housed under conditions
of controlled temperature of 22 2 °C and normal diet. Mice
were inoculated subcutaneously with 0.1 mL of cell suspension
of BMG-1 (1 × 106 cells) in the right front flank under
sterilized condition. When the tumor volume reached ∼400
mm3 (after 3−4 weeks of inoculation), the BMG-1 tumor-
bearing mice were used for biodistribution and imaging studies.
Blood Kinetics Study. The blood kinetics was carried out in
male albino New Zealand rabbit weighing approximately 2.6−
3.0 kg to study the clearance of the radiolabeled complex from
the blood circulation. 37.0 MBq of radiolabeled compound was
injected intravenously through the dorsal ear vein of the rabbit
and blood samples (0.3 mL) were taken from the other ear vein
at different time interval starting from 10 min until 240 min,
and counts were monitored on a gamma counter. Whole body
blood was considered to an amount equal to 7.3% of the body
weight of the rabbit. Data were expressed as percent
administered dose at different time intervals.
Statistical Analysis. Results are depicted as the mean
standard error of the mean (SEM). Statistical analysis was
performed using a Student’s t test. P < 0.05 was considered
significant.
RESULTS
■
Synthesis, Radiolabeling, Partition Coefficient, and in
Vitro Stability. The potential of anthraquinone to control
cellular proliferation through DNA binding was utilized for
tumor targeting by conjugating it with macrocyclic, 1,4,7,10-
tetraazacyclododecane based chelating system. The synthesis of
DO3A-Act-AQ involves the conjugation of DO3A unit by
means of a linker to an anthraquinone moiety. 2-Chloroacetyl
chloride was chosen as source of linkage and was reacted with
1-aminoanthraquinone to give the α-chloramide, 2-chloro-N-
(9,10-dioxo-9,10-dihydroanthracen-1-yl)-acetamide, 2, in 68.6%
yield. 1,4,7-Tris(carboxymethyl)-1,4,7,10-tetraazacyclodode-
cane, 1 (80%), was synthesized by reaction of tert-
butylbromoacetate on 1,4,7,10-tetraazacyclododecane. The
fourth unsubstituted arm of DO3A was conjugated with 2-
chloro-N-(9,10-dioxo-9,10-dihydro-anthracen-1-yl)-acetamide
in the presence of DIPEA to yield compound 3 in 80% yield.
The progress of the reaction steps was checked through TLC in
solvent system 9.5:0.5 (DCM−CH3OH). The compound 3 was
1
characterized by NMR spectroscopy wherein the H spectrum
data clearly shows the entire seven protons of anthraquinone in
aromatic region of 7.8−8.9 ppm. While the 24 methylene
protons in the region of 2.1−4.1 ppm along with the 27
protons of methyl group at 1.5 ppm confirms the conjugation.
Later, tertiary butyl groups of compound 3 were cleaved
using (1:1) TFA−DCM to obtain the desired compound,
2,2′,2″-(10-(2-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino)-
2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
acid in 76% yield after purification. The precursors and final
compounds were characterized by 1H, 13C, and high resolution
mass spectrometry (Figures S1−S12 in the Supporting
Information). 68Ga labeling was performed in a straightforward
method with a preparation time for 68Ga-DO3A-Act-AQ of 20
min at 90 °C with a good radiochemical yield (84%) and a
specific activity of 4.62 MBq/nmol. The maximum radio-
chemical yield was observed at pH 4.0 using HEPES buffer,
after heating for 10 min at 90 °C. The radiochemical purity was
greater than 95%, with DO3A-Act-AQ binding to Ga3+
quantitatively to provide a single radiolabeled product as
monitored by radio-TLC using 50% aqueous acetonitrile
solvent as shown in Figure 1. After radiolabeling traces of
free Ga3+ from the reaction mixture were removed by passing
the labeled compound on a C-18 cartridge followed by washing
with water. 68Ga-DO3A-Act-AQ was eluted from the C-18
cartridge with H2O−C2H5OH (7:3) which further enhanced on
increasing the elution volume from 100 to 200 μL. The
partition coefficient, log P (pH 7.4), of complex was
determined by the “shake-flask” method using 1-octanol-to-
Ex vivo Biodistribution Study. The biodistribution of 68Ga-
DO3A-Act-AQ was studied in six weeks old female athymic
nude mice weighing approximately 20 g. An intravenous
injection of 68Ga-DO3A-Act-AQ in a volume of 100 μL (9.25
MBq) was injected through the tail vein, and mice were
sacrificed at 1, 2, 3, and 4 h time intervals post injection. Blood
was collected by cardiac puncture, and all other different
organs, heart, lung, liver, spleen, kidneys, stomach, and muscle
were removed and washed with normal saline to remove
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Figure 1. Radio-TLC of 68Ga-DO3A-Act-AQ in the ACN−water (1:1)
solvent system.
Figure 2. Colorimetric estimation for the cytotoxicity of DO3A-Act-
AQ with respect to time (4, 24, and 48 h) and concentration (0.5, 1,
10, 50, 100, 500 μM) in HEK and BMG-1 cell lines.
PBS and was found to be −1.67 0.2. To check the stability of
the complex at physiological conditions, the complex was
incubated with PBS buffer (pH 7.4). The probe was stable up
to 2 h at physiological condition as no free 68Ga was detected
by the radio TLC. For in vitro serum stability analysis, the probe
was challenged with the proteins present in serum (albumin
and transferrin). More than 92% of the radioactivity was found
to be associated with the conjugate even after 2 h incubation at
37 °C. After 2 h 68Ga-DO3A-Act-AQ exhibited 13.7%
transcomplexation in human serum which increased to 14.3%
after 4 h of challenge suggesting serum stability up to 2 h
(Figure S13 in the Supporting Information).
Cell Viability Assay. The cytotoxic effect of DO3A-Act-AQ
was determined on two cell lines, BMG-1 and HEK cells, by an
MTT assay. Time and concentration dependence on
cytotoxicity was analyzed by cell viability data (surviving
fraction) at different concentrations (0.05−0.5 mM) and time
intervals (4, 24, and 48 h). At lower concentrations no sign of
cytotoxicity was seen in either cell lines. HEK cells showed
nonsignificant cell death after 24 h of treatment (0.05 mM),
late apoptotic cell population after 4 h was noticed in HEK cells
which decreased nonsignificantly at late time point of 24 h.
After 4 h, a necrotic BMG-1 cell population showed a 47-fold
increase which was 2.3-fold in HEK cells.
Cell Cycle Analysis. Cell cycle analysis was performed on
HEK and BMG-1 cells after 4 and 24 h on treatment with
DO3A-Act-AQ (0.1 mM). A measurable change at various
phases of cell cycle was observed post 4 h treatment. BMG-1
cells in the G1 phase decreased to 53.27% from 58.47%, while
an increase in HEK cell population in the G1 phase from 34.2%
to 59.31% was observed. A significant increase in cellular
population was observed in the S phase of BMG-1 cells (6.09%
in control to 17.77% in 4 h), but HEK cell population
decreased from 16.59% to 6.66%.
The distribution of cells in different phases post 24 h
treatment suggested increase in BMG-1 cells in S phase to
26.94% with concomitant increase of 8.88% of HEK cells
(Figures 5 and 6). In G1 phase population of BMG-1 cells came
down to 35.32%, while an unsubstantial change was seen in
HEK cells. BMG-1 cells also exhibited 8-fold increases in sub
G1 phase cell population (0.84% in control to 6.41% after 24
h); however, the apoptotic HEK cell population decreased with
time and reaches to 0.24% (after 24 h) compared to 1.09% in
control.
and the surviving fraction decreased from 0.971
0.01% to
0.930 0.03%, whereas BMG-1 cells on incubation with
DO3A-Act-AQ (0.05 mM) exhibited a significant reduction in
survival (59.0 0.5%) after 4 h which further decreased to 43.0
0.11% on increasing the concentration to 0.5 mM. DO3A-
Act-AQ displayed a time- and dose-dependent toxicity toward
BMG-1 cells as shown in Figure 2.
Phosphatidylserine Externalization. The ability of DO3A-
Act-AQ to induce apoptosis in BMG-1 and HEK cells was
ascertained through flow cytometry. Phosphatidylserine ex-
ternalization/apoptosis was examined after 4 and 24 h of
treatment with DO3A-Act-AQ (0.1 mM) as shown in
flowcytometric plot in Figure 3. Here, Q1 quadrant represents
cells having high affinity for annexin V, simultaneous binding of
annexin V, and propidium iodide represents Q2 quadrant,
indicating cells in late apoptosis; Q3 represents live cells, and
Q4 are solely stained with PI and corresponds to necrotic cells.
Treatment of DO3A-Act-AQ on BMG-1 cells resulted in an
increase in early apoptotic cells (FITC+/PI−) to 23.8% (4 h) in
comparison to 0.1% in control which reached to 26.3% after 24
h of treatment, whereas late apoptotic cell population (FITC+/
PI+) displayed a significant increase from 0.2% at 0 h to 16.9%
at 24 h as shown in Figure 4. However, in HEK cells, cells
undergoing early apoptosis in FITC+/PI+ fraction showed a
marginal increase from 0.4% in control cells to 1.3% after 4 h
and 2.0% after 24 h of treatment. Also, a 3-fold increase fold in
Photophysical Results. UV−vis and fluorescence spectros-
copy studies were performed on DO3A-Act-AQ at different
concentration ranges (0.01−0.07 mM). The concentration
range of DO3A-Act-AQ was optimized for performing
fluorescence and absorption studies, and the suitable window
for the studies was found in the region 0.01 mM. Absorbance
and fluorescence bands were observed which enhanced on
progressive increase in concentration (hyperchromic shift. The
λabsorption‑max was found at 393 nm, while λexcitation‑max and
λemission‑max were observed at 415 nm and 543 nm, respectively
(Figure S14 in the Supporting Information).
DNA Binding Study Using Fluorescence and CD Spectros-
copy. The binding of DO3A-Act-AQ with CT-DNA was
studied by evaluating the fluorescence emission intensity of the
ethidium bromide−DNA system upon successive addition of
the DO3A-Act-AQ which acted as quencher. The fluorescence
quenching profile of EB bound to DNA on addition of DO3A-
Act-AQ is shown in Figure 7 in which the fluorescence intensity
at 610 nm (λexcitation‑max 518 nm) of EB in the bound form was
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Figure 3. Flow cytometric plots of BMG-1 (a, b, c) and HEK (d, e, f) cells in the presence of annexin-V/propidium iodide (PI) after treatment with
DO3A-Act-AQ (0.1 mM) at 4 and 24 h.
reported value of DOTA; however, with Cu(II) it was found to
be less.22
Blood Kinetics Study, Ex Vivo Biodistribution, and PET
Imaging. The blood clearance studies of 68Ga-DO3A-Act-AQ
performed in normal rabbits displayed a biphasic clearance
pattern from blood circulation and 70.3% of injected activity
washed out from the circulation at 30 min post injection as
shown in Figure 8. The percentage of residual activity in blood
circulation at 2 h was 13.61 0.01% which further decreased to
9.42 0.04% at 4 h p.i. Thus, blood clearance of complex from
circulation followed a biphasic mode with activity rapidly
clearing in the initial phase followed by a slow phase later.
Figure 4. Apoptosis in HEK and BMG-1 cells at different time
Biological half-lives t1/2(fast) and t1/2(slow) were found to be
intervals (4 and 24 h) after treatment with DO3A-Act-AQ (0.1 mM).
40
0.3 min and 3 h 40 min
0.1 min, respectively. The
PET/CT studies were carried out using an integrated PET/CT
scanner on athymic nude mice xenografted with BMG-1 cells at
different time intervals. The mice injected with 68Ga-DO3A-
Act-AQ exhibited accumulation at the site of tumor within 1 h
which reached maximum in 2 h (Figure 9). Prominent
abdominal uptake was observed; however, a quick washout
from liver was also seen. 68Ga-DO3A-Act-AQ was checked ex
vivo for their pharmacokinetics on athymic nude mice
xenografted with BMG-1 cells. The biodistribution studies
were performed at various time intervals between 30 and 240
min. The study was useful for the screening of biological profile
and to assess the potential of complex in achieving the
objective. Data from these studies are shown in the graphical
form in Figure 10. The accumulation of 68Ga-DO3A-Act-AQ in
kidney and liver was 6.21 0.03 and 3.6 0.01 at 30 min p.i.
The localization in stomach and intestine was lower than 1.2%
after 1 h p.i. The study reveals good accumulation of the
complex in tumor which reached maximum after 2 h with value
of 1.85 0.03%. After 3 h, localization of activity was second
highest in tumor (1.2 0.32) after kidney (2.31 0.21). The
accumulation in tumor decreased to 0.98 0.34 after 4 h. The
ratio of tumor to soft tissue (muscle) was found to be 1.72 at
30 min which increased to 8.40 after 2 h of administration.
plotted against the compound concentration and found to be in
agreement with a Stern−Volmer plot of Io/I vs [DO3A-Act-
AQ] (Figure S15 in the Supporting Information). The
quenching constant was found to be 2.4157 × 106 L mol−1.
The apparent binding constant Kapp was determined to be 1.25
× 106 M−1. The CD spectrum of CT-DNA after addition of
DO3A-Act-AQ in a ratio of (1:1) demonstrated a decrease in
the intensity of both the positive band at 275 nm and the
negative band at 248 nm as shown in Figure S16 in the
Supporting Information.
Protonation and Thermodynamic Stability Constant. The
protonation and stability constant of complex of DO3A-Act-
AQ with Cu(II), Ga(III), and therapeutic metals like Sm(III)
and Lu(III) was determined by potentiometric titration over
the pH range 2−11.5. A pH-potentiometric titration of the
ligand DO3A-Act-AQ gave three protonation constants at 9.66,
8.07, and 5.87 which were likely to be due to the three
carboxylic acid groups present in the structure. The stability
constant of DO3A-Act-AQ for Sm3+, Lu3+, Ga3+, and Cu2+ were
found to be 19.5, 18.5, 19.3, and 11.6, respectively, and shown
in Tables 1 and 2. The values obtained for protonation constant
were found to be in accordance of the previously reported value
of DOTA. The stability constant with Ga(II) and therapeutic
metals Sm(III) and Lu(III) were in accordance of previously
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Figure 5. (a−f) Cell cycle analysis of BMG-1 and HEK cells treated with DO3A-Act-AQ (0.1 mM) at different time intervals (4 and 24 h).
Figure 6. Cell cycle distribution of HEK and BMG-1 cells on different stages following treatment of DO3A-Act-AQ (0.1 mM) after 4 and 24 h. The
percentage of cells in different phases of the cell cycle was analyzed using Mod fit software.
cytotoxic behavior and specificity toward tumor was studied
DISCUSSION
■
and compared with known derivatives. The synthesis of probe
Anthraquinone-based compounds that inserts between the two
base pairs present in the DNA double helix and culminates to
cell death have demonstrated amazing potential in cancer
chemotherapy.23,24 Similarly, 1,4,7,10-tetraazacyclododecane-
1,4,7,10-tetraacetic acid (DOTA) forms high in vitro and in
vivo stable radiometal (M3+) labeled complexes.25,26 Thus it was
thought worthwhile to utilize the DNA intercalating properties
of anthraquinone in the detection and treatment of tumors. A
facile synthetic strategy was used where 1,4,7-tris-
(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A) is
conjugated with anthraquinone derivative, 2-chloro-N-(9,10-
dioxo-9,10-dihydro-anthracen-1-yl)-acetamide to finally obtain
2,2′,2″-(10-(2-(9,10-dioxo-9,10-dihydroanthracen-1-ylamino)-
2-oxoethyl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic
acid (DO3A-Act-AQ) as the product, and its cytostatic/
1
DO3A-Act-AQ was confirmed through H NMR by presence
of peaks in the region of 7.8−8.1 and 3.2−4.2 ppm
corresponding to 7 H of aromatic anthraquinone moiety and
24 H of the DO3A unit, and the [M + Na]+ peak at 632.2347
further supported the successful formation of DO3A-Act-AQ.27
The three protonation constant values (9.66, 8.07, and 5.87)
corresponding to three carboxylic acid groups present in probe
were in range of other known derivatives of DOTA further
supported the synthesis of DO3A-Act-AQ an suggested no
effect of anthraquinone on the chemical properties of DO3A.
68Ga is a 68Ge/68Ga generator system β+ emitting radionuclide
with a half-life of 68 min and is a promising candidate for
application in positron emission tomography (PET) requiring
no on-site cyclotron facility, making it economic. Unlike other
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Figure 7. Emission spectra of EB (5.0 μM) bound to CT−DNA (50
μM) in the absence and presence of compound, (r = 0, 0.2, 0.4, 0.6,
0.8, 1.0, 1.2, and 1.4), r = [compounds]/[CT−DNA]. Arrows show
the intensity changes upon the addition of increasing concentrations of
DO3A-Act-AQ, respectively. λexcitation‑max = 518 nm.
Figure 9. PET/CT images of athymic nude mice (xenografted with
BMG-1 cells on front right flank) after 2 h showing coronal and
transaxial images.
Table 1. Protonation Constant (log K) of DOTA, DTPA,
and DO3A-Act-AQ in NMe4Cl (0.1 M) at 298 K
DOTA
DTPA
DO3A-Act-AQ
log K1H
log K2H
log K3H
11.14
9.69
4.85
10.49
8.37
4.09
9.66
8.07
5.87
Table 2. Stability Constant (log β) of Metal Complexes of
DOTA and DO3A-Act-AQ (298 K, μ = 0.1 M, NMe4Cl)
DOTA
DO3A-Act-AQ
log βGaL
log βCuL
log βSmL
log βLuL
21.3
22.2
23.0
25.4
19.3
11.6
19.5
18.5
Figure 10. Biodistribution of 68Ga-DO3A-Act-AQ performed on
female athymic nude mice bearing BMG-1 tumor on the right front
flank at different time intervals (30, 60, 120, 180, and 240 min).
buffer HEPES. A higher pH resulted in inferior radiolabeling,
probably due to the formation of hydroxide species. Similarly, a
temperature lower than 90 °C reduced the radiochemical yield
to almost 40% at 55 °C as higher reaction temperatures
enhance the complexation of less reactive Ga3+ species, that is,
partly oxygenated species.28 Radio-TLC analysis suggested
formation of 68Ga-DO3A-Act-AQ as the major species (>95%)
which was suitable enough to proceed for further studies.
DOTA derivatives are known for stable gallium complex
formation, which was further confirmed by high stability
constant value (21.3) with Ga3+.29 In vitro stability of 68Ga-
DO3A-Act-AQ was established in PBS (pH 7.4) with >94%
intact radiotracer as no significant detachment from the
radiolabeled complex was observed on ITLC even after 4 h
incubation at physiological condition. Since the title compound
was designed for tumor thus consequently, investigation on its
tumor binding affinity was of prime importance along with its
pharmacokinetics. Strong serum protein binding, that is,
68Ga3+-transferrin complex in comparison to 68Ga-labeled
complex, often leads to a delay in blood clearance, resulting
in a low target-to-background ratio; besides human plasma
enzyme also tends to decompose the exogenous compounds, so
Figure 8. Blood clearance of 68Ga-DO3A-Act-AQ (37.0 MBq) activity
administered through ear vein in normal New Zealand rabbit.
PET radioisotopes like 11C or 18F, ionic Ga3+ cannot be bound
covalently to targeting biological vectors but must be
complexed by a ligand which is conjugated to the vector giving
an advantage that labeling can be performed just prior to in vivo
evaluation, and loss of activity is thus kept to a minimum. Thus
DO3A-Act-AQ with DO3A backbone was utilized for PET
imaging post labeling with 68Ga. 68Ga-DO3A-Act-AQ was
prepared in high radiochemical purity (>94%) and radio-
chemical yield (84%) with a specific activity of 4.62 MBq/nmol.
The optimum complexation yield for 68Ga3+ with DO3A-Act-
AQ was achieved in the pH range of 3.5−4.0 using nonionic
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a binding study with human serum protein is a requirement for
the correct prediction of the biological importance of the
probe.30 In vitro serum stability of DO3A-Act-AQ was assessed
and found to be more than 90% after 2 h of incubation with
human serum at physiological pH, making it appropriate to
avoid background activity due to dissociation of the metal ion.
MTT assay on HEK cell line predicts limited toxicity on normal
cells inducing 10% toxicity after 24 h of treatment (0.1 mM)
ensuring its suitability for imaging purpose. In vivo blood
clearance exhibited a biphasic clearance pattern in blood
kinetics data of 68Ga-DO3A-Act-AQ which could be con-
tributed to low lipophilicity of probe. Biological distribution
studies were performed in athymic nude mice, and the
percentage of injected dose associated with each organs was
calculated based on the activity measured/g organ or tissue.
The biodistribution study suggested prominent uptake of 68Ga-
DO3A-Act-AQ in kidneys at all measured time points
suggesting renal excretion route of the probe. As anthraquinone
derivatives are mainly metabolized through liver, the activity
due to the marginal G1 and S and G2 phase arrest after 4 and 24
h. These results were found to be better than 1,3-substituted
anthraquinone derivatives which were inducing G2/M phase
arrest in NTUB1 cells (a human bladder carcinoma cell line),41
thus ensuring retained anticancer property of the compound as
evident by apoptosis induction along with S phase arrest in
cancer cells after 24 h of treatment. The therapeutic behavior of
anthraquinone derivatives are known by DNA binding nature.
The efficacy of the developed molecule was checked by
performing DNA binding studies with fluorescence and CD
spectroscopic techniques under physiological conditions. It is
already well-known that the emission intensity of ethidium
bromide dye enhances upon DNA intercalation.42 A reduction
in emission intensity was observed upon progressive addition of
DO3A-Act-AQ (10−70 μM) due to the substitution of bound
ethidium bromide from the EB-DNA system with DO3A-Act-
AQ. The Stern−Volmer quenching constant, Ksv, and apparent
binding constant, Kapp, of DO3A-Act-AQ with CT-DNA were
found to be 2.4157 × 106 L mol−1 and 1.25 × 106 M−1,
respectively. These values were compared with Stern−Volmer
quenching constant of 2-aminoanthraquinone with CT-DNA
which was 1.246 × 103 L mol−1 demonstrating that compound
have strong binding affinity toward CT-DNA.43 The Stern−
Volmer quenching constant of DO3A-Act-AQ was found to be
higher than reported for other macrocyclic moiety like 1,4,7-
triazacyclononane (TACN, 0.053 0.010).44 CD spectrum of
CT-DNA exhibits a positive absorption band at 282 nm due to
the base stacking and a negative absorption band at 240 nm due
to the helicity of B-DNA. The intensity of both bands
decreased in the presence of DO3A-Act-AQ (1:1, CT-DNA-
DO3A-Act-AQ) signifying unwinding of the double helix DNA
after binding with the conjugate, leading to disturbing the
secondary structure of DNA. Results obtained from fluo-
rescence and CD spectroscopic studies concluded that
compound strongly binds to DNA.45 To increase the
therapeutic potential of DO3A-Act-AQ, it can be complexed
with β-emitting radionuclides; thus its complexation behavior
with 153Sm(III) and 177Lu(III) was studied.46 The essential
requirement for DO3A-Act-AQ to be suitable for therapeutic
purpose is the stable complex formation with metal ions along
with the cytotoxicity toward cancer cells. A highly stable and
strong complex formation was observed with both samarium
and lutetium as found by high log K values obtained after
titration, suggesting that the probe can be used with therapeutic
metals for the treatment of cancer.
was found high (3.6
0.01%) in liver at 30 min which got
significantly reduced to 1.4 0.2% within 1 h. The uptake in
liver decreased faster than kidney with time and no prominent
accumulation was observed in other organs like the lungs,
spleen, heart, stomach, and intestine. The accumulation of
68Ga-DO3A-Act-AQ at the site of tumor is seen at an early time
point (1.2 0.01%, 1 h) which reached to a maximum (1.85
0.03%) at 2 h and remained significant up to 3 h. The tumor-to-
muscle ratio was 8.4-fold which further demonstrated the
potential of this hybrid compound as a tumor-seeking agent.
The imaging studies further provide strong support to the
tumor targeting ability of the developed probe.
The well-known DNA intercalating property (cytostatic/
cytotoxic nature) of anthraquinone, which is being exploited in
cancer chemotherapy to control cellular proliferation, was
investigated on DO3A-Act-AQ.31−34 The in vitro toxicity of
DO3A-Act-AQ toward neuroblastoma cells was compared with
healthy nephritic cells. The probe (0.1 mM) induced cellular
death in BMG-1 cells with only 47% cell viability was observed
after 24 h; however, no significant sign of cellular death was
seen in HEK cells, corroborating its specificity toward cancer
cells. The IC50 of simple 1,4-diamido anthraquinone derivative
on C6 (glioma) cells (>10 μM) were related with probe’s
toxicity toward BMG-1 cells (0.1 mM).35 The high IC50 value
of the conjugate was probably due to the conjugation of DO3A
moiety to the anthraquinone.35−39 As reported in literature,
anthraquinone derivatives induce apoptosis in cancer cells at
the micromolar range.40 Thus flow cytometry studies
performed on BMG-1 cells at IC50 showed an increase of
263-fold in early apoptosis cells, whereas HEK cells displayed a
nonsignificant increase of 5-fold after 24 h of treatment with
probe as compared to gastric cancer cell line after 24 h, which
showed 4.2-fold increase on treatment with 25 ng/mL of
triazole derived anthraquinone derivatives.31 BMG-1 cells
displayed an approximate 8-fold increase in sub-G1 level
which is an indicator of apoptotic cell population. However, a
decrease of 4.5% was observed in the sub-G1 phase of HEK
cells after 24 h. A significant increase in the cell population
from 6.09% to 26.94% after 24 h in the S phase revealed that
BMG-1 cells could arrest cell population in the S phase with
time. However a transient G1 phase arrest was seen initially in
HEK cells, which released at a later time point. Cell cycle
results were found to be supporting annexin V apoptosis
results, as the necrotic cell population in HEK cells could be
CONCLUSIONS
■
In conclusion, the synthetic strategy presented here demon-
strates a facile way for the design of a macrocyclic ligand with
DNA binding ligand like anthraquinone. The significant tumor
targeting property makes it a candidate of choice for PET
imaging. The cytotoxicity, induction of apoptosis in cancer
cells, formation of highly stable complex with therapeutic
metals like samarium and lutetium and preferential strong
binding with CT-DNA can be used to understand the
mechanism of anticancer nature and further expands its
application from diagnosis to therapy.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H, 13C NMR, high resolution mass spectra and HPLC profiles
of tert-butyl 2,2′,2″-(1,4,7,10-tetraazacyclododecane-1,4,7 triyl)-
triacetate (1), 2-chloro-N-(9,10-dioxo-9,10-dihydroanthracen-
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Molecular Pharmaceutics
Article
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pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*Tel.: +91-11-23905123, 23905117. E-mail: anupama@inmas.
drdo.in.
■
(15) Huang, H. S.; Chiu, H. F.; Lee, A. R.; Guo, C. L.; Yuan, C. L.
Synthesis and structure-activity correlations of the cytotoxic bifunc-
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12, 6163−6170.
*E-mail: akmishra63@gmail.com.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
This work was made possible by a financial contribution from
DRDO, project INM-311.3.1. We thank Dr. R. P. Tripathi,
Director, Institute of Nuclear Medicine and Allied Sciences,
Defence Research and Development Organization for provid-
ing excellent research facilities.
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