Luminescent iridium(iii) complexes as COX-2-specific imaging agents in cancer cells

Article abstract of DOI:10.1039/c6cc08109f

Two luminescent iridium(iii) complexes, 1 and 2, were synthesized and evaluated for their ability to probe COX-2 in human cancer cells. This is the first application of iridium(iii) complexes as imaging agents for COX-2. We demonstrate that complex 1 diff

Full text of DOI:10.1039/c6cc08109f

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Marilyn M. Olmstead, Alan L. Balch, Josep M. Poblet, Luis Echegoyenet al.
Reactivity differences of Sc3N@C2n (2n 68 and 80). Synthesis of the
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Luminescent iridium(III) complexes as COX-2-specific imaging
agents in cancer cells†
Chenfu Liu,‡a Chao Yang,‡b Lihua Lu,‡ac Wanhe Wang,‡a Weihong Tan,*de Chung-Hang Leung*b and
Dik-Lung Ma*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
Two luminescent iridium(III) complexes, 1 and 2, were synthesized such as COX-2. Recently, Uddin et al. reported
a CF3-
and evaluated for their ability to probe COX-2 in human cancer fluorocoxib A-based fluorescence molecule for COX-2-specific
cells. This is the first application of iridium(III) complexes as imaging.10 Marnett and co-workers studied the structure-
imaging agents for COX-2. We demonstrate that complex 1 activity relationship (SAR) of COX-2-specific imaging dyes.11
differentiates cancer cells from normal cells with high stability and Peng and co-workers developed several fluorescence probes
low cytotoxicity.
targeting COX-2 in cancer cells,12 while Zhang and co-workers
prepared a naphthalene-based two-photon optical probe for
real-time bioimaging of COX-2 in living biosystems.13 However,
to the best of our knowledge, no metal-based imaging agent of
COX-2 has been previously reported.
Cancer has become one of the greatest causes of death in
humans worldwide.1 Early diagnosis of cancer is particularly
important to minimize cancer mortality. Consequently, efforts
have intensified over the last decade to develop imaging
agents and inhibitors to diagnose and treat cancers.2
Molecular fluorescence imaging has attracted particular
attention for cancer cell imaging by its high sensitivity, high cell
permeability, and noninvasive nature.3
In recent years, luminescent transition metal complexes
have found significant use as biological and chemical imaging
agents and probes by virtue of their desirable photophysical
and structural properties, including, for example, (i) adjustable
emission and excitation spectra, (ii) long phosphorescent
lifetimes, (iii) relatively high luminescent quantum yields, and
(iv) well-defined three-dimensional structures that interact
specifically with biomolecules.14 As a result, transition metal
complexes have emerged as promising alternative choices to
organic dyes as diagnostic imaging agents for diseases.15
Cancer cells often overexpress enzymes, which therefore
act as biomarkers for cancer cell detection.4 For example, γ-
glutamyltranspeptidase (GGT),5 membrane type 1-matrix
metalloproteinase (MT1-MMP),3a quinone oxidoreductase
isozyme 1 (NQO1),6 and cyclooxygenase-2 (COX-2)7 are all
overexpressed in cancerous cells. In particular, COX-2 is highly
expressed in stomach, colon, pancreas, and other cancers.8
Importantly, although COX-2 is barely expressed in normal
cells, clinical data show that the level of COX-2 increases as
cancer progresses.9
Chemical probes can be used to monitor cancer biomarkers,
Fig. 1. Chemical structure of Ir(III) complexes 1, 2 and 3 and compounds
4 and 5.
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Fig. 2. Time-resolved emission spectra of complex 1 in the presence of
coumarin 460 fluorescent media. (a) t < decay (b) t > decay. Excitation
wavelength = 355 nm.
from strong autofluorescence. To validate this hypothesis, we
used the fluorescent organic dye coumarin 460 (Cm-460) as a
model matrix interference. For TRES measurement, the time
gate was set after the complete fluorescence decay of Cm-460.
When the time gate was shorter than the decay time, results
showed that Cm-460 exhibited a strong emission peak at 455
Scheme 1. Syntheses of Ir(III) complexes 1 and 2. Reagents and
conditions: (a) 4-chlorobenzyl chloride, NaH, DMF, 0 to 25 °C, 2 h, 4:
nm and that the peak of
1 was partially obscured by the
52% yield; Compound
5 is commercially available. (b) 1,10-
trailing edge of the Cm-460 peak (Fig. 2a). However, when the
time gate was set to be longer than the decay time,
fluorescence of Cm-460 was eliminated, and the emission of
phenanthrolin-5-amine, oxalyl chloride, DMF, DCM, room temperature,
overnight, 4a: 55% yield, 5a: 42% yield; (c) [Ir(ppy)2Cl]2, DCM/MeOH
(1:1, v/v), 25 °C, overnight, then NH4PF6, 2 h, 1: 53% yield, 2: 65% yield.
DMF = N,N-dimethylformamide, DCM = dichloromethane.
complex
In consideration of the promising luminescent behavior
shown by complexes and , we employed and as imaging
1 became more obvious (Fig. 2b).
In this study, we designed two novel Ir(III) complexes,
1
1
2
1
2
and
complex
Compound
indol-3-yl)acetic acid, a structural analogue of the well-known
COX-2 inhibitor indomethacin (compound
).16 Therefore,
complexes and can be considered to consist of a “signal
unit” conjugated to a “binder unit” or via an amide bond
2,
through conjugating an initial luminescent Ir(III)
with compound or , respectively (Fig. 1).
is 2-(1-(4-chlorobenzyl)-5-methoxy-2-methyl-1H-
agents to detect COX-2 in living cells. A Western blotting assay
was first employed to determine the expression of COX-2 in
two normal human cell lines, human embryonic kidney 293
(HEK293) and human liver cell (LO2) cells, and two human
cancer cell lines, human breast cancer (MCF-7) and human
cervical cancer (HeLa) cells. The results showed that COX-2 was
strongly expressed in both cancer cell lines, but not in the two
normal cell lines (Fig. S3).12, 16a Subsequently, HeLa cells and
3
4
5
4
5
1
2
3
4
5
linkage. We anticipated that such design could generate
suitable probe molecules to sense COX-2 and still retain
specific binding to the target enzyme.
LO2 cells were incubated with complex 1, 2 or DMSO for 4 h.
Fluorescence imaging of the cells showed that the HeLa cells
exhibited a strong and stable florescence upon excitation at
405 nm, while LO2 cells showed negligible fluorescence
The structures of complexes
intermediates, were characterized by 1H NMR, 13C NMR and
HRMS spectrometry. The photophysical properties of and
were then investigated. Complex shows maximum
emission wavelength at 575 nm with excitation at 295 nm,
while complex exhibits a maximum emission at 580 nm at an
1 and 2, as well as all
1
2
towards complexes
indicate that complexes
between cancer and normal cells. However, complex
lower solubility in the cell imaging assays. As seen in Figure S4,
insoluble particles, presumably from complex , were found
when HeLa cells were stained with complex . Thus, in further
experiments, complex was used a as a model to demonstrate
1
and
2
(Figs. 3a and 3b). These results
could potentially differentiate
showed
1
a
1
and
2
2
2
excitation wavelength of 290 nm. Both metal complexes
possess large Stokes shifts of around 280 nm based their
metal-to-ligand charge-transfer (MLCT) states, which are
2
2
1
significantly higher than those of organic dyes. Complexes
1
the imaging ability of Ir(III) complexes for COX-2. We presume
that the mechanism of the differential luminescence
enhancement in the imaging experiments is due to the binding
and were stable at 298 K in a DMSO-d6/D2O (9:1, v/v)
2
solution for at least seven days, according to 1H NMR
spectroscopy (Fig. S1). They are also stable in acetonitrile/H2O
(9:1, v/v) solution at 298 K for at least seven days, as shown by
of complex
1
to COX-2 in the cells. In normal cells, COX-2
would not accumulate
expression is low. Hence, complex
1
UV/Vis spectroscopy (Fig. S2). Moreover, complexes
1 and 2
within normal cells and no luminescence enhancement would
be observed. On the other hand, cancer cells exhibit strong
displayed long lifetimes of ca. 4.56 μs and 4.28 μs, respectively
(Table S1), virtually on the same order as that exhibited by
other Ir(III) complexes,17 whereas organic chemosensors
usually show nanosecond lifetimes. Employing time-resolved
emission spectroscopy (TRES), it was shown that the long-lived
phosphorescence of transition metal complexes, such as that
expression of COX-2. Complex
1 is therefore able to bind to
COX-2 and is accumulated within the cells, thus generating a
high level of luminescence in cancer cells. We also tested the
luminescence of complex 1
in the presence of H+, K+, Na+, Mg2+,
amino acids, bovine serum albumin (BSA) and human serum
of complexes
1 and 2, allowed emission to be differentiated
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curcumin treatment, HeLa cells were incubated for another 4 h
with complex (1 μM). Cell imaging showed that the
1
fluorescence intensity of the HeLa cells decreased with
increasing concentration of curcumin (Fig. 4b). Taken together,
this evidence shows that the luminescence of complex
1
is
linked to COX-2 expression, suggesting, in turn, that complex
could specifically recognize COX-2 in human cancer cells.
1
We then performed a dose-response experiment to study
the staining ability of complex The results of the
1.
fluorescence imaging experiment showed that the
luminescence intensity of the cancer cell line HeLa, after
incubation with complex
concentration of complex
1 for 2 h, increased with the
over the range of 0.3–30 µM (Fig.
1
5a). On the contrary, normal cell line LO2 showed only
negligible luminescence, even with the highest concentration
of complex
cell imaging was evaluated. The luminescence intensities of
complex -treated HeLa cells increased with incubation time
1 (Fig. S6). Next, the effect of incubation time on
1
(Fig. 5b). On the other hand, no luminescence was observed in
Fig. 3. Living cells stained by (a) complex 1 and (b) complex 2 (1.0
μM). (a, c, e, g, j, l, n, p) HeLa cells and (b, d, f, h, k, m, o, q) LO2 cells.
The upper row is luminescence imaging, and the lower row is bright
filed imaging. Excitation wavelength = 405 nm.
albumin (HSA) in the absence of cells. The experiment
demonstrated that these ions, amino acids, BSA and HSA did
not dramatically affect the luminescence of complex
S5a and b).
1 (Figs.
In order to confirm that complex
1 could specifically target
COX-2, the expression of COX-2 was blocked by treatment with
curcumin, which specifically inhibits the expression of COX-
2.16a, 18 After treatment of HeLa cells with curcumin (0–40 μM)
for 24 h, Western blotting showed that the expression of COX-
2 in the cells was markedly reduced (Fig. 4a). After 24 h of
Fig. 5. (a) Cancer cell line (HeLa) stained by different
concentrations of complex 1 (0.3, 1, 3, 10 and 30 μM). (b) Cancer
cell line (HeLa) stained by complex 1 (1.0 μM) at different
incubation time (0.5, 1, 2, 4, 8, 24 and 48 h).
LO2 cells, even up to the maximum incubation time of 48 h (Fig.
S7).
It is noteworthy that Ir(III) complex
and photostability. HeLa cells stained by complex
their luminescence intensity for at least 24 h (Fig. S8). This
indicates that complex could potentially detect and image
1
showed high stability
1
preserved
1
COX-2 in living cells over relatively longer time scales, which
contrasts favorably with reported organic fluorescence
probes.13
The cytotoxicity of complexes
1 and 2 was examined by
using the (2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-
Fig. 4. (a) Immunoblotting analysis of the effect of curcumin treatment
tetrazolium-5-carboxanilide) (XTT) assay (Fig. S9). HEK293 cells,
LO2 cells, MCF-7 cells and HeLa cells were incubated with
on HeLa cells after 24 h. Densitometry analysis revealed that curcumin
inhibited
dehydrogenase (GAPDH) was used as control enzyme. (b) HeLa cells
stained by complex (1.0 μM) in the presence of different
COX-2
expression.
Glyceraldehyde-3-phosphate
different concentrations of Ir(III) complexes
1 and 2 for 72 h,
and cell viability was examined. The IC50 value of complex
1
1
was estimated to be over 100 µM after exposure for 72 h. The
estimated IC50 values against all four cell lines were higher
concentration of curcumin. The upper row is luminescence imaging, and
the lower row is bright filed imaging. Excitation wavelength = 405 nm.
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6
7
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2 inhibited the
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and containing indomethacin analogue or
1
2
,
(
4
5
)-
functionalized N^N ligands, were explored as imaging probes
for COX-2 based on the unique molecular structures
8
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1
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of China (21575121), the Guangdong Province Natural Science
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16/03), the Science and Technology Development Fund,
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