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Mitochondria-targeted half-sandwich iridium(iii)-Cp*-arylimidazophenanthroline complexes as antiproliferative and bioimaging agents against triple negative breast cancer cells MDA-MB-468.
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Mitochondria-targeted half-sandwich iridium(III)Cp*-arylimidazophenanthroline complexes as
antiproliferative and bioimaging agents against
triple negative breast cancer cells MDA-MB-468†
Ashaparna Mondal, ‡a Shanooja Shanavas, ‡b Utsav Sen, b Utpal Das,
Nilmadhab Roy, a Bipasha Bose *b and Priyankar Paira *a
a
To reduce the side effects of marketed cancer drugs against triple negative breast cancer cells we have
reported mitochondria targeting half-sandwich iridium(III)-Cp*-arylimidazophenanthroline complexes for
MDA-MB-468 cell therapy and diagnosis. Out of five Ir(III) complexes (IrL1–IrL5), [iridium(III)-Cp*-2(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]phenanthroline]PF6 (IrL1) has exhibited the best cytoselectivity
against MDA-MB-468 cells compared to normal HaCaT cells along with excellent binding efficacy with
DNA as well as serum albumin. The subcellular localization study of the complex revealed the
localization of the compound in cytoplasm thereby pointing to a possible mitochondrial localization and
consequent mitochondrial dysfunction via MMP alteration and ROS generation. Moreover, the IrL1
Received 16th February 2022
Accepted 28th March 2022
complex facilitated a substantial G1 phase cell-cycle arrest of MDA-MB-468 cells at the highest tested
concentration of 5 mM. The study verdicts support the prospective therapeutic potential of the IrL1
complex in the treatment and eradication of triple negative breast cancer cells. These results validate
DOI: 10.1039/d2ra01036d
that these types of scaffolds will be fairly able to exert great potential for tumor diagnosis as well as
rsc.li/rsc-advances
therapy in the near future.
Introduction
Nowadays, breast cancer prevails over most of the deadly life
threats towards women from all over the world. 11.7% of new
cases of cancer diagnosed in 2020 were female breast cancer
which is the highest number of cases among all types of cancer
reported.1 This disease is heterogenous at the molecular level
but, as a consequence of diligent research over past decades,
chances of healing have been increased by 70–80% in breast
cancer patients when the cancer is non-metastatic and treated
in the early stage. Over the years of research a few categorisations of tumours were rened based on the alterations at the
molecular level.2 Current clinical practices involve an alternate
classication of 5 types that includes triple negative breast
cancer (TNBC) without expression of ER, PR or HER2. TNBC is
adenoid cystic and metaplastic in nature with a poor prognosis
a
Department of Chemistry, School of Advanced Sciences, Vellore Institute of
Technology, Vellore-632014, Tamilnadu, India. E-mail: priyankar.paira@vit.ac.in
b
Department Stem Cells and Regenerative Medicine Centre, Institution Yenepoya
Research Centre, Yenepoya University, University Road, Derlakatte, Mangalore
575018, Karnataka, India. E-mail: bipasha.bose@gmail.com
† Electronic supplementary information (ESI) available: 1H, 31P and 19F NMR,
LCMS, IR, UV and uorescence spectra of all compounds. See
https://doi.org/10.1039/d2ra01036d
‡ Equal contribution.
© 2022 The Author(s). Published by the Royal Society of Chemistry
prole.2 A thorough investigation of medical literature reveals
that the term, “triple negative breast cancer”, was rst
mentioned in 2005 by Brenton and coworkers.3 However, in the
present situation 12–17% of all reported breast cancer cases
have been recognised under this class.
It has been challenging to create an effective treatment
regime for TNBC patients as this aggressive subtype does not
respond to the HER2 targeting drugs or hormonal therapy.
Consequently, chemotherapy remains as the main systemic
treatment option even though TNBC develops resistance easily
to existing targeted medicines like trastuzumab. Potential
molecular targets for TNBC may include EGFR (a surface
receptor), PARP1 (poly ADP-ribose polymerase 1) and DNA. The
phenotypic similarity of TNBC to BRCA-1 associated malignancy
helped researchers to develop few targeted cytotoxic agents
currently which may lead to a new horizon of TNBC therapeutics. In 2020 FDA approved trodelvy (sacituzumab govitecanhziy) which is a topoisomerase inhibitor conjugate antibody
directed to Trop-2 receptor for metastatic TNBC and for the
rst-time improved progression of overall survival was witnessed. Triple negative breast cancer caused by BRCA1 and
BRCA2 mutation can be a potential target of DNA damaging
chemotherapeutics and a number of clinical data leads to the
suggestion that involving platinum-based chemotherapeutics
may be use in standard treatment regime of early stage as well
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as advanced TNBC.4 As reported in earlier scientic research,
platinum salts like cisplatin, carboplatin or oxaliplatin are able
to initiate a platinum–DNA adduct formation followed by DNA
damage in cancer cells leading towards apoptosis of the targeted cancer cells. BRCA1 decient breast cancer showed
sensitivity towards platinum and gemcitabine neoadjuvant
treatment but with poor chance of progression free survival.5 A
clinical study, reported by von Minckwitz in 2013 involving 315
early stage TNBC patients, let out the fact that a neoadjuvant
treatment regime comprising doxorubicin and carboplatin
achieved 59% pathologic complete response rate.6 However,
a combination therapy of gemcitabine/carboplatin given to
advanced metastatic TNBC patients in a trial scored 34%
objective response rate and progression free survival of 5.1
months.7 Although cisplatin and its analogical platinum
complexes showed slight prospect in TNBC treatment they have
exerted some major drawbacks like toxicity, chemoresistance
and narrow activity window.8 This fact motivated broader area
of investigation on efficacy of other metal complexes to target
metastasis and defective cell proliferation. A handful of gold,
iron, copper, ruthenium, rhodium and iridium complexes
happened to be synthesized and tasted against breast cancer
cell lines in vitro or in vivo and found to manifest excellent
outcomes.9 Of late, Ir(III) based metal complexes have been
fascinating the mind of the investigators for being reconnoitered as highly active anticancer agents with their excellent
photoluminescence property possessing high photostabilities,
long-lived excited triplet states aer quick singlet to triplet
intersystem crossing (ISC), larger Stokes' shi associated with
high quantum yields.10,11 Furthermore, the higher oxidation
state of iridium metal, ability to display a wide range of ligand
substitution, considerable redox properties, exible structural
features and stability towards cancer cell environments has
enabled them to be used in preparing anticancer metal
complexes.10,11 Instead, bioactivities of metal complexes are also
reliant on the structure of the ligands. Arylimidazophenthroline
compounds are well known as pertinent probes of DNA structure having the capability of disentangling the double stranded
DNA through strong intercalative interaction.12 Encouraged by
Fig. 1
Paper
our previous work with ruthenium and arylimidazophenthroline herein we have intended to design ve iridium(III)-Cp* N^N
metal complexes with planar and p-extended arylimidazophenanthroline moiety as ligand having strong metal binding
capacity (Fig. 1).13–18 and evaluated their antiproliferative activity
against MDA-MB-468 TNBC cells that are EGFR +ve, TGF alpha
+ve, high in Ki67. These complexes exhibited dual properties
like (i) killing of cancer cells via DNA damage (ii) mitochondrial
dysfunction by ROS production.19 The intrinsic phosphorescence property of these Ir(III) complexes are also helpful for
cellular imaging and tracking of drug accumulation in subcellular organelles.20
Results and discussion
Chemistry
Synthesis and characterization. A series of imidazo[4,5-f]
[1,10]phenanthroline ligands (L1–L5) was prepared by treating
an equimolar mixture of 1,10-phenanthroline-5,6-dione and
different aromatic carboxaldehydes (1–5) in the presence of
ammonium acetate and glacial acetic acid, following the same
procedure as mentioned in our previous communication.21
Further to prepare Ir(III)-Cp*-imidazophenanthroline complexes
(IrL1–IrL5), [(C5(CH3)5IrCl2)]2 was added to the prepared ligands
(L1–L5) in a 1 : 2 ratio in methanol and stirred at room
temperature for 2 h. Aer a change in colour from light yellow to
orange, 2.5 equivalents of NH4PF6 were added and stirred again
for 90 min (Scheme 1). The complexes [Cp*Ir(L1–L5)Cl]PF6
labelled as IrL1–IrL5 were obtained in good yield (92–95%). The
structures of all the complexes (IrL1–IrL5) were analysed via 1H,
13
C, 19F and 31P NMR, and mass spectroscopy. The complex IrL1
displayed a characteristic singlet peak at 1.74 ppm, corresponding to the ve methyl groups of pentamethylcyclopentadiene. The protons of complex IrL1 experienced
a considerable downeld effect upon attachment to the iridium
Cp* precursor. In the 13C NMR spectrum, the ligand carbons
appeared at around d 125.8–153.2 ppm. The aliphatic CH3
carbons peaks were observed at d 8.7 ppm and aromatic CH
carbons peaks were observed at d 89.6 ppm. In the 19F NMR
Design of half-sandwich iridium(III)-Cp*-arylimidazophenanthroline complexes.
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Scheme 1
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Synthesis of (h5-Cp*)iridium(III)-imidazophenanthroline complexes.
spectrum, characteristic peaks of six uorines appeared at
d 69.2 and 71.08 ppm. The characteristic septate of phosphorous was observed in the range of d 135.41 to 157.37 ppm
in the 31P NMR spectrum. The ESI-MS peak at m/z: 709.17 [M]+
and isotopic pattern of iridium conrmed the formation of
complex IrL1. Similarly, clear differences in peak values in the
NMR, FT-IR and ESI-MS between the other complexes (IrL2–
IrL5) were observed.
Electronic absorption (UV-visible) and uorescence study.
The absorption and emission spectra of all the complexes (IrL1–
IrL5) at 298 K were recorded in a DMSO–water (1 : 1) solvent
system, as shown in Fig. 2. The photophysical data is summarized in Table 1. The characteristic intraligand (p–p*) transitions (N^N ligands) appeared at 250–350 nm and metal to
ligand charge transfer (1MLCT) at 360–400 nm.22,23 Among the
complexes, we observed the maximum absorption in the 1MLCT
region for the benzothiazole derivative (IrL5). In the emission
spectra, we observed the MLCT emission of all the complexes in
the range of 350–525 nm (Fig. 2). Similar to the absorption
spectra, the emission for the anthracene derivative is the most
intense because of its strong p conjugation. Using the emission
spectral data, the quantum yield of these complexes was
Fig. 2
calculated. These complexes didn't show remarkable quantum
yield, complex IrL1 showed moderate quantum yield (0.002)
though for the MLCT transition (Table 1).
Solubility, lipophilicity and conductivity study. Both hydrophilicity and lipophilicity studies were performed to determine
the tumour-inhibiting potential of the metal complexes. These
complexes were highly soluble in DMSO and moderately soluble
in H2O, MeOH, EtOH and CH3CN. Furthermore, they were
soluble in the range of 0.6–0.8 mg per mL of 10% DMSO in
DMEM, 10 : 90 v/v (comparable to cell media) at 25 C (Table 1).
The lipophilicity of these complexes was determined by performing an n-octanol/water partition coefficient (log Po/w, where
Po/w ¼ the octanol/water partition coefficient) study using the
shake ask method (Table 1).24 The experimental log Po/w values
of these complexes were determined to be in the range of 0.27–
0.34 (Table 1). Complex IrL2 exhibited the highest log Po/w due
to the hydrophobic nature of its anthracene group. The lowest
log Po/w value was observed for compound IrL3 because it's
hydrophilic chromone group. The iridium complexes IrL1–IrL5
exhibited molar conductance values in the range of 7–9 S m2
M1 in pure DMSO. Furthermore, their molar conductance
increased in 10% DMSO (22–36 S m2 M1, Table 1), suggesting
(a) UV-vis spectra (b) emission spectra of IrL1–IrL5 in DMSO–water (1 : 1) at RT.
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Photophysical characterization, solubility, lipophilicity and conductivity study of the complexes (IrL1–IrL5)
Table 1
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LMg (ms)
Samples
lmaxa (nm)
lfb (nm)
Stoke's
shi
3c (M1 cm1)
(ff)d
Solubilitye (M)
log Pf
DMSO
10%
DMSO
IrL1
IrL2
IrL3
IrL4
IrL5
Quinine sulfate
284
375
397
278
336
350
409
437
462
360
380
452
125
62
65
82
44
102
15 800
6850
2200
2200
2350
—
0.002
—
—
—
—
0.57
0.0007
0.0006
0.0012
0.0009
0.0007
—
0.27 0.07
0.34 0.07
0.23 0.2
0.02 0.05
0.29 0.08
—
8
6
7
9
9
—
22
27
27
30
36
—
a
Absorption maxima. b Maximum emission wavelength. c Extinction coefficient. d Quantum yield. e DMSO-10% DMEM medium (1 : 99 v/v,
comparable to cell media). f n-Octanol/water partition coefficients. g Conductance in DMSO and 10% aq. DMSO (IrL1–IrL5; 3 105 M).
their 1 : 1 and 1 : 2 electrolytic nature in pure DMSO and 10%
DMSO, respectively.25–27 This change in the electrolytic behaviour of the complexes from 1 : 1 to 1 : 2 can be attributed to the
dissociation of the Ir–Cl bond and subsequent aquation of the
complexes.
Stability study of the complexes by UV-vis spectroscopy. The
stability studies of complex IrL1 were conducted in six
different solvents, i.e. aqueous DMSO (H2O : DMSO ¼ 9 : 1)
and aqueous GSH medium (Fig. 3) respectively in presence or
absence of different concentrations of NaCl. It is essential
that the complexes remain stable in the biological environments of cells, and thus the stability studies were performed.
Fig. 3
The obvious change in absorbance (15–30% decrease aer
24 h) with time in aqueous DMSO clearly revealed the
moderate dissociation of the –Cl ligand from the Ir(III)
complexes followed by aqua complex formation, which was
also quantitatively determined based on the observed molar
conductivity of the complexes (IrL1–IrL5) in aqueous DMSO
and it favours DNA covalent binding as well. It has been reported that many cancer cells become resistant to various
drugs by increasing their cellular glutathione level.28 Hence,
to determine the effect of GSH on the reported complexes,
a stability study was performed in the presence of excess (10
eq.) glutathione (GSH) via time-dependent UV spectroscopy.
Stability study of IrL1 in (a) 10% DMSO media, (d) aqueous GSH media and in presence of 12N and 24N NaCl in each solution (b), (c), (e) and
(f).
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However, GSH didn't have much impact on the stability of
metal complex and hence resistivity may not be induced
against this complex in cancer cells containing high level of
GSH.
Cyclic voltammetry. The CV response of ten-continuous cycle
shows a well-dened reversible redox peak at E0 ¼ 0.485
0.005 V versus Ag/AgCl with a peak separation potential of DEp
Fig. 4 Cyclic voltammetry response of (a) Au modified IrL1 and (b)
bare Au electrode in PBS buffer.
Fig. 5
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value is 0.121 0.005 (DEp ¼ Epa Epc). Peak current (ipa and
ipc) of the redox peak was found to increasing linearly which
ensures the high stability and co-ordination of the metal–ligand
complex (Fig. 4).
Biology
In vitro cytotoxicity study. The in vitro cytotoxicity of
complexes IrL1–IrL5 and cisplatin were investigated using the
typical
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay protocol against a panel of triple negative cancer cell line MDA-MB-468. The cells were initially incubated with the all test compounds at concentrations ranging
from 0.5–50 mM for 48 h in vitro.
All the complexes exhibited signicant cytotoxicity with
IC50 values of 3.673 mM, 20.35 mM, 20.27 mM, 5.202 mM, 4.412
mM for IrL1, IrL2, IrL3, IrL4 and IrL5 respectively (Fig. 4). The
dose dependent cytotoxic effects of these complexes were
evident on MDA-MB-468 with IrL1 exhibiting the most potent
effects thereby exhibiting the lowest IC50 value of 3.67 mM
(Fig. 5). Consequently, the IrL1 complex doses of 1 mM, 3 mM
and 5 mM were selected for further experimental analysis.
Furthermore, the possible cytotoxic effects of IrL1 on normal
cell line were also examined on the immortalized human
keratinocyte cell line HaCaT. The IC50 value of IrL1 on HaCaT
Cytotoxicity study of IrL1 against TNBC and normal cells.
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Fig. 6
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DNA binding plot of complex IrL1; inset: [DNA]/(3a 3f) vs. [DNA] linear plots of IrL1.
is found to be 11.42 mM, which is signicantly 3 times higher
than the IC50 value of the complex on the breast cancer cell
line MDA-MB-468, suggesting the low cytotoxic effects of the
same on normal cells and safety implications to use the
compound for selective therapeutics at lower concentrations
(Fig. 4).
DNA binding studies
UV absorption method. To design effective chemotherapeutic drugs, it is essential to explore the interactions of metal
complexes with DNA. Complex IrL1 displayed strong absorption
band at 297 nm. DNA base pairs such as purine (adenine and
guanine) and pyrimidine (cytosine and thymine) analogues are
Fig. 7 Fluorescence quenching plot of complex IrL1; inset: I0/I vs. [complex] linear plots of IrL1.
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responsible for electronic transitions. Upon the addition of CTDNA in increasing concentration from 5 mM to 60 mM, we
observed a hyperchromic shi in p–p* region and hypochromic
shi at the MLCT region resulting in the appearance of an
isosbestic point, which indicates the prevalence of covalent
interaction of the complex with DNA together with the intercalative mode of interaction (Fig. 6).
EtBr quenching study. The competitive binding of
compounds IrL1 to CT-DNA was studied via uorescence
spectroscopy using ethidium bromide (EtBr) as the uorophore. We clearly observed a gradual decrease in the uorescence intensity of the EtBr bound DNA in the presence of
the complexes since they displaced EtBr from DNA, and
consequently got bound between the base pairs of the DNA,
suggesting the intercalative binding mode of action, as
observed in Fig. 7. Intrinsic binding constant (Kb), Stern–
Volmer quenching constant (Ksv) and apparent binding
constant (Kapp) were highlighted in Table 2.
The higher value of binding constant and lower values of
Stern–Volmer's quenching constant and apparent binding
Table 2
constant is evidence of the fact that IrL1 complex interacts with
DNA via covalent bond and not via intercalation mode.
BSA binding study. Upon excitation at 295 nm, the emission
intensity of BSA at lem ¼ 350 nm decreased gradually on
increasing the complex concentration, which conrmed that
the interaction between complex IrL1 with BSA had occurred, as
observed in Fig. 8. The Stern–Volmer quenching constant of
these complexes with BSA (KBSA) was calculated using the Stern–
Volmer equation and the corresponding Stern–Volmer plots
(Fig. 8, inset). The binding affinity (K) of the complexes was
calculated from Scatchard plot analysis (Fig. 8, inset).
The complexes showed strong binding propensity with BSA,
which is required for the transport of protein-bound complexes
in biological systems. KBSA for complex IrL1 was found to be 5
104 M1 the K value was 6.3 106 M1. The value of bimolecular
quenching constant (kq) calculated from KSV and s0 (1 108 s)
was observed to be 5 1012 M1 S1. These values are higher
than the maximum possible value for dynamic quenching (2.0
1010 L mol1 s1),29,30 suggesting the involvement of static
quenching mechanism by the present iridium(III) complexes.
Binding parameters for complex IrL1 with CT-DNA
Complex
lmax (nm)
Change in absorbance
D3a (%)
Kbb (M1)
Ksvc (M1)
Kappd (M1)
IrL1
250
305
Hyperchromism
Hypochromism
60
25
—
0.11 106
—
2.2 104
—
2.5 103
a
% change in hypochromism or hyperchromism. b Kb, intrinsic DNA binding constant from UV-visible absorption titration. c Ksv, Stern–Volmer
quenching constant. d Kapp, apparent DNA binding constant from competitive displacement.
Fig. 8 Fluorescence quenching of BSA by IrL1; inset: I0/I vs. [complex] linear plots for stern Volmer's constant and Scatchard plot of log([I0 I]/I)
vs. log[complex] for BSA.
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The non-linearity of the Stern–Volmer plot of IrL1 is due to the
formation of a ground state complex between the Ir(III) complex
and BSA.
Co-localization study. To identify the subcellular localization
of the IrL1 complex, the cells were treated with the complex and
stained with Hoechst and further explored using uorescence
microscopy. The complex exhibited cytoplasmic localization at
3 mM concentration with a green uorescence emission (Fig. 9).
Mitochondria are one of the key targets for several anticancer
drugs. Consequently, further experimental validation involved
mitochondrial membrane potential (MMP) analysis by JC-1
staining and detection of ROS generation by DCFDA staining.
Mitochondrial membrane dysfunction study. The IrL1
complex demonstrated mitochondrial depolarization of MDAMB-468 cells in a dose dependent manner. The cytoplasmic
localization of the IrL1 complex as evidenced by sub-cellular
localization study impelled us to explore the effect of the
complex on cytoplasmic mitochondria. Consequently, the
alterations in mitochondrial membrane potential (MMP, DJm)
and associated mitochondrial dysfunction was demonstrated by
JC-1, a cationic carbocyanine dye which exhibits potentialdependent accumulation in mitochondria. The cells treated
with the mitochondrial uncoupler CCCP (carbonyl cyanide mchlorophenylhydrazone), which mediates the dissipation of
mitochondrial membrane potential, served as positive control
for the detection of mitochondrial dysfunction.
The ow cytometric quantication of JC-1-stained cells
revealed a normal mitochondrial function in control cells
devoid of any treatment exhibiting 94% JC-1 aggregates (red+
green uorescing healthy mitochondria). Impaired mitochondrial function was observed in only 3.30% of control cells with
JC-1 monomers indicating green uorescence (Fig. 10B).
Unstained control represented in Fig. 10A has been used for
gating purposes. Conversely, the positive control with CCCP
treatment, displayed a high percentage of cells (79%) with
damaged mitochondria indicating the presence of green uorescing JC-1 monomers against 20% cells retaining healthy
Fig. 9
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mitochondria with red + green uorescing JC-1 aggregates
(Fig. 10C).
Treatment of the MDA-MB-468 cells with IrL1 has ensued
a dose dependent increase in JC-1 monomer expressing/green
uorescing cells indicating damaged mitochondria (Fig. 10D–
F). IrL1 complex at 1 mM concentration resulted in damaged
mitochondria in 27.86% cells (Fig. 10D). However, the higher
concentrations of the IrL1 complex at 3 and 5 mM induced
alterations of mitochondrial membrane potential and associated damage in high percentage 47.52% and 67.02% respectively in MDA-MB-468 breast cancer cells (Fig. 10E and F). The
proportion of green uorescing JC-1 monomers induced by
high concentrations of the complex at 3 and 5 mM thereby
indicates alteration in the MMP and mitochondrial depolarization and associated mitochondrial dysfunction leading to
a dose dependent possible cell death meditated by the IrL1
complex comparable to the CCCP treated positive control cells
(Fig. 10C).
ROS generation studies. The IrL1 complex exhibited a dose
dependent reactive oxygen species (ROS) generation in the
MDA-MB-468 breast cancer cells. The MDA-MB-468 breast
cancer cells exhibited ROS generation suggestive of cellular
stress leading towards cell death at elevated ROS levels.
Our results indicated a dose dependent increase in ROS
production in the MDA-MB-468 cells treated with IrL1 concentrations 1 mM and 3 mM. The extent of ROS production is indicated by the 20 ,70 -dichlorouorescein (DCF) formed by the
deacetylation and subsequent oxidation of 20 ,70 -dichlorouorescin diacetate (DCFDA) through cellular esterases and
ROS. 1 mM concentration of the complex has resulted in 55.01%
DCF positive cells which has increased up to 70.67% DCF
positive cells at 3 mM concentrations. ROS production was
absent in untreated control. However, in the positive control,
treated with H2O2, ROS production was high which is 99%
(Fig. 11).
Cell cycle analysis. The IrL1 complex exerted a G0/G 1 cell
cycle arrest at the highest tested concentration of 5 mM. The
Representative image of the cytoplasmically localized IrL1 complex in MDA-MB-468 cells, co-stained with Hoechst. Scale bar 75 mM.
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Fig. 10 Image representing the flow cytometric quantification of JC-1-stained MDA-MB-468 breast cancer cells for mitochondrial membrane
potential assessment (A) unstained control for gating purposes (B) untreated control (C) positive control (CCCP treated cells) (D) 1 mM treatment
(E) 3 mM treatment (F) 5 mM treatment (G) graph representing the percentage of JC-1 monomers/aggregates.
Fig. 11 Reactive oxygen species (ROS) assessment as a measure of cellular damage after treating the MDA-MB468 breast cancer cells with
various concentrations of IrL1 compound and the positive control H2O2.
effects of various concentrations of IrL1 complex on the cell
cycle of breast cancer cell line MDA-MB-468 cells were
further explored by cell cycle analysis. The untreated control
breast cancer cells demonstrated high S phase (35.16%),
followed by a G 2 /M phase with 33.18% cells, and 31.66% of
G 0/G 1 cells (Fig. 12). Conversely, the treatment of IrL1
complex resulted in a decrease of G 2/M phase cells upto
18.37% and a decrease of S phase cells upto 28.32% with
© 2022 The Author(s). Published by the Royal Society of Chemistry
the highest concentration of the complex i.e., 5 mM.
Remarkably, 5 mm concentration considerably increased G 0/
G 1 phase cells (53.32%), indicative of a substantial G1 arrest
of MDA-MB-468 breast cancer cells mediated by the IrL1
complex.
Annexin FITC/PI assay. The Annexin FITC/PI assay has
demonstrated signicant apoptotic initiation of IrL1 treated
MDA-MB-468 breast cancer cells. At 3 mM concentration of
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Analysis of apoptotic MDA-MB-468 cells treated with IrL1 complex by Annexin V-FITC/PI assay. The 3 mM concentration of the drug (IC50) has resulted in 34.89% early apoptotic cells, 9.41% of late apoptotic cells and 4.33% necrotic cells. The viable cell population was found to be
51.37%.
Fig. 12
the complex, 34.89% of cells were found to be displaying
early apoptotic phase and 9.49% cells exhibited late
apoptotic phase (Fig. 13). The viable cell population constituted 51.37% which supports the IC50 concentration of
3.673 mM demonstrated by IrL1 on MDA-MB-468 cells
thereby corroborating the IC50 data at this particular
concentration.
Experimental section
Materials and methods
In all the experiments, the reagents and solvents used were of
the highest grade and best commercial quality. All organic
solvents used throughout the chemical synthesis and chromatography procedures were of analytical grade and used without
Fig. 13 Cell cycle analysis of untreated and IrL1 treated (1 mM, 3 and 5 mM) MDA-MB-468 cells and representative graph of the cell cycle phases
up on treatment with IrL1 complex. IrL1 mediates G1 arrest of MDA-MB-468 cells at 5 mM concentration. The p value of <0.0001 (***) was
considered statistically significant. Error bar represents the standard error of mean (SEM).
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further purication as received from E. Merck (India). Pentamethylcyclopentadienyl iridium(III) chloride dimer, 1,10phenanthroline-5,6-dione, a-naphthaldehyde, 9-anthraldehyde,
chromone-3-carboxaldehyde, indole-3-carboxaldehyde and
benzothiazole-2-carboxaldehyde were procured from Sigma
Aldrich Chemical Ltd, Merck and Spectrochem. Thin layer
chromatography was performed on pre-coated silica gel 60 F254
aluminium sheets (E. Merck, Germany) and the solvent system
was an ethyl–acetate–methanol mixture.
1
H NMR, 13C NMR, 19F NMR and 31P NMR spectra were
recorded on a 400 MHz Advance Bruker DPX spectrometer with
tetramethylsilane (TMS) as the internal standard. The chemical
shis were reported in ppm units. Abbreviations are as
follows: s, singlet; d, doublet; dd, double doublet; t, triplet; and
m, multiplet. The melting points of the complexes were
measured on an Elchem Microprocessor-based DT apparatus
using an open capillary tube. The mass spectra of the synthesized compounds were recorded on a Shimadzu ESI-mass
spectrometer having a 4000 triple quadrupole MS, using
methanol as the solvent. UV-visible spectra were recorded on
a JASCO V-730 spectrometer using a 1 cm quartz cell and uorescence spectra on Hitachi F7000 uorescence spectrophotometer equipped with a xenon lamp. A PerkinElmer
instrument was used for the elemental analysis. The conductivity and viscosity study were performed using a conductivityTDS meter-307 and Ostwald viscometer, respectively.
For the cytotoxicity (MTT) assay and imaging study, an Elisa
reader, 96-well plate and Olympus CX41 uorescence microscope were used. Bovine serum albumin (BSA) was purchased
from Sigma Aldrich Chemical Limited. The MDA-MB-468 and
HaCaT cell lines were purchased from NCCS, Pune.
Synthetic procedure
Synthesis of [Ir(III)-Cp*-(arylimidazophenanthroline)Cl]PF6
complexes [IrL1–IrL5]: 30 mg (0.037 mmole, 1 eq.) of pentamethylcyclopentadienyl iridium dichloride dimer, [(C5(CH3)5IrCl2)]2, was dissolved in about 10 ml of methanol in a 50 ml
round bottomed ask and was stirred continuously for 5–
10 min to dissolve the reactant. To the completely dissolved
solution, 2.1 equivalents of the previously synthesized ligands
(L1–L5) were added and stirred at room temperature. Aer
90 min, 2.5 equivalents of ammonium hexauorophosphate
(NH4PF6) (13.3 mg, 0.082 mmol) was added as ligand exchange
salt in order to increase crystallinity and hence, purity of the
product and again the reaction mixture was stirred for 90 min
more at room temperature. The progress of reaction was
conrmed by TLC. Aer complete conversion of the starting
materials to the desired product, the solvent was evaporated
under reduced pressure. The crude product was washed with
hexane and further recrystallized from diethyl ether/methanol
(1 : 1) solvent system. Finally, the complexes (IrL1–IrL5) were
obtained as light brown crystals with high yield (90–92%).
Characterization data for complexes (IrL1–IrL5)
[Iridium(III)-Cp*-2-(naphthalen-1-yl)-1H-imidazo[4,5-f][1,10]
phenanthroline]PF6 (IrL1). 60 mg (0.0702 mmol, 95%); Mr
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(C33H29ClF6IrN4P) ¼ 854.25 g mol1; anal. calcd for C33H29ClF6IrN4P: C 46.40, H 3.42, N 6.56; found: C 46.12; H 3.09; N
6.23; Rf (100% methanol): 0.2; mp: >200 C; IR (cm1): n 3612,
3350, 3100, 1605, 1403, 1363, 1089, 836, 743, 566; 1H NMR
(DMSO-d6, 400 MHz): d 1.74 (s, 15H, Cp* aliphatic-CH3); 7.63–
7.78 (m, 3H, ligand ArH); 8.08 (d, 1H, J ¼ 7.6 Hz ligand ArH);
8.16–8.22 (dd, 2H, J ¼ 8.0 Hz, ligand ArH); 8.27 (m, 2H, ligand
ArH); 8.44 (m, 2H, ligand ArH); 9.08 (d, 1H, J ¼ 8.2 Hz, ligand
ArH); 9.34 (d, 2H, J ¼ 5.2 Hz, ligand ArH); 9.55 (d, 1H, J ¼
10.8 Hz, ligand ArH); 13C NMR (DMSO-d6, 100 MHz): d 8.7 (Cp*aliphatic Me), 89.63–90 (Cp*-aromatic carbon), 125.81 (2C,
ArC), 127.07 (2C, ArC), 127.95 (4C, ArC), 128.96 (2C, ArC), 130.8
(2C, ArC), 131.2 (2C, ArC), 133.5 (2C, ArC), 134.08 (2C, ArC),
150.58 (2C, ArC), 153.24 (2C, ArC); 19F NMR (DMSO-d6, 376
MHz): d 71.07 to 69.18 (6F, PF6); 31P NMR (DMSO-d6, 162
MHz): d 152.98 to 135.43 (PF6); ESI-MS (MeOH): m/z ¼ 709.17
[M]+.
[Iridium(III)-Cp*-2-(anthracen-1-yl)-1H-imidazo[4,5-f][1,10]
phenanthroline]PF6 (IrL2). 63 mg (0.069 mmol, 93%); Mr (C37H31ClF6IrN4P) ¼ 904.31 g mol1; anal. calcd for C37H31ClF6IrN4P: C 49.14, H 3.46, N 6.20; found: C 49.35; H 3.63; N 6.02; Rf
(100% methanol): 0.2; mp: >200 C; IR (cm1): n 3626, 3056,
1613, 1450, 1136, 835, 741, 556; 1H NMR (DMSO-d6, 400 MHz):
d 1.76 (s, 15H, Cp* aliphatic-CH3); 7.53 (t, 2H, J ¼ 8.0 Hz, ligand
ArH); 7.62 (t, 2H, J ¼ 8.0 Hz ligand ArH); 7.75 (brs, 2H, ligand
ArH); 8.28 (d, 4H, J ¼ 8.0 Hz ligand ArH); 8.96 (s, 1H, ligand
ArH); 9.17 (d, 1H, J ¼ 8.0 Hz, ligand ArH); 9.31 (d, 1H, J ¼ 8.0 Hz,
ligand ArH); 9.37 (s, 1H, ligand ArH); 13C NMR (DMSO-d6, 100
MHz): d 8.7 (Cp*-aliphatic Me), 89.68 (Cp*-aromatic carbon),
124.75 (3C, ArC), 125.75 (4C, ArC), 126.34 (4C, ArC), 127.85 (2C,
ArC), 128.07 (3C, ArC), 129.16 (2C, ArC), 130.12 (3C, ArC), 131.1
(4C, ArC), 131.2 (4C, ArC), 133.3 (1C, ArC); 144.6 (2C, ArC); 150.5
(2C, ArC); 151.2 (1C, ArC); 19F NMR (DMSO-d6, 376 MHz):
d 71.07 to 69.18 (6F, PF6); 31P NMR (DMSO-d6, 162 MHz):
d 152.98 to 135.43 (PF6); ESI-MS (MeOH): m/z ¼ 759.19 [M]+.
[Iridium(III)-Cp*-3-(1H-imidazo[4,5-f][1,10]phenanthrolin-2yl)-4H-chromen-4-one]PF6 (IrL3). 61 mg (0.07 mmol, 95%); Mr
(C32H27ClF6IrN4P) ¼ 872.23 g mol1; anal. calcd for C32H27ClF6IrN4P: C 44.07, H 3.12, N 6.42; found: C 44.32; H 3.45; N
6.12; Rf (100% methanol): 0.2; mp: >200 C; IR (cm1): n 3251,
1640, 1462, 1375, 1143, 1027, 836, 759, 555; 1H NMR (DMSO-d6,
400 MHz): d 1.73 (s, 15H, Cp* aliphatic-CH3); 7.66 (t, 2H, J ¼
7.6 Hz, ligand ArH); 7.85 (d, 1H, J ¼ 8.2 Hz, ligand ArH); 7.95 (t,
1H, J ¼ 7.6 Hz, ligand ArH); 8.24–8.31 (m, 4H, ligand ArH); 9.32
(s, 2H, ligand ArH); 9.43 (s, 1H, ligand ArH); 13C NMR (DMSOd6, 100 MHz): d 8.6 (Cp*-aliphatic Me), 89.7 (Cp*-aromatic
carbon), 92.7 (1C, ArC), 114.47 (1C, ArC), 119.05 (2C, ArC),
123.50 (1C, ArC), 125.59 (1C, ArC), 126.94 (1C, ArC), 127.81 (2C,
ArC), 133.52 (1C, ArC), 135.49 (1C, ArC), 144.43 (2C, ArC), 147.01
(2C, ArC), 150.41 (2C, ArC), 155.82 (1C, ArC), 158.58 (1C, ArC),
174.75 (1C, ArC); 19F NMR (DMSO-d6, 376 MHz): d 71.07 to
69.18 (6F, PF6); 31P NMR (DMSO-d6, 162 MHz): d 152.98 to
135.43 (PF6); ESI-MS (MeOH): m/z ¼ 727.15 [M]+.
[Iridium(III)-Cp*-2-((3aR,7aR)-3a,7a-dihydro-1H-indol-3-yl)1H-imidazo[4,5-f][1,10]phenanthroline]PF6 (IrL4). 57 mg
(0.068 mmol, 92%); Mr (C31H28ClF6IrN5P) ¼ 843.23 g mol1;
anal. calcd for C31H28ClF6IrN5P: C 44.16, H 3.35, N 8.31; found:
RSC Adv., 2022, 12, 11953–11966 | 11963
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C 44.31; H 3.59; N 8.67; Rf (100% methanol): 0.2; mp: >200 C; IR
(cm1): n 3112, 1635, 1448, 1393, 1090, 837, 727, 559; 1H NMR
(DMSO-d6, 400 MHz): d 1.73 (s, 15H, Cp* aliphatic-CH3); 7.54–
7.57 (q, 1H, J ¼ 2.8 Hz ligand ArH); 8.22–8.25 (q, 3H, J ¼ 4.4 Hz
ligand ArH); 8.58 (s, 1H, ligand ArH); 8.7 (s, 1H, ligand ArH);
9.29 (d, 3H, J ¼ 5.2 Hz, ligand ArH); 9.4 (brs, 2H, ligand ArH);
11.84 (s, 1H, ligand NH); 13C NMR (DMSO-d6, 100 MHz): d 8.62
(Cp*-aliphatic Me), 41.69 (1C, ArC), 89.64 (Cp*-aromatic
carbon), 92.80 (3C, ArC), 106.21 (2C, ArC), 121.32 (1C, ArC),
123.24 (2C, ArC), 125.26 (1C, ArC), 127.02 (1C, ArC), 127.58 (2C,
ArC), 133.02 (2C, ArC), 136.94 (1C, ArC), 143.85 (2C, ArC), 149.86
(2C, ArC), 151.67 (1C, ArC); 19F NMR (DMSO-d6, 376 MHz):
d 71.07 to 69.18 (6F, PF6); 31P NMR (DMSO-d6, 162 MHz):
d 152.98 to 135.43 (PF6); ESI-MS (MeOH): m/z ¼ 698.17 [M]+.
[Iridium(III)-Cp*-2-((3aR,7aR)-3a,7a-dihydrobenzo[b]thiophen-2-yl)-1H-imidazo[4,5-f][1,10]phenanthroline]PF6
(IrL5).
58 mg (0.068 mmol, 92%); Mr (C31H27ClF6IrN4PS) ¼ 860.28 g
mol1; anal. calcd for C31H27ClF6IrN4PS: C 43.28, H 3.16, N 6.51;
found: C 43.47; H 3.41; N 6.72; Rf (100% methanol): 0.2; mp:
>200 C; IR (cm1): n 3120, 2926, 1604, 1448, 1393, 1090, 837,
727, 559; 1H NMR (DMSO-d6, 400 MHz): d 1.72 (s, 15H, Cp*
aliphatic-CH3); 7.47 (s, 2H, ligand ArH); 8.01–8.08 (dt, 2H, J ¼
6.7 Hz ligand ArH); 8.23–8.26 (m, 2H, ligand ArH); 8.34 (s, 1H,
ligand ArH); 9.23 (d, 2H, J ¼ 8.0 Hz, ligand ArH); 9.3 (d, 2H, J ¼
5.2 Hz, ligand ArH); 13C NMR (DMSO-d6, 100 MHz): d 8.67 (Cp*aliphatic Me), 49.06 (1C, ArC), 89.67 (Cp*-aromatic carbon),
92.63 (1C, ArC), 123.22 (2C, ArC), 124.13 (1C, ArC), 125.11 (1C,
ArC), 125.76 (1C, ArC),126.52 (1C, ArC), 127.96 (1C, ArC), 133.29
(2C, ArC), 139.98 (2C, ArC), 140.09 (2C, ArC), 144.56 (2C, ArC),
150.54 (2C, ArC); 19F NMR (DMSO-d6, 376 MHz): d 71.07 to
69.18 (6F, PF6); 31P NMR (DMSO-d6, 162 MHz): d 152.98 to
135.43 (PF6); ESI-MS (MeOH): m/z ¼ 715.13 [M]+.
Cytotoxicity studies
Cytotoxicity study of all the synthesized compounds against
triple negative breast cancer cells MDA-MB-468 and immortalized human keratinocyte cell line HaCaT were done with a drug
incubation period of 48 h by following standard procedure that
were mentioned in ESI.† Preliminary studies were done to
understand the mode of action of the complex to induce cytotoxicity using ow cytometric methods.
Mitochondrial membrane dysfunction assay
Alterations in mitochondrial membrane potential (MMP,
DJm) and associated mitochondrial dysfunction was demonstrated by JC-1, a cationic carbocyanine dye. The cells were
treated with the mitochondrial uncoupler CCCP (carbonyl
cyanide m-chlorophenylhydrazone), which mediates the
dissipation of mitochondrial membrane potential, served as
positive control for the detection of mitochondrial dysfunction. The ow cytometric quantication of JC-1 stained cells
revealed a normal mitochondrial function in control cells and
the results obtained from the quantied JC-1 stained cells
among IrL1 treated cells were compared with the same of the
control.
11964 | RSC Adv., 2022, 12, 11953–11966
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Imaging studies
Colocalization study and ROS generation studies were performed using live cell lines in their log phase.
Statistical analysis
As the study had more than one group, one way ANOVA was
used for statistical analysis. The p value <0.05 was considered as
signicant.
Conclusions
Five Ir(III)-imidazophenanthroline complexes were synthesized
and characterized successfully. Our study has demonstrated the
cytotoxic potential of IrL1 complex on MDA-MB-468 breast
cancer cells in a dose dependent manner. The subcellular
localization study of the complex revealed the localization of the
compound in cytoplasm thereby pointing to a possible mitochondrial localization and consequent mitochondrial dysfunction. Subsequent analysis to demonstrate the alterations in
MMP and ROS generation mediated by the IrL1 complex has
revealed a signicant increase in mitochondrial dysfunction
and a resultant increase in ROS production suggestive of the
mitochondrial targeting potential of the IrL1 complex. The
Annexin V-FITC/PI assay has validated the cytotoxic potential,
along with the IC50 dose of the complex on MDA-MB-468 breast
cancer cells by initiating apoptotic pathway probably due to the
cellular energetic stress triggered by elevated ROS levels.
Furthermore, the IrL1 complex mediated a substantial G1 phase
cell-cycle arrest of MDA-MB-468 cells at the highest tested
concentration of 5 mM. The study ndings support the
prospective therapeutic potential of the IrL1 complex in the
treatment and eradication of triple negative breast cancer cells.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Authors are grateful to Department of Science and Technology,
Government of India for supporting the work through the DSTSERB CRG project grant (CRG/2021/002267) and ICMR-SRF
fellowship and contingency grant. The authors are grateful to
VIT University for providing VIT SEED funding. We acknowledge DST, New Delhi, India for DST-FIST project.
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