DOI: 10.1002/ejic.

201501105

Full Paper

Photoactive Anticancer Compounds

Mitochondria-Targeting Iron(III) Catecholates for Photoactivated

Anticancer Activity under Red Light
Uttara Basu,[a] Ila Pant,[b] Paturu Kondaiah,*[b] and Akhil R. Chakravarty*[a]
Abstract: Iron(III) catecholates [Fe(R-bpa)(R-dopa)Cl] (1, 2)
with a triphenylphosphonium (TPP) moiety, where R-bpa
is 2-(TPP-N,N-bis((pyridin-2-yl)methyl)ethanamine) chloride
(TPPbpa) and R-dopa is 4-{2-[(anthracen-9-yl)methylamino]ethyl}benzene-1,2-diol (andopa, 1) or 4-{2-[(pyren-1-yl)methylamino]ethyl}benzene-1,2-diol (pydopa, 2), were synthesized and their photocytotoxicity studied. Complexes 3 and 4
with [phenyl-N,N-bis(pyridin-2-yl)methyl]methanamine (phbpa)
were used as controls. The catecholate complexes showed an
absorption band near 720 nm. The 5e paramagnetic complexes showed a FeIII/FeII irreversible response near 0.45 V and

a quasi-reversible catechol/semiquinone couple near 0.5 V versus saturated calomel electrode (SCE) in DMF/0.1 M tetrabutylammonium perchlorate. They showed photocytotoxicity in red/
visible light in HeLa, HaCaT, MCF-7, and A549 cells. Complexes
1 and 2 displayed mitochondrial localization, reactive oxygen
species (ROS) generation under red light, and apoptotic cell
death. Control complexes 3 and 4 exhibited uniform distribution throughout the cell. The complexes showed DNA photocleavage under red light (785 nm), forming hydroxyl radicals as
the ROS.

Introduction

phology selectively against cancer cells both in vitro and in vivo

by many lipophilic cations.[11] In this regard, Neamati and coworkers developed TPP-appended chlorambucil molecules for
mitochondria-targeted cytotoxicity in breast and pancreatic
cancer cells.[12] A similar strategy was adopted by Dhar et al. to
synthesize a mitochondria-targeting cisplatin analog, Platin-M,
and a nanoparticle-based zinc(II) phthalocyanine photosensitizer that was shown to disrupt the mitochondrial genome instead of the nuclear DNA.[13,14] Guo and co-workers developed
a cation-appended copper complex for mitochondria targeting
that was cytotoxic against cisplatin-resistant tumor cells
through multiple mechanisms of action.[15,16]

Mitochondria being the cellular organelle responsible for oxidative damage, calcium metabolism, and cell death are actively
involved in several human diseases resulting from its dysfunction or mutation of genes.[13] Mitochondrial disruption affects
the metabolic pathway from glucose oxidation to glycolysis
(Warburg effect), which is an indication of malignant cells.[4]
The alterations in cancer cells that result in evasion from apoptosis can be targeted to inhibit proliferation. Thus, it is imperative to target and deliver bio-active molecules to the mitochondria to address mitochondrial dysfunctions associated with several human diseases including cancer. Lipophilic cations can accumulate in the mitochondria of cells owing to the higher (negative) mitochondrial membrane potential.[58] Smith et al. developed a strategy to target bio-active molecules to the mitochondria by attaching a lipophilic triphenylphosphonium cation
(TPP, PPh3+) with an alkyl linker.[9,10] These molecules were
shown to rapidly permeate the lipid bilayers because of the
large negative mitochondrial membrane potential (180
200 mV) and accumulate inside the mitochondria in cultured
cells. Chen and co-workers showed the disruption of cell mor[a] Inorganic and Physical Chemistry Department, Indian Institute of Science,
Bangalore 560012, Karnataka, India
E-mail: arc@ipc.iisc.ernet.in
http://ipc.iisc.ernet.in/arc.html
[b] Department of Molecular Reproduction Development and Genetics, Indian
Institute of Science,
Bangalore 560012, Karnataka, India
E-mail: paturu@mrdg.iisc.ernet.in
www.mrdg.iisc.ernet.in/PK.htm
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/ejic.201501105.
Eur. J. Inorg. Chem. 2016, 10021012

Photodynamic therapy (PDT) is a relatively new mode of cancer treatment, which depends on the retention of the photosensitizers in the tumor cells followed by their selective activation under red light in the presence of molecular
oxygen.[1720] Photosensitizers are light-sensitive compounds
that cause localized oxidative damage within the target cells
upon irradiation. Several organic molecules, including porphyrins, texaphyrins, and phthalocyanines, have been reported as
efficient PDT agents. However, their potential usage in cancer
treatment is limited as a result of skin sensitivity and acute
hepatotoxicity on prolonged use. Metal complexes with their
varied structural, photophysical, and photochemical properties
are thought to be ideal alternatives to the organic chromophores as they could circumvent the shortcomings of the organic drugs.[21] Redox-active metal complexes may provide a
photoredox pathway for the generation of different reactive
oxygen species (ROS), leading to tumor damage as an alternative to the generation of singlet oxygen. Research in this direction has resulted several 3d5d metal complexes that show in
vitro photocytotoxicity in visible or UV-A light.[2225] However,

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the examples of 3d transition-metal complexes that can be
photo-excited within the PDT window of 620800 nm for better
tissue penetration are rare in the literature.[26,27]
We have recently reported a class of iron(III) catecholates
that showed red light induced cytotoxicity with the complexes
primarily localizing in the nucleus of HeLa and HaCaT cells.[28,29]
Considering the drug resistance associated with nuclear targeting drugs such as cisplatin or its analogs as a result of the
extensive repair of the drugDNA adducts by the nucleotide
excision repair (NER) pathway,[30,31] we have attempted to
change the cellular localization mode of these iron(III) complexes from the nucleus to the mitochondria of the cells. To
achieve this objective, we have designed and synthesized new
iron(III) catecholates with a cationic TPP moiety as a pendant in
[Fe(R-bpa)(R-dopa)Cl] (1, 2), where R-bpa is 2-{TPP-N,Nbis[(pyridin-2-yl)methyl]ethanamine} chloride (TPPbpa) and Rdopa is functionalized dopamine, that is, 4-{2-[(anthracen-9yl)methylamino]ethyl}benzene-1,2-diol (andopa, 1) or 4-{2[(pyren-1-yl)methylamino]ethyl}benzene-1,2-diol (pydopa, 2),
and studied their altered cellular activity (Figure 1). The positively charged lipophilic TPP cation was incorporated to drive
the complexes to the mitochondria. To retain the photocytotoxic behavior of the complexes, the dopamine unit was functionalized with planar, aromatic, and photoactive anthracenyl
and pyrenyl groups. Moreover, to establish the role of the TPP
unit, two similar complexes in which TPPbpa was substituted by
[phenyl-N,N-bis(pyridin-2-yl)methyl]methanamine (phbpa) (3, 4)
were synthesized and used as controls. Herein, we report the
photocytotoxicity under red light and cellular localization of the
complexes 14.

Figure 1. Schematic drawing of the structures of the iron(III) complexes 14.

Results and Discussion

Synthesis and General Aspects
Ligand TPPbpa was synthesized in three steps. Pyridine-2-carbaldehyde and 2-aminoethanol were stirred for 3 d in the presence of sodium tris-acetoxyborohydride and glacial acetic acid
to give 2-{bis[(pyridin-2-yl)methyl]amino}ethanol. This was followed by chlorination with thionyl chloride and subsequent
heating with triphenylphosphine at 100 C in the presence of a
trace amount of potassium iodide in n-butanol for 4 d to isolate
the ligand as a brown precipitate. The product was washed with
chloroform and diethyl ether, dried in vacuo and characterized
by 1H NMR spectroscopy (Scheme S1, Figure S1 in the Supporting Information). Ligand phbpa was synthesized by following
reported literature procedures.[32] The catecholate ligands were
synthesized in three steps starting from the Schiff bases by reaction with 2-(3,4-dimethoxyphenyl)ethanamine and anthracene-9-carbaldehyde or pyrene-1-carbaldehyde in ethanol. This
was followed by the reduction of the Schiff bases with sodium
borohydride in methanol. The ether linkage was finally deprotected with boron tribromide (1 M solution in CH2Cl2) to afford
the required ligand, andopa or pydopa, as a yellow precipitate
(Scheme S2). Both the ligands were characterized by 1H NMR
spectroscopy (Figure S2 and Figure S3). The complexes were
synthesized by a general reaction starting from one equivalent
of anhydrous ferric chloride and one equivalent of dipicolylamine ligand in methanol, which was stirred for an hour. A
methanolic solution of one equivalent of the catecholate ligands previously deprotonated with two equivalents of triethylamine was added to the reaction mixture. Stirring for 3 h
afforded violet precipitates of the complexes 14 (Figure 1).
The complexes were characterized by physicochemical and
analytical data. Selected data are given in Table 1. The ESI-MS
of the complexes recorded in methanol showed a single molecular ion peak for 1 and 2, corresponding to the species [M
2Cl]2+ whereas the peaks for complexes 3 and 4 corresponded
to the species [M Cl]+ (Figures S4S7). The IR spectra are characteristic of complexes showing CO stretches of the catecholate within the range 12701260 cm1 (Figure S8). The UV/Visible
spectra of the complexes in DMF showed a broad and intense
absorption band around 700750 nm that originated from the
charge transfer from the catecholate orbital to the iron(III)
d* orbital (Figure 2, a and Figure S9).[33] The hump near
450 nm was also due to charge transfer from catechol to

Table 1. Selected physicochemical and calf thymus (ct)-DNA binding data for the complexes 14.
Parameters

Complex 1

Complex 2

Complex 3

Complex 4

ESI-MS [m/z][a]
[nm] ( [M1 cm1])[b]
em [nm] ()[c]
[S m2 M1][d]
eff [B][e]
Kb [M1][f ]

442.12
748 (1030)
425 (0.03)
147
5.81
1.8 0.3 105

454.32
685 (3850)
395 (0.04)
153
5.83
2.4 0.5 105

686.19
745 (885)
420 (0.04)
67
5.82
1.3 0.4 105

710.24
675 (4850)
400 (0.04)
73
5.85
2.0 0.6 105

[a] In methanol. [b] In DMF. [c] In DMSO. Excitation wavelengths are 390 nm for complexes 1 and 3 and 340 nm for 2 and 4. Quantum yield calculated by
using anthracene in ethanol and quinine sulfate in sulfuric acid as standards. [d] Molar conductance in DMF. [e] Magnetic moment values at 298 K. [f] Intrinsic
binding constants of the complexes to ct-DNA in 5 % DMF-Tris-HCl buffer medium.
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iron(III). The molar conductivity values of complexes 1 and 2 in
DMF were approximately 150 S m2 M1, in accord with their 1:2
electrolytic behavior. Complexes 3 and 4 behaved as 1:1 electrolytes under identical conditions, giving values around
70 S m2 M1. This reaffirms our previous observations in which
the chloride ion coordinated to the metal center is displaced
by solvent molecules.[18] Anthracene-based emission was observed for complexes 1 and 3 near 420 nm, whereas 2 and 4
emitted at 400 nm owing to the presence of the pyrene group
(Figure 2, b and Figure S9). Magnetic moment values of approximately 5.8 B at room temperature are in agreement with their
five-electron paramagnetic nature. The redox active complexes
displayed an irreversible metal-based redox response near
0.45 V versus saturated calomel electrode (SCE) in DMF/0.1 M
tetrabutylammonium perchlorate and a quasi-reversible redox
response near 0.5 V, which is assignable to the catechol/semiquinone couple (Figures S10 and S11).[29,34]

calculations were conducted to assign the electronic transitions

of the complexes, which tallied reasonably well with the experimental results (Table S1). The visible absorption bands near
750 nm and 420 nm were assigned to the catecholate () to
iron(III) (*) charge-transfer transitions.[29] The electronic transitions below 400 nm were assigned to intra-ligand charge-transfer (ILCT) transitions involving the anthracenyl or the pyrenyl
groups.[38] The orbitals involved in the LMCT transitions that
result in the rich photophysical properties of the complexes are
shown in Figure 3 (b, and Figure S12).

Stability Studies
The stability of the complexes 14 under physiological conditions was assessed by using UV/Visible spectral scans at different time intervals for 24 h. The spectra were recorded with
solutions of the complexes in 1 % DMSO/Dulbecco's phosphate
buffered saline (DPBS) and remained unaltered with respect to
the peak positions and their intensities (Figure S13). Hence, the
complexes showed stability in the buffer medium and were
deemed suitable for biological assays.

Cytotoxicity

Figure 2. (a) The UV/Vis spectrum of complex 2 in DMF. (b) The excitation
(dotted line) and emission (solid line) spectra of complex 2 (10 M) in DMSO.

Theoretical Studies
The molecular structures of the complexes 1 and 2 were optimized by using density functional theory (DFT) (Figure 3, a and
Figure S12).[3537] The initial coordinates were obtained from
the crystal structure of a previously reported complex
[Fe(andpa)(cat)(NO3)] and were used for further optimization.[28,29] Time-dependent density functional theory (TD-DFT)

Figure 3. (a) The energy-optimized structure of complex 2, showing the labeling of the metal and the heteroatoms. (b) The frontier molecular orbitals of
complex 2.
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The anti-proliferative activities of the complexes 14 to inhibit

cellular growth and induce cell death upon excitation with red
light (600720 nm, 50 J cm2) and broad band visible light
(400700 nm, 10 J cm2) in various mammalian cell lines, that
is, HeLa (human cervical cancer), MCF-7 (human breast cancer),
A549 (human lung cancer), and HaCaT (human skin keratinocyte), were investigated by using MTT assay (Figure 4). The complexes were found to be photocytotoxic with IC50 values between 825 M in broad band visible light (400700 nm) and
1634 M in red light (600720 nm). Complexes 1 and 2 with
the triphenylphosphonium cation were found to be more cytotoxic than complexes 3 and 4. The anthracene or the pyrene

Figure 4. The cell viability plots of 14 in HeLa (a), MCF-7 (b), A549 (c), and
HaCaT (d) cells upon exposure to visible light (400700 nm, 10 J cm2, green
bars) for 1 h and red light (600720 nm, 50 J cm2, red bars) for 30 min or
kept in the dark (black bars).

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Table 2. Cytotoxicity values (IC50 [M]) of the complexes 14 in different cell lines.
Cell lines
HeLa
MCF-7
A549
HaCaT

Complex 1
Light[a]

Dark[b]

Complex 2
Light[a]

Dark[b]

Complex 3
Light[a]

Dark[b]

Complex 4
Light[a]

Dark[b]

10.8
16.4
13.4
10.2

66.3
63.2
63.8
61.4

8.0 [16.6]
13.9 [17.0]
10.7 [14.4]
10.8 [17.4]

66.4
68.8
70.9
61.8

15.5
24.9
14.3
14.5

86.0
83.9
86.1
81.5

15.3
22.0
13.5
11.8

85.7
85.9
82.6
86.0

[25.0]
[26.4]
[18.9]
[19.8]

[32.3]
[30.7]
[27.8]
[27.2]

[27.4]
[30.2]
[27.5]
[23.3]

[a] IC50 values in M corresponding to the cells treated with the respective complex and exposed to broad band visible light (400700 nm, 10 J cm2) for 1 h.
The values in parenthesis correspond to the IC50 values in M under red light (600720 nm, 50 J cm2) for an exposure time of 30 min. [b] IC50 values in M
corresponding to the cells treated with the complexes and kept in the dark. The error in the values varied between 0.5 M in light to 2.7 M in the dark.

photoactive moieties in complexes 1 and 2 (or their respective

controls; 3 and 4) behaved similarly and no significant difference in their activities was observed. The dark toxicities were
near 60 M for the TPP-appended complexes 1 and 2 and
>80 M for 3 and 4 (Table 2, Figures S14S17). The dark toxicity
of 1 and 2 might be due to the inherent toxicity of the TPP
cation as reported previously.[39] The free ligand TPPbpa gave
an IC50 value of approximately 50 M under both light and dark
conditions.

treated with the dye showed a minor shift in the cell population
corresponding to the basal cellular ROS. A similar shift in cell
population was also noted when the cells treated with the complexes were incubated in the dark. However, when the experiment was repeated in presence of red light (600720 nm,
50 J cm2), a major shift in the cell population was noted, corresponding to a positive signal for DCF; see Figure 5 (a and b).
This establishes the role of the ROS in cell death, species that
are produced from the complexes only with photo-irradiation
but not in the dark.

Generation of Reactive Oxygen Species

Photocytotoxic iron(III) complexes are known for their ability to
generate reactive oxygen species (ROS), especially radicals such
as hydroxyl and superoxide, when exposed to light irradiation.[40] These are powerful oxidizing agents that can oxidize
non-fluorescent species, that is, 2,7-dichlorofluorescein diacetate (DCFDA) to a green fluorescent entity 2,7-dichlorofluorescein (DCF).[41] The cell permeable fluorogenic probe undergoes
hydrolysis of the ester bonds, eventually getting oxidized to
DCF in presence of ROS. Thus, we performed a DCFDA assay in
HeLa cells for complexes 1 and 2 by using flow cytometry. Cells
alone did not show any signal for DCF fluorescence. The cells

Figure 5. Flow cytometric analysis (FACS) for ROS generation by complex 2

using DCFDA dye in (a) HeLa and (b) HaCaT cells. The ROS generation is
evident from the shift in the cell population showing a positive signal for
DCF when treated with the complex and irradiated with red light (600
720 nm, 50 J cm2) for 30 min compared with the cells alone and on treatment with 2 but kept in the dark [D, dark; L, light]. (c) Formation of the DNA
ladder isolated from the HeLa cells on treating with complex 2 for 4 h followed by exposure to red light (600720 nm, 50 J cm2, L) for 30 min or kept
in the dark (D). M denotes a marker of 100 base pairs.
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Cell Death Pathway

Cell death generally proceeds through apoptosis or necrosis.[42]
Whereas apoptosis is a programmed cell death pathway, necrosis occurs as a result of gross injury to the cell and is often
accompanied by inflammation. To assess the cell death pathway, we performed a DNA fragmentation assay, which relies on
the activation of endonucleases with the onset of apoptosis
and subsequent cleavage of chromatin DNA into internucleosomal fragments of roughly 180 base pairs (bp) and its multiples.
Thus, when the DNA is run on a gel, it gives a ladder-like pattern. HeLa cells were treated with the TPP complex 2 for 4 h
and exposed to red light irradiation (600720 nm, 50 J cm2). An
identical experiment was also performed in the dark to serve
as a control. The DNA was extracted and run on agarose gel.
The results are shown in Figure 5 (c). There was no visible ladder-like pattern for the sample kept in the dark. However, the
light irradiated sample showed a distinct ladder formation as
seen by comparing with the DNA marker (M). This affirms that
complex 2 induces apoptotic cell death in HeLa cells.
Apoptosis in cells can be quantified by using an annexin-VFITC/PI assay.[43] Annexins are a group of homologous proteins
that bind to phosphatidylserine (a phospholipid) in a calciumdependent manner. Annexins tagged with the fluorophore fluorescein isothiocyanate (FITC) give a quantitative measure of the
cell population undergoing apoptosis. As membrane flipping is
one of the early phenomena in the apoptotic pathway, this assay can be used to distinguish between early and late apoptotic
cells. Propidium iodide stains the DNA of the cell population
for which the membrane has been totally compromised. Thus,
a flow cytometric study was performed by using the complexes
14 in HeLa cells under different experimental conditions. In
one set of samples, the cells were treated with the complexes
for 4 h and exposed to red light (600720 nm, 50 J cm2). A
second set was prepared similarly except that the samples were

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kept in the dark throughout. The results are tabulated in
Table S2. There were almost three to five fold increases in the
percentage of apoptotic cell population upon irradiation compared with the dark controls. The results of this experiment
suggest an apoptotic pathway for cell death.

and the cytotoxicity is not a consequence of a remnant amount

present in the media. The cytometry results showed that the
complexes were internalized by a significant percentage of the
cell population (ca. 6080 %) in 4 h (Figure 7, Figures S20 and
S21). This study prompted us to explore the localization pattern
of the complexes inside the HeLa and HaCaT cells, which is
discussed in the subsequent sections.

Cell Cycle Analysis

The effect of the complexes 14 on the progression of the cell
cycle under different experimental conditions was studied to
determine the impact of light on the toxicity and estimate the
apoptotic cell population. The study was carried out in HeLa
and HaCaT cells and the results were consistent (Figure 6). The
premise of several DNA binding dyes, such as propidium iodide
(PI), is that the process occurs stoichiometrically. Thus, cells that
are in the S phase will have more DNA than cells in the G1
phase and will proportionally take up more dye and will fluoresce more brightly until they have doubled their DNA content.
The cells in the G2 phase will be approximately twice as bright
as cells in G1 as DNA replication occurs in the S phase. When
the cells were incubated with the complexes and kept in the
dark for 4 h, there was no significant change in the population
of the various phases of the cell cycle compared with the control set where cells alone were used. Again, when similarly
treated cells were exposed to red light (600720 nm,
50 J cm2), a major population of the cells was observed below
the G1 phase. The presence of this sub-G1 phase in the cell
cycle indicates that the complexes are rather harmless in the
dark but become cytotoxic in the presence of light (Figures S18
and S19). The percentage of the sub-G1 population was again
higher in the case of cells treated with the TPP complexes 1 or
2 compared with those treated with 3 or 4. This again is in
agreement with the higher anti-proliferative activities of the
two complexes as discussed in the preceding section.

Figure 6. The percentage of total cell population in the sub-G1 phase of the
cell cycle in HeLa (a) or HaCaT (b) cells alone or when treated with complexes
14 and exposed to red light (600720 nm, 50 J cm2, grey bars) for 30 min
or kept in the dark (black bars). The control sample corresponding to cells
alone in the x axis is shown by 0.

Cellular Internalization Studies

The passage of the complexes inside HeLa and HaCaT cells was
observed by using a flow cytometer. The fluorescence of the
pendant anthracenyl or the pyrenyl group was used to estimate
the cell population, which showed a positive signal for the compound uptake. Though not quantitative, this study enabled us
to ensure that the complexes are accumulated inside the cells
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Figure 7. The percentage of total cell population showing a positive signal

for the fluorescence of the complexes 14 after incubation for 4 h in dark in
HeLa (a) or HaCaT cells (b). The control sample corresponding to cells alone
in the x axis is shown by 0.

Cellular and Sub-Cellular Localization

The cellular uptake profile of a complex is indicative of its bioavailability and stability under physiological conditions. To investigate the precise pattern of localization of the complexes
14 inside HeLa or HaCaT cells, we performed confocal microscopy experiments using nucleus staining dye PI, which has red
emission (em: 620 nm). The complexes are blue fluorescent
(em = 410420 nm), which allowed us to perform a dual staining assay. The merged images of the blue and the red fluorescence for complexes 1 and 2 showed that they did not really
overlap with each other. There was rather a distinct blue color
beyond the periphery of the red region, which denotes the
nucleus. Thus, it was concluded that both 1 and 2 predominantly accumulate in the cytoplasm (Figure S22). In contrast,
complexes 3 and 4 showed both cytoplasmic as well as nuclear
localization (Figure S23).
The TPP moiety was appended in the design of the complexes 1 and 2 to direct them to the mitochondria of the cells.
Hence, we probed the mitochondria-targeting properties of the
four complexes by using Mitotracker Deep Red (MTR). Cells
were treated similarly and were visualized under a confocal
scanning microscope. The merged images for the complexes 1
and 2 showed a distinct pink color arising from the exact overlap of the red and blue fluorescence of MTR and the complexes,
respectively, as can be seen in the panels i(d) and iii(d) of Figure 8 (Figure S24). Complexes 3 and 4 did not show such precise overlap of their fluorescence signals with MTR, as shown in
the panels ii(d) and iv(d) of Figure 8 (Figure S24). This study
substantiates the role of the TPP moiety, which drives the complexes solely to the mitochondria owing to the fine balance of
its lipophilicity and the positive charge.
To further verify our observation, another co-localization
study was done with an endoplasmic reticulum (ER) marker,
that is, ER Tracker Green (ERTG, em: 510 nm), and complex 2
in HeLa and HaCaT cells. It was observed from the merged images, as shown in the panels (d) and (h) in Figure S25, that the

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treated with MTR or DCFDA, then mounted on slides. The
merged images showed that there was exact overlap of the
fluorescence of MTR, or DCF and the complexes, giving a bluish
white color, as can be seen in the panels (h) and (p) of Figure 9
(Figure S26). This suggests that the DCF was generated inside
the mitochondria of the cells as a consequence of ROS generation. Cell death occurs as a result of oxidative stress occurring
inside the mitochondria. No ROS were detected in the absence
of light as evident from the panels (d) and (l) of Figure 9 (Figure S26).

DNA Binding Studies

Figure 8. Confocal microscopy images of complexes 2 and 4 in HaCaT (i, ii)

and HeLa (iii, iv) cells showing their sub-cellular localization pattern after
incubation for 4 h in the dark. Panels (a) correspond to the bright field images
of the cells, panels (b) denote the emission of Mitotracker Deep Red, panels
(c) are the fluorescence of the complexes, and panels (d) correspond to the
merged images of panels (b) and (c). The scale bar corresponds to 10 m.

green and the blue fluorescence signals of the ER marker and

the complex did not coalesce accurately. This points to the fact
that the TPP-appended complex does not accumulate significantly in the ER of the cells, but inside the mitochondria.

Detection of ROS in Mitochondria

The generation of ROS inside the cells was also evidenced by
using a confocal microscope. Our aim was to observe the locale
of ROS generation inside the cell. HeLa or HaCaT cells were
treated with the complexes 1 and 2 for 4 h and then subjected
to light irradiation (600720 nm, 50 J cm2). The cells were then

Figure 9. Confocal images of complex 2 in HaCaT cells [(a)(h)] and HeLa cells
[(i)(p)] after 4 h incubation followed by exposure to red light (600720 nm)
for 30 min or kept in the dark for 1 h. Panels (a), (e), (i), and (m) show the
fluorescence of complex 2. Panels (b), (f), (j), and (n) are the fluorescence
images of the Mitotracker Deep Red (MTR). Panels (c), (g), (k), and (o) show
fluorescence of DCF. Panels (d), (h), (l), and (p) are the merged images of the
complex, MTR, and DCF. Scale bar: 10 m.
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The mitochondria-targeting TPP complexes (1, 2) and their controls (3, 4) were tested for their ability to bind to calf thymus
(ct)-DNA. DNA is one of the potent targets inside the cells that
might be affected by the complexes. Thus, UV/Visible titrations
were carried out with a fixed concentration of the complexes
by varying the concentration of DNA. The hypochromic effect
observed with increasing DNA concentrations was fitted by using the McGheevon Hippel (MvH) equation and the intrinsic
DNA binding constant was found to be in the order of
105 M1.[44,45] The binding constants of the pyrene-appended
complexes 2 and 4 were marginally higher than those of 1 and
3 with an anthracenyl moiety. This observation is a consequence of the higher planarity of the pyrene group, which interacts well with the DNA base pairs.[46] Again, the TPP-appended
complexes showed greater DNA binding affinity than the control complexes. The DNA binding order is: 2 > 4 > 1 > 3 (Figure 10 and Figure S27). Measuring the change in the viscosity
of DNA before and after addition of the complexes offered another method to study the interaction of DNA with the binding
molecule. As the complexes bind between the DNA base pairs,
there is an increase in the contour length, which leads to an
increase in its viscosity. Thus, the viscosities of DNA alone, in
presence of the complexes 14, in presence of the classical DNA
intercalator ethidium bromide (EB), or Hoechst dye as a DNA
groove binder were measured. The plot of relative viscosity
(/0)1/3 versus [complex]/[DNA] is shown in Figure 10. The DNA
binding ability order followed the trend 2 > 4 > 1 > 3, which
is consistent with the DNA binding data.

Figure 10. (a) Spectral traces of complex 2 showing the effect of addition of
ct-DNA (250 M) in 5 % DMF/Tris-HCl buffer (pH 7.2). (b) Plots showing the
effect of addition of an increasing quantity of the complexes 14, ethidium
bromide (EB as the DNA intercalator), and Hoechst dye (as the DNA groove
binder) on the relative viscosity of ct-DNA at 37.0 C.

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DNA Cleavage Studies
The cleavage of supercoiled (SC) pUC19 DNA to its nicked circular (NC) or linear form in presence of the complexes 14 was
studied under different conditions in the light or dark and in
presence of various additives. A solution of complex 2 when
added to DNA and incubated in the dark resulted in partial
cleavage (ca. 25 %) to its NC form. This hydrolytic cleavage is a
consequence of the labile coordination site occupied by the
chloride ion in the solid state. A similar experiment conducted
after irradiation with a continuous-wave (CW) diode laser light
at 785 nm for 2 h showed approximately 80 % cleavage of SC
DNA to its NC form (Figure 11, a and Figure S28). The other
complexes also showed similar DNA photocleavage potential.
There was no significant photocleavage of DNA under an argon
atmosphere, which led us to believe that oxygen species were
involved in the DNA photocleavage reactions. To probe the
mechanistic aspects of DNA photocleavage, various additives
were used as the reactive oxygen species quenchers/scavengers
(Figure 11, b). By using singlet oxygen quenchers such as TEMP,
DABCO (1,4-diazabicyclo[2.2.2]octane), and L-histidine did not
suppress the photocleavage property of complex 2. The
hydroxyl and superoxide radical scavengers, that is, KI, DMSO,
superoxide dismutase (SOD), and catalase, inhibited the photocleavage by more than 50 %. Thus, hydroxyl and superoxide
radicals are established as the ROS in the DNA photocleavage
reactions.

toxins to other cellular organelles may have a better impact for

the development of more active anticancer agents. Moreover,
mitochondrial dysfunction is implicated in tumorigenesis that
renders it a popular target for the development of various cytotoxins. Appendage of a lipophilic triphenylphosphonium (TPP)
cation has enabled the effective internalization of the complexes 1 and 2 primarily into the mitochondria of the HeLa and
HaCaT cells rather than the nuclei. Thus, in the presence of light,
the complexes can photocleave mitochondrial DNA and not nuclear DNA. It has further been substantiated from the confocal
microscopy studies that in the absence of the TPP group, a
general diffused distribution of the complexes takes place
throughout the cell without showing any selectivity. The anthracene or pyrene groups, conjugated to the dopamine unit,
have served as fluorescent markers for tracing the complexes
inside the cells by cellular imaging studies. The generation of
ROS precisely inside the mitochondria of the cells could be the
cause of the apoptotic cell death. Photocytotoxicity studies
have shown that the iron(III) catecholates bearing either an anthracene or a pyrene photoactive moiety behave similarly and
could be activated by using red light owing to the presence
of an intense LMCT-type catecholate to iron(III) charge transfer
absorption band near 750 nm. Such examples of 3d metal complexes that can be selectively activated by low energy light are
rare in the literature.[4749] The present series of near-IR light
active photocytotoxic iron(III) complexes with their good DNA
binding, DNA photocleavage, and cellular organelle targeting
properties opens up the relatively unexplored area of metalbased PDT agents for cancer management and cure.

Experimental Section
Materials and Methods

Figure 11. (a) Bar diagram showing the percentage of nicked circular DNA
formation in presence of the complexes 14 (25 M) after photo-exposure
for 2 h using a CW diode laser (785 nm, 100 mW, bars 16): bar 1, DNA alone;
bar 2, DNA + 2 (under argon); bars 36, DNA + 14, respectively. (b) Gel
electrophoresis diagram showing the percentage of NC DNA formation with
complex 2 in the presence of various ROS quenching/scavenging agents as
additives: lane 1, DNA alone; lane 2, DNA + 2 + TEMP; lane 3, DNA + 2 +
DABCO; lane 4, DNA + 2 + L-histidine; lane 5, DNA + 2 + KI; lane 6, DNA + 2
+ DMSO (4 L); lane 7, DNA + 2 + catalase (4 units); lane 8, DNA + 2 + SOD
(4 units).

Conclusions
We have been successful in synthesizing mitochondria-targeting iron(III) catecholates by suitable ligand modification. They
show remarkable PDT activity under red light with low toxicity
in the dark. The complexes are based on suitable modification
of the previously reported iron(III) catecholates that were seen
to localize primarily in the nucleus of different cells.[17,18] As NER
mechanisms are operative in the nucleus to rectify and revive
damaged DNA, it has been hypothesized that delivering cytoEur. J. Inorg. Chem. 2016, 10021012

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The chemicals were purchased from S. D. Fine Chemicals, India, and

Sigma Aldrich, USA. The solvents were procured from commercial
sources and dried by using standard procedures. Dulbecco's modified eagle medium (DMEM), Dulbecco's phosphate buffered saline
(DPBS), fetal bovine serum (FBS), 2,7-dichlorofluorescein diacetate
(DCFDA), propidium iodide (PI), and annexin-V-FITC/PI kits were obtained from Sigma Aldrich (USA). Mitotracker Deep Red (MTR) FM
(Cat. no. M22426) was purchased from Invitrogen (USA).
A Thermo Finnigan Flash EA 1112 CHNS analyzer was used for elemental analysis. Electrospray ionization mass spectrometry (ESI-MS)
data were obtained from an Agilent 6538 Ultra High Definition
(UHD) Accurate Mass-Q-TOF (LC-HRMS) instrument. 1H NMR spectra
were obtained with a Bruker 400 MHz NMR spectrometer. The infrared and UV/Visible spectra were obtained with a Bruker Alpha or
PerkinElmer Spectrum 750 spectrophotometer, respectively. Emission spectra were recorded by using a PerkinElmer LS 55 spectrophotometer. The molar conductivity of the complexes was measured with a Control Dynamics (India) conductivity meter. Electrochemical studies were performed by using an EG&G PAR Model 253
VersaStat potentiostat/galvanostat with electrochemical analysis
software 270. A three-electrode setup consisting of a glassy carbon
electrode (working electrode), a platinum wire (auxiliary electrode),
and a saturated calomel electrode (SCE) as reference was used. Tetrabutylammonium perchlorate (TBAP, 0.1 M) was used as a supporting electrolyte in DMF. Magnetic susceptibility measurements of the
complexes at 300 K were recorded with solid samples by using

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MPMS SQUID VSM (Quantum Design, USA). Light irradiation was
performed by using Waldmann 1200L PDT instrument as a red light
source (600720 nm) or a Luzchem Photoreactor (Model LZC-1, Ontario, Canada, fitted with eight fluorescent Sylvania white tubes,
cool white, 4100 K) as a source for broad band visible light (400
700 nm). The photoreactor consists of an inbuilt cooling device to
maintain an ambient temperature inside the chamber. MTT assay
readings were obtained by using a Molecular Devices Spectra Max
M5 plate reader. Fluorescence assorted cell sorting experiments
were performed by using a FACS Verse instrument (BD Biosciences).
Confocal microscopy images were acquired by using a Leica microscope (TCS, SP5) with oil immersion lens with a magnification of
63.
Syntheses
The ligands and the complexes were synthesized by using the procedures detailed below.
2-{TPP-N,N-bis[(pyridin-2-yl)methyl]ethanamine} chloride
(TPPbpa)[5052]
Sodium tris-acetoxyborohydride (16.9 g, 80 mmol) and glacial acetic
acid (3.4 mL, 60 mmol) were added to a mixture of 2-aminoethanol
(1.2 g, 20 mmol) and pyridine-2-carbaldehyde (4.3 g, 40 mmol) in
dry tetrahydrofuran (THF, 50 mL) and stirred under a nitrogen atmosphere at room temperature for 72 h. The solvent was removed;
the residue was dissolved in dichloromethane and neutralized by
the addition of saturated sodium hydrogen carbonate solution. The
organic fractions were separated and dried with sodium sulfate. The
removal of the solvent under reduced pressure afforded a yellow
oil as the desired product, that is N,N-bis(2-pyridylmethyl)-2-aminoethanol (yield 70 %), which was used without further purification
for the next step.
N,N-Bis(2-pyridylmethyl)-2-aminoethanol (2.4 g, 10 mmol) was dissolved in dry dichloromethane (50 mL) and thionyl chloride (3.5 g,
30 mmol) was added slowly over a time span of 2 h. The mixture
was stirred and heated at reflux for 1 h. The resulting solution was
cooled and excess thionyl chloride was destroyed by dropwise addition of methanol. The solvent was evaporated under reduced pressure to afford a yellow oil as the desired product, that is, 2-chloroN,N-bis[(pyridin-2-yl)methyl]ethanamine (yield 65 %).
2-Chloro-N,N-bis[(pyridin-2-yl)methyl]ethanamine (2.6 g, 10 mmol)
was dissolved in n-butanol (25 mL) and triphenylphosphine (PPh3,
5.2 g, 20 mmol) was added. A pinch of KI was added and the reaction mixture was heated at 100 C for 4 d. A brown precipitate
formed, which was washed thoroughly with chloroform and ether,
dried in vacuo, and used without further purification (yield 30 %).
The compound was characterized from the 1H NMR spectrum in
D2O. 1H NMR (D2O, 400 MHz): = 8.89 (d, J = 8.0 Hz, 2 H), 8.74
8.60 (m, 2 H), 8.46 (d, J = 8.0 Hz, 2 H), 8.238.18 (m, 2 H), 7.917.41
(m, 15 H), 4.084.02 (m, 4 H), 3.24 (t, J = 8.0 Hz, 2 H), 1.84 ppm (t,
J = 8.0 Hz, 2 H) (Figure S1 in the Supporting Information).
4-{2-[(Anthracen-9-yl)methylamino]ethyl}benzene-1,2-diol (andopa) and 4-{2-[(pyren-1-yl)methylamino]ethyl}benzene-1,2diol (pydopa)[53]
An ethanol solution of anthracene-9-carbaldehyde (2.0 g, 10 mmol)
or pyrenecarbaldehyde (2.3 g, 10 mmol) was added to 2-(3,4-dimethoxyphenyl)ethanamine (1.8 g, 10 mmol) dissolved in ethanol
(20 mL) and stirred at room temperature for 2 h. The resulting precipitate of the respective Schiff base was filtered, dried in air, and
recrystallized from ethanol. The Schiff base (5 mmol) was dissolved
in methanol (50 mL) and cooled in an ice bath. Sodium borohydride
(1 g, excess) was slowly added and the reaction mixture was stirred
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at room temperature for 10 h. The solvent was evaporated and

water (100 mL) was added to dissolve the inorganic side products.
The product was extracted with CH2Cl2, dried with sodium sulfate,
and evaporated to obtain an oily product. Drying in a desiccator
afforded a dark-yellow solid of 2-(3,4-dimethoxyphenyl)-N-[(anthracene-9-yl)methyl]ethanamine or 2-(3,4-dimethoxyphenyl)-N[(pyren-1-yl)methyl]ethanamine as the product.
The reduced Schiff base (5 mmol) was dissolved in CH2Cl2 (previously dried with P4O10) under a nitrogen atmosphere and cooled
in an ethyl acetate/liquid nitrogen bath. Boron tribromide (BBr3, 1 M
in CH 2 Cl 2 , 20 mmol) was added and the reaction mixture was
warmed to room temperature. The solution was stirred for 24 h
under a nitrogen atmosphere with occasional sonication. The resulting suspension was again cooled in an ethyl acetate/liquid nitrogen
bath and carefully quenched with methanol. The solution was
warmed to room temperature, followed by addition of water
(10 mL). The solvent CH 2 Cl 2 was evaporated and more water
(50 mL) was added to the resulting suspension. Stirring for an hour
afforded the desired product (andopa or pydopa) as a dark-yellow
solid, which was filtered, washed thoroughly with water, and dried
in air, yield ca. 60 %. 1H NMR for andopa ([D6]DMSO, 400 MHz): =
8.53 (s, 1 H), 8.37 (d, J = 8.0 Hz, 2 H), 8.07 (d, J = 8.0 Hz, 2 H), 7.55
7.48 (m, 4 H), 6.836.71 (m, 3 H), 4.65 (s, 2 H), 3.00 (t, J = 8.0 Hz, 2
H), 2.75 ppm (t, J = 8.0 Hz, 2 H) (Figure S2 in the Supporting Information). 1H NMR for pydopa ([D6]DMSO, 400 MHz): = 8.588.13
(m, 9 H, pyrene), 6.706.50 (m, 3 H, aromatic), 4.97 (s, 2 H), 3.05 (t,
J = 8.0 Hz, 2 H), 2.84 ppm (t, J = 8.0 Hz, 2 H) (Figure S3 in the
Supporting Information).
Complexes 14
The metal complexes were synthesized by a general reaction starting from anhydrous ferric chloride (0.16 g, 1.0 mmol) in methanol
(5 mL) and TPPbpa (0.52 g, 1.0 mmol for 1, 2) or phbpa (0.29 g,
1.0 mmol for 3, 4) also in methanol (10 mL) and stirred for an hour.
A methanol solution of andopa (0.34 g, 1.0 mmol for 1, 3) or pydopa
(0.37 g, 1.0 mmol for 2, 4) previously deprotonated with triethylamine (0.2 g, 2.0 mmol) was added to the reaction mixture. Stirring
for 3 h afforded a violet precipitate of the complex. The solid was
washed thoroughly with ether and dried with P4O10 in vacuo.
[Fe(TPPbpa)(andopa)Cl]Cl (1): Yield ca. 60 %. Elemental analysis
calcd (%) for C55H50Cl2FeN4O2P (956.75): C 69.05, H 5.27, N 5.86;
found: C 68.83, H 5.52, N 5.62. ESI-MS in MeOH (m/z): 442.1195 [M
2Cl]2+. FTIR: = 3040 (w, CH, aromatic), 2950 (w, CH, alkyl), 1610
(m), 1575 (m), 1480 (s), 1425 (w), 1260 (m, CO), 1080 (m), 1040 (s),
950 (m), 870 (m), 855 (m), 790 (m), 740 (s), 620 (m), 600 (m), 515
(m), 480 (m), 410 cm1 (m) (s, strong; m, medium; w, weak). UV/
Visible spectra in DMF: max () = 748 (1030), 468 (1480), 398
(12,540), 369 (13,920), 351 (10,700), 337 nm (7450 M1 cm1). Emission spectrum in DMSO: em (ex, ) = 425 nm (390 nm, 0.03). Molar
conductance in DMF (M): 147 S m2 M1. Magnetic moment eff :
5.81 B at 298 K.
[Fe(TPPbpa)(pydopa)Cl]Cl (2): Yield ca. 50 %. Elemental analysis
calcd (%) for C57H50Cl2FeN4O2P (980.77): C 69.80, H 5.14, N 5.71;
found: C 71.09, H 5.86, N 5.80. ESI-MS in MeOH (m/z): 454.3191 [M
2Cl]2+. FTIR: = 3060 (w, CH, aromatic), 2600 (w, CH, alkyl), 1670
(m), 1495 (s), 1335 (s), 1260 (m, CO), 1050 (w), 820 (w), 750 (w),
725 (w), 605 (w), 500 cm1 (w). UV/Visible spectrum in DMF: max
() = 685 (3850), 462 (5110), 340 (25480), 325 (20680), 274 (26390),
264 nm (19960 M1 cm1). Emission spectrum in DMSO: em (ex, ):
395 nm (340 nm, 0.04). M olar conduc tance in DMF ( M):
153 S m2 M1. Magnetic moment eff : 5.83 B at 298 K.
[Fe(phbpa)(andopa)Cl] (3): Yield ca. 80 %. Elemental analysis calcd
(%) for C42H38ClFeN4O2 (722.09): C 69.86, H 5.30, N 7.76; found: C

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70.03, H 5.22, N 7.68. ESI-MS in MeOH (m/z): 686.1936 [M Cl]+.
FTIR: = 3080 (w, CH, aromatic), 2955 (w, CH, alkyl), 1630 (m),
1575 (m), 1480 (s), 1435 (w), 1280 (m, CO), 1060 (m), 940 (m), 875
(m), 800 (m), 745 (s), 620 (m), 600 (m), 510 (m), 460 (m), 420 cm1
(m). UV/Visible spectra in DMF: max (): 745 (885), 467 (1280), 389
(12340), 369 (13375), 351 (10610), 336 nm (7260 M1 cm1). Emission
spectrum in DMSO: em (ex, ): 420 nm (390 nm, 0.04). Molar conductance in DMF (M): 67 S m2 M1. Magnetic moment eff : 5.82 B
at 298 K.
[Fe(phbpa)(pydopa)Cl] (4): Yield 75 %. Elemental analysis calcd (%)
for C44H38ClFeN4O2 (746.11): C 70.83, H 5.13, N 7.51; found: C 71.09,
H 5.05, N 7.59. ESI-MS in MeOH (m/z): 710.2404 [M Cl]+. FTIR: =
3040 (w, CH, aromatic), 2650 (w, CH, alkyl), 1650 (m), 1485 (s),
1340 (s), 1270 (m, CO), 1040 (w), 840 (w), 770 (w), 725 (w), 600 (w),
505 cm 1 (w). UV/Visible spectra in DMF: max (): 675 (4850),
469 (6440), 340 (31910), 324 (25490), 274 (32980), 264 nm
(24950 M1 cm1). Emission spectrum in DMSO: em (ex, ): 400 nm
(340 nm, 0.04). Molar conductance in DMF (M): 73 S m2 M1. Magnetic moment eff : 5.85 B at 298 K.
Theoretical Calculations: The geometries of the complexes 1 and
2 were optimized by density functional theory (DFT) methods using
the B3LYP/LanL2DZ level as implemented in the Gaussian 09 program.[3537] The electronic transitions and transition probabilities
were obtained from linear response time-dependent density functional theory (TD-DFT). Selected electronic transitions for the complexes 1 and 2 are listed in Table S1.
MTT Assay: Cytotoxicity experiments were done to measure the
anti-proliferative activities of complexes 14 in four different cell
lines, that is, HeLa (human cervical carcinoma), HaCaT (human keratinocyte), MCF-7 (human breast cancer), and A549 (human lung adenocarcinoma) cells. They were maintained in reconstituted Dulbecco's Modified Eagle's Medium (DMEM) supplemented with fetal
bovine serum (FBS, 10 %), penstrep (100 g mL1), and glutamax
(2 mM) at 37 C in an incubator at a CO2 level of 5 %. The adherent
cultures were grown as a monolayer and were passaged once in
5 d by trypsinizing with trypsinEDTA (0.25 %). Approximately 8000
cells were used for plating. Different concentrations of the complexes (dissolved in DMSO) were added to the cells, making the
final DMSO concentration as 1 %. The cell plates were covered with
aluminum foil to avoid light exposure and incubated for 4 h. This
was followed by irradiation with red or visible light in DPBS medium
(200 L). A dark control was also used where the media was discarded and replaced with fresh media (200 L). After light treatment, the DPBS was removed and replaced with fresh media
(200 L). Incubation was continued for another 20 h, after which
MTT (5 mg mL1 in DPBS) was added. After 34 h, the media was
removed carefully and DMSO (200 L) was added into each of the
wells. The absorbance reading of formazan was made at 540 nm.
The cytotoxicity of the complexes was measured as the percentage
ratio of the absorbance of the treated cells to the untreated controls. The IC50 values of the complexes were determined by a nonlinear regression analysis by using GraphPad Prism 5.1.
DCFDA Assay: The generation of ROS by complex 2 was studied
by a dichlorofluorescein diacetate (DCFDA) assay in HeLa and HaCaT
cells. Cells were treated with complex 2 (10 M) and incubated for
4 h in the dark. Subsequently, one of the plates was exposed to red
light (600720 nm, 50 J cm2) for 30 min. Cells were washed with
DPBS, quickly trypsinized, and harvested. DCFDA (1 M) was added
to the cell suspensions, incubated for 15 min in ice, and the fluorescence distribution was studied by using a FACS Verse machine (BD
Biosciences).
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DNA Fragmentation Experiment: About 3 105 HeLa cells were

grown in 6-well tissue culture plates for 24 h in DMEM with 10 %
FBS. Cells were treated with complex 2 for 4 h and one of the plates
was irradiated with red light (600720 nm) for 30 min in the DPBS
medium. After irradiation, DPBS was removed and the cells were
further allowed to grow for another 10 h in fresh DMEM supplemented with 10 % FBS. Eventually, the media was discarded; cells
were washed with DPBS, trypsinized, and suspended in lysis buffer
(0.4 mL, 10 mM Tris-HCl, pH 8.0, 20 mM EDTA, 0.2 % triton-X 100)
for 20 min on ice. The cells were centrifuged for 20 min at
13000 rpm and the supernatant was collected. It was treated with
phenol and chloroform to remove all protein content. The supernatant was precipitated by using sodium acetate (3 mM, pH = 5.8) and
ethanol at 20 C overnight. The DNA pellet formed was washed
with 70 % ethanol and suspended in Tris-EDTA (TE) containing
RNAse (1X Tris-EDTA with 100 mg mL1 RNAse) for 2 h at 37 C. The
samples were loaded in 1.5 % agarose gel and run for about 3 h at
70 V. A 100 bp marker was used as a control. They were photographed in UV light.
Annexin-V-FITC/PI Assay: This assay was performed in HeLa cells
by using 14 to quantify the cell population showing features of
apoptosis. About 3 105 HeLa cells were cultured in 6-well plates
for 24 h in 10 % FBS/DMEM medium. The cells were treated with
14 for 4 h in the dark. Controls including untreated cells and cells
treated with the dyes alone were also used. One of the plates was
exposed to red light (600720 nm, 50 J cm2) for 30 min in DPBS
medium. Following irradiation, DPBS was removed and the cells
were allowed to grow for another 10 h in 10 % FBS/DMEM medium.
The media was discarded; the cells were washed with DPBS and
trypsinized. They were suspended in binding buffer (400 L, 1X).
Annexin-V-FITC (1 L) and PI (2 L) were added to the cell suspensions and incubated for 20 min in the dark. The fluorescence was
immediately recorded by using a flow cytometer and gating of the
cell population was performed based on the cells stained by the
dyes alone.
Cell Cycle Analysis Experiment: This was done to investigate the
effect of the complexes 14 on the cell cycle progress. Approximately 1.0 106 HeLa or HaCaT cells were plated per well in 6-well
tissue culture plates with DMEM reconstituted with 10 % FBS. They
were allowed to grow for 24 h at 37 C in a CO2 incubator. DMSO
solutions of the complexes (10 M) were added to the cells and
incubation was continued in the dark for 4 h. Subsequently, DMEM
was replaced with DPBS in one of the plates, which was exposed
to red light irradiation (600720 nm) for 30 min. After irradiation,
DPBS was again replaced with fresh DMEM containing 10 % FBS,
and the cells were incubated for another 10 h. The other plate was
treated with the complexes similarly for 4 h, after which the media
was replaced with fresh reconstituted DMEM. Eventually, they were
processed as reported earlier.[29] Flow cytometry analysis was performed by using a FACS Verse machine (BD Biosciences) at FL2
channel (595 nm) and the distribution of cells in the various phases
of the cell cycle was assessed from the histogram generated by
Cell Quest Pro software (BD Biosciences). Data analysis for the
percentage of cells in each cell cycle phase was performed by using
WinMDI 2.9.
Cellular Internalization Experiments: To estimate the cell population that internalize the complexes, flow cytometry analysis was
performed using 14. HeLa or HaCaT cells (1.0 106) were plated
per well in a 6-well tissue culture plate with DMEM containing 10 %
FBS. After 24 h of incubation at 37 C in a CO2 incubator, complexes
14 (10 M) were added to the cells and incubated for 4 h in the
dark. The media was discarded and cells were washed three times

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with DPBS. They were trypsinized, collected in eppendorf vials, and
centrifuged. The supernatant was discarded and the cells were resuspended in DPBS. Flow cytometry analysis was performed by using a FACS Verse machine (BD Biosciences) using the Pacific blue-A
filter.
Confocal Microscopy: The localization of the complexes inside
HeLa and HaCaT cells was studied by using a Leica microscope (TCS,
SP5) with oil immersion lens with a magnification of 63. Cell plating was done in 12-well tissue culture plates with cover slips at a
seeding density of 1 105 cells in 1.5 mL of the culture medium
for 24 h. Complexes 14 (10 M) were added to the cells and incubated in the dark for 4 h. The cells were fixed and permeabilized
by using chilled methanol for 5 min at 20 C. After discarding the
methanol, the cells were washed with DPBS and incubated with
propidium iodide (1 mg mL1). The cover slips were mounted on
slides, attached with nail enamel, and the cells were visualized with
the microscope. To probe the sub-cellular localization, a similar
treatment was performed. However, the cells were not fixed but live
cells were stained with either Mitotracker Deep Red (MTR) or ER
Tracker Green (250 nM) and incubated for 30 min at room temperature. To investigate the precise locale of ROS generation inside
HeLa or HaCaT cells, confocal microscopy experiments were conducted by using complexes 1 and 2 along with MTR and DCFDA
dyes. Cells were plated in 12-well tissue culture plates on cover slips
as described earlier. The cells were subsequently treated with the
complexes for 4 h in the dark. One of the plates was exposed to
red light (600720 nm) irradiation for 40 min, the media was removed and cells were washed with DPBS. This was followed by
treatment with MTR for 30 min at room temperature and DCFDA
for 20 min. The cover slips were mounted on slides, attached with
nail enamel, and visualized under a Leica microscope (TCS, SP5)
with oil immersion lens with a magnification of 63. Images were
processed by using LAS AF Lite software.
DNA Binding and Cleavage Experiments
To calculate the intrinsic binding constants of the complexes 14
to calf thymus (ct)-DNA, UV/Visible titrations were performed in TrisHCl buffer medium (5 mM, pH 7.2) with known concentrations of
the complexes (30 M) and increasing the concentrations of ct-DNA.
DNA (250 M) in the buffer medium gave a ratio of UV absorbance
at 260 and 280 nm of 2:1, indicating its protein-free nature. The
concentration of ct-DNA in nucleobases was calculated from its absorption intensity at 260 nm with = 6600 M1 cm1. The data fitting
was done by using the McGheevon Hippel equation.[44,45] Viscometric titrations were carried out by using a Schott AVS. 310 Automated Viscometer. Initially, the concentration of ct-DNA was kept
at 150 M while the flow times were measured with an automated
timer. Data were represented as (/0)1/3 versus [complex]/[DNA],
where is the viscosity of DNA in the presence of the complex and
0 is that of ct-DNA alone. Viscosity values were calculated from the
observed flow time of DNA containing solutions (t) of the complexes corrected for the flow time of the buffer alone (t0), where
= (t t0)/t0.
DNA cleavage experiments were performed by using supercoiled
pUC19 DNA (0.2 g, 30 M) and the complexes (20 M) under various experimental conditions. A diode laser of 785 nm wavelength
[100 mW, Model: LQC785100C, Newport Corporation, LD module,
continuous wave (CW) circular beam, power 100 mW] was used. In
a typical experiment, the total volume of 20 L, consisting of DNA
(1 L), NaCl (50 mM, 1 L), complex (20 M), and Tris-HCl buffer
(50 mM, pH 7.2), was incubated for 1 h at 37 C followed by irradiation for 2 h at room temperature. Following further incubation for
1 h, the samples were loaded in 1 % agarose gel containing ethidEur. J. Inorg. Chem. 2016, 10021012

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ium bromide (1 g mL1) using loading dye (25 % bromophenol

blue, 0.25 % xylene cyanol, and 30 % glycerol, 2 L). The gel was
run in TAE (Tris-acetate-EDTA) buffer for 2 h at 60 V. The gel was
photographed by using a UVITECH Gel documentation system. The
mechanistic aspects of DNA photocleavage studies were conducted
by using various additives, that is, KI (0.5 mM), DMSO (4 L), SOD (4
units), catalase (4 units), TEMP (0.5 mM), DABCO (0.5 mM), and Lhistidine (0.5 mM), which were added prior to addition of the complex. The procedures followed are described elsewhere.[29]

Acknowledgments
The authors thank the Indian Institute of Science (IISc), Department of Science and Technology (DST), Government of India
(grant number SR/S5/MBD-02/2007 and J. C. Bose Fellowship to
A. R. C.) and the Council of Scientific and Industrial Research
(CSIR), New Delhi (grant number 01(2559)/12/EMR-II/2012) for
financial support. The authors also thank the Alexander von
Humboldt (AvH) Foundation, Germany, for an electrochemical
system. The authors thank Ms. Koushambi Mitra for helping
with the theoretical calculations, Mr. Vashista K. for the FACS
data, and Dr. Santosh Poddar for the confocal microscopy images.
Keywords: Bioinorganic chemistry Medicinal chemistry
Anticancer agents Iron Mitochondrial localization

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Received: September 28, 2015

Published Online: February 2, 2016

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