BODIPY-Appended 2-(2-Pyridyl)benzimidazole Platinum(II) Catecholates for Mitochondria-Targeted Photocytotoxicity

Article abstract of DOI:10.1002/cmdc.201600320

Platinum(II) complexes of the type [Pt(L)(cat)] (1 and 2), in which H₂cat is catechol and L represents two 2-(2-pyridyl)benzimidazole ligands with 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) pendants, were synthesized to achieve mitochondria-targeted photocytotoxicity. The complexes showed strong absorptions in the range λ=510–540 nm. Complex 1 exhibited intense emission at λ=525 nm in 1 % DMSO/water solution (fluorescence quantum yield of 0.06). Nanosecond transient absorption spectral features indicated an enhanced population of the triplet excited state in di-iodinated complex 2. The generation of singlet oxygen by complex 2 upon exposure to visible light, as evidenced from experiments with 1,3-diphenylisobenzofuran, is suitable for photodynamic therapy because of the remarkable photosensitizing ability. The complexes resulted in excellent photocytotoxicity in HaCaT cells (half maximal inhibitory concentration IC₅₀≈3 μm, λ=400–700 nm, light dose=10 J cm^?2), but they remained non-toxic in the dark (IC₅₀>100 μm). Confocal microscopy images of 1 and Pt estimation from isolated mitochondria showed colocalization of the complexes in the mitochondria. Complex 2 displayed generation of reactive oxygen species induced by visible light, disruption of the mitochondrial membrane potential, and apoptosis.

Full text of DOI:10.1002/cmdc.201600320

DOI: 10.1002/cmdc.201600320
Full Papers
BODIPY-Appended 2-(2-Pyridyl)benzimidazole Platinum(II)
Catecholates for Mitochondria-Targeted Photocytotoxicity
Koushambi Mitra,[a] Srishti Gautam,[b] Paturu Kondaiah,*[b] and Akhil R. Chakravarty*[a]
Platinum(II) complexes of the type [Pt(L)(cat)] (1 and 2), in
which H2cat is catechol and L represents two 2-(2-pyridyl)ben-
zimidazole ligands with 4,4-difluoro-4-bora-3a,4a-diaza-s-inda-
cene (BODIPY) pendants, were synthesized to achieve mito-
chondria-targeted photocytotoxicity. The complexes showed
strong absorptions in the range l=510–540 nm. Complex 1 ex-
hibited intense emission at l=525 nm in 1% DMSO/water so-
lution (fluorescence quantum yield of 0.06). Nanosecond transi-
ent absorption spectral features indicated an enhanced popu-
lation of the triplet excited state in di-iodinated complex 2.
The generation of singlet oxygen by complex 2 upon exposure
to visible light, as evidenced from experiments with 1,3-diphe-
nylisobenzofuran, is suitable for photodynamic therapy be-
cause of the remarkable photosensitizing ability. The com-
plexes resulted in excellent photocytotoxicity in HaCaT cells
(half maximal inhibitory concentration IC50 ꢀ3 mm, l=400–
700 nm, light dose=10 JcmÀ2), but they remained non-toxic in
the dark (IC50 >100 mm). Confocal microscopy images of 1 and
Pt estimation from isolated mitochondria showed colocaliza-
tion of the complexes in the mitochondria. Complex 2 dis-
played generation of reactive oxygen species induced by visi-
ble light, disruption of the mitochondrial membrane potential,
and apoptosis.
Introduction
Cisplatin and its analogues are the currently used major che-
motherapeutic drugs.[1–3] The mechanism of action of these
platinum drugs generally points toward the formation of cross-
links with nuclear DNA, which inhibits DNA replication and
transcription processes.[4] The present platinum-based drugs
suffer from two major disadvantages: a) inherent/acquired
drug resistance can occur and b) the drugs cause associated
side effects. Thus, for the resurgence of platinum-based anti-
cancer agents to occur, these shortcomings need to be miti-
gated. Enhanced DNA repair by the nucleotide excision repair
(NER) mechanism and increased efflux of the drug are respon-
sible for acquired chemoresistance.[5,6] One possibility for over-
coming this drug resistance is to direct the platinum(II) com-
plexes to cellular organelles other than the nucleus. Mitochon-
dria are organelles that regulate metabolic energy, metal ho-
meostasis, apoptotic machinery, and oxidative stress.[7,8] Mito-
chondria dysfunction is related to cancer progression, so
breaking down of the mitochondrial integrity could lead to cel-
lular death. Moreover, mitochondrial DNA (mt-DNA) can serve
as a better target for platinum-based drugs because mitochon-
dria lack the NER system and accelerate DNA mutation.[9,10] In
fact, cisplatin is also known to deactivate the mitochondrial
enzyme thioredoxin reductase and thereby decrease the levels
of reduced nicotinamide adenine dinucleotide phosphate
(NADPH) and increase cellular reactive oxygen species (ROS)
levels.[11]
The side effects also result from uncontrolled hydrolysis of
cisplatin en route to the tumor. Various strategies have been
adopted to control the pharmacokinetics by altering the li-
gands and tuning the redox potential of platinum.[1,12,13] It was
observed that oxaliplatin and carboplatin reduced the adverse
effects and showed improved antiproliferative effects relative
to those of cisplatin.[1,2] Thus, replacement of chlorides with bi-
dentate O,O-donor ligands reduced the lability of the metal–
donor bonds. Based on these underlying principles, we de-
signed and synthesized platinum(II) complexes that can target
mt-DNA in a controlled fashion. We have chosen 4,4-difluoro-
4-bora-3a,4a-diaza-s-indacene (BODIPY) as the mitochondria-
targeting unit because many BODIPY conjugates are well
documented to target the mitochondria of cancer cells.[14–16]
Moreover, their robust photostability and structural integrity in
the tumor microenvironment contributes to their ideal in vitro
fluorogenic properties.[15,17] The introduction of heavy atoms
like iodine into the BODIPY core results in excellent photosen-
sitizing abilities.[18–20] Thus, iodinated BODIPY dyes have been
explored in photodynamic therapy (PDT) for their ability to
generate singlet oxygen in the presence of visible light and
molecular oxygen.[20,21] The advantage of PDT as compared
with other therapies lies in the photosensitized generation of
singlet oxygen selectively in the irradiated cancer cells, which
reduces any damage to the non-irradiated normal cells.[22–24] Al-
[a] Dr. K. Mitra, Prof. A. R. Chakravarty
Department of Inorganic and Physical Chemistry,
Indian Institute of Science, Bangalore 560012, Karnataka (India)
Fax: (+91)80-23600683
E-mail: arc@ipc.iisc.ernet.in
[b] S. Gautam, Prof. P. Kondaiah
Department of Molecular Reproduction, Development and Genetics,
Indian Institute of Science, Bangalore 560012, Karnataka (India)
Fax: (+91)80-23600999
E-mail: paturu@mrdg.iisc.ernet.in
Supporting information for this article can be found under http://
dx.doi.org/10.1002/cmdc.201600320.
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though the research groups of Burgess, Nagano, and Ziessel
synthesized myriads of BODIPY dyes and reported their diverse
photophysical properties,[25–30] BODIPY-tethered metal com-
plexes as theranostic agents remain virtually unexplored.[31–34]
With an aim to enhance the efficacy of platinum-based drugs,
we have designed and synthesized new BODIPY-appended
platinum(II) complexes for achieving mitochondria-targeted
dual-action DNA crosslinking and PDT effects (Figure 1). The
sired (Scheme S1 in the Supporting Information). A BODIPY
precursor (A) containing 2,4-dimethylpyrrole as the core with
a chloromethylphenyl substituent at the meso position was
coupled with the NÀH group of 2-(2-pyridyl)benzimidazole to
yield ligand L1 in moderate yield (ꢀ42%). Iodination of L1 at
the alpha position of the BODIPY core with N-iodosuccinimide
was performed to obtain L2 (yieldꢀ54%) as a photosensitizer
(PS) for PDT activity. By using these ligands, the dichloro pre-
cursor complexes (1a–3a) of the type [Pt(L)Cl2] were synthe-
sized from [Pt(DMSO)2Cl2]. The chlorides were then replaced
with catecholate to yield the desired complexes in varying
yields (55–88%; Scheme S2 in the Supporting Information).
The complexes of formulation [Pt(L)(cat)], in which H2cat is cat-
echol and L is L1 in 1 and L2 in 2, were characterized and stud-
ied for cellular imaging and for their photocytotoxic properties
(Figure 1). A non-BODIPY analogue [Pt(L3)(cat)] (3), in which L3
is 1-benzyl-2-(2-pyridyl)benzimidazole, was prepared and used
as a control to study the role of the BODIPY unit in the anti-
cancer activity of 1 and 2.
The ligands and complexes were characterized by various
analytical and spectroscopic methods (Table 1, Figure 2, Fig-
ure S1–S16 in the Supporting Information). Mass spectral data
indicated the formation of the ligands (Figure S1 and S2 in the
Supporting Information). The H NMR spectrum of L1 showed
1
Figure 1. Structures of complexes 1–3.
the characteristic peaks of the BODIPY unit at d=5.8 ppm for
the alpha proton and at d=2.5 and 1.2 ppm for the methyl
groups (Figure S3a in the Supporting Information). The meth-
ylene group proton peaks appeared at d=6.4 ppm. The aro-
matic proton signals appeared in the region d=7.0–9.5 ppm.
The replacement of the alpha protons by iodine was evident
from the absence of the peak at d=5.8 ppm in the proton
NMR spectrum of L2 (Figure S4a in the Supporting Informa-
tion). The 13C NMR spectrum of L1 showed aromatic carbon
atom peaks in the region d=112–160 ppm (Figure S3b in the
Supporting Information). Signals for the aliphatic carbon atoms
of the methylene and methyl groups were observed at d=49
and 15 ppm, respectively. Two new peaks at d=29 and
87 ppm were observed in the 13C NMR spectrum of L2 and cor-
respond to the carbon atoms attached to the iodine atoms
(Figure S4b in the Supporting Information). The precursor plat-
inum(II) chloro complexes were isolated and characterized by
BODIPY unit linked to 2-(2-pyridyl)benzimidazole binding plati-
num in a bidentate mode can direct the complexes to the mi-
tochondrial site. After accumulation of the complex, the slow
release of the active platinum species inside the cells leads to
crosslink formation with the mt-DNA. Based on the previous
knowledge, we have chosen catecholate as the O,O-donor
ligand to achieve controlled release of the active moieties.
Results and Discussion
Synthesis and general properties
The synthetic versatility of BODIPY was used to design new bi-
dentate ligands and tune their photophysical properties as de-
Table 1. Selected physicochemical data for complexes 1–3 and ligands L1 and L2.
1
2
3
L1
L2
lmax [nm] (ꢁ10À4
[mÀ1 cmÀ1])[a]
lem [nm] (Ff)[b]
tf [ns][c]
e
510 (1.6), 325 (1.0)
550 (1.37), 525 (1.1), 394 (0.55),
315 (0.93)
–
–
530 (0.28), 335 (1.1),
314 (1.2)
–
–
–
35
505 (2.21), 310
(1.58)
505 (0.12)
27.3
–
–
550 (2.16), 388 (0.88),
315 (1.5)
–
–
505 (0.06)
29.6
–
tT [ms] (FT)[d]
2.62Æ0.01 (0.15)
3.83Æ0.06 (0.27)
LM [Sm2 mÀ1
]
35
32
–
[e]
Ef [V] (DE [mV])[f]
1.09, 0.48 (65), À1.13 1.10, 0.50 (70), À1.3, À0.82
1.03, 0.51 (90), À1.26
À1.09 (90)
À0.82 (100)
(190)
(90)
[a] In 10% DMSO/DPBS. [b] In 1% DMSO/DPBS (Fluorescence quantum yield with respect to fluorescein, excitation wavelength=480 nm). [c] Fluorescence
lifetimes measured in 1% DMSO/DPBS (pH 7.2). [d] Triplet-state lifetime obtained from nanosecond time-resolved transient difference absorption spectra
in deaerated DMF solutions. [e] Molar conductivity in DMF at T=258C. [f] The potentials are versus a saturated calomel electrode (SCE) in DMF/0.1m TBAP
at a scan rate of 100 mVsÀ1
.
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studies (Figure 2b). Complex 1 with a fluorescence quantum
yield (Ff) value of 0.06 mimicked the emission properties of
the free ligand L1 (Ff =0.12). There was a loss in emission in-
tensity in 1, which is possibly a result of partial quenching by
platinum(II). The small Stokes shift observed for 1 is character-
istic of the fluorescent BODIPY dyes.[15,16] The fluorescence life-
times of complex 1 and ligand L1 in 1% DMSO/DPBS solution
were found to be 29.6 and 27.3 ns, respectively. In contrast,
complex 2 showed no emission band, which suggests that the
presence of the heavy atom iodine promotes efficient intersys-
tem crossing that results in complete quenching of the fluores-
cence intensity (Figure 2b).[20,21]
Redox chemistry and stability
The redox chemistry of the complexes was studied by cyclic
voltammetry with complexes and ligands of 1 mm concentra-
tion in DMF solutions and by using 0.1m tetrabutylammonium
perchlorate (TBAP) as the supporting electrolyte (Figure 3, Fig-
ure S14–S16 in the Supporting Information). The non-BODIPY
Figure 2. a) Absorption spectra of complexes 1–3 (25 mm) and b) emission
spectra (lex =480 nm) of complexes 1 and 2 and ligand L1 in 1% DMSO/
DPBS solution (pH 7.2).
both NMR spectroscopy and mass spectral methods (Fig-
ure S5–S8 in the Supporting Information). Complexes 1–3 dis-
played additional peaks in the proton NMR spectra at d=6.0–
6.5 ppm that were assignable to the hydrogen atoms of the
catecholate moiety (Figure S9–S11 in the Supporting Informa-
tion). The peaks in complexes 1 and 2 within the range d=
6.1–9.2 ppm correspond to the protons of the BODIPY frag-
ment that are not affected by the presence of the metal be-
cause they were separated from the platinum coordination
sphere by the benzyl spacer moiety. Thus, the BODIPY part was
expected to retain its photophysical properties in the com-
plexes. Complex 3 showed peaks only in the aromatic region
and for the methylene protons (Figure S11 in the Supporting
Information).
Figure 3. Cyclic voltammograms for complex 2, showing the anodic re-
sponse involving the metal-bound catechol moiety, in DMF/0.1m TBAP at
The observed peaks in the mass spectra at m/z 835.22,
1085.98, and 589.12 for complexes 1–3 are in agreement with
the calculated m/z values for the [M+H]+ species. The isotopic
distribution suggested the presence of platinum in the frag-
ments (Figure S12 in the Supporting Information). The purity
of the complexes was confirmed from elemental and induc-
tively coupled plasma mass spectrometry (ICP-MS) analytical
data. The complexes showed spectral properties similar to
those of the ligands (Figure 2a, Figure S13 in the Supporting
Information). Complexes 1 and 2 displayed strong absorption
bands in 1% DMSO/Dulbecco’s phosphate-buffered saline
(DPBS; pH 7.2) at l=510 (e=1.61ꢁ104 mÀ1 cmÀ1) and 550 nm
(e=1.37ꢁ104 mÀ1 cmÀ1), respectively, which were assignable to
the electronic transitions involving the BODIPY unit. The con-
trol complex 3 showed a relatively weaker and broad absorp-
tion band at around l=530 nm (e=0.28ꢁ104 mÀ1 cmÀ1). This
was a result of charge transfer from the catecholate to the
N,N-bound platinum(II) center. Complex 1 showed a strong
emission signal at l=505 nm (lexc =480 nm) even in 1%
DMSO/DPBS medium, which makes it ideal for cellular imaging
scan rates of 50, 100, and 200 mVsÀ1
.
ligand L3 was found to be redox inactive within the potential
window of the solvent. However, the dichloro precursor plati-
num(II) complex 3a displayed a quasireversible cathodic re-
sponse at a potential of E=À1.15 V and an irreversible anodic
peak at E=1.08 V. These oxidation and reduction processes,
therefore, involved the phenyl-substituted 2-(2-pyridyl)benzimi-
dazole ligand coordinated to the platinum(II) center. The cyclic
voltammogram of the BODIPY ligand L1 was dominated by
a quasireversible cathodic peak at E=À1.09 V, which is attrib-
uted to a reduction involving the BODIPY phenyl-substituted
2-(2-pyridyl)benzimidazole moiety. In the case of the iodinated
ligand L2, the reduction peak was found to be at E=À0.82 V.
The observed positive shift of 270 mV is a result of the iodina-
tion of the BODIPY core.[35,36] The BODIPY-containing dichloro
precursor complexes 1a and 2a showed reduction peaks simi-
lar to those of free ligands L1 and L2. Thus, it was concluded
that platinum(II) coordination did not affect the electrogenerat-
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ed radical species of the BODIPY core because the introduction
of a spacer phenyl ring broke the conjugation. Additional irre-
versible redox responses were found in 1a and 2a at E=
À1.20 and 1.10 V and were similar to that found in the precur-
sor complex 3a. The catecholate complexes 1–3 exhibited an
additional reversible response near E=0.5 V relative to the
redox profile of the respective dichloro precursor complexes
(Figure 3). Therefore, this couple was assigned to the catecho-
late and semiquinone radical species; this is a well-known
characteristic of the catecholate complexes.[37,38]
320 and 340 nm. Broad absorption bands were observed
within the range l=600–700 nm. These excited-state spectral
features indicate an enhanced triplet-state population, which is
localized on the BODIPY part.[39–41] The diiodinated ligand L2
gave transient absorption profiles similar to those of complex
2. Under the same experimental conditions, neither complex
1 nor L1 displayed significant excited-state absorption intensi-
ties. The result indicates that iodination of the BODIPY core in-
creased the lifetime of the excited states and, hence, the triplet
manifold was easily accessible in the nanosecond timescale.
The triplet-state lifetimes obtained from exponential fitting of
the decay curves were (2.62Æ0.01) and (3.83Æ0.06) ms for
complex 2 and ligand L2, respectively. From the difference ab-
sorbance values of the ground state and the excited state, the
triplet-state quantum yields were found to be 15 and 27%, re-
spectively, for complex 2 and ligand L2.
The complexes were readily soluble in polar solvents like
MeOH, MeCN, DMSO, and DMF. The stability of the complexes
was studied (25 mm in 1% DMSO/DPBS, pH 7.2) by monitoring
the changes in the absorption spectra with time. It was ob-
served that the complexes were stable in the dark for 48 h.
There was no apparent change in their absorption spectra
upon photoirradiation for 1 h (l=400–700 nm, light dose=
10 JcmÀ2), which indicated their photostability (Figure S17 in
the Supporting Information).
Singlet oxygen generation
ROS generation is an essential feature of PDT agents.[22–24] It is
well documented that BODIPY dyes with iodination or bromi-
nation at the core can generate singlet oxygen (1O2) upon light
exposure. Thus, trap experiments were performed with the flu-
orescent dye 1,3-diphenylisobenzofuran (DPBF) to detect any
generation of singlet oxygen by the diiodinated analogue,
complex 2.[42] DPBF (50 mm), if treated with complex 2 (5 mm) in
an air-saturated DMF medium and exposed to visible light (l=
400–700 nm), showed significant attenuation in both emission
and absorption intensities, which indicated photoinduced gen-
eration of singlet oxygen (Figure 5, Figure S19 in the Support-
ing Information). The singlet oxygen quantum yield for this
complex was 0.23, whereas that for the free ligand L2 was 0.48.
The complex, as expected, failed to generate any singlet
Transient absorption spectra
We next studied the nanosecond time-resolved transient differ-
ence absorption spectra in order to elucidate the dynamics of
the triplet excited states of the complexes and ligands
(Figure 4, Figure S18 in the Supporting Information). The diio-
dinated BODIPY complex 2 exhibited a bleaching band at l=
535 nm upon nanosecond pulsed laser excitation; this band
was a result of depletion of the ground-state population of the
BODIPY unit upon excitation. Significant bleaching was also
observed for the band at l=388 nm, which was assignable to
the BODIPY moiety. A strong transient absorption band at l=
480 nm was noted, along with weak absorption bands at l=
Figure 4. a) Nanosecond time-resolved transient difference absorption spec-
tra and b) first-order exponential decay curve showing the difference in ab-
sorbance versus time for complex 2 in aerated DMF with pulsed laser excita-
tion at l=532 nm at room temperature.
Figure 5. Decrease in a) absorption and b) emission intensity of DPBF
(50 mm) in the presence of complex 2 (5 mm) in DMF solution upon photoir-
radiation (l=400–700 nm). Data recorded at 5 min time intervals of photo-
exposure.
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oxygen species in the absence of light. In contrast, the non-
BODIPY analogue complex 3 showed no apparent change in
the DPBF intensity under the same photoirradiation conditions
(Figure S19 in the Supporting Information).
Theoretical studies
Detailed computational studies were carried out to gain in-
sights into the photophysical properties of the BODIPY com-
plexes 1 and 2 and the non-BODIPY analogue 3. We employed
the B3LYP functional with LanL2DZ as the basis set for all
atoms and computation was done with the Gaussian 09
suites.[43–45] The bond lengths and bond angles in the energy-
minimized structures were in good agreement with those
found in the crystal structures of known platinum catecholates
and BODIPY derivatives separately (Table S1 and Figure S20 in
the Supporting Information).[46,47] The platinum(II) center
adopted a square-planar geometry with PtÀN and PtÀO bond
lengths of approximately 2 ꢂ in these complexes. It was ob-
served that the BODIPY core preferred a perpendicular orienta-
tion with the phenyl ring at the meso position (Figure S20a,b
in the Supporting Information). In such a configuration, the
free rotation of the phenyl ring is prohibited because of steric
hindrance of the aromatic proton with the adjacent protons of
the 1,7-dimethyl groups. This restricts the extended p conjuga-
tion and diminishes the probability of non-radiative decay of
the excited states of the BODIPY. The calculated HOMO–LUMO
energy gaps for complexes 1 and 2 were 1.40 and 1.09 eV, re-
spectively. The decrease in the HOMO–LUMO energy gap in
complex 2 is a result of the incorporation of iodine and ex-
plains the redshift observed in the absorption spectra of com-
plex 2. The lower HOMO–LUMO energy difference is in accord-
ance with the varying reduction potentials of the BODIPY-cen-
tered reductions in complexes 1 and 2, as observed in the
cyclic voltammetric studies. These energy-minimized structures
were further subjected to time-dependent DFT (TDDFT) calcu-
lations with the B3LYP/LANL2DZ level of theory to rationalize
the nature of the transitions in the visible region and the orbi-
tal contributions. Selected singlet transitions and their oscilla-
tor strength (f) values are tabulated in Table S2 in the Support-
ing Information. It was deduced that the BODIPY-containing
complexes 1 and 2 showed transitions (f=0.7) at l=600 nm
originating from ligand-to-metal charge transfer that involved
orbitals localized on the catecholate and platinum(II). A strong
transitions with f=0.34 was seen at l=465 nm, which was as-
signed to intraligand charge transfer characterized by chief or-
bital contributions localized on the BODIPY unit (Figure 6, Fig-
ure S21a–c in the Supporting Information). Increased electron
densities of the molecular orbitals involved in transitions were
found in 2 relative to those in the non-iodinated analogue 1.
The orbitals localized on the iodine atoms, which suggests
they have vital roles in the excited-state behavior of the mole-
cules (Figure S21b,c in the Supporting Information). Interest-
ingly, complex 3, with no BODIPY pendant, showed very weak
transitions in the visible region (f=0.01; Figure S21d in the
Supporting Information). The calculated results therefore indi-
cate that the introduction of the BODIPY moiety resulted in
Figure 6. Frontier molecular orbitals involved in the transitions in the visible
region for complexes 1 (a and c) and 2 (b and d), as obtained from DFT and
TDDFT calculations with the B3LYP/LANL2DZ level of theory. Color codes:
Pt: brown; I: pink; F: green; O: red; N: blue; C: black; B: yellow. Hydrogen
atoms are omitted for brevity.
strong absorption bands in the visible region of complexes
1 and 2. The electronic structures of the triplet excited states
for complex 2 were obtained by TDDFT studies based on the
energy-minimized ground-state geometry. It is clear that the
low-lying triplet states are characterized by maximum orbital
contributions from the HOMO-8 and LUMO (Figure S22 in the
Supporting Information). Electron density analysis of these mo-
lecular orbitals concluded that the triplet states are localized
on the BODIPY unit, which explains the experimental findings
in the nanosecond transient absorption spectral studies.
MTT assay
The light-induced antiproliferative activities of the complexes
were investigated in tumor cell cultures by in vitro experi-
ments. HaCaT human skin keratinocyte cells were chosen for
our studies because photoinduced therapies are relevant for
easily accessible exposed tissues like skin. The cells were pre-
treated with the complexes for a short incubation time of 4 h
and the cytotoxicities were evaluated in the light and the dark
by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay (Table 2, Figure 7). MTT gets reduced by in-
tercellular oxidoreductase enzymes into insoluble formazan,
the level of which directly correlates with the cell viability.[48]
Complex 2 showed a prominent dose-dependent loss in the
survival rate in HaCaT cells only when illuminated, with a low
Table 2. IC50 values in HaCaT cells and mitochondrial Pt uptake of the
complexes 1–3.
[c]
Complex
IC50 [mm][a]
Light[b]
Pt [ng(ng mitochondria)À1
]
Dark
1
2
3
35.0Æ5.0
2.9Æ0.3
>100
>100
>100
1.05Æ0.35
1.29Æ0.21
–
>100
[a] HaCaT cells treated with complexes for 4 h in the dark. [b] Visible light
(l=400–700 nm, light dose=10 JcmÀ2). [c] HaCaT cells were treated with
complexes (100 mm) and the platinum content was determined by ICP-
MS methods.
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Figure 7. Cell viability plots as obtained from the MTT assay in HaCaT cells treated with complexes 1 (a), 2 (b), and 3 (c) for a 4 h incubation period. Cells were
either exposed to light (light gray symbols, l=400–700 nm, light dose=10 JcmÀ2) or kept in the dark (black symbols).
half-maximal inhibitory concentration (IC50) value of approxi-
mately 3 mm in visible light (l=400–700 nm, light dose=
10 JcmÀ2) and with no apparent toxicity in the dark (IC50 >
100 mm in the dark). Interestingly, the emissive complex 1 gave
a high IC50 value of approximately 35 mm in visible light under
similar treatment conditions. This reflects the crucial role of the
iodine on the BODIPY core in augmenting the photosensitizing
effect. The low toxicity of 1 in the light and dark made it more
suitable for cellular imaging. The non-BODIPY complex 3
showed no cellular toxicity even at a higher concentration of
100 mm, which highlights the essential role of the diiodinated
BODIPY unit as a photosensitizer in complex 2. Complexes 1–3
showed substantial toxicity in the dark (IC50 values of approxi-
mately 20 mm) in HaCaT cells that were preincubated with the
complexes for a longer incubation period of 24 h (Figure S23
in the Supporting Information). The cellular toxicity exhibited
by the non-BODIPY analogue 3 was found to be similar to that
of the Pt–BODIPY conjugates 1 and 2. This indicated slow dis-
sociation of the PtÀO bonds in the cellular medium, which ulti-
mately led to the formation of lethal Pt–DNA adducts that
caused cell death even in the absence of light.[1,2]
Figure 8. a) Confocal microscopy images of 1 (10 mm) in HaCaT cells after 4 h
incubation in the dark. Top row for propidium iodide and second row for
Mito-Tracker Deep Red. Merged panels show mitochondrial localization of
complex 1. b) Confocal microscopy images showing generation of ROS at
the mitochondrial site by complex 2 upon photoexposure. The formation of
DCF is seen from the green intensity in the second column and mitochon-
drial localization is observed in the merged panels. Scale bar=25 mm.
Confocal imaging and mitochondrial platinum uptake
The observed PDT effect of 2 made us keen to investigate the
mechanism of action. The emissive property of complex 1 in
1% DMSO/DPBS buffer was used to track its key cellular tar-
gets. HaCaT cells were treated with 1 (10 mm) and incubated
for 4 h in the dark prior to a confocal microscopy study. Propi-
dium iodide (PI) was used as the nuclear staining dye. The cy-
toplasmic distribution of 1 is clearly illustrated by the merged
images in Figure 8a (top row). To further evaluate the exact
subcellular colocalization, different cell organelle trackers,
namely Mito-Tracker Deep Red (MTR), ER-Tracker Red, and
Lyso-Tracker Deep Red, were used. The Z-stack confocal image
of the complex overlaid with that of the MTR revealed pre-
dominant colocalization of complex 1 within the mitochondria
(Figure 8a, second row). The presence of complex 1 in the en-
doplasmic reticulum was indicated by the yellow color of the
overlapped images of the complex and ER-Tracker (first row in
Figure S24 in the Supporting Information). However, the green
emission of 1 showed no overlap with the Lyso-Tracker Deep
Red (last row in Figure S24 in the Supporting Information).
The platinum content in isolated mitochondria of HaCaT
cells pretreated with the complexes was estimated in order to
understand the intercellular fate of the complexes. HaCaT cells
were treated with complexes 1–3 (100 mm) and incubated for
4 h in the dark. After the treatment, the mitochondria were iso-
lated and the quantity of platinum was estimated by the ICP-
MS method. The results are listed in Table 2.[49] It was observed
that complexes 1 and 2 both showed substantial platinum
content in the mitochondria (1.05 and 1.30 ng of platinum per
ng of mitochondria), whereas no platinum was detected for
complex 3 in the isolated mitochondria. This indicates the es-
sential role of the BODIPY units for mitochondrial accumulation
of the complexes. The combined results from the cellular imag-
ing and platinum estimation studies increased the probability
that intact complexes are localizing in the mitochondria.
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Dichlorofluorescein diacetate (DCFDA) assay and mitochon-
drial ROS
membrane potential with flow cytometry.[55] Membrane-poten-
tial-driven accumulation of TMRE in mitochondria results in
a strong red fluorescence. Reduction of the emission intensity
of TMRE correlates to mitochondrial apoptosis or oxidative-
stress-induced depolarization of the mitochondrial membrane.
HaCaT cells treated with complex 2 and exposed to light dis-
played a significant decrease in the TMRE intensity of approxi-
mately 350 and 250 a.u., respectively, relative to the treated
cells kept in the dark or untreated cells (Figure 9). Valinomycin
(25 mm) caused a decrease of 200 a.u. in the TMRE intensity
under similar conditions. There was no change in the TMRE in-
tensity in cells treated with complex 3, which is in agreement
with the earlier experimental data.
The complexes showed light-induced generation of singlet
oxygen, as evident from the DPBF studies. Thus, we estimated
the cellular ROS amounts in photoexposed HaCaT cells prein-
cubated with the complexes 2 and 3 by a DCFDA assay.
The assay is based on the oxidative production of the
fluorescent dye dichlorofluorescein (DCF, lem =525 nm), the
emission intensity of which quantifies the cellular ROS levels.[50]
Complex 2 (10 mm) showed significant enhancement in the
mean DCF intensity (ꢀ150 a.u.) in light (l=400–700 nm,
light dose=10 JcmÀ2) in the exposed set of cells relative to
that of the identical set kept in the dark (Figure S25 in the
Supporting Information). However, complex 3 showed similar
ROS levels in the dark and light to that in the untreated cells,
which confirms its inability to act as a photosensitizer in visible
light.
Mitochondria were the key targets of these complexes, so
we used confocal imaging to map the subcellular sites of ROS
generation. HaCaT cells were treated with complexes 2 and 3
and exposed to light (l=400–700 nm, light dose=10 JcmÀ2)
or kept in the dark. The cells were stained with DCFDA dye
(1 mm) for 5 min and images were captured with a Zeiss confo-
cal microscope. Substantial green emission of DCF was ob-
served only in cells treated with complex 2 and subjected to
light treatment. Interestingly, the green emission of the pro-
duced DCF coincided exactly with the mitochondrial distribu-
tion inside the cells (Figure 8b). This showed selective genera-
tion of ROS in the mitochondria, which in turn signifies the ac-
cumulation of the complexes solely in the mitochondria. The
non-BODIPY analogue 3 failed to generate any distinct DCF in-
tensity in both dark and light conditions (Figure S26 in the
Supporting Information).
Figure 9. Bar diagram presenting the emission intensity of TMRE in HaCaT
cells treated with 2 and 3 for 4 h and then either exposed to light (l=400–
700 nm; yellow cylinders) or kept in the dark (black cylinders). “Cells” refers
to untreated controls and “val” refers valinomycin (25 mm) used as a positive
control.
Apoptotic study and cell-cycle assay
It is well established that mitochondrial function governs the
intrinsic apoptotic pathway in mammalian cells.[56] The fact
that our complexes are perturbing the mitochondrial mem-
brane potential elevates the probability of triggering apoptosis
through mitochondrial damage. We examined such a possibility
with an annexin-V/fluorescein isothiocyanate (FITC)/propidium
iodide (PI) assay with HaCaT cells treated with the complexes
under different conditions. The basic principle is selective per-
meability of the annexin-V/FITC dye toward early apoptotic
cells.[57] The percentage population of early apoptotic cells can
be inferred from the lower right quadrant of the dot plots in
Figure 10a (Figure S28 in the Supporting Information). It was
observed that complex 2 showed approximately 50% annexin-
V/FITC positive cells only in the presence of light. The complex
in the dark resulted in a negligible (ꢀ5%) early apoptotic cell
population, which is similar to the result with the untreated
controls or cells treated with complex 3 in the dark and light.
The sub-G1 cell population also relates to the apoptotic pop-
ulation. This cell-cycle phase is characterized by a low DNA
content; this DNA can be stained with PI and quantified by
a fluorescence-activated cell sort (FACS) analysis.[58] The bar dia-
gram in Figure 10b, obtained from the statistical analysis of
the histograms (Figure S29 in the Supporting Information),
shows a 59% increase in the sub-G1 cell population for HaCaT
cells treated with complex 2 that were photoirradiated (l=
400–700 nm) relative to the population in those unexposed to
Mitochondrial membrane potential
The mitochondrial ROS formed by complex 2 upon light expo-
sure could lead to alterations in the mitochondrial transmem-
brane potential (Dym).[51,52] The cell-permeant JC-1 dye assem-
bles in mitochondria to form concentration-dependent red-flu-
orescent J aggregates (lem =590 nm). Mitochondrial dysfunc-
tion causes a decrease in the Dym value and consequent leak-
age of JC-1 dye into the cytoplasmic matrix, where it exists as
a green-emissive monomer (lem =530 nm). This potential-de-
pendent dual emissive property renders JC-1 as a ratiometric
probe to monitor any loss of mitochondrial potential.[53] Com-
plex 2 reduced the red-fluorescence intensity with a concomi-
tant increase in the green signal of the JC-1 dye in light-ex-
posed HaCaT cells (Figure S27 in the Supporting Information).
Valinomycin (25 mm), which is well documented to depolarize
mitochondria, was used as a positive control, with which only
the green signal of JC-1 was observed.[54] Complex 3 showed
no such changes in cellular images recorded under identical
experimental conditions.
Tetramethylrhodamine ethyl ester (TMRE) was employed as
a monochromatic probe for measuring the mitochondrial
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Experimental Section
Chemicals and experimental methods: Potassium tetrachloropla-
tinate was purchased from Arora Matthey, India. Mito-Trackerꢃ
Deep Red FM, ER-TrackerTM Red, and Lyso-Tracker Deep Red were
purchased from Invitrogen (USA). All other chemicals and reagents
are obtained from S. D. Fine Chemicals, India, and Sigma–Aldrich,
USA. TBAP (0.1m) was obtained by treating tetrabutylammonium
bromide with perchloric acid. [Caution! This reaction must be car-
ried out with proper safety measures.] Solvents were purified in ac-
cordance with earlier literature reports.[59] Ligand L3, precursor com-
pound A, and Pt(DMSO)2Cl2 were prepared in accordance with ear-
lier reports.[60–62] Platinum(II) catecholates were synthesized by
a modified version of a method reported earlier.[63] Elemental analy-
sis was carried out with a Thermo Finnigan Flash EA 1112 CHNS an-
alyzer. NMR spectra were recorded by using a Bruker Avance NMR
spectrometer. Mass spectral data were acquired with an Agi-
lent 6538 Ultra High Definition Accurate Mass-Q-TOF (LC-HRMS) in-
strument. IR, UV/Vis, and emission spectra were recorded with
Bruker Alpha and Perkin–Elmer Spectrum 750 spectrophotometers
and a HORIBA Jobin Yvon IBH TCSPC fluorimeter (fitted with Fluo-
roHub software analysis). The fluorescence lifetimes were mea-
sured with an IBH DataStationHub fluorimeter and fitted by using
HORIBA Jobin Yvon decay software. Cyclic voltammetric studies
were performed with an EG&G PAR Model 253 VersaStat potentio-
Figure 10. a) Dot plots obtained from the annexin-V/FITC/PI assay with
HaCaT cells treated with complex 2 and exposed to light (l=400–700 nm)
or kept in the dark. The percentage cell population is given in the respective
quadrants: Lower left: live cells; lower right: early apoptotic cells; upper
right: late apoptotic/necrotic cells; upper left: dead cells. b) Cell-cycle analy-
sis with propidium iodide staining in HaCaT cells incubated with complexes
2 and 3 for 4 h and exposed to light (l=400–700 nm). The bar diagram rep-
resents the percentage cell population in different phases of cell-cycle pro-
gression as indicated in the figure. D: dark; L: light (l=400–700 nm). Errors
are within Æ5%.
stat/galvanostat consisting of
a three-electrode setup (glassy
carbon electrode as the working electrode, a platinum wire as the
auxiliary electrode, and a SCE as the reference). Ferrocene was
used as a standard. TBAP (0.1m) was used as a supporting electro-
lyte. All experiments requiring light exposure were carried out with
broad-band white light from a Luzchem Photoreactor (Model LZC-
1, Ontario, Canada) fitted with eight fluorescent Sylvania white
tubes (l=400–700 nm). Cytotoxic data were obtained with
a TECAN microplate reader and fitted by using GraphPad Prism 5
software. Flow cytometric experiments were performed by using
a FACS Verse instrument (BD Biosciences) fitted with a MoFLo XDP
cell sorter and analyzer with three lasers (l=488, 365, and 640 nm)
and ten-color parameters. Confocal microscopy images were ac-
quired by using a Leica microscope (TCS, SP5) with an oil immer-
sion lens with magnification of 63ꢁ. Images were processed by
using LAS AF Lite software.
light. Complex 3, because of its non-toxic nature, showed a be-
havior that was similar to that of the untreated controls.
Conclusions
In this work, we have successfully exploited the BODIPY unit to
direct bifunctional platinum(II) complexes to target mitochon-
dria. The choice of bidentate catecholate as an O,O-donor
ligand ensured slow release of the active platinum(II) species
that are capable of forming crosslinks with mt-DNA. The
strong emissive properties of complex 1 upon cellular uptake
and the high mitochondrial Pt content confirmed accumula-
tion of the complexes in the mitochondria. Incorporation of
two iodine atoms in the BODIPY moiety of complex 2 led to
an enhanced triplet population in the photoexcited state,
which resulted in higher singlet oxygen production in visible
light. The photosensitizing ability of complex 2 resulted in a re-
markably low IC50 value of approximately 3 mm in HaCaT cells
in visible light (l=400–700 nm) relative to that with complex
1. Importantly, these complexes were completely ineffective in
the dark and so fulfill the essential criteria for PDT agents. The
detailed mechanistic studies in HaCaT cells revealed light-de-
pendent generation of ROS leading to mitochondrial mem-
brane depolarization and the intrinsic apoptotic pathway. This
work highlights the tuning of photophysical properties of
BODIPY-appended platinum(II) complexes for achieving photo-
induced mt-DNA damage. The results are of importance be-
cause paradigm shifts from targeting nuclear DNA to mt-DNA
and of spatiotemporal control over tumor destruction are nec-
essary to counter drug resistance and minimize the side effects
of an anticancer agent.
Syntheses: The ligands and the complexes were synthesized by
using the procedures that are detailed below (Scheme S1 and S2
in the Supporting Information).
Ligand L1: A mixture of compound A (500 mg, 1.34 mmol), K2CO3
(370 mg, 2.68 mmol, 2 equiv), and KI (445 mg, 2.68 mmol, 2 equiv)
was placed in aqueous acetonitrile (1:2 v/v, 30 mL) and purged
with nitrogen for 15 min. 2-(2-Pyridyl)benzimidazole (209 mg,
1.07 mmol, 0.9 equiv) was added and the solution was heated at
reflux at T=1008C for 24 h under a nitrogen atmosphere. The solu-
tion was then cooled to ambient temperature, diluted with di-
chloromethane, and washed with water and brine. The organic
phase was collected, dried over sodium sulfate, filtered, and evapo-
rated with a rotary evaporator. The crude product thus obtained
was purified by silica gel column chromatography by eluting with
chloroform/ethyl acetate (9:1 v/v) to yield the desired orange-col-
ored product. Yield=300 mg (0.56 mmol, 42%); 1H NMR (CDCl3,
400 MHz): d=9.19–9.17 (m, 2H), 8.75–8.69 (m, 1H), 8.33 (t, J1 =
4.4 Hz, J2 =3.6 Hz, 1H), 8.10 (d, J=8 Hz, 1H), 7.96 (br, 1H), 7.55 (m,
3H), 7.31 (t, J1 =8 Hz, J2 =2.8 Hz, 1H), 6.99 (m, 2H), 6.41 (d, J=
2.4 Hz, 2H), 5.94 (s, 2H), 2.528 (s, 6H), 1.19 ppm (s, 6H); 13C NMR
(100 MHz, CDCl3): d=156, 149, 143, 137, 129, 128, 127, 124, 121,
111, 49, 15 ppm; UV/Vis (10% DMSO/DPBS at pH 7.2): lmax (e)=
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505 nm (2.2ꢁ104 mÀ1 cmÀ1); emission spectrum (10% DMSO/DPBS
at pH 7.2): lem (lex, Ff)=505 nm (480 nm, 0.12); elemental analysis:
calcd for C32H28BF2N5: C 72.33, H 5.31, N 13.18; found: C 72.10, H
5.07, N 13.06; molecular weight=531.41 gmolÀ1; MS (MeOH): m/z
532.24 [M+H]+.
Preparation of [Pt(L)(cat)] (1–3): The appropriate dichloroplatinum
complex [Pt(L)Cl2] was mixed with catechol (1 equiv) and cesium
carbonate (2 equiv) in methanol (20 mL) and heated at reflux for
6 h in the dark under a nitrogen atmosphere. The solvent was then
evaporated to dryness and the residue was dissolved in DMF
(2 mL). The desired complex was precipitated by adding excess di-
ethyl ether and was repeatedly washed with water and diethyl
ether to remove any unreacted starting materials.
Ligand L2: Ligand L1 (0.70 gm, 1.31 mmol) was placed in dry di-
chloromethane (50 mL) and degassed with bubbling nitrogen. An
excess of N-iodosuccinimide (1.80 gm, 8 mmol, 6 equiv) was added
and the mixture was stirred for 12 h under a nitrogen atmosphere.
The red solution was diluted with dichloromethane and washed
with water. The organic phase was dried over sodium sulfate, fil-
tered, and evaporated with a rotary evaporator. The crude product
was subjected to silica gel column chromatography by using
chloroform/ethyl acetate (9:1 v/v) to afford a pink-red solid. Yield=
550 mg (0.7 mmol, 54%); 1H NMR (CDCl3, 400 MHz): d=9.19 (m,
2H), 8.74 (m, 1H), 8.34 (d, J=8.0 Hz, 1H), 8.14 (t, J1 =8.0 Hz, J2 =
8 Hz, 2H), 7.65–7.59 (br, 3H), 7.57 (d, J=4.0 Hz, 1H), 7.25 (m, 2H),
6.42 (s, 2H), 2.63 (d, J=4 Hz, 6H), 1.22 ppm (s, 6H); 13C NMR
(100 MHz, [D6]DMSO/CDCl3 (1:1)): d=156, 149, 145, 141, 139, 138,
133, 131, 128, 125, 124, 119, 112, 87, 49, 29, 16 ppm; UV/Vis (10%
DMSO/DPBS at pH 7.2): lmax (e)=550 (2.17ꢁ104), 515 (1.75ꢁ104),
388 nm (0.9ꢁ104 mÀ1 cmÀ1); elemental analysis: calcd for
C32H26BF2I2N5: C 49.07, H 3.35, N 8.94; found: C 48.90, H 3.56, N
8.90; molecular weight=783.21 gmolÀ1; MS (MeOH): m/z 784.04
[M+H]+.
Complex [Pt(L1)(cat)] (1): [Pt(L1)Cl2] (100 mg, 0.12 mmol), catechol
(17 mg, 0.15 mmol), Cs2CO3 (90 mg, 0.25 mmol); yield=50%
(brown solid, 50 mg, 0.06 mmol); 1H NMR ([D6]DMSO, 400 MHz):
d=9.62 (d, J=7.4 Hz, 2H), 9.08 (d, J=8 Hz, 1H), 8.23 (d, J=7.6 Hz,
1H), 8.11–8.04 (m, 2H), 7.77 (t, J1 =7.2 Hz, J2 =6.8 Hz, 3H), 7.64–
7.32 (m, 1H), 7.33 (d, J=5.6 Hz, 1H), 6.51 (d, J=3.2 Hz, 2H), 6.32–
6.29 (m, 4H), 6.13 (s, 2H), 2.48 (s, 6H), 1.18 ppm (s, 6H); UV/Vis
(10% DMSO/DPBS at pH 7.2): lmax (e)=510 (1.6ꢁ104), 325 nm (1.1ꢁ
104 mÀ1 cmÀ1); emission spectrum (1% DMSO/DPBS at pH 7.2): lem
(lex, Ff)=505 nm (480 nm, 0.06); elemental analysis: calcd for
C38H32BF2N5O2Pt: C 54.69, H 3.68, N 8.39; found: C 54.22, H 3.82, N
8.25; molecular weight=834.59 gmolÀ1; MS (MeOH): m/z 835.59
[M+H]+.
Complex [Pt(L2)(cat)] (2): [Pt(L2)Cl2] (125.9 mg, 0.12 mmol), cate-
chol (17 mg, 0.15 mmol), Cs2CO3 (90 mg, 0.25 mmol); yield=44%
1
(violet solid, 55 mg, 0.05 mmol); H NMR ([D6]DMSO, 400 MHz): d=
9.65 (d, J=4.9 Hz, 1H), 9.30 (d, J=5.1 Hz, 1H), 9.07 (d, J=8.2 Hz,
1H), 8.46–8.44 (m, 1H), 8.31–8.22 (m, 1H), 8.14–8.06 (m, 1H), 7.97
(d, J=8.8 Hz, 1H), 7.69–7.53 (m, 2H), 7.37–7.34 (m, 1H), 7.21–7.20
(d, J=7.2 Hz, 2H), 6.61–6.55 (m, 2H), 6.33 (s, 2H), 6.16 (2H, s) 2.54
(s, 6H), 1.18 ppm (s, 6H); UV/Vis (10% DMSO/DPBS at pH 7.2): lmax
(e)=550 (1.37ꢁ104), 525 nm (1.10ꢁ104 mÀ1 cmÀ1); elemental analy-
sis: calcd for C38H30BF2I2N5O2Pt: C 42.01, H 2.78, N 6.45; found: C
41.89, H 2.62, N 6.33; molecular weight=1049.19 gmolÀ1; MS
(MeOH): m/z 1048.94 [M+H]+.
Preparation of [Pt(L)Cl2] (1a–3a): The precursor complex
[Pt(DMSO)2Cl2] was mixed with the appropriate ligand (1 equiv) in
degassed acetonitrile (30 mL) and the reaction was heated at
reflux for 24 h under a nitrogen atmosphere. Upon cooling of the
reaction mixture to 08C, a precipitate was obtained, which was fil-
tered and washed thoroughly with hexane and diethyl ether.
Complex [Pt(L1)Cl2] (1a): [Pt(DMSO)2Cl2] (78 mg, 0.188 mmol), L1
(100 mg, 0.188 mmol); yield=132 mg (0.16 mmol, 88%); 1H NMR
([D6]DMSO, 400 MHz): d=9.63 (d, J=6 Hz, 2H), 9.08 (d, J=8.4 Hz,
1H), 8.24 (t, J1 =8.8 Hz, J2 =7.6 Hz, 1H), 8.11–8.04 (dd, J1 =8.4 Hz,
J2 =8.8 Hz, J3 =8.0 Hz, 1H), 7.77 (t, J1 =6.4 Hz, J2 =7.2 Hz, 2H),
7.64–7.49 (m, 2H), 7.36–7.29 (m, 3H), 6.32 (s, 2H), 6.17 (d, J=
16.4 Hz, 2H), 1.35 (s, 6H), 1.18 ppm (s, 6H); elemental analysis:
calcd for C32H28BCl2F2N5Pt: C 48.20, H 3.54, N 8.78; found: C 48.06,
H 3.72, N 8.52; molecular weight=797.40 gmolÀ1; MS (MeOH): m/z
819.12 [M+Na]+, 761.17 [MÀCl]+.
Complex [Pt(L2)Cl2] (2a): [Pt(DMSO)2Cl2] (132 mg, 0.32 mmol), L2
(250 mg, 0.32 mmol); yield=130 mg (0.12 mmol, 50%); 1H NMR
([D6]DMSO, 400 MHz): d=9.65 (d, J=5.6 Hz, 1H), 9.08 (d, J=8.4 Hz,
1H), 8.27 (t, J1 =J2 =8.0 Hz, 1H), 8.07 (t, J1 =8.4 Hz, J2 =5.2 Hz, 1H),
7.81 (t, J1 =8.4 Hz, J2 =6.8 Hz, 1H), 7.56 (t, J1 =8 Hz, J2 =7.6 Hz, 1H),
7.41–7.34 (m, 6H), 6.33 (s, 2H), 1.19 ppm (m, 12H); elemental anal-
ysis: calcd for C32H26BCl2F2I2N5Pt: C 36.63, H 2.50, N 6.68; found: C,
36.40, H 2.63, N 6.32; molecular weight=1049.19 gmolÀ1; MS
(MeOH): m/z 1071.93 [M+Na]+, 1012.96 [MÀCl]+.
Complex [Pt(L3)(cat)] (3): [Pt(L3)Cl2] (275 mg, 0.5 mmol), catechol
(65 mg, 0.5 mmol), Cs2CO3 (358 mg, 1.1 mmol); yield=60% (violet
solid, 175 mg, 0.3 mmol); 1H NMR ([D6]DMSO, 400 MHz): d=9.29 (d,
J=5.6 Hz, 2H), 8.45 (t, J1 =2.4 Hz, J2 =6.8 Hz, 1H), 8.22 (d, J=
7.6 Hz, 1H), 8.12 (d, J=8 Hz, 1H), 7.97–7.95 (m, 4H), 7.68–7.19 (m,
2H), 7.61–7.55 (m, 2H), 6.25–6.22 (m, 2H), 6.15 (s, 2H), 5.94 ppm (s,
2H); UV/Vis (10% DMSO/DPBS at pH 7.2): lmax (e)=530 (0.3ꢁ104),
335 nm (1.1ꢁ104 mÀ1 cmÀ1); elemental analysis: calcd for
C25H19N3O2Pt: C 51.02, H 3.25, N 7.14; found: C 50.84, H 3.36, N
7.10; molecular weight=588.53 gmolÀ1; MS (MeOH): m/z 589.12
[M+H]+.
Nanosecond time-resolved transient absorption spectroscopy:
The difference absorption spectra were recorded by using a laser
flash photolysis spectrometer (LKS.60 Applied Photophysics). DMF
solutions of the complexes and ligands (20 mm) were purged with
argon for 15–30 min prior to the measurements. The solutions
were excited with a nanosecond-pulsed laser (532 nm, pulse width
of 5–8 ns) and the transient signals were acquired and digitalized
with an oscilloscope (54520 A, Hewlett–Packard). The quantum
yield was calculated with zinc(II) phthalocyanine (ZnPc) as the ref-
erence compound (FT =0.65).
Complex [Pt(L3)Cl2] (3a): [Pt(DMSO)2Cl2] (333 mg, 0.792 mmol), L3
(225 mg, 0.792 mmol); yield=365 mg (0.66 mmol, 83%); 1H NMR
([D6]DMSO, 400 MHz): d=9.63 (d, J=6.0 Hz, 1H), 9.04 (d, J=8.4 Hz,
1H), 8.30 (t, J1 =J2 =8.0 Hz, 1H), 8.15 (d, J=7.6 Hz, 1H), 7.94 (d, J=
8.4 Hz, 1H), 7.77 (t, J1 =6.4 Hz, J2 =6.8 Hz, 1H), 7.59–7.50 (m, 2H),
7.37–7.28 (m, 3H), 7.19 (d, J=7.2 Hz, 2H), 6.18 ppm (s, 2H); ele-
mental analysis: calcd for C19H15Cl2N3Pt: C 41.39, H 2.74, N 7.62;
Detection and quantum yield for singlet oxygen: DPBF showed
1
poor sensitivity for O2 in aqueous solutions. It was also prone to
photobleaching in halogenated solvents. However, it was photo-
stable in DMF. Therefore, the singlet oxygen quenching experi-
ments were carried out with DMF solutions. The non-emissive
complexes 2 and 3 (5 mm) were treated with DPBF (50 mm) in DMF
and the emission spectra were recorded at an excitation wave-
found: C 41.19, H 2.86, N 7.51; molecular weight=551.33 gmolÀ1
MS (MeOH): m/z 573.01 [M+Na]+, 515.06 [MÀCl]+.
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length of 405 nm. Either irradiation was performed with 400–
700 nm visible light for different time intervals or the samples were
kept in the dark. The time-dependent absorption spectral experi-
ments with DPBF and complexes 2 and 3 in DMF were performed
in a similar fashion. The quantum yield of singlet oxygen genera-
DCFDA assay: The photoinduced generation of ROS by complexes
2 and 3 (in 1% DMSO/DMEM) in HaCaT was quantified by using
the DCFDA assay. Cells were incubated with the complexes 2
(5 mm) and 3 (20 mm) for 4 h in complete darkness. One of the
plates was irradiated (l=400–700 nm, light dose=10 JcmÀ2) for
1 h in phenol red free medium, whereas the other was kept in the
dark. Cells were then washed with DPBS, treated with trypsin (1ꢁ),
and collected by centrifugation. Cells were resuspended in DPBS,
treated with DCFDA (1 mm), and incubated for 15 min at T=08C.
Samples were then taken for reading with a FACS Verse machine
(BD Biosciences). Untreated controls were kept as references and
experiments were done in duplicate. The DCFDA assay was per-
formed by using confocal microscopy for complexes 2 and 3 (in
1% DMSO/DPBS) in HaCaT cells. To visualize the subcellular site of
ROS production, Mitotrackerꢃ Deep Red (MTR, 50 nm) and DCFDA
(1 mm) were used. Cells were plated in 12-well tissue culture plates
on cover slips in the usual way for microscopic experiments. Cells
were then incubated in the dark for 4 h with complexes 2 (5 mm)
and 3 (20 mm). Cells were either photoexposed (l=400–700 nm,
t=30 min in phenol red free media) or kept in the dark. The cells
were stained with MTR and DCFDA for 20 min at room tempera-
ture. The cover slips were mounted and tethered to slides. Images
were captured by using a Leica microscope (TCS, SP5) with an oil
immersion lens with a magnification of 63ꢁ. Multiple images were
recorded and experiments were done in duplicate to confirm the
results.
ref
tion (FD) was calculated by using the equation: FD =FD (k/
kref)(Iaref/Ia), in which FD is the singlet oxygen quantum yield for
ref
the reference (Rose Bengal was used as the standard, FD =0.76), k
and kref are the photobleaching rate constants in the presence of
the respective samples and the standard, and Ia and Iaref are the ab-
sorption intensities at the irradiation wavelength (510 nm) of the
samples and reference, respectively.[64]
Theoretical calculations: The energy-minimized geometries of
complexes 1–3 were obtained by the DFT method by employing
the B3LYP functional (LANL2DZ basis sets for all atoms) as imple-
mented in the Gaussian 09 program.[43–45] Linear-response TDDFT
performed on the optimized structures revealed the energy of
transitions along with the oscillator strengths and orbital contribu-
tions.
MTT assay: Cytotoxicity studies were done with complexes 1–3 in
HaCaT (human keratinocyte) cells. Cells were incubated with vari-
ous concentrations of 1–3 (5, 10, 15, 20, 30, 50, and 100 mm in 1%
DMSO/Dulbecco’s modified Eagle’s medium (DMEM)) for 4 h in
complete darkness. One set of cells was exposed to visible light
(l=400–700 nm, light dose=10 JcmÀ2), whereas the other was
kept in the dark for 1 h. Data were obtained by using three inde-
pendent sets of experiments done in triplicate for each concentra-
tion. Ligands and precursor dichloroplatinum(II) complexes were
not studied because they were insoluble in 1% DMSO/DPBS
medium (see the Supporting Information for details).
Mitochondrial membrane potential JC-1 assay: The dual emissive
nature of the JC-1 dye, the differential intensities of which depend
on the mitochondrial membrane potential, was employed to fur-
ther confirm the mitochondrial membrane depolarization. Com-
plexes 2 (5 mm) and 3 (20 mm) in 1% DMSO/DMEM were added to
HaCaT cells and incubated for 4 h in the dark. The cells were ex-
posed to light (l=400–700 nm, t=1 h) or kept in the dark in
phenol red free media. The confocal images were captured in both
red and green channels by using a Leica microscope. Untreated
cells served as negative controls, whereas cells treated with valino-
mycin (25 mm, t=1 h) served as a positive control for the experi-
ments (see the Supporting Information for details).
Confocal microscopy and mitochondrial Pt estimation: The intra-
cellular localization of complex 1 was investigated by using a Leica
microscope (TCS, SP5) with an oil immersion lens with a magnifica-
tion of 63ꢁ. HaCaT cells were incubated with the complex (10 mm
in 1% DMSO/DMEM) in the dark for 4 h. Cells were stained with PI
(1 mgmLÀ1) for 5 min. Further information on the subcellular locali-
zation was obtained by using similar treatment procedures but
without any prior fixation of cells by using methanol. Live cells
were stained with Mito-trackerꢃ Deep Red (50 nm), ER-TrackerTM
Red (50 nm), or Lyso-Tracker Deep Red (50 nm), incubated for
20 min at room temperature, and subsequently visualized under
the microscope. Multiple images were recorded and experiments
were done in duplicate to confirm the results (see the Supporting
Information for details). Approximately 106 HaCaT cells were incu-
bated with complexes 1–3 (100 mm) for 4 h in the dark. After treat-
ment, the cells were collected and resuspended in mitochondrial
isolation buffer (10 mL of 0.1m tris(hydroxymethyl)aminomethane
(Tris)/3-(N-morpholine)propanesulfonic acid and 1 mL of ethylene
glycol-bis(b-aminoethyl ether)-N,N,N’,N’-tetraacetic acid/Tris to
20 mL of 1m sucrose and volume made up to 100 mL (pH 7.4)).
Cells were then homogenized at T=48C by using a Teflon pestle
(1600 rpm) and the cell suspensions were stroked gently in a glass
potter (initially cooled in an ice bath to maintain the mitochondrial
integrity). The homogenate was centrifuged again at 7000 g at 48C
for 10 min and the pellet containing the mitochondria was collect-
ed. One such set was used for measuring the concentration by
Biuret methods and for examining the separation of mitochondria
by gel electrophoresis. Another identical set was used for Pt esti-
mation by ICP-MS after dilution in 2% HNO3/DPBS solutions (see
the Supporting Information for details).
Mitochondrial membrane potential TMRE assay: To assess the
mitochondrial dysfunction, TMRE, a fluorescent dye (lem =690 nm)
that exhibits a mitochondrial membrane potential dependent accu-
mulation in mitochondria, was used. HaCaT cells were incubated
with complexes 2 (5 mm) and 3 (20 mm) in 1% DMSO/DMEM for
4 h. Cells were either kept in the dark or irradiated with visible
light for 1 h in phenol red free medium. Untreated cells and cells
treated with valinomycin (25 mm, t=1 h), which is known to depo-
larize mitochondria, were kept as controls. The FACS analysis of
these samples gave a quantitative measure of TMRE intensity (see
the Supporting Information for details).
Annexin-V/FITC/PI assay: The early apoptotic cell population was
determined for complexes 2 (5 mm) and 3 (20 mm) in 1% DMSO/
DMEM. Approximately 3ꢁ105 HaCaT cells were seeded in six-well
plates and cultured for 24 h. The cells were incubated with 2 and 3
for 4 h in the dark and then exposed to light (l=400–700 nm,
light dose=10 JcmÀ2) in phenol red free media or kept in com-
plete darkness. Cells were then kept for another 14 h in DMEM/
10% fetal bovine serum (FBS) medium in the dark, after which the
medium was discarded and the cells were trypsinized and resus-
pended in binding buffer (300 mL, 1ꢁ). Annexin-V/FITC (0.5 mL) and
PI (1 mL) were added to the cell suspensions and incubated for
5 min. Readings were taken with the FACS instrument. Gating of
cell population was performed based on the untreated controls.
&
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Full Papers
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Acknowledgements
The authors thank the Department of Science and Technology
(DST), Government of India, for financial support (grant numbers
SR/S5/MBD-02/2007 and EMR/2015/000742). The authors also
thank the Alexander von Humboldt (AvH) Foundation, Germany,
for an electrochemical system. A.R.C. thanks the DST for a J. C.
Bose Fellowship. P.K.’s research group is funded by DBT-IISc
grants. K.M. is a recipient of a BMS fellowship. We thank Urvashi
and Deepika for FACS data, Saima and Santosh for confocal mi-
croscopy images, and Sudeeksh for help in the transient absorp-
tion studies. We thank Dr. R. Chakrabarti from CEaS, IISc, for the
ICP-MS data.
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Keywords: antitumor agents
BODIPY · platinum · singlet oxygen
· bioinorganic chemistry ·
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Received: June 24, 2016
Published online on && &&, 0000
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Light on target: Platinum(II) catecho-
lates with 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene (BODIPY)-appended
pyridylbenzimidazole ligands specifically
target mitochondria. The complexes ex-
hibit remarkable photocytotoxicity in
visible light in HaCaT cells by causing
apoptotic cell death through singlet
oxygen species, yet they are non-toxic
in the dark. The emisssive BODIPY com-
plex was used for cellular imaging,
whereas the diiodo-BODIPY analogue
showed remarkable photodynamic ther-
apy effects.
K. Mitra, S. Gautam, P. Kondaiah,*
A. R. Chakravarty*
&& – &&
BODIPY-Appended 2-(2-
Pyridyl)benzimidazole Platinum(II)
Catecholates for Mitochondria-
Targeted Photocytotoxicity
ChemMedChem 2016, 11, 1 – 13
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