Maloplatin-B, a Cisplatin-Based BODIPY-Tagged Mito-Specific chemo-PDT Agent Active in Red Light

Article abstract of DOI:10.1021/acs.inorgchem.1c00124

Maloplatin-B, a cisplatin-based complex, namely [Pt(A-BOD)(NH3)2](NO3) (Pt-A-BOD) with a pendant boron-dipyrromethene (BODIPY) moiety, where HA-BOD is a methyl malonyl chloride derived monostyryl BODIPY ligand, was designed and developed as near-IR light (600-720 nm) organelle-targeting photodynamic therapy agent. The complex [Pt(acac)(NH3)2](NO3) (Pt-Ac) was used as a control. Pt-A-BOD displayed an absorption band at 616 nm (? = 2.9 × 104 M-1 cm-1) in 10% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (DMSO/DMEM, pH 7.2). This complex displayed a broad emission band within 650-850 nm with a λem value of 720 nm in 10% DMSO-DMEM (pH 7.2) upon excitation (λex) at 615 nm with a large Stokes shift. The fluorescence quantum yield (φF) value for Pt-A-BOD is 0.032 and for the ligand HA-BOD is 0.24. The BODIPY complex and ligand showed the formation of singlet oxygen as the ROS (reactive oxygen species) on irradiation with near-IR red light of 660 nm, as evidenced from a 1,3-diphenylisobenzofuran (DPBF) assay. The complex displayed remarkable apoptotic NIR light-induced PDT activity with half-maximum inhibitory concentration values (IC50) of 1.6-2.4 μM in A549 lung and HeLa cervical cancer cells, while it was less active in the dark. The cellular ROS generation by the complex in red light was ascertained by a DCFDA (2′,7′-dichlorofluorescein diacetate) assay. Cellular imaging showed its localization primarily in the mitochondria of A549 cancer cells. The JC1 and Annexin-V FITC/PI assays carried out for A549 cancer cells treated with the BODIPY complex showed the alteration of mitochondrial membrane potential and apoptotic cell death on near-IR red light (600-720 nm) irradiation, respectively.

Full text of DOI:10.1021/acs.inorgchem.1c00124

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Maloplatin-B, a Cisplatin-Based BODIPY-Tagged Mito-Specific
“Chemo-PDT” Agent Active in Red Light
Vanitha Ramu, Paramita Kundu, Paturu Kondaiah,* and Akhil R. Chakravarty*
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ABSTRACT: Maloplatin-B, a cisplatin-based complex, namely
[Pt(A-BOD)(NH3)2](NO3) (Pt-A-BOD) with a pendant boron-
dipyrromethene (BODIPY) moiety, where HA-BOD is a methyl
malonyl chloride derived monostyryl BODIPY ligand, was designed
and developed as near-IR light (600−720 nm) organelle-targeting
photodynamic therapy agent. The complex [Pt(acac)(NH3)2](NO3)
(Pt-Ac) was used as a control. Pt-A-BOD displayed an absorption
band at 616 nm (ε = 2.9 × 104 M−1 cm−1) in 10% dimethyl sulfoxide/
Dulbecco’s Modified Eagle’s Medium (DMSO/DMEM, pH 7.2).
This complex displayed a broad emission band within 650−850 nm
with a λem value of 720 nm in 10% DMSO−DMEM (pH 7.2) upon
excitation (λex) at 615 nm with a large Stokes shift. The fluorescence
quantum yield (ΦF) value for Pt-A-BOD is 0.032 and for the ligand
HA-BOD is 0.24. The BODIPY complex and ligand showed the formation of singlet oxygen as the ROS (reactive oxygen species) on
irradiation with near-IR red light of 660 nm, as evidenced from a 1,3-diphenylisobenzofuran (DPBF) assay. The complex displayed
remarkable apoptotic NIR light-induced PDT activity with half-maximum inhibitory concentration values (IC50) of 1.6−2.4 μM in
A549 lung and HeLa cervical cancer cells, while it was less active in the dark. The cellular ROS generation by the complex in red
light was ascertained by a DCFDA (2′,7′-dichlorofluorescein diacetate) assay. Cellular imaging showed its localization primarily in
the mitochondria of A549 cancer cells. The JC1 and Annexin-V FITC/PI assays carried out for A549 cancer cells treated with the
BODIPY complex showed the alteration of mitochondrial membrane potential and apoptotic cell death on near-IR red light (600−
720 nm) irradiation, respectively.
drugs showed reduced therapeutic activity in comparison to
the bifunctional CP analogues.15,16
INTRODUCTION
■
The serendipitous discovery of cisplatin (CP) has led to the
foundation of the chemistry of metal-based anticancer agents,
and this has propelled the later discovery of two other
platinum-based drugs: namely, carboplatin and oxaliplatin.1−4
The impetus to search for new Pt-based drugs originates from
the major side effects associated with CP due to rapid
intracellular Pt−Cl bond dissociation kinetics thus necessitat-
ing the design and synthesis of new CP analogues having the
Pt−Cl bond replaced by other ligands, with the complex
generating the active platinum species at a slower rate in
comparison to CP, thus enhancing the efficacy of the drug.3,5−8
For example, oxaliplatin and carboplatin having O,O-donor
dianionic ligands release the active platinum species at a slower
rate in comparison to CP and display better anticancer
properties.5−8 A few other CP analogues such as nedaplatin,
heptaplatin, lobaplatin, aroplatin, spiroplatin, enloplatin,
zeniplatin, miboplatin, picoplatin, ormaplatin, iproplatin, etc.
have been reported to show promising activity to make them
suitable for the treatment of cancer.9−14 In a parallel
development, Lippard and co-workers have enriched the
chemistry of monofunctional platinum-based drugs, but such
Subsequently, to address the specificity and efficacy of CP
and its analogues, apparently inactive platinum(IV) complexes
have been developed and reported by researchers in recent
years.17−19 Two axial ligands in the pseudo-octahedral
geometry derived from the CP core have tumor-targeting
properties. These complexes are suitable for targeted chemo-
therapy, where the axial bonds can be released on cellular
reduction of Pt(IV) by thiols to Pt(II), thus generating active
platinum(II) species. In a different approach, Sadler and co-
workers have developed platinum(IV) complexes as prodrug
molecules which can generate the active chemotherapeutic
drug CP or its analogue on UV light irradiation.5 Photo-
activated chemotherapy (PACT) has been developed as an
Received: January 15, 2021
Published: April 12, 2021
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effective modality to selectively activate a drug in the cancer
cells by light irradiation while the noncancerous cellular
environment is protected.20−22 A simultaneous development
has occurred with the use of transition-metal complexes in the
photodynamic therapy (PDT) of cancer.23−25 PDT was
developed before to CP as an alternate therapeutic modality
different from chemotherapy and radiation therapy but got
FDA (Food and Drug Administration) approval after CP. In
the conventional PDT, a photosensitizer, namely Photofrin, in
the cancer cell is photoirradiated with red light in the presence
of molecular oxygen (3O2) to generate highly cytotoxic singlet
oxygen (1O2) that kills the photoexposed cancer cells, thus
leaving the unexposed healthy cells unaffected.25−27
A combination of PDT- and CP-based chemotherapy can be
achieved by designing a new generation of cisplatin-derived
platinum(II) complexes having a dissociable ligand tagged with
a photosensitizer as a “chemo-PDT” agent that can generate
singlet oxygen on irradiation using near-IR red light.28−30 The
light within the PDT spectral window has the advantage of the
highest penetration to reach the cancer cells in deep tissues for
better efficacy, and red-light-absorbing tissue chromophores
are generally absent in the human body. Although Photofrin is
the FDA-approved PDT drug, it suffers from selectivity, post-
treatment skin sensitivity, and hepatotoxicity.31−34 Reports
from our group have shown that boron-dipyrromethene-
appended transition-metal complexes can be used as photo-
sensitizers for generating singlet oxygen in high yield on light
activation.35−40 BODIPY dyes have good photostability, and
their core is amenable for tuning the absorption and emission
properties within a broad spectral window ranging from the
visible to the near-IR region.41,42 In addition, BODIPY dyes
can be tuned to localize into different cellular organelles, thus
specifically enhancing their therapeutic potential.43−46 We have
recently reported a diplatinum(II) catecholate, [{Pt-
(dach)}2(μ-Dcrb)] (DP), where dach is 1,2-diaminocyclohex-
ane and Dcrb is a tetraanionic morpholine-conjugated
BODIPY-linked dicatecholate base, as a lysosome-targeting
PDT agent in red light.47 This complex acts more as an
organelle-targeting PDT agent localizing in lysosomes than as a
chemotherapeutic agent with IC50 values of 0.6 μM in HeLa
cells in red light (600−720 nm) and 105 μM in the dark with a
remarkable PI (phototoxic index) value. The present work
stems from our interest in improving the model by using
{Pt(NH3)2}2+ of CP bonded to an O,O-donor chelating ligand
having an appended red-light-active BODIPY moiety. The
O,O-donor ligand derived from the methyl malonyl chloride
(methyl chloroformylacetate) framework is a suitable replace-
ment for oxalic or dicarboxylic acid in comparison to aromatic
catecholate base.48 Herein, we present a platinum(II) complex,
[Pt(A-BOD)(NH3)2](NO3) (Pt-A-BOD), having a red-light-
activatable BODIPY ligand that could release an active
platinum(II) species for chemotherapeutic action while it
initiates PDT activity involving the monostyryl BODIPY unit
in red light (Figure 1). The complex Pt-A-BOD as a “chemo-
PDT” agent has a cisplatin-like backbone structure with a
chelating methyl malonate tagged to a monostyryl BODIPY.
The use of a malonyl-based O,O-donor ligand in a platinum
complex is to ensure slow release of the active platinum species
{Pt(NH3)2}2+ that can possibly bind to DNA (nuclear and/or
mitochondrial) and act as a transcription inhibitor. Significant
results of this study include high PDT activity of the complex
in red light, giving submicromolar IC50 values, while being
essentially nontoxic in the dark, mitochondrial localization, a
Figure 1. Chemical structures of the ligand HA-BOD, [Pt(A-
BOD)(NH3)2](NO3) (Pt-A-BOD) as an active platinum(II)
complex, and [Pt(acac)(NH3)2](NO3) (Pt-Ac) as a control species.
high yield of singlet oxygen generation, and apoptotic cellular
death promoted by reactive oxygen species (ROS). In addition,
the ligand HA-BOD alone is significantly less toxic than the
complex, justifying the importance of coordination to the
heavy metal platinum that is needed to promote facile
intersystem crossing (ISC) in a type II process for the
activation of molecular oxygen. The complex Pt-A-BOD,
denoted Maloplatin-B, derived from a cisplatin framework
exemplifies a rare chemo-PDT agent due to its dual activity.
EXPERIMENTAL SECTION
■
Materials and Methods. All of the chemicals and reagents were
obtained from commercial sources (see the Supporting Information).
The ligand HA-BOD and complexes Pt-A-BOD and Pt-Ac were
prepared by following literature reports with some modifications.37,48
1H, 11B, and 13C NMR spectra were recorded using a Bruker Avance
400 MHz NMR spectrometer. An Agilent Model 6538 Ultra High
Definition Accurate Mass-Q-TOF (LC-HRMS) instrument was used
to obtain mass spectral data. A Thermo Finnigan Flash EA 1112
CHNS analyzer was employed for elemental analysis. The platinum
complex showed a marginally lower carbon percentage in the
elemental analysis possibly due to poor or insufficient combustion
of the sample under the standard conditions used. Bruker Alpha and
PerkinElmer Spectrum 750 spectrophotometers were used for
recording UV−visible spectra. A HORIBA Jobin Yvon IBH TCSPC
fluorimeter (fitted with FluoroHub analysis software) was used to
record emission spectra. The geometries of the platinum complexes
were optimized using the B3LYP/LANL2DZ level of theory. By
standard protocols, a red-light photoreactor (Waldmann PDT 1200
L) was used for the red-light experiments. Cytotoxic data were
obtained from a TECAN microplate reader by using GraphPad Prism
6 software. Flow cytometry experiments were done using a Becton
Dickinson fluorescence-activated cell sorting (BD-FACS) Verse
instrument (BD Biosciences). It was configured with a MoFLo
XDP cell sorter, three lasers of wavelengths 488, 365, and 640 nm,
and 10-color parameters. FACS data acquisition and analysis were
done using the Windows 7 operating system and BD-FACS suite
software. Confocal microscopy images were obtained from a Zeiss
LSM 880 with Airyscan confocal microscope. It had an oil immersion
lens with a magnification of 63×. The images were processed by using
Zeiss software.
Synthesis. Ligand HA-BOD, namely 4-(4-(3-((E)-4-
(dimethylamino)styryl)-5,5-difluoro-1,7,9-trimethyl-5H-4l4,5l4-
dipyrrolo[1,2-c:2′,1′-f ][1,3,2]diazaborinin-10-yl)phenyl)but-3-yn-1-yl
(E)-3-hydroxy-3-methoxyacrylate, and the platinum complexes,
namely Pt-A-BOD and Pt-Ac, were synthesized by following the
literature procedures.37,48,49 The steps involved in the synthesis of the
ligand HA-BOD and the complexes are provided as Schemes S1 and
S2 in the Supporting Information. The detailed procedures employed
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in the synthesis of the ligand and the complexes along with the
characterization data are given below.
in one set of the cells and washed with DPBS buffer, and the cells
were then subjected to red-light irradiation for 15 min (λ = 600−720
nm, light dose 30 J cm−2, Waldmann PDT 1200 L). Meanwhile, the
DMEM-buffer-containing compounds were replaced with fresh
DMEM from the other set of cells. After irradiation the PBS buffer
was replaced with fresh DMEM in the irradiated cells and both sets of
cells were further incubated for 16 h in dark. Data were obtained by
carrying out experiments with three independent sets. For each
concentration, the experiment was done in triplicate and the IC50
(half maximum inhibitory concentration) values were obtained by
using Graph Pad Prism 6 and employing a nonlinear regression
analysis. The DNA binding experiments were performed in Tris-HCl
buffer (5 mM, pH 7.2) using a DMF solution of the complex and calf
thymus DNA (complete experimental details are provided in the
Supporting Information).
For the confocal laser scanning microscopy (CLSM) experiments,
A549 cells were cultured in Dulbecco’s modified Eagle’s medium
supplemented with 10% FBS under a 5% CO2 atmosphere at 37 °C.
Approximately 1 × 105 cells were seeded onto 35 mm round glass
bottom dishes. After 24 h, A549 cells were incubated with the
compounds HA-BOD and Pt-A-BOD (2 μM) in 1% DMSO/DMEM
for 4 h. Before imaging, the cells were washed with DPBS and were
treated with Mitotracker Green (MTG, 200 nM, 15 min) and 4′,6-
diamidino-2-phenylindole (DAPI) as respective mitochondria- and
nucleus-selective trackers. The CLSM images were recorded in fresh
DMEM by maintaining a 5% CO2 atmosphere at 37 °C in the dark by
using a Zeiss LSM 880 with Airyscan microscope with an oil
immersion lens having a magnification of 63×. For the cells incubated
with the compounds HA-BOD and Pt-A-BOD, the CLSM images
were captured with a band path of 650−750 nm upon excitation at
633 nm. The fluorescence of Mito Tracker Green in A549 cells was
captured with a band path of 520−560 nm upon excitation at 508 nm,
and for DAPI, the emission detection band path was set to 420−460
nm with an excitation of 408 nm. To confirm the results, multiple
CLSM images were obtained and the experiments were carried out in
duplicate.
For the detection of singlet oxygen generation inside the cells, the
cells were treated with Pt-A-BOD (2 μM) in 1% DMSO/DMEM for
4 h. Further, the cells were rinsed with DPBS and were incubated with
singlet oxygen sensor green (SOSG) (10 μM, 30 min), washed with
DPBS before red-light irradiation (λ = 600−720 nm, light dose 30 J
cm−2) for the light case and subjected to CLSM for the dark set of
cells. For the determination of mitochondrial membrane potential
disruption in A549 cells, the cells were treated with Pt-A-BOD (2
μM) in 1% DMSO/DMEM for 4 h. Further, the cells were rinsed
with DPBS, incubated with JC1 (5,5,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimidazoylcarbocyanine iodide) dye (100 μM, 4 h),
washed with DPBS before red-light irradiation (λ = 600−720 nm,
light dose 30 J cm−2 in the case of light), and subjected to CLSM for
the dark set of cells. For an analysis of the extent of bleaching of the
complex Pt-A-BOD in A549 cells, the cells were treated with Pt-A-
BOD (2 μM) in 1% DMSO/DMEM for 4 h and subjected to CLSM
to record images at time intervals of 30 s for 20 min. The live cellular
uptake of the complex Pt-A-BOD (1 μM) in 1% DMSO/DMEM was
determined under a 5% CO2 atmosphere at 37 °C in the dark.
The flow cytometry analysis experiments were performed with
approximately 2 × 105 A549 cells seeded in six-well plates using
DMEM supplemented with 10% FBS maintained under a 5% CO2
atmosphere at 37 °C. The generation of reactive oxygen species
(ROS) inside A549 cells was detected by a 2′,7′-dichlorofluorescein
diacetate (DCFDA) assay. A549 cells were treated with the
compounds Pt-A-BOD and HA-BOD (2 μM) in 1% DMSO/
DMEM for 4 h in the dark. After incubation, the medium was
removed, the cells were harvested by trypsinization, and a single cell
suspension was prepared. The cells were subsequently treated with 1
μM DCFDA (solution prepared with DMSO) in the dark for 10 min
at room temperature. The distribution of DCFDA-stained A549 cells
was obtained by flow cytometry in the FL-1 channel by a BD-FACS
Verse instrument. Cellular uptake experiments were carried out for
the fluorescent compounds Pt-A-BOD and HA-BOD (2 μM) in 1%
HA-BOD. The precursor BODIPY compound mBOD-OL, namely
4-(4-(3-(4-(dimethylamino)styryl)-5,5-difluoro-1,7,9-
trimethyldipyrrolo[1,3,2]diazaborinin-10-yl)phenyl)but-3-yn-1-ol
(212 mg, 0.5 mmol, 1 equiv), was dissolved in 10 mL of freshly
distilled dichloromethane. Under an N2 atmosphere, pyridine (11 mg,
0.14 mmol) was added. After the resulting mixture was cooled in an
ice bath for 15 min, methyl malonyl chloride (17 mg, 0.13 mmol) in 5
mL of distilled dichloromethane was added dropwise. The resulting
mixture was further warmed to room temperature followed by
continuous stirring for 12 h. The reaction mixture was filtered and
extracted with dichloromethane. It was concentrated using a rotavap,
and the product was isolated by silica gel column chromatography
using dichloromethane/hexane as eluent in the ratio of 65/35 (v/v).
Blue solid (158 mg). Yield: ∼54%. Anal. Calcd for C36H36BF2N3O4
(Mw: 623.508): C, 69.35; H, 5.82; N, 6.74. Found: C, 68.92; H, 5.50;
N, 6.68. ESI-MS m/z: calcd, 624.2749; found [M + H]+, 624.2849.
1H NMR (400 MHz, DMSO-d6): δ (ppm) 7.57 (s, 2 H), 7.53 (s, 2
H), 7.32 (s, 1 H), 7.28 (s, 1 H), 7.24 (s, 1 H), 7.12 (s, 1 H), 6.72 (s, 1
H), 6.70 (s, 1 H), 6.62 (s, 1 H), 5.99 (s, 1 H), 5.04 (s, 1 H), 4.40 (t, 2
H, 4 Hz), 3.63 (s, 3 H), 3.05 (s, 6 H), 2.85 (t, 2 H, 4 Hz), 1.47 (s, 3
H), 1.44 (s, 6H) (s, singlet; d, doublet; t, triplet). 13C NMR (100
MHz, DMSO-d6): δ (ppm) 152.38, 139.73, 138.55, 135.67, 138.27,
132.71, 129.04, 125.07, 124.32, 123.95, 118.33, 116.35, 114.74,
112.48, 63.70, 53.04, 41.71, 35.19, 34.30, 32.10, 29.40, 23.16, 20.30,
15.06, 14.61. UV−vis (10% DMSO/DMEM at pH 7.2): λmax, nm (ε,
M
−1 cm−1) 614 (4.4 × 104). Emission spectrum (10% DMSO/DPBS
at pH 7.2): λem (λex, ΦF) 720 nm (615 nm, 0.24).
[Pt(A-BOD)(NH3)2](NO3), (Pt-A-BOD). cis-[Pt(NH3)2Cl2] (cis-
platin, 190 mg, 0.5 mmol, 1 equiv) was treated with AgNO3 (162 mg,
0.98 mmol) in 5 mL of distilled water and stirred at room temperature
for 24 h in the dark and then filtered twice to remove AgCl. The
yellowish filtrate was added to a solution of the ligand HA-BOD
(165.5 mg, 0.25 mmol, 1 equiv) and 1 mL of freshly distilled
triethylamine in a dropwise manner and stirred for 4 h in the dark at
room temperature in methanol (30 mL). Using a rotavap the
methanol solvent was removed and the solid was dissolved in DMF (2
mL). The desired complex was precipitated by adding excess diethyl
ether. To eliminate any unreacted starting materials, the precipitated
complex was repetitively treated with water and diethyl ether to
obtain the pure product.
Dark blue solid (172 mg). Yield: ∼54%. Anal. Calcd for
C36H41BF2N6O7Pt (Mw: 914.657): C, 47.27; H, 4.63; N, 9.29.
Found: C, 45.29, H, 4.44; N, 9.50. ESI-MS m/z: calcd, 852.2900;
1
found, 852.2616 [M − NO3]+. H NMR (400 MHz, DMSO-d6): δ
(ppm) 7.56 (s, 2 H), 7.49 (s, 1 H), 7.45 (s, 2 H), 7.37 (s, 1 H), 7.36
(s, 2 H), 7.24 (s, 1 H), 7.20 (s, 1 H), 6.91 (s, 1 H), 6.12 (s, 1 H), 5.30
(s, 1 H), 4.76 (s, 3 H), 4.66 (s, 3 H), 4.26 (s, 2 H), 3.16 (s, 3 H), 2.98
(s, 6 H), 2.81 (s, 2 H), 1.40 (s, 3H), 1.33 (s, 6 H). 13C NMR (100
MHz, DMSO-d6): δ (ppm) 167.43, 167.01, 143.18, 138.25, 132.56,
129.52, 129.15, 123.95, 123.85, 118.89, 112.98, 112.60, 63.22, 52.65,
46.24, 41.37, 31.70, 29.39, 22.52, 14.86, 14.39, 9.03. UV−vis (10%
DMSO/DMEM at pH 7.2): λmax, nm (ε, M−1 cm−1) 616 (2.9 × 104).
Emission spectrum (10% DMSO/DPBS at pH 7.2): λem (λex, ΦF) 720
nm (615 nm, 0.032). ΛM in DMF: 76 S m2 mol−1.
Cellular Experiments. HeLa (human cervical cancer cell line),
A549 (human lung adenocarcinoma cell line), and HPL1D
(immortalized human lung epithelial cell line) were cultured in
Dulbecco’s modified Eagle’s medium (DMEM) supplemented with
10% fetal bovine serum (FBS) under a 5% CO2 atmosphere at 37 °C.
A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay was carried out to determine the photocytotoxicities
of the complexes in red light. Approximately 8000 cells of HeLa,
A549, and HPL1D were plated separately in two different 96-well
culture plates. After ∼24 h, the cells were treated with various
concentrations of the compounds, namely HA-BOD, Pt-A-BOD, and
Pt-Ac, from 0.195 to 100 μM in 1% DMSO/DMEM and the cells
were further incubated for 4 h under a 5% CO2 atmosphere at 37 °C
in the dark. The DMEM-buffer-containing compounds were removed
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DMSO/DMEM in A549 cells. The distribution of the compounds in
stained cells was observed in the APC channel. The mode of cell
death induced by the compounds in A549 cells was determined by an
AnnexinV-FITC (fluorescein isothiocyanate) experiment. Two sets of
cells were treated with both compounds (2 μM) in 1% DMSO/
DMEM for 4 h. One of the plates was exposed to red-light
photoirradiation (λ = 600−720 nm, light dose 30 J cm−2) in DPBS
with subsequent addition of fresh media. The cells were further
incubated for 19 h, trypsinized, and washed twice with DPBS. The
cells were resuspended in 400 μL of 1X binding buffer, and 1.0 μL of
Annexin V-FITC and 1.0 μL of PI (propidium iodide) were added to
each cell suspension. These tubes were then incubated at room
temperature for 10 min in the dark and were further subjected to a
FACS analysis.
Figure 2. (a) Electronic absorption and (b) emission (λex = 615 nm)
spectra of the complexes Pt-Ac (nonemissive) and Pt-A-BOD and the
ligand HA-BOD in 10% DMSO−DMEM (pH 7.2).
RESULTS AND DISCUSSION
■
A-BOD in more polar solvents was observed, suggesting a
weak charge transfer character (Figure S9 in the Supporting
Information). Such high values of the absorption coefficients
for the absorption bands within the PDT spectral window are
conducive for a desirable photosensitizing effect. The presence
of platinum as a heavy metal in Pt-A-BOD makes this complex
suitable as a photosensitizer for PDT activity. The control
complex Pt-Ac, lacking any visible band, is not a PDT-active
species. Both Pt-A-BOD and HA-BOD upon excitation at 615
nm displayed a broad emission band within 650−850 nm with
a maximum at 720 nm in pH 7.2 10% DMSO−DMEM (Figure
2b). A partial charge transfer effect, observed in BODIPY
moieties having dimethylamino groups as styryl units, resulted
in large Stokes shifts in the emission spectra of Pt-A-BOD and
HA-BOD. The complex Pt-A-BOD gave a fluorescence
quantum yield (ΦF) value of 0.032. This is much lower in
comparison to the ΦF value of the ligand (ΦF = 0.24). This
reduction is attributed to the presence of platinum(II) bonded
to the ligand facilitating ISC. The ligand and complex both
generated singlet oxygen under red-light irradiation with a
singlet oxygen quantum yield (ΦΔ) value of 0.19 for the ligand
and 0.48 for the complex. Time- and temperature-dependent
UV−visible measurements were also done to ascertain the
stability of the BODIPY complex and its ligand in 10%
DMSO/DMEM, 1% DMSO/DMEM, and also DMSO alone
for a time period of up to 48 h in dark. The temperature-
dependent UV−visible absorption spectra of Pt-A-BOD kept
at 4 and 37 °C for 4 h displayed no significant change in
absorbance from that kept at room temperature. The results
displayed a very negligible decrease in the absorbance of Pt-A-
BOD in 10% DMSO/DMEM and DMSO, but a small
decrease was observed in 1% DMSO/DMEM (Figure S10 in
the Supporting Information). Both the complex and ligand
were thus found to be stable.
Synthesis and General Properties. The ligand HA-BOD
and its platinum(II) complex [Pt(A-BOD) (NH3)2](NO3)
(Pt-A-BOD), as a cisplatin analogue, were synthesized as
stable species in moderate yields. The compounds were
characterized with NMR and mass spectral data (Figures S1−
S8 in the Supporting Information). The purity of the
1
compounds was ascertained from the H NMR spectral and
elemental analysis data. The complex Pt-A-BOD displayed a
peak in the mass spectrum at m/z 852.2616 in methanol along
with the characteristic isotopic distribution of platinum (Figure
1
S2 in the Supporting Information). The H NMR spectra of
the complex Pt-A-BOD and ligand HA-BOD displayed peaks
characteristic of monosubstituted styryl BODIPY (Figures S3
and S4 in the Supporting Information). The characteristic
1
peaks for the ammine protons were observed in the H NMR
spectra of the complex Pt-A-BOD within the range 4.6−4.8
ppm, and the aromatic protons were shifted downfield from
6.6−7.6 ppm to 6.9−7.6 ppm, which indicated the formation
of the platinum(II) complex. The 13C NMR spectra of both
the complex and ligand showed peaks corresponding to the
carbon atoms in the monosubstituted styryl BODIPY (Figures
S5 and S6 in the Supporting Information). The 11B NMR
spectra of both the complex and ligand displayed respective
characteristic triplet peaks at 0.84 and 0.927 ppm correspond-
ing to a BF2 unit in a monosubstituted styryl boron-
dipyrromethene (BODIPY) moiety (Figures S7 and S8 in
the Supporting Information). The solution conductivity
measurements gave a molar conductivity value of ∼76 S m2
M−1 in dimethylformamide, suggesting a 1/1 electrolytic
behavior of the complex. Both the complex and ligand are fairly
soluble in solvents such as as chloroform, dichloromethane,
methanol, dimethylformamide (DMF), and dimethyl sulfoxide
(DMSO).
The UV−visible spectroscopic study was done in pH 7.2
10% DMSO/DMEM buffer (v/v). HA-BOD and Pt-A-BOD
displayed absorption bands at 614 nm (ε = 4.4 × 104 M−1
cm−1) and 617 nm (ε = 2.9 × 104 M−1 cm−1), respectively
(Figure 2a). The lowest energy absorption bands were
assigned to electronic transitions involving a monostyryl
BODIPY unit having a dimethylamino group; the attachment
of two 4-(dimethylamino)phenylethynyl moieties results in the
shift in the absorption and emission bands toward the red
region from the core BODIPY. The UV−visible absorption
spectrum of Pt-A-BOD was recorded in methanol and
acetonitrile solvents, and the extent of the shift was analogous
with that observed for 4-(dimethylamino)phenylethynyl
BODIPY analogues (Figures S9 and S10 in the Supporting
Information). The broadening of the absorption spectra of Pt-
The energy -optimized structure of the complex Pt-A-BOD
was obtained by density functional theory (DFT) by using the
B3LYP/LANL2DZ level of theory (Figure 3 and Tables S1
and S2 in the Supporting Information).50,51 For the atoms, the
electronic charge density distributions pertaining to the
frontier molecular orbitals were obtained through DFT. The
energy-minimized structure of Pt-A-BOD showed a square-
planar geometry with platinum(II) bonded to two ammine
groups in a cis disposition and the O,O-donor methyl
malonate moiety of the monoanionic A-BOD ligand bonded
to the metal in a chelating mode. The HOMO (highest
occupied molecular orbital) and LUMO (lowest unoccupied
molecular orbital) are shown in Figure 3. The HOMO of the
complex Pt-A-BOD was found to be on the BODIPY-
appended methyl malonate unit, while the LUMO was
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Figure 3. Energy-minimized structure (a) and the FMOs, namely the HOMO (b) and LUMO (c), of the ligand HA-BOD and complex Pt-A-BOD
using density functional theory.
localized on the Pt−O bonds. Both the HOMO and LUMO of
the ligand HA-BOD were localized on monostyryl BODIPY.
The DFT data were useful toward an understanding of the
spectral properties of the compounds.
Photocytotoxicity in Red Light. The complex Pt-A-
BOD, ligand HA-BOD, and control Pt-Ac were studied by an
MTT assay for their photoinduced cytotoxicity in red light
(600−720 nm) with a 4 h preincubation of the compounds in
lung cancer cells (A549), an immortalized lung epithelial cell
line (HPL1D), and human cervical cancer cells (HeLa)
followed by 20 h postincubation. The results of the MTT assay
in the form of IC50 values are given in Table 1 (Figures S11
μM in the cancer cells with low dark cytotoxicity (IC50 = 79−
85 μM). The ligand HA-BOD in comparison showed low
cytotoxicity in the cancer cells in red light (λ = 600−720 nm,
light dose 30 J cm−2), giving IC50 values within 40−52 μM,
possibly due to lower singlet oxygen production. The
chemotherapeutic activity of the complex Pt-A-BOD, assumed
to be occurring due to the presence of the active “cisplatin”
unit, was determined from an MTT assay, and the IC50 value
was 44 μM for 24 h of incubation with the complex. The IC50
value of the ligand HA-BOD was >100 μM for 24 h incubation
in the dark. The possibility of any interaction of the complex
Pt-A-BOD with DNA was studied by UV−visible absorption
spectral studies using calf thymus (ct) DNA. The ct-DNA
binding constant (Kb) for Pt-A-BOD was found to be ∼3.9 ×
106 M−1 in 5% DMF-Tris buffer (pH 7.2), implying that the
complex interacts with DNA through groove binding and/or
an intercalative mode (Figure S13 in the Supporting
Information).52 The results indicate the utility of platinum in
Pt-A-BOD for singlet oxygen generation and the remarkable
PDT effect along with a DNA binding interaction with the
possibility of inducing a chemotherapeutic effect. The complex
Pt-A-BOD and ligand HA-BOD both showed lower
cytotoxicity in immortalized human lung epithelial HPL1D
cells in the dark and with light (red light, λ = 600−720 nm,
light dose 30 J cm−2).
Cellular Uptake. The mode of cellular uptake and the
time-dependent cellular internalization ability of a drug being
of importance, the time-dependent cellular uptake of the
complex was analyzed from a fluorescence-activated cell
sorting (FACS) analysis in A549 cells. The cells were
incubated with the fluorescent compounds Pt-A-BOD and
HA-BOD (2 μM) for different time periods of incubation:
namely, 2, 4, and 6 h. The cellular fluorescence of the complex
and ligand was detected through the APC channel (λex = 633
nm). There was a large shift in the histogram of both types of
treated cells in comparison to the histogram of untreated cells,
clearly indicating the cellular uptake of these compounds. For
the complex Pt-A-BOD, the fluorescence slightly increased as
the time of incubation increased from 2 to 4 h but there was no
significant difference in the fluorescence emission between 4
Table 1. MTT Assay Data (IC50/μM) of the Complexes Pt-
Ac and Pt-A-BOD and the Ligand HA-BOD
cell line
HeLa
conditions
Pt-Ac
>100
>100
>100
>100
72.0 0.2
92.1 1.1
>100
Pt-A-BOD
HA-BOD
a
L
2.4 0.2
79.0 3.2
1.6 0.2
85.0 0.4
44.0 0.4
33.4 0.4
101.0 1.2
40.0 0.2
102.0 3.2
52.0 0.2
97.0 0.4
>100
b
D
a
A549
L
b
D
c
D
a
HPL1D
L
63.0 0.6
>100
b
D
a
IC50 values are in μM with 4 h preincubation in dark with
subsequent to red light exposure (L, 600−720 nm, light dose of 30 J
cm−2 using Waldmann PDT 1200 L). Post incubation period was of
b
c
19 h. IC50 values are in μM for samples in the dark. On 24 h
incubation under dark.
and S12 in the Supporting Information). The values for the
complex Pt-Ac in red light (λ = 600−720 nm, light dose 30 J
cm−2) and in the dark for an incubation time of 24 h were
above 100 μM in these cells. In contrast, the same complex
displayed noticeable dark toxicity with an IC50 value of 72 μM
for 24 h of incubation in A549 cells, indicating its
chemotherapeutic action due to the presence of the platinum-
(II) center (Figure S11 in the Supporting Information). The
complex Pt-A-BOD having a monostyryl BODIPY moiety
displayed remarkable near-IR light (λ = 600−720 nm, light
dose 30 J cm−2) photocytotoxicity with IC50 values of 1.6−2.4
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and 6 h (Figure 4a). This indicated the maximum uptake of Pt-
A-BOD (2 μM) at 2 h. However, for the ligand, the
to confocal laser scanning microscopy (CLSM) following
standard protocols. Cells treated with both the complex and
ligand showed almost no fluorescence emission, indicating no
apparent cellular uptake on incubation at 4 °C, whereas
fluorescence emission was observed on incubation at 37 °C.
The results suggest that the complex and ligand follow an
energy-dependent pathway of cellular uptake (Figure 4b).
Further, even after a prolonged incubation of the complex and
ligand for 4 h, no increase in emission intensity was found in
the treated cells incubated at 4 °C (Figure S14 in the
Supporting Information).
The red fluorescence emission property at 720 nm from the
complex Pt-A-BOD in 1% DMSO/DMEM enabled us to carry
out real-time imaging in live cells. This property is useful to
understand the cellular uptake mechanism of a drug in live cells
by recording pictures constantly at different time intervals or at
no time intervals. The cellular entry of the complex Pt-A-BOD
(1 μM) in 1% DMSO/DMEM was visualized in A549 cells
through live cell imaging by maintaining a 5% CO2 atmosphere
at 37 °C. Images were recorded immediately after treatment
with the complex Pt-A-BOD (1 μM) with no time interval and
a band path of 660−750 nm by using λex = 633 nm. The entry
of the complex was observed within 100 s of incubation from
fluorescence emission and complete localization, and the
maximum uptake was reached 750 s after the treatment.
Further, the cells did not show any major increase in
fluorescence or varied localization (Figure S15 in the
Supporting Information). The CLSM images also indicated
punctate fluorescence, suggesting that the uptake pathway is
possibly through endocytosis. The results indicated a rapid
cellular uptake of the BODIPY complex in A549 cells,
suggesting a high cell membrane permeability of the complex.
Singlet Oxygen as a Cytotoxic Agent. The objective of
appending a monostyryl BODIPY moiety on {Pt(NH3)2}2+
coordinated to a O,O-donor ligand was to generate reactive
oxygen species (ROS) on red-light irradiation. The complex
Figure 4. (a) Quantitative analysis of cellular uptake of the
compounds by flow cytometry upon incubating A549 cells with Pt-
A-BOD (2 μM) at 37 °C at different time intervals along with
untreated cells used as a control. (b) Confocal images of HeLa (left
panels) and A549 (right panels) cells incubated with Pt-A-BOD (2
μM) at two different temperatures, namely 37 and 4 °C, for 4 h. Scale
bar: 20 μm.
fluorescence gradually increased with increasing time from 2
to 6 h. To determine the uptake pathway, A549 cells were
treated with the complex and ligand (2 μM) in 1% DMSO/
DMEM at the two temperatures 37 and 4 °C. The compounds
were incubated for two time periods of 2 and 4 h and subjected
Figure 5. (a) Cellular ROS generation by a DCFDA assay in Pt-A-BOD (2 μM, 4 h)-treated A549 cells under red light of 600−720 nm (L, light
dose 30 J cm−2) and in the dark (D). (b) Spectral variations of DPBF at 417 nm on treatment with the ligand HA-BOD and the complex Pt-A-
BOD in DMF at 5 s intervals under red light irradiation (λ = 600−720 nm). (c) Cellular detection of 1O2 generated by Pt-A-BOD (2 μM, 4h) in
A549 cells in red light of 600−720 nm (light dose 30 J cm−2) and in the dark by an SOS analysis kit (SOS is a singlet oxygen sensor).
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Figure 6. Confocal images of A549 cells incubated with red-emitting Pt-A-BOD (2 μM) for 4 h at 37 °C along with Mito Tracker Green (MTG).
DAPI (4′,6-diamidino-2-phenylindole) was used as a blue -emitting nuclear staining dye. Scale bar: 20 μm. Lower panels are the expanded region of
the highlighted portion (see box) of the top panels.
Pt-A-BOD and ligand HA-BOD were developed as novel NIR-
PDT photosensitizers for therapeutic applications. To
determine the formation of any reactive oxygen species
under red-light photoirradiation (600−720 nm) in a cellular
environment, a DCFDA (2′,7′-dichlorofluorescein diacetate)
assay in A549 cells was carried out. DCFDA is a non-
fluorescent compound that has the ability to penetrate the cell
membrane and react with any ROS to form green fluorescent
2′,7′-dichlorodihydrofluorescein with a λem value of 525 nm
(λex = 488 nm) upon oxidation. Hence, any generation of ROS
can be determined using a flow cytometry analysis by
measuring the green fluorescence of 2′,7′-DCF in the FL-1
channel, which would indicate the cell population generating
ROS. Two sets of A549 cells were treated with the complex Pt-
A-BOD and the ligand HA-BOD at their IC50 concentrations
(Pt-A-BOD, 2 μM; HA-BOD, 40 μM) for 4 h followed by
irradiation with red light (λ = 600−720 nm, light dose 30 J
cm−2) for one set and samples kept in the dark for the other
set. Both sets of cells were further incubated with DCFDA (1
μM, 10 min) and subjected to a FACS analysis. The cells
treated with only DCFDA and the cells treated with the
complex Pt-A-BOD but kept in the dark showed only a small
shift in the histogram. In contrast, Pt-A-BOD-treated cells on
irradiation with red light displayed a large shift in fluorescence,
indicating the ability of Pt-A-BOD to generate cytotoxic
reactive oxygen species (Figure 5a). A similar observation was
made for the cells treated with HA-BOD at its IC50
concentration (Figure S16 in the Supporting Information).
BODIPY dyes as photosensitizers are well-known to generate
singlet oxygen as the ROS upon photoexcitation via a type II
process in which the photosensitizer in its triplet state transfers
energy to triplet oxygen to generate singlet oxygen. To
ascertain the nature of ROS generated from Pt-A-BOD and
HA-BOD in red light, a 1,3-diphenylisobenzofuran (DPBF)
assay was done in DMF. The absorption spectral intensity of
DPBF as a singlet oxygen scavenger in DMF in the presence of
the complex and ligand at 417 nm was examined by employing
a 660 nm continuous-wave diode laser as the excitation source.
At intervals of 5 s, a gradual decrease in the absorbance of
DPBF treated with the complex Pt-A-BOD at 417 nm was
observed in the UV−visible spectral assay. A similar experi-
ment with the ligand HA-BOD with 10 s intervals of light
irradiation did not show any significant changes. The results
indicate much a higher efficacy of singlet oxygen generation by
the complex in comparison to the ligand (Figure 5b and Figure
S16 in the Supporting Information). The singlet oxygen
generation ability of the complex Pt-A-BOD inside the cellular
medium was determined in A549 cells using a singlet oxygen
specific dye: namely, singlet oxygen sensor green (SOSG).
SOSG is a dye that displays weak blue fluorescence inside the
cellular medium, but in the presence of singlet oxygen the
fluorescence emission shifts from blue to green (λem = 525 nm,
λex = 488 nm). Two sets of A549 cells were incubated with the
complex Pt-A-BOD (2 μM) for 4 h followed by treatment with
SOSG dye (100 μM) for 20 min. After the removal of the
buffer containing SOSG, one set of cells was subjected to
irradiation using red light (λ = 600−720 nm, light dose 30 J
cm−2), while the other set was kept in the dark. The cells were
subjected to CLSM. The complex-treated cells without red
light irradiation displayed a weak blue fluorescence inside the
cells, but no fluorescence was observed in the green region
(Figure 5c). In contrast, the cells treated with the complex and
irradiated under red light (λ = 600−720 nm, light dose = 30 J
cm−2) displayed bright green fluorescence in the green channel
but no blue fluorescence. The results unequivocally showed
the cellular generation of singlet oxygen by the complex Pt-A-
BOD in red light.
Mitochondrial Localization. The appended BODIPY
moiety enabled us to employ complex Pt-A-BOD for cellular
imaging as a NIR fluorophore that would inhibit the
intervention of background signals occurring due to tissue
autofluorescence. To determine the target organelle of the
complex (2 μM), A549 cells were incubated for 2 h at 37 °C in
the dark and were later subjected to confocal laser scanning
microscopy. The cells retained good morphology after 24 h of
incubation, and the CLSM images of the complex-incubated
cells were captured with a band path of 650−750 nm upon
excitation at 633 nm. A study on the localization of the
compounds using their fluorescence along with trackers,
namely Mito Tracker Green (MTG) and the nucleus-specific
dye 4′,6-diamidino-2-phenylindole (DAPI), was carried out for
A549 cells. The CLSM merged images of the complex and
DAPI clearly indicated its localization in the cytoplasm rather
than the nucleus. The red fluorescence emission caused by the
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Figure 7. AnnexinV-FITC and propidium iodide (PI) staining of A549 cells that undergo apoptosis/necrosis caused by the compounds Pt-A-BOD
(2 μM) and HA-BOD (40 μM) in red light of 600−720 nm (L, light dose 30 J cm−2) and in the dark (D) by FACS analysis. The percent cell
population can be viewed in the respective quadrants: lower left for live cells, lower right for early apoptotic cells, upper right for late apoptotic cells,
and upper left for dead cells.
complex merged with the green fluorescence of MTG, showing
a strong overlapping with an overlap coefficient value of 93%.
This indicated significant localization of the complex in the
mitochondria of A549 lung cancer cells (Figure 6 and Figure
S17 in the Supporting Information). Similar results were
obtained with the merged images of the ligand HA-BOD and
the trackers MTG and DAPI, with a clear indication that the
complex Pt-A-BOD is localized in the mitochondria due to the
presence of the coordinated ligand A-BOD (Figure S18 inthe
Supporting Information). The mitochondrial localization
achieved by both the ligand and the complex can be attributed
as well to the presence of N,N-dimethylaniline as a monostyryl
unit in the BODIPY ligand HA-BOD. For a fluorophore to be
employed for cellular imaging, the bleaching of the dye should
be at a minimum with negligible effects on the cellular
morphology. A549 cells were incubated with the complex Pt-
A-BOD (2 μM) for 2 h and then subjected to CLSM. A frame
of the red fluorescence image of the complex was fixed, images
were recorded constantly at time intervals of 1 min for a period
of 20 min. With no signs of morphological changes in the cells,
we observed a minor decrease in the fluorescence intensity
from the first recorded image to the last image, clearly
indicating negligible bleaching of complex Pt-A-BOD as a dye
inside the live cellular medium under 5% CO2 at 37 °C (Figure
S19 in the Supporting Information). This property of the
complex can be effectively employed for the intracellular
imaging and tracking of a drug to gain complete insight into
the mechanism of drug action.
stabilized to a minimum level. The results showed that
∼74% of the cells in the Pt-A-BOD-treated set of cells in light
(L) were in an early apoptotic phase and ∼15% of the cells
were in a late apoptotic phase with no cells being found to have
a necrotic death pathway (Figure 7). For the Pt-A-BOD-
treated set of cells in the dark (D), ∼17% of the cells were in
an early apoptotic phase, while ∼8.3% of the cells were in the
late apoptosis phase and ∼4% cells in the necrotic death
pathway. In contrast, for HA-BOD-treated set of cells in the
light (L), ∼1% of the cells were in an early apoptosis phase,
∼21% of the cells were in a late apoptotic phase, and
remarkably ∼47% of the cells were in a necrotic death pathway
(Figure 7). There was not much change in the results of the
ligand HA-BOD-treated set of cells in the dark (D) in
comparison to the set of cells that were untreated. The results
clearly suggest apoptotic cellular death induced by complex Pt-
A-BOD in light, while the pathway is predominantly necrotic
for the ligand HA-BOD.
Mitochondrial Membrane Potential. Complex Pt-A-
BOD in A549 cells induced an apoptotic pathway of cell death
on irradiation with red light. The confocal imaging results
indicated specific mitochondrial localization. To further
determine the possible damage that would occur in the
mitochondria of A549 cells treated with this complex, a JC1
assay was performed to study the mitochondrial membrane
integrity. Two sets of A549 cells were treated with the complex
(2 μM) at its IC50 value and incubated for 4 h in the dark. One
set of the cells after removal of the complex was kept in the
dark (D), while the other set of cells was irradiated with red
light of 600−720 nm (L, light dose 30 J cm−2). JC1 dye is
known to show red fluorescence inside the cells when the
mitochondrial membrane potential remains intact. The dye,
however, displays green fluorescence when the mitochondrial
membrane potential is altered. The complex-treated set of cells
in the light (L) exhibited bright green fluorescence of the JC1
dye with negligible red fluorescence (Figure 8). However, the
complex-treated set of cells kept in the dark (D) showed red
fluorescence of the JC1 dye with negligible green fluorescence,
Cellular Apoptosis. The cellular death pathway induced
by the complex Pt-A-BOD and the ligand HA-BOD under
red-light photoirradiation was determined by an AannexinV-
FITC/PI assay in A549 cells. Two sets of A549 cells were
treated with the complex (2 μM) and the ligand (40 μM) at
their IC50 values and incubated for 4 h in the dark. One set of
cells after removal of the compounds was kept in the dark (D),
while the other set of cells was irradiated with red light of
600−720 nm (L, light dose 30 J cm−2). With the untreated
cells, the FITC fluorescence and PI fluorescence were
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namely, near-IR-light cellular imaging and PDT activity. This
work exemplifies a rare cisplatin analogue having monostyryl
BODIPY as a near-IR-light PDT agent localizing in
mitochondria and causing cellular apoptosis on singlet oxygen
generation.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.inorgchem.1c00124.
■
sı
Reaction schemes, ESI-mass, NMR, and IR spectra,
stability plots, MTT assay, cellular uptake, DCFDA
assay, confocal images, Cartesian coordinates, and bond
distances/angles of the ligand HA-BOD and complex
Pt-A-BOD (PDF)
AUTHOR INFORMATION
Corresponding Authors
■
Paturu Kondaiah − Department of Molecular Reproduction,
Development and Genetics, Indian Institute of Science,
Bangalore 560012, India; Phone: +91-80-22932688;
Email: paturu@iisc.ac.in; Fax: +91-80-23600999
Akhil R. Chakravarty − Department of Inorganic and Physical
Chemistry, Indian Institute of Science, Bangalore 560012,
India; orcid.org/0000-0002-6471-9167; Phone: +91-
80-22932533; Email: arc@iisc.ac.in; Fax: +91-80-
23600683
Figure 8. Confocal microscopic images of the JC-1 assay showing the
fluorescence images of JC-1 dye in both red and green channels in
A549 cells incubated with the complex Pt-A-BOD (2 μM) for 4 h at
37 °C in the dark and on red-light photoirradiation (λ = 600−720
nm; light dose 30 J cm−2). The scale bar is 20 μm in all of the panels.
clearly indicating that the complex did not alter the
mitochondrial membrane potential in the dark but altered it
on irradiation with red light.
Authors
Vanitha Ramu − Department of Inorganic and Physical
Chemistry, Indian Institute of Science, Bangalore 560012,
India; orcid.org/0000-0003-4438-1071
Paramita Kundu − Department of Molecular Reproduction,
Development and Genetics, Indian Institute of Science,
Bangalore 560012, India; orcid.org/0000-0003-4538-
4573
CONCLUSIONS
■
A platinum(II) complex, [Pt(A-BOD)(NH3)2](NO3) (Pt-A-
BOD, Maloplatin-B), as a cisplatin analogue having mono-
anionic O,O-donor methyl malonyl moiety-conjugated to
monostyryl BODIPY ligand (HA-BOD) was designed,
synthesized, and exemplified as an efficient near-IR red-light-
active photodynamic therapy (PDT) agent against cancer cells,
while it is dormant against immortalized human lung epithelial
cells. The non-BODIPY complex [Pt(acac)(NH3)2](NO3)
(Pt-Ac) was used as a control, which showed no significant
activity in the dark or light. The complex Pt-A-BOD showed
rapid cellular uptake in lung cancer A549 cells through an
energy-dependent pathway. It predominantly localized in the
mitochondria of the cancer cells and produced singlet oxygen
(1O2) as the ROS, targeting this specific organelle under NIR
light irradiation (600−720 nm). In HeLa and A549 cancer
cells, the BODIPY complex induced remarkable photo-
cytotoxicity in the PDT spectral window of 650−850 nm
with IC50 values within 1.6−2.4 μM while being effectively
nontoxic in dark. The PDT activity is based on the high-yield
generation of singlet oxygen inside the cells on red-light
irradiation. The ligand HA-BOD, due to its lower singlet
oxygen quantum yield and lower cellular uptake in comparison
to its complex, gave relatively higher IC50 values, thus
indicating its reduced photocytotoxicity. The ligand followed
a necrotic pathway of cell death, while the complex primarily
showed the desirable apoptotic pathway. The complex caused
significant changes in the mitochondrial membrane potential,
leading to apoptotic cell death in A549 cancer cells. It showed
higher stability with lower bleaching properties, thus making it
a potential therapeutic agent suitable for dual application:
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.inorgchem.1c00124
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
A.R.C. thanks the Department of Science and Technology
(DST), Government of India, New Delhi, for financial support
(SR/S5/MBD-02/2007, EMR/2015/000742, CRG/2018/
000081) and for a J. C. Bose national fellowship (SR/S2/
JCB-26/2007). P.K.’s research group is funded by DBT-IISc
grants. We are grateful to Ms. Leepika for help in recording
FACS data and Ms. Saima and Ms. Divya for help in acquiring
confocal microscopic images from the Division of Biological
Sciences, IISc Bangalore.
REFERENCES
■
(1) Alderden, R. A.; Hall, M. D.; Hambley, T. W. The Discovery and
Development of Cisplatin. J. Chem. Educ. 2006, 83, 728−734.
(2) Wilson, J. J.; Lippard, S. J. Synthetic Methods for the Preparation
of Platinum Anticancer Complexes. Chem. Rev. 2014, 114, 4470−
4495.
(3) Lebwohl, D.; Canetta, R. Clinical Development of Platinum
Complexes in Cancer Therapy: An Historical Perspective and an
Update. Eur. J. Cancer 1998, 34, 1522−1534.
6418
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Inorg. Chem. 2021, 60, 6410−6420

Inorganic Chemistry
pubs.acs.org/IC
Article
(4) Wang, X.; Wang, X.; Guo, Z. Functionalization of Platinum
Complexes for Biomedical Applications. Acc. Chem. Res. 2015, 48,
2622−2631.
(5) Johnstone, T. C.; Suntharalingam, K.; Lippard, S. J. The Next
Generation of Platinum Drugs: Targeted Pt(II) Agents, Nanoparticle
Delivery, and Pt(IV) Prodrugs. Chem. Rev. 2016, 116, 3436−3486.
(6) Kelland, L. The Resurgence of Platinum-Based Cancer
Chemotherapy. Nat. Rev. Cancer 2007, 7, 573−584.
(7) Alcindor, T.; Beauger, N. Oxaliplatin: A Review in the Era of
Molecularly Targeted Therapy. Curr. Oncol. 2011, 18, 18−25.
(8) Pavelka, M.; Lucas, M. F. A.; Russo, N. On the Hydrolysis
Mechanism of the Second-Generation Anticancer Drug Carboplatin.
Chem. - Eur. J. 2007, 13, 10108−10116.
(9) (a) Ndagi, U.; Mhlongo, N.; Soliman, M. E. Metal Complexes in
Cancer Therapy − an Update from Drug Design Perspective. Drug
Des., Dev. Ther. 2017, 11, 599−616. (b) Farrell, N. P. Multi-Platinum
Anti-Cancer Agents. Substitution-Inert Compounds for Tumor
Selectivity and New Targets. Chem. Soc. Rev. 2015, 44, 8773−8785.
(10) Choi, C. H.; Cha, Y. J.; An, C. S.; Kim, K. J.; Kim, K. C.; Moon,
S. P.; Lee, Z. H.; Min, Y. D. Molecular Mechanisms of Heptaplatin
Effective against Cisplatin-Resistant Cancer Cell Lines: Less
Involvement of Metallothfionein. Cancer Cell Int. 2004, 4, 6.
(11) Zhou, J.; Kang, Y.; Chen, L.; Wang, H.; Liu, J.; Zeng, S.; Yu, L.
The Drug-Resistance Mechanisms of Five Platinum-Based Antitumor
Agents. Front. Pharmacol. 2020, 11, 1−17.
(12) Shah, N.; Dizon, D. S. New-Generation Platinum Agents for
Solid Tumors. Future Oncol. 2009, 5, 33−42.
(13) Wheate, N. J.; Walker, S.; Craig, G. E.; Oun, R. The Status of
Platinum Anticancer Drugs in the Clinic and in Clinical Trials. Dalton
Trans. 2010, 39, 8113−8127.
(19) Zhao, J.; Hua, W.; Xu, G.; Gou, S. Biotinylated Platinum(IV)
Complexes Designed to Target Cancer Cells. J. Inorg. Biochem. 2017,
176, 175−180.
(20) Bonnet, S. Why Develop Photoactivated Chemotherapy?
Dalton Trans. 2018, 47, 10330−10343.
(21) Farrer, N. J.; Salassa, L.; Sadler, P. J. Photoactivated
Chemotherapy (PACT): The Potential of Excited-State d-Block
Metals in Medicine. Dalton Trans. 2009, 44, 10690−10701.
(22) Shi, H.; Imberti, C.; Sadler, P. J. Diazido Platinum(IV)
Complexes for Photoactivated Anticancer Chemotherapy. Inorg.
Chem. Front. 2019, 6, 1623−1638.
(23) Dąbrowski, J. M.; Arnaut, L. G. Photodynamic Therapy (PDT)
of Cancer: From Local to Systemic Treatment. Photochem. Photobiol.
Sci. 2015, 14, 1765−1780.
(24) Castano, A. P.; Mroz, P.; Hamblin, M. R. Photodynamic
Therapy and Anti-Tumour Immunity. Nat. Rev. Cancer 2006, 6, 535−
545.
(25) Doherty, R. E.; Sazanovich, I. V.; McKenzie, L. K.; Stasheuski,
A. S.; Coyle, R.; Baggaley, E.; Bottomley, S.; Weinstein, J. A.; Bryant,
H. E. Photodynamic Killing of Cancer Cells by a Platinum(II)
Complex with Cyclometallating Ligand. Sci. Rep. 2016, 6, 1−9.
(26) Raza, M. K.; Gautam, S.; Howlader, P.; Bhattacharyya, A.;
Kondaiah, P.; Chakravarty, A. R. Pyriplatin-Boron-Dipyrromethene
Conjugates for Imaging and Mitochondria-Targeted Photodynamic
Therapy. Inorg. Chem. 2018, 57, 14374−14385.
(27) Heinemann, F.; Karges, J.; Gasser, G. Critical Overview of the
Use of Ru(II) Polypyridyl Complexes as Photosensitizers in One-
Photon and Two-Photon Photodynamic Therapy. Acc. Chem. Res.
2017, 50, 2727−2736.
(28) Mao, J.; Zhang, Y.; Zhu, J.; Zhang, C.; Guo, Z. Molecular
Combo of Photodynamic Therapeutic Agent Silicon(Iv) Phthalocya-
nine and Anticancer Drug Cisplatin. Chem. Commun. 2009, 8, 908−
910.
(14) Zhong, L. Z.; Xu, H. Y.; Zhao, Z. M.; Zhang, G. M.; Lin, F. W.
Comparison of Efficacy and Toxicity between Nedaplatin and
Cisplatin in Treating Malignant Pleural Effusion. OncoTargets Ther.
2018, 11, 5509−5512.
(29) (a) Mitra, K.; Lyons, C. E.; Hartman, M. C. T. A Platinum(II)
Complex of Heptamethine Cyanine for Photoenhanced Cytotoxicity
and Cellular Imaging in Near-IR Light. Angew. Chem., Int. Ed. 2018,
57, 10263−10267. (b) Naik, A.; Rubbiani, R.; Gasser, G.; Spingler, B.
Visible-Light-Induced Annihilation of Tumor Cells with Platinum-
Porphyrin Conjugates. Angew. Chem., Int. Ed. 2014, 53, 6938−6941.
(30) Raza, M. K.; Gautam, S.; Garai, A.; Mitra, K.; Kondaiah, P.;
Chakravarty, A. R. Monofunctional BODIPY-Appended Imidazopla-
tin for Cellular Imaging and Mitochondria-Targeted Photocytotox-
icity. Inorg. Chem. 2017, 56, 11019−11029.
(15) (a) Imran, M.; Ayub, W.; Butler, I. S.; Zia-Ur-Rehman.
Photoactivated Platinum-Based Anticancer Drugs. Coord. Chem. Rev.
2018, 376, 405−429. (b) Imran, M.; Zia-Ur-Rehman; Kondratyuk,
T.; Belanger-Gariepy, F. New Ternary Platinum(II) Dithiocarba-
mates: Synthesis, Characterization, Anticancer, DNA Binding and
DNA Denaturing Studies. Inorg. Chem. Commun. 2019, 103, 12−20.
(c) Kashif Amir, M.; Hogarth, G.; Khan, Z.; Imran, M.; Zia-ur-
Rehman. Inorg. Chim. Acta 2020, 512, 119853−119863. (d) Lovejoy,
K. S.; Serova, M.; Bieche, I.; Emami, S.; D'Incalci, M.; Broggini, M.;
Erba, E.; Gespach, C.; Cvitkovic, E.; Faivre, S.; Raymond, E.; Lippard,
S. J. Anticancer Activity of Pyriplatin, A Monofunctional Cationic
Platinum(II) Compound, in Human Cancer Cells. Mol. Cancer Ther.
2011, 10, 1709−1719.
(16) Park, G. Y.; Wilson, J. J.; Song, Y.; Lippard, S. J.
Phenanthriplatin, a Monofunctional DNA-Binding Platinum Anti-
cancer Drug Candidate with Unusual Potency and Cellular Activity
Profile. Proc. Natl. Acad. Sci. U. S. A. 2012, 109, 11987−11992.
(17) (a) Hall, M. D.; Mellor, H. R.; Callaghan, R.; Hambley, T. W.
Basis for design and development of platinum(IV) anticancer
complexes. J. Med. Chem. 2007, 50, 3403−3411. (b) Raynaud, F. I.;
Boxall, F. E.; Goddard, P.; Barnard, C. F.; Murrer, B. A.; Kelland, L. R.
Metabolism, Protein Binding and In Vivo Activity of the Oral
Platinum Drug JM216 and its Biotransformation Products. Anticancer
Res. 1996, 16, 1857−1862. (c) Gibson, D. Platinum(IV) Anticancer
Prodrugs-Hypotheses and Facts. Dalton Trans. 2016, 45, 12983−
12991. (d) Guo, Z.; Sadler, P. J. Metals in Medicine. Angew. Chem.,
Int. Ed. 1999, 38, 1512−1531.
(31) Kou, J.; Dou, D.; Yang, L. Porphyrin Photosensitizers in
Photodynamic Therapy and Its Applications. Oncotarget 2017, 8,
81591−81603.
(32) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. The Role of
Porphyrin Chemistry in Tumor Imaging and Photodynamic Therapy.
Chem. Soc. Rev. 2011, 40, 340−362.
(33) Sharman, W. M.; Allen, C. M.; Van Lier, J. E. Photodynamic
Therapeutics: Basic Principles and Clinical Applications. Drug
Discovery Today 1999, 4, 507−517.
(34) Zhu, S.; Yao, S.; Wu, F.; Jiang, L.; Wong, K. L.; Zhou, J.; Wang,
K. Platinated Porphyrin as a New Organelle and Nucleus Dual-
Targeted Photosensitizer for Photodynamic Therapy. Org. Biomol.
Chem. 2017, 15, 5764−5771.
(35) Mitra, K.; Gautam, S.; Kondaiah, P.; Chakravarty, A. R.
BODIPY-Appended 2-(2-Pyridyl)Benzimidazole Platinum(II) Cat-
echolates for Mitochondria-Targeted Photocytotoxicity. ChemMed-
Chem 2016, 11, 1956−1967.
(36) Paul, S.; Kundu, P.; Bhattacharyya, U.; Garai, A.; Maji, R. C.;
Kondaiah, P.; Chakravarty, A. R. Ruthenium(II) Conjugates of
Boron-Dipyrromethene and Biotin for Targeted Photodynamic
Therapy in Red Light. Inorg. Chem. 2020, 59, 913−924.
(37) Ramu, V.; Gautam, S.; Garai, A.; Kondaiah, P.; Chakravarty, A.
R. Glucose-Appended Platinum(II)-BODIPY Conjugates for Tar-
geted Photodynamic Therapy in Red Light. Inorg. Chem. 2018, 57,
1717−1726.
(18) (a) Chin, C. F.; Wong, D. Y. Q.; Jothibasu, R.; Ang, W. H.
Anticancer platinum(IV) prodrugs with novel modes of activity. Curr.
Top. Med. Chem. 2011, 11, 2602−2612. (b) Ma, J.; Wang, Q.; Huang,
Z.; Yang, X.; Nie, Q.; Hao, W.; Wang, P. G.; Wang, X. Glycosylated
Platinum(IV) Complexes as Substrates for Glucose Transporters
(GLUTs) and Organic Cation Transporters (OCTs) Exhibited
Cancer Targeting and Human Serum Albumin Binding Properties
for Drug Delivery. J. Med. Chem. 2017, 60, 5736−5748.
6419
https://doi.org/10.1021/acs.inorgchem.1c00124
Inorg. Chem. 2021, 60, 6410−6420

Inorganic Chemistry
pubs.acs.org/IC
Article
(38) Garai, A.; Gandhi, A.; Ramu, V.; Raza, M. K.; Kondaiah, P.;
Chakravarty, A. R. Photochemotherapy of Infrared Active BODIPY-
Appended Iron(III) Catecholates for in Vivo Tumor Growth
Inhibition. ACS Omega 2018, 3, 9333−9338.
(39) Bhattacharyya, A.; Dixit, A.; Mitra, K.; Banerjee, S.; Karande, A.
A.; Chakravarty, A. R. BODIPY Appended Copper(II) Complexes of
Curcumin Showing Mitochondria Targeted Remarkable Photo-
cytotoxicity in Visible Light. MedChemComm 2015, 6, 846−851.
(40) Mukherjee, N.; Podder, S.; Banerjee, S.; Majumdar, S.; Nandi,
D.; Chakravarty, A. R. Targeted Photocytotoxicity by Copper(II)
Complexes Having Vitamin B6and Photoactive Acridine Moieties.
Eur. J. Med. Chem. 2016, 122, 497−509.
(52) (a) Paitandi, R. P.; Mukhopadhyay, S.; Singh, R. S.; Sharma, V.;
Mobin, S. M.; Pandey, D. S. Anticancer Activity of Iridium(III)
Complexes Based on a Pyrazole-Appended Quinoline-Based
BODIPY. Inorg. Chem. 2017, 56, 12232−12247. (b) Amir, M. K.;
Khan, S. Z.; Hayat, F.; Hassan, A.; Butler, I. S.; Zia-Ur-Rehman.
Anticancer Activity, DNA-Binding and DNA-Denaturing Aptitude of
Palladium(II) Dithiocarbamates. Inorg. Chim. Acta 2016, 451, 31−40.
(41) (a) Liu, Y.; Li, Z.; Chen, L.; Xie, Z. Near Infrared BODIPY-
Platinum Conjugates for Imaging, Photodynamic Therapy and
Chemotherapy. Dyes Pigm. 2017, 141, 5−12. (b) Loudet, A.;
Burgess, K. BODIPY Dyes and Their Derivatives: Syntheses and
Spectroscopic Properties. Chem. Rev. 2007, 107, 4891−4932.
(42) Chen, K.; Dong, Y.; Zhao, X.; Imran, M.; Tang, G.; Zhao, J.;
Liu, Q. Bodipy Derivatives as Triplet Photosensitizers and the Related
Intersystem Crossing Mechanisms. Front. Chem. 2019, 7, 1−14.
(43) Turksoy, A.; Yildiz, D.; Akkaya, E. U. Photosensitization and
Controlled Photosensitization with BODIPY Dyes. Coord. Chem. Rev.
2019, 379, 47−64.
(44) Kowada, T.; Maeda, H.; Kikuchi, K. BODIPY-Based Probes for
the Fluorescence Imaging of Biomolecules in Living Cells. Chem. Soc.
Rev. 2015, 44, 4953−4972.
(45) Kue, C. S.; Ng, S. Y.; Voon, S. H.; Kamkaew, A.; Chung, L. Y.;
Kiew, L. V.; Lee, H. B. Recent Strategies to Improve Boron
Dipyrromethene (BODIPY) for Photodynamic Cancer Therapy: An
Updated Review. Photochem. Photobiol. Sci. 2018, 17, 1691−1708.
(46) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.;
Burgess, K. BODIPY Dyes in Photodynamic Therapy. Chem. Soc. Rev.
2013, 42, 77−88.
(47) Ramu, V.; Gautam, S.; Kondaiah, P.; Chakravarty, A. R.
Diplatinum(II) Catecholate of Photoactive Boron-Dipyrromethene
for Lysosome-Targeted Photodynamic Therapy in Red Light. Inorg.
Chem. 2019, 58, 9067−9075.
(48) (a) Stacey, O. J.; Ward, B. D.; Coles, S. J.; Horton, P. N.; Pope,
S. J. A. Chromophore-Labelled, Luminescent Platinum Complexes:
Syntheses, Structures, and Spectroscopic Properties. Dalton Trans.
̈
2016, 45, 10297−10307. (b) Unlu, H.; Okutan, E. Preparation of
̈
BODIPY- Fullerene and Monostyryl BODIPY-Fullerene Dyads as
Heavy Atom Free Singlet Oxygen Generators. Dyes Pigm. 2017, 142,
340−349.
(49) Mitra, K.; Gautam, S.; Kondaiah, P.; Chakravarty, A. R. The
Cis-Diammineplatinum(II) Complex of Curcumin: A Dual Action
DNA Crosslinking and Photochemotherapeutic Agent. Angew. Chem.,
Int. Ed. 2015, 54, 13989−13993.
(50) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.;
Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci,
B.; Petersson, G. A.; Nakatsuji, H.; Caricato, M.; Li, H. P.; Hratchian,
A. F.; Izmaylov, J.; Bloino, G.; Zheng, J. L.; Sonnenberg, M.; Hada, M.
E.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.;
Honda, Y.; Kitao, O.; Nakai, H.; Vreven, T.; Montgomery, J. A., Jr.;
Peralta, J. E.; Ogliaro, F.; Bearpark, M.; Heyd, J. J.; Brothers, E.;
Kudin, K. N.; Staroverov, V. N.; Kobayashi, R.; Normand, J.;
Raghavachari, K.; Rendell, A.; Burant, J. C.; Iyengar, S. S.; Tomasi, J.;
Cossi, M.; Rega, N.; Millam, J. M.; Klene, M.; Knox, J. E.; Cross, J. B.;
Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.;
Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.;
Martin, R. L.; Morokuma, K.; Zakrzewski, V. G.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Dapprich, S.; Daniels, A. D.; Farkas,
̈
O.; Foresman, J. B.; Ortiz, J. V.; Cioslowski, J.; Fox, D. J. Gaussian 09,
Rev. A.1; Gaussian, Inc.: Wallingford, CT, 2009.
(51) (a) Becke, A. D. Density-Functional Exchange-Energy
Approximation with Correct Asymptotic Behavior. Phys. Rev. A: At.,
Mol., Opt. Phys. 1988, 38, 3098−3100. (b) Becke, A. D. Density-
Functional Thermochemistry. III. The Role of Exact Exchange. J.
Chem. Phys. 1993, 98, 5648.
6420
https://doi.org/10.1021/acs.inorgchem.1c00124
Inorg. Chem. 2021, 60, 6410−6420