Iron(III) complexes of a pyridoxal Schiff base for enhanced cellular uptake with selectivity and remarkable photocytotoxicity

Article abstract of DOI:10.1021/ic5027625

Iron(III) complexes of pyridoxal (vitamin B6, VB6) or salicylaldehyde Schiff bases and modified dipicolylamines, namely, [Fe(B)(L)](NO₃) (1-5), where B is phenyl-N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1), (anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine (pybpa in 3, 5) (H₂L¹ is 3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-methylpyridine (1-3) and H₂L² is 2-[(2-hydroxyphenyl-imino)methyl]phenol), were prepared and their uptake in cancer cells and photocytotoxicity were studied. Complexes 4 and 5, having a non-pyridoxal Schiff base, were prepared to probe the role of the pyridoxal group in tumor targeting and cellular uptake. The PF₆ salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate FeN₄O₂ core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible Fe(III)/Fe(II) response near -0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in various cancer cells with IC₅₀ values ranging from 0.4 to 5 μM with 10-fold lower dark toxicity. The cell death proceeded by the apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display 3-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5 are distributed throughout the cells without any specific localization pattern.

Full text of DOI:10.1021/ic5027625

Article
pubs.acs.org/IC
Iron(III) Complexes of a Pyridoxal Schiff Base for Enhanced Cellular
Uptake with Selectivity and Remarkable Photocytotoxicity
Uttara Basu, Ila Pant, Akhtar Hussain, Paturu Kondaiah,* and Akhil R. Chakravarty*
†
‡
†
,‡
,†
†
‡
Department of Inorganic and Physical Chemistry and Department of Molecular Reproduction, Development and Genetics, Indian
Institute of Science, Bangalore 560012, Karnataka, India
*
S Supporting Information
ABSTRACT: Iron(III) complexes of pyridoxal (vitamin B6,
VB6) or salicylaldehyde Schiff bases and modified dipicolyl-
amines, namely, [Fe(B)(L)](NO ) (1−5), where B is phenyl-
3
N,N-bis((pyridin-2-yl)methyl)methanamine (phbpa in 1),
(
(
anthracen-9-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine
anbpa in 2, 4) and (pyren-1-yl)-N,N-bis((pyridin-2-yl)-
1
methyl)methanamine (pybpa in 3, 5) (H L is 3-hydroxy-5-
2
(
hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-
2
methylpyridine (1−3) and H L is 2-[(2-hydroxyphenyl-
2
imino)methyl]phenol), were prepared and their uptake in
cancer cells and photocytotoxicity were studied. Complexes 4
and 5, having a non-pyridoxal Schiff base, were prepared to
probe the role of the pyridoxal group in tumor targeting and
cellular uptake. The PF salt (1a) of complex 1 is structurally characterized. The complexes have a distorted six-coordinate
6
FeN O core where the metal is in the +3 oxidation state with five unpaired electrons. The complexes display a ligand to metal
4
2
charge transfer band near 520 and 420 nm from phenolate to the iron(III) center. The photophysical properties of the complexes
are explained from the time dependent density functional theory calculations. The redox active complexes show a quasi-reversible
Fe(III)/Fe(II) response near −0.3 V vs saturated calomel electrode. Complexes 2 and 3 exhibit remarkable photocytotoxicity in
various cancer cells with IC values ranging from 0.4 to 5 μM with 10-fold lower dark toxicity. The cell death proceeded by the
5
0
apoptotic pathway due to generation of reactive oxygen species upon light exposure. The nonvitamin complexes 4 and 5 display
-fold lower photocytotoxicity compared to their VB6 analogues, possibly due to preferential and faster uptake of the vitamin
3
complexes in the cancer cells. Complexes 2 and 3 show significant uptake in the endoplasmic reticulum, while complexes 4 and 5
are distributed throughout the cells without any specific localization pattern.
INTRODUCTION
(PDT) of cancer, is also known for its therapeutic action in red
light of 633 nm involving disruption of the mitochondria.
■
12−17
A novel strategy to design and develop new generation of
chemotherapeutic or photo-chemotherapeutic agents is to
direct them to other cellular organelles instead of the nucleus
in order to circumvent the drawbacks associated with nuclear
Sadler et al. discussed the disruption of mitochondrial function
as an attractive target for anticancer drug design in a recent
18
article.
1
−5
While targeting mitochondria provides a novel pathway for
apoptotic cell death, another equally useful strategy could be to
target the endoplasmic reticulum (ER) and induce stress in the
DNA targeting anticancer drugs.
The resistance of cisplatin
or its analogues can be partially explained by the extensive
repair of the cisplatin-DNA and related adducts by the
nucleotide excision repair (NER) pathway which reduces the
19−21
ER of the cells which is associated with protein folding.
6
,7
The ER stress which can occur as a consequence of exogenous
or endogenous effects of an anticancer agent could disrupt the
folding capacity of the ER. Severe or prolonged stress could
result in toxic signals that cause intrinsic apoptosis through ER
efficacy of the drug. Gasser et al. have explored the biological
effects of ruthenium complexes of 2-pyridyl-2-pyrimidine-4-
carboxylic acid showing excellent anti-proliferative property in
various cell lines with predominant localization in the
mitochondria, disrupting the mitochondrial membrane poten-
2
2,23
stress response (ERSR).
Cytotoxicity arising from ERSR
8
involving the reactive oxygen species (ROS) formed on
photoactivation of the photosensitizer at specific sites of cancer
cells inducing tumor damage is a novel way to target cancer
cells. Development of such tumor specific delivery agents to the
tial in HeLa cells. Neamati and co-workers have reported a
selective mitochondria targeting chlorambucil with remarkable
cytotoxicity in breast and pancreatic cancer cells. Dhar et al.
recently reported a mitochondria targeting cisplatin analogue,
namely, Platin-M that disrupts the mitochondrial genome
instead of its conventional target, the nuclear DNA.
9
10,11
The
Received: November 17, 2014
FDA approved drug Photofrin, used in photodynamic therapy
©
XXXX American Chemical Society
A
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Inorganic Chemistry
Article
ER could improve the efficacy of an anticancer agent by
distinguishing normal cells from the tumor cells. PDT which
has evolved as a minimally invasive treatment procedure for
cancer relies on the production of ROS upon photoactivation
Chart 1. Schematic Representation of the Ternary Iron(III)
Complexes 1−5
24−26
of a photosensitizer (PS).
The PS on activation with light
of suitable wavelength goes to its singlet excited state from
where it may reach a low lying triplet excited state by
intersystem crossing (ISC). This is followed by the energy
1
transfer to molecular oxygen to form singlet oxygen ( O ) via
2
type-II process. The alternate pathway is an electron transfer
•
•−
reaction to produce hydroxyl (HO ) and/or superoxide (O2
)
radicals which is called the photoredox pathway. These
cytotoxic ROS are responsible for cell death. Thus, the delivery
vehicle should consist of a tumor targeting group appended to
ROS generator for the desired localized photocytotoxicity.
We have recently reported ternary iron(III) complexes
having photosensitizing moieties showing significant photo-
27
cytotoxicity in red light. These complexes, however, show
significant nuclear localization and target the nuclear DNA for
their cytotoxic activity. With an objective to target other cellular
organelle, we have chosen vitamin B6 (VB6) in our ligand
design to prepare new iron(III) complexes with the knowledge
of ER targeted photocytotoxicity by an oxovanadium(IV)
of the metal from the complex in a biological medium. The
N,N,N-donor tridentate dipicolylamine base is functionalized
with pendant aromatic groups like phenyl (1), anthracenyl (2,
4
) and pyrenyl (3, 5) for better photocytotoxic activity.
Complex 1 without a photoactive group is used as a control.
The anthracenyl and pyrenyl groups as fluorescent tags are used
to trace the molecule inside the cells from an imaging study.
The photocytotoxicity of the complexes, their differential
uptake properties inside the cells, and their cellular localization
are investigated. The notable results of this work include
remarkable photocytotoxicity in visible light (400−700 nm) of
the VB6 derived complexes and apoptotic cell death via
generation of ROS in cancer cells by predominantly affecting
the ER as compared to the nonvitamin complexes.
2
8
complex of VB6. VB6 is a biologically important molecule
which plays a major role in various metabolic, physiological,
29,30
and developmental processes.
It is an important cofactor in
various cellular processes like DNA biosynthesis for cell growth
and proliferation. Besides, VB6 plays important role as a
protective factor in various cancers, and VB6 derivatives
decrease cell proliferation and angiogenesis and it is implicated
31
in reducing tumorigenesis. Though the molecular mechanism
of VB6 remains elusive, it is known that most of VB6 is
transported to the liver and taken up by facilitated diffusion
through specific VB6 transporting membrane carriers (VTC)
EXPERIMENTAL SECTION
Materials and Methods. The chemicals were procured from
various commercial sources (s.d. Fine Chemicals, India; Sigma-Aldrich,
U.S.A.) and used without further purification. The solvents were dried
or distilled using standard procedures. 2′,7′-Dichlorofluorescein
diacetate (DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide (MTT), propidium iodide (PI), Dulbecco’s modified
eagle medium (DMEM), Dulbecco’s phosphate buffered saline
■
3
2−35
into the cells.
Serine hydroxymethyltransferase (SHMT) is
an enzyme with a profound role in DNA biosynthesis which
depends on VB6. The activity of this enzyme increases during
DNA duplication in the proliferating tumor cells as a result of
which the consumption of VB6 is also increased. Thus, to meet
the increased VB6 requirement in the cancer cells, it is
absorbed from the neighboring cells. Since tumor cells have a
higher demand for VB6, the VB6 conjugated molecules can
accomplish improved entry into tumor cells. As VB6 or its
conjugates are known to show higher cellular uptake inside the
cancer cells, the iron(III) complex of the Schiff base ligand
derived of this molecule is likely to increase the cellular uptake
3
7
(
DPBS), Annexin-V/PI kit and fetal bovine serum (FBS) were
obtained from Sigma (U.S.A.). ER tracker green was procured from
Invitrogen U.S.A.
The elemental analysis was conducted with a Thermo Finnigan
Flash EA 1112 CHNS analyzer. The infrared and electronic spectra
were obtained using Bruker Alpha and PerkinElmer Spectrum 650
spectrophotometers, respectively. Emission spectra were recorded
using a PerkinElmer LS 55 spectrophotometer. Molar conductivities of
the complexes were measured with a Control Dynamics (India)
conductivity meter. Electrochemical studies were performed using
EG&G PAR model 253 VersaStat potentiostat/galvanostat with
electrochemical analysis software 270 and a three electrode setup. A
glassy carbon electrode was used as the working electrode, a platinum
wire was used as an auxiliary electrode, and a saturated calomel
electrode (SCE) served the purpose of a reference electrode.
Tetrabutylammonium perchlorate (TBAP, 0.1 M), used as a
supporting electrolyte, was prepared from dropwise addition of
36
and selectivity of the complex.
Combining the aforementioned strategies, we have synthe-
sized new ternary iron(III) complexes, namely, [Fe(B)(L)]-
(
NO ) (1−5) of dipicolylamine bases (B: phbpa, anbpa,
3
pybpa) and tridentate Schiff base ligands (H L) derived from
2
1
pyridoxal (VB6, H L in complexes 1−3) or salicylaldehyde
2
2
(H L in 4, 5) (Chart 1). The pyridoxal moiety is anticipated to
2
facilitate the diffusion of the complexes inside the cells ensuring
faster cellular uptake and hence better cytotoxicity. To
substantiate our hypothesis, control complexes 4 and 5 having
a Schiff base ligand of salicylaldehyde are prepared, and their
various biological properties are studied and compared. The
Schiff base ligands having phenolic groups are envisaged to bind
strongly to iron(III) to furnish an intense charge transfer (CT)
band in visible light which is used for inducing photo-
cytotoxicity. The tridentate ligands are also expected to stabilize
iron in its +3 oxidation state and to avoid any possible leaching
perchloric acid (HClO ) to tetrabutylammonium bromide in water
4
with vigorous stirring followed by filtration of the white solid and
thorough washing with water to remove the acid. Care has to be taken
during production and handling of TBAP as contact with combustible
material may cause fire. It should not be heated (Caution!).
Electrospray ionization mass spectrometry (ESI-MS) data were
obtained using an Agilent 6538 Ultra high definition (UHD) accurate
1
Mass-Q-TOF (LC-HRMS) instrument. H NMR spectral measure-
ments were done with a Bruker 400 MHz NMR spectrometer.
B
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Article
2
Fluorescence assorted cell sorting experiments were done using FACS
Verse instrument (BD Biosciences). Magnetic susceptibility measure-
ments of the complexes at 300 K were done with solid samples using
MPMS SQUID VSM (Quantum Design, USA). Confocal microscopy
images were acquired using Leica microscope (TCS, SP5) with oil
immersion lens having magnification of 63×.
[Fe(anbpa)(L )](NO ) (4). Yield, ∼74% (0.53 g). Anal. Calcd for
3
C H FeN O (Mw: 718.56): C, 66.86; H, 4.49; N, 9.75. Found: C,
66.71; H, 4.41; N 9.83. ESI-MS in MeOH (m/z): 656.2350
[M−NO ] . Molar conductance in DMF (Λ ): 70 S m M . FT-
IR: 3052 w, 2350 w, 1597 s, 1527 w, 1470 m, 1380 s, 1295 vs, 1145 w,
4
0
32
5
5
+
2
−1
3
M
1020 m, 830 m, 745 s, 600 w, 530 w, 470 w. UV−visible in DMF
−
1
−1
Synthesis of the Ligands. The dipicolylamine derivatives were
[λ /nm (ε/M cm )]: 501 (2140, sh), 408 (8710), 389 (10680),
max
38
synthesized following reported procedures. The Schiff base ligands
were synthesized by refluxing an ethanol solution of pyridoxal
hydrochloride (neutralized with 1 equiv of triethylamine) or
369 (8280), 350 (8280). μ (298 K) = 5.85 μB.
eff
2
[Fe(pybpa)(L )](NO ) (5). Yield, ∼71% (0.52 g). Anal. Calcd for
3
C H FeN O (Mw: 742.58): C, 67.93; H, 4.34; N, 9.43. Found: C,
4
2
32
5
5
salicylaldehyde and 2-aminophenol for 1−3 h (Scheme S1, Supporting
67.78; H, 4.26; N 9.51. ESI-MS in MeOH (m/z): 680.1948
[M−NO ] . Molar conductance in DMF (Λ ): 73 S m M . FT-
IR: 3052 w, 2350 w, 1597 s, 1530 w, 1470 m, 1380 s, 1295 vs, 1150 w,
1
+
2
−1
Information). To prepare the VB6 Schiff base (H L ), pyridoxal
2
3
M
hydrochloride (0.21 g, 1 mmol) was dissolved in dry ethanol (10 mL)
and was neutralized with triethylamine (0.10 g, 1 mmol). 2-
Aminophenol (0.11 g, 1 mmol) also dissolved in dry ethanol (5
mL) was added to the reaction mixture and was refluxed for 3 h. The
orange precipitate was filtered, washed thoroughly with ether, and
1
[
3
025 w, 845 s, 760 vs, 605 w, 540 w, 480 w. UV−visible in DMF
−1 −1
λ
/nm (ε/M cm )]: 507 (2760, sh), 415 (16590), 345 (33390),
max
30 (27790), 315 (21040), 300 (19240). μeff (298 K) = 5.81 μB.
X-ray Crystallographic Procedures. The crystal structure of
complex 1 was obtained by single crystal X-ray diffraction method as
its PF salt (1a). The slow evaporation of a methanol−acetonitrile (3:2
v/v) solution of the complex resulted in the formation of dark red
color crystals. Details of the crystallographic procedures are given as
Supporting Information. The CCDC deposition number is 1027849.
Theoretical Study. The geometries of the complexes 1−3 were
optimized by density functional theory (DFT) using B3LYP level of
crystallized from ethanol to give yellow orange crystals (yield: ∼90%).
2
The other Schiff base (H L ) was prepared by reacting salicylaldehyde
2
6
(
0.12 g, 1 mmol) in dry ethanol (5 mL) with 2-aminophenol (0.11 g, 1
mmol) also in dry ethanol (5 mL) on stirring at room temperature for
h. The orange precipitate was filtered and washed with ether. It was
crystallized from ethanol to give bright orange crystals (yield: ∼95%).
1
38
1
1
Characterization data for H L : H NMR (DMSO-d , 400 MHz) δ
2
6
(
ppm): 14.702 (s, 1H), 10.011 (s, 1H), 9.234 (s, 1H), 7.953 (s, 1H),
theory and LanL2DZ basis set as implemented in Gaussian 09
7
6
.480 (m, 1H), 7.482−7.458 (m, 1H), 7.224−7.182 (m, 1H), 7.022−
39,40
program.
The initial coordinates were obtained from the crystal
.999 (m, 1H), 6.946−6.905 (m, 1H) 5.408 (t, J = 5.2, 1H), 4.764 (d, J
structure of 1a which was used for the optimization of the structures of
the complexes. The electronic transitions with their transition
probability were obtained using linear response time dependent
density functional theory (TDDFT). The coordinates of the energy
minimized structures and selected transitions in the visible region are
listed in Tables S1 and S2 (Supporting Information).
Cellular Measurements. The photocytotoxicity of the complexes
was studied using an MTT assay by following experimental procedures
=
5.6, 2H), 2.542 (s, 3H) (s, singlet; t, triplet; m, multiplet; J, coupling
+
constant). ESI-MS (m/z): 259.1082 [M + H] . Characterization data
2
1
for H L : H NMR (DMSO-d , 400 MHz) δ (ppm): 13.787 (s, 1H),
41
2
6
9
2
.747 (s, 1H), 8.959 (s, 1H), 7.619−7.596 (m, 1H), 7.404−7.343 (m,
H), 7.146−7.104 (m, 1H), 6.966−6.857 (m, 4H), 3.374 (s, 3H).
1
2
Synthesis of [Fe(phbpa/anbpa/pybpa)(L /L )](NO ) (1−5).
3
The complexes were synthesized by a general procedure in which
Fe(NO ) ·9H O (0.41 g, 1 mmol) was dissolved in methanol (5 mL)
38
3
3
2
that have been reported earlier (see Supporting Information).
to which was added the dipicolylamine base [phbpa for 1, anbpa for 2
and 4, and pybpa for 3 and 5, 1 mmol] also dissolved in methanol (5−
Cellular incorporation experiments were done to estimate the cellular
uptake of the complexes 2−5 by flow cytometry in HeLa and HaCaT
cells. Details of the experimental procedures are given in the
1
0 mL) (Scheme S2, Supporting Information). The reaction mixture
1
was stirred for 1 h. The Schiff base ligand H L (for 1−3, 0.26 g, 1
mmol) or H L (for 4 and 5, 0.21 g, 1 mmol) was dissolved in
38
2
2
Supporting Information. The experiment was repeated with cells
2
that were pretreated with pyridoxal hydrochloride (2 mM) 4 h prior to
treatment with the complexes. Dose-dependent estimation of cellular
uptake study was done to see the effect of varying concentrations of
methanol (6 mL) and treated with 2 equiv of triethylamine (0.20 g, 2
mmol). The solution having a dianionic Schiff base was added to the
reaction mixture and stirred at room temperature for 3 h. The brown
precipitate was isolated by filtration followed by thorough washing
with diethyl ether and drying in a vacuum over P O .
6
VB6 on the cellular uptake using flow cytometry. About 1 × 10 HeLa
cells were plated per well of a six-well tissue culture plate in DMEM
containing 10% FBS. After 24 h of incubation at 37 °C in a CO2
incubator, varying concentrations of VB6 (2 mM, 1.5 mM, 1.0 mM,
4
10
1
[
3
Fe(phbpa)(L )](NO ) (1). Yield, ∼75% (0.49 g). Anal. Calcd for
C H FeN O (Mw: 661.46): C, 59.92; H, 4.42; N, 12.71. Found: C,
3
3
29
6
6
0
.5 mM, 0.25 mM, and 0.1 mM) were added and incubated in the dark
5
9.81; H, 4.31; N 12.63. ESI-MS in MeOH (m/z): 599.1650
+
2
−1
for 4 h. It was followed by addition of complexes 3 or 5 (10 μM) and
kept for another 4 h in the dark after which the supernatant was
discarded by gently inverting the tube, and the cell pellet was
suspended in 200 μL of DPBS. Flow cytometric analysis was
performed using a FACS Verse machine (BD Biosciences) using the
Pacific blue A filter. Cell cycle analysis was carried out to investigate
the effect of complexes 1−5 on the progression of the cell cycle. HeLa
or HaCaT cells were incubated with the complexes (10 μM) for 4 h
followed by light irradiation (400−700 nm). Similarly, treated cells
were also kept in the dark to serve as controls. Post irradiation cell
processing was performed using standard procedures (see Supporting
[
M−NO ] . Molar conductance in DMF (Λ ): 74 S m M . FT-
3
M
IR: 3054 w, 1575 m, 1460 s, 1420 s, 1380 s, 1290 vs, 1254 vs, 1146 s,
1
043 m, 1005 s, 872 w, 820 m, 740 s, 576 m, 524 m (vs, very strong; s,
−1
strong; m, medium; w, weak). UV−visible in DMF [λmax/nm (ε/M
cm )]: 576 (4420), 420 (19080). μ (298 K) = 5.84 μ .
−
1
eff
B
1
[Fe(anbpa)(L )](NO ) (2). Yield, ∼70% (0.54 g). Anal. Calcd for
3
C H FeN O (Mw: 761.58): C, 64.66; H, 4.37; N, 11.03. Found: C,
4
1
33
6
6
6
4.79; H, 4.46; N 11.21. ESI-MS in MeOH (m/z): 699.2209
+
2
−1
[
M−NO ] . Molar conductance in DMF (Λ ): 71 S m M . FT-
3
M
IR: 3052 w, 1578 m, 1440 m, 1390 s, 1290 vs, 1160 m, 1020 m, 850 w,
8
M
20 m, 742 s, 650 w, 547 w, 472 w. UV−visible in DMF [λmax/nm (ε/
3
8
−1
−1
Information for details). Annexin-V-FITC/PI assay experiment was
performed to estimate the population of apoptotic cells by standard
cm )]: 517 (2520, sh), 413 (8960, sh), 389 (16460), 370
(
16700), 350 (13950), 335 (11080) (sh, shoulder). μeff (298 K) = 5.80
3
8
μ .
[
reported procedures (see Supporting Information). A DCFDA assay
was done to study the formation of ROS in HeLa or HaCaT cells
treated with the photoactive complexes 2 or 3 under different
conditions using 2′,7′-dichlorofluorescein diacetate (DCFDA). Post
treatment processing of the cells was done following standard
procedures (see Supporting Information). The cellular localization
pattern of the fluorescent complexes 2−5 (10 μM) inside HeLa and
HaCaT cells was visualized using a confocal microscope (Leica, TCS,
SP5). Standard experimental procedures were adopted for plating the
B
1
Fe(pybpa)(L )](NO ) (3). Yield, ∼70% (0.56 g). Anal. Calcd for
3
C H FeN O (Mw: 785.60): C, 65.74; H, 4.23; N, 10.70. Found: C,
4
1
33
6
6
6
5.59; H, 4.31; N 10.76. ESI-MS in MeOH (m/z): 723.2215
+
2
−1
[
M−NO ] . Molar conductance in DMF (Λ ): 77 S m M . FT-
3
M
3
8
IR: 3052 w, 2352 w, 1600 s, 1385 s, 2490 w, 1590 w, 1295 vs, 1190 m,
1
015 m, 840 m, 755 m, 552 w, 490 w. UV−visible in DMF [λmax/nm
−1 −1
(ε/M cm )]: 519 (3060, sh), 403 (16450), 345 (48870), 330
40960), 315 (25820), 300 (21220). μeff (298 K) = 5.80 μB.
(
C
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Table 1. Selected Physicochemical Data of the Complexes 1−5
a
b
−1
λ/nm (ε/M−1 cm−1
c
d
e
2
−1
f
g
complex
ESI-MS (m/z)
IR /cm
)
λem /nm (φ)
ΛM /S m M
E /V (ΔE/mV)
μeff
f
1
2
3
4
5
599.1650
699.2209
723.2215
656.2350
680.1948
1380
1390
1385
1380
1380
576 (4420, sh)
517 (2520, sh)
519 (3060, sh)
501 (2140, sh)
74
71
77
70
73
−0.26 (88)
−0.25 (86)
−0.26 (100)
−0.19 (90)
5.84
5.80
5.80
5.85
5.81
423 (0.04)
396 (0.06)
426 (0.03)
395 (0.05)
507 (2760, sh)
−0.20 (96)
d
a
+
b
c
Molecular ion peak corresponding to [M-NO ] in MeOH. Nitrate stretching in solid phase. In DMF. sh, shoulder. Emission of the complexes
3
recorded in DMSO with the quantum yield values in parentheses with respect to anthracene and quinine sulfate. The fluorescence lifetimes are 9.89
−
9
−9
e
×
10 s and 8.47 × 10 s for 2 and 3 respectively. The excitation wavelengths for 2−5 are 371, 346, 378, 341 nm, respectively. Molar conductivity
−1
f g
values in DMF at 25 °C. Formal potential of the Fe(III)/Fe(II) redox couple vs SCE in DMF-0.1 M TBAP at a scan rate of 50 mV s . Effective
magnetic moment of solid samples at 25 °C.
Figure 1. (a) UV−visible spectra of the complexes 1−5 in DMF in different color codes: black, 1; red, 2; green, 3; blue, 4; dark cyan, 5. (b) Emission
spectra of the complexes 2−5 in DMSO in color codes: red, 2; dark cyan, 3; blue, 4; green, 5 [excitation wavelength (λ ) for 2−5 are 371, 346, 378,
ex
and 341 nm, respectively].
cells and processing them after treatment with the complexes (see
Supporting Information).
appears that the cyclization occurs in the reaction mixture in
the presence of Fe3+ which acts as a Lewis acid since the ligand
alone did not show any trace of cyclized product. This is also
corroborated from the ESI-MS data of the complexes 1−3
which show the molecular ion peak two units less than what
would have been expected of a noncyclized Schiff base ligand
38
RESULTS AND DISCUSSION
■
Synthesis and General Aspects. The tridentate Schiff
1
base (H L ) of pyridoxal hydrochloride as yellow orange
2
(
Figures S5−S9, Supporting Information). The IR spectra of
crystalline solid was prepared by reacting it with 2-amino-
the complexes 1−3 also suggest the absence of any free
hydroxyl group (Figure S10, Supporting Information). The
Schiff base ligands bear two phenolic groups which stabilize the
metal in its +3 oxidation state and also generate an intense
ligand to metal charge transfer (LMCT) band near 520 and 420
nm from phenolate π orbital to the dπ* orbital of iron (Figure
phenol. A similar reaction of salicylaldehyde with 2-amino-
2
phenol gave the Schiff base H L (Scheme S1, Supporting
2
1
Information). The ligands were characterized from H NMR in
DMSO-d6 and ESI-MS data (Figures S1−S4, Supporting
Information). Ternary iron(III) complexes [Fe(B)(L)](NO )
3
(
(
(
1−5) were prepared from a general reaction of Fe-
4
5,46
1
a).
The LMCT bands enabled us to study the photo-
NO ) .9H O with 1 equiv of the dipicolylamine derivative
3
3
2
1
2
cytotoxicity of the complexes in visible light. The anthracene
and pyrene appended complexes 2−5 were emissive in the blue
region near 400−420 nm upon excitation near 370 nm (Figure
B) and the Schiff base (H L /H L ) in methanol in the
2
2
presence of triethylamine (Chart 1, Scheme S2). The
complexes were characterized from their physicochemical data
1
b). The quantum yield values were in the range of 0.03−0.05
with respect to pure anthracene and quinine sulfate as standards
Table 1). The complexes were 1:1 electrolytes in DMF giving
(Table 1, Figures S5−S9, Supporting Information). Complex 1
having a pendant phenyl group on the dipicolylamine moiety
was used as a control complex to explore the role of the
anthracenyl and pyrenyl fluorophore moieties in showing the
photocytotoxic effect. These groups enabled us to track the
complexes inside the cells by cellular imaging. Complexes 4 and
(
2
−1
molar conductivity values of ∼75 S m M . The complexes
were redox active showing quasi-reversible Fe(III)−Fe(II)
couple near −0.3 V in DMF-0.1 M TBAP (Figure S11,
Supporting Information). The complexes gave magnetic
5
having the salicylaldehyde Schiff base were also used as
controls to ascertain the importance of the VB6 entity in
complexes 1−3 in cellular uptake, localization, and photo-
cytotoxicity.
moment values of ∼5.7 μ at 25 °C which is in accord with
B
the high spin state of the iron(III) center.
X-ray Crystal Structure. Complex 1 was crystallized as a
The structure of the complexes was initially elucidated from
PF
group P1
6
salt (1a) and the crystals belonged to the triclinic space
with two molecules in the unit cell. Selected
the crystal structure of 1 as a PF salt (1a) where the pyridoxal
̅
6
moiety was seen to undergo cyclization. The hydroxyl group
forms a five-membered ring with the CN moiety, which is
discussed in a subsequent section. Such cyclization reactions are
crystallographic parameters are given in Table 2. An ORTEP
view of the molecule is shown in Figure 2 (Figure S12,
Supporting Information, for unit cell packing diagram).
Selected bond distances and angles are given in Table 3. The
42−44
known to occur in the presence of protic or Lewis acids.
It
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Table 2. Selected Crystallographic Data for
structure showed a distorted octahedral FeN O core with the
4
2
1
[
Fe(phbpa)(L )](PF ) (1a)
tridentate ligands coordinating to the metal in the facial mode.
The pyridoxal Schiff base coordinated through two phenolic
oxygen atoms and a nitrogen atom. The Fe(1)−O(1) and
Fe(1)−O(2) bond lengths were 1.926(3) and 1.920(3) Å,
respectively. The Fe−N distances varied from 2.128(3) to
6
parameters
values
empirical formula
FW
C H F FeN O P
3
3
29
6
5
3
744.43
crystal system
space group
a (Å)
triclinic
2
.222(3) Å. The Fe(1)−N(1) distance is longer compared to
P1
̅
the other Fe−N bond lengths. The hydroxyl group of the
pyridoxal moiety was seen to undergo cyclization forming a
five-member ring (Figure S13, Supporting Information). The
pyridoxal Schiff base binding to other metal ions does not show
10.357(2)
11.440(3)
14.297(4)
99.748(11)
95.099(11)
101.668(10)
1621.7(7)
2
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
V (Å3)
28,47
3+
a similar cyclization process.
Thus, it appears that Fe
being a strong Lewis acid promotes such in situ cyclization of
the ligand. This is corroborated by similar observation as
mentioned above where Lewis acids or protic acids promote
Z
42−44
such novel cyclization reactions.
T (K)
ρcalcd (g cm−3
296 (2)
1.525
Theoretical Studies. DFT calculations were carried out
using B3LYP level theory with LanL2DZ basis set with
Gaussian 09 programs to get further insights on the excited
state properties of complexes 1−3. The initial coordinates were
obtained from the crystal structure of 1a and were used for
optimization (Table S1, Supporting Information). TDDFT
calculations were carried out on the geometrically optimized
structures of 1−3 to assign the transitions obtained in the UV−
visible spectra. The dipole allowed transitions along with the
oscillator strengths for all three complexes are listed in Table S2
(see Supporting Information). A strong LMCT was observed
theoretically near 590 nm which corresponds to the transition
from the phenolate π* orbitals to the dπ orbitals of the metal in
)
λ (Å) (Mo Kα)
0.71073
0.592
μ (mm−1
)
data/restraints/parameters
F(000)
5659/0/442
762
goodness of fit
1.084
a
b
R (F ) , I > 2σ(I)/wR (F )
0.0592/0.1588
0.0866/0.1735
1.770, −0.334
o
o
R (all data)/wR (all data)
largest diff peak and hole (e Å−3)
a
R = Σ∥F | − |F ∥/Σ|F |. wR = {Σ[w(F − F ) ]/Σ[w(F ) ]}1/2. w =
b
2
o
2
c
2
2
o
c
o
o
2
2
−1
2
2
[
(σF ) + (AP) + BP] , where P = (F + 2F )/3.
o o c
1
. Additionally, in complexes 2 and 3, the anthracenyl and
pyrenyl groups also contribute strongly to this LMCT band.
This could explain the excellent photocytotoxicity of 2 and 3
but not 1. The other transitions above 400 nm also correspond
mainly to LMCT transitions involving the orbitals of phenolate
and the iron. Transitions below 400 nm correspond to
intraligand charge transfer (ILCT) transitions involving both
the dipicolylamine and the Schiff base ligands. In fact, the
HOMOs of complexes 2 and 3 primarily involved the electron-
rich anthracenyl or pyrenyl groups but not so in the case of the
phenyl analogue in complex 1. The LUMO was based on the
dipicolylamine moiety in complexes 2 and 3 (Figure 3).
Solubility and Stability. Complexes 1−3 were soluble in
water, methanol, ethanol, dichloromethane, dimethylforma-
mide, and dimethyl sulfoxide. They were insoluble in
hydrocarbon solvents such as hexane, xylene, or toluene.
Complexes 4 and 5 were sparingly soluble in water. To examine
the biological activity of the complexes, it is a prerequisite that
Figure 2. ORTEP diagram of the cationic complex in [Fe(phbpa)-
1
(
L )](PF ) (1a) showing the thermal ellipsoids at 50% probability and
6
the labeling of the metal and heteroatoms. The hydrogen atoms are
omitted for clarity. Color codes: metal, red; nitrogen, green; oxygen,
blue; carbon, white.
1
Table 3. Selected Bond Lengths and Bond Angles of [Fe(phbpa)(L )](PF ) (1a)
6
bond
bond length (Å)
bond
bond angle (deg)
Fe(1)−O(1)
Fe(1)−O(2)
Fe(1)−N(1)
Fe(1)−N(2)
Fe(1)−N(3)
Fe(1)−N(4)
bond
1.927(3)
O(1)−Fe(1)−N(4)
O(2)−Fe(1)−N(1)
O(2)−Fe(1)−N(2)
O(2)−Fe(1)−N(3)
O(2)−Fe(1)−N(4)
N(1)−Fe(1)−N(2)
N(1)−Fe(1)−N(3)
N(1)−Fe(1)−N(4)
N(2)−Fe(1)−N(3)
N(2)−Fe(1)−N(4)
N(3)−Fe(1)−N(4)
90.37(13)
102.63(11)
90.78(11)
91.24(11)
90.63(13)
76.11(11)
166.03(11)
74.97(11)
105.74(11)
150.63(13)
103.55(12)
1.922(3)
2.222(3)
2.128(3)
2.144(3)
2.137(3)
bond angle (deg)
169.55(11)
87.68(11)
93.44(11)
78.41(11)
O(1)−Fe(1)−O(2)
O(1)−Fe(1)−N(1)
O(1)−Fe(1)−N(2)
O(1)−Fe(1)−N(3)
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Figure 3. Geometrically optimized structures of complexes 1−3 obtained from DFT calculations using B3LYP level and LanL2DZ basis set and the
frontier molecular orbitals for the same obtained from TDDFT calculations.
Figure 4. Bar diagrams showing the cytotoxicity of the complexes 1−5 in different cells lines upon irradiation with visible light (400−700 nm, 10 J
−2
cm , green bars) and in dark (black bars): HeLa (a), HaCaT (b), MCF-7 (c), A549 (d), and HPL1D (e).
they are stable under physiological conditions. The stability of
the complexes was thus monitored in DMSO−DPBS (1:1 v/v)
for 24 h using UV−visible spectral scans. There was no
significant shift of the absorption bands or appearance of any
new peaks (Figure S14, Supporting Information). The intensity
of the bands also remained essentially similar, which rules out
the possibility of any degradation of the complexes in
physiological conditions or leaching of the metal ion in the
solution phase.
IC50 values of the complexes, ligands along with few related
2
7,48−50
compounds are listed in Table 4.
HaCaT cells were
chosen as a model for superficial cells, and HPL1D served as
the control normal cell line. The cells were incubated with
different concentrations of the complexes or the ligands for 4 h
in dark. Complex 1 did not show any significant photo-
cytotoxicity when irradiated with visible light (400−700 nm).
However, pyridoxal appended complexes 2 and 3 were
remarkably active with IC50 values ranging from 0.4 to 5 μM
in different cells under identical experimental conditions in
HeLa, HaCaT, MCF-7, and A549 cells. The corresponding
control complexes 4 and 5 were rather less cytotoxic with IC50
values >20 μM. The complexes did not show any pronounced
dark toxicity with IC values >50 μM. Again, complexes 2 and
Cytotoxicity. The cytotoxicity of complexes 1−5 and the
ligands was examined in different cell lines, namely, HeLa
(human cervical cancer), MCF-7 (human breast cancer),
HaCaT (human keratinocytes), A549 (lung adenocarcinoma),
and HPL1D (nontransformed human epithelial lung cells)
50
(Figure 4, Figures S15−S20, Supporting Information). The
3 were 6-fold less cytotoxic in normal lung cell line HPL1D
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with the IC50 value in light being >30 μM (Table 4). A
comparison of the IC50 values in cancerous and normal cell
lines of the same origin (A549 and HPL1D) shows the distinct
selectivity with which the molecules affect the cancer cells.
Thus, the selective photocytotoxicity of the complexes having
pyridoxal derivative toward cancer cells over normal cells is a
manifestation of the better uptake properties of the VB6 group,
which adds a new dimension to the PDT agents under study.
Again, the anthracene or pyrene conjugated dipicolylamine
ligands showed moderate cytotoxicity and photocytotoxicity
under identical experimental conditions in HeLa cells (Table 4,
Figure S20, Supporting Information). Similarly, the Schiff base
ligands were nontoxic in HeLa cells irrespective of the presence
or absence of light (Figure S20, Supporting Information). The
broad band 400−700 nm light source (Luzchem Photoreactor)
was used due to the presence of the LMCT band of the
complexes in the green light region.
Cellular Incorporation Studies. The percent cell
positivity of the complexes 2−5 in HeLa and HaCaT cells
were thus monitored using FACS analysis. After the cells were
incubated for 4 h with the complexes in the dark, complexes 2
and 3 displayed nearly 90% of the cells showing positive signal
for complex uptake, while only about 30% of the cells showed a
positive response upon treatment with 4 and 5 (Figure 5). Our
hypothesis is that the internalization of the complexes of
pyridoxal derivative (2 and 3) is more facile as compared to
complexes 4 and 5 which lack such a group.
In another experiment, the cells were incubated with
pyridoxal hydrochloride for 4 h prior to addition of the
complexes, and the cellular internalization was monitored. The
FACS analysis showed significant lowering of the fluorescence
signal (∼15%) for complexes 2 and 3, which corresponds to
fewer number of cells taking up the complexes. Complexes 4
and 5 also showed a reduced fluorescence signal albeit much
less. This effect was again more pronounced in HeLa cells
compared to the HaCaT cells. Hence it can be inferred that the
presence of a VB6-like group in complexes 2 and 3 somehow
enhances the cellular uptake properties especially in the cancer
cells, which results in better cytotoxicity after light irradiation.
This is of paramount importance where the VB6 tagged
complexes are preferentially internalized in cancer cells which
induce a higher degree of selectivity to the PDT agent as
compared to the conventional PDT drugs where the selectivity
is induced only with light irradiation.
Dose-Dependent Cellular Uptake. A dose-dependent
cellular uptake of 3 and 5 was monitored in HeLa cells by
varying the concentration of VB6 (pyridoxal hydrochloride).
HeLa cells were treated with various concentrations of
pyridoxal hydrochloride (2, 1.5, 1.0, 0.5, 0.25, and 0.1 mM)
for 4 h in the dark followed by addition of either of the
complexes. It was observed that with the lowering of the
concentration of VB6, there was a stark increase in the percent
positivity of cells to which complex 3 was added. However,
there was no such marked dose-dependent increase in the mean
fluorescence of the cells that were treated with complex 5. This
observation can be related with the presence of the pendant
group resembling pyridoxal, which probably assists in cellular
uptake of the complexes (Figure S21, Supporting Information).
Cell Cycle Analysis. The effect of the photocytotoxic
complexes on the progression of the cell cycle was studied by
FACS analysis in HeLa and HaCaT cells treated with the
complexes 1−5 in light of 400−700 nm or in the dark. Before
analysis, the cells were treated with PI that stains the DNA
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Figure 5. Percent cell positivity upon treatment with the complexes 2−5 in HeLa (a) and HaCaT (b) cells with (black bars) or without VB6
derivative (white bars) indicating the internalization of the complexes inside the cells.
quantitatively. Hence, the fluorescence intensity of the stained
cells correlates with the amount of DNA. The sub G1
population of the cells corresponds to those whose membrane
has been compromised and have undergone cell death.
Complex 1 did not alter the cell cycle progression in light or
in the dark in either HeLa or HaCaT cells. It was seen that both
2
and 3 induced significant sub G1 population compared to 4
and 5 in HaCaT cells upon exposure to light. The percentage of
sub G1 population induced by 2 and 3 in HeLa cells was nearly
30%. Treatment of cells with complexes 4 or 5 followed by
irradiation resulted in the arrest of the G2/M phase (Figure 7,
Figures S21 and S22, Supporting Information). The complexes
of pyridoxal base thus have enhanced cellular uptake as
evidenced from the cell cycle profiling analysis.
Figure 7. Annexin-V-FITC/PI assay showing the percent population
of early apoptotic cells stained by annexin-V-FITC alone (lower right
quadrant), dead cells stained by propidium iodide alone (upper left
quadrant), or late apoptotic cells stained by both annexin-V-FITC and
PI (upper right quadrant) in HeLa cells alone or treated with
complexes 2−5 in the dark (D) or after exposure to visible light (400−
7
00 nm, L).
apoptosis in ∼25% of the cells when irradiated with visible light
compared to ∼8% for complexes 4 and 5 under identical
experimental conditions. A prominent fraction of cells showed
features of late apoptosis for all four complexes (Figure 8).
DCFDA Assay. 2′,7′-Dichlorofluorescein diacetate
(
DCFDA) assay was done to detect generation of any cellular
ROS. The percentage of cell population generating ROS can be
determined by flow cytometry analysis. To ascertain the
formation of ROS inside the cells, HeLa or HaCaT cells were
incubated with 2 and 3 for 4 h in the dark, and then one of the
plates was exposed to light irradiation (400−700 nm) for 1 h.
The cells treated with these complexes and light showed a
significant shift in the cell population showing fluorescence as
compared to those kept in the dark (Figure 9, Figure S23,
Supporting Information). This indicates the formation of ROS
which is believed to be responsible for the cell death only upon
photoirradiation but not in the dark. Hydrogen peroxide was
used as a positive control for the experiment. When cytotoxicity
assay was performed in the presence of N-acetyl cysteine
(NAC) as a ROS scavenger, there was a significant increase in
the IC50 values for complexes 2−5 in HeLa cells in identical
Figure 6. Cell cycle progression in cells alone or after treatment with
the complexes 2−5 in the dark (D) or upon irradiation with visible
−2
light (400−700 nm, 10 J cm , L) in HeLa (a) and HaCaT (b) cells
showing the percentage of cells in different phases of the cell cycle.
Annexin-V-FITC/PI Assay. The cells in the early stages of
apoptosis undergo morphological changes including the
translocation of phosphatidylserine to the external portion of
the lipid bilayer. To estimate the number of cells undergoing
apoptosis, we performed the Annexin-V-FITC/PI assay using
complexes 2−5 in HeLa cells. Cells were incubated with the
complexes for 4 h in dark followed by light irradiation (400−
700 nm). Complexes 2 and 3 induced features of early
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Figure 8. Increase in fluorescence of DCF in HeLa (above) or HaCaT
(
(
below) cells upon treatment with complex 3 on light irradiation
−2
400−700 nm, 10 J cm ) or in dark with H O used as a positive
2
2
control.
Figure 10. Confocal microscopy images of HaCaT (panels (i) and
(ii)) and HeLa cells (panels (iii) and (iv)) treated with ER tracker
green as the endoplasmic reticulum (ER) marker and complexes 3 or 5
as labeled. Panels labeled (a) show the bright field images of the cells,
panels labeled (b) show the fluorescence of ER tracker green, panels
labeled (c) show the fluorescence of either complex 3 or 5, and panels
labeled (d) show the merged images of the ER tracker green and
complex 3 or 5. The scale bars are 14.6 μm.
With the knowledge that the ER forms channel-like
structures at the nuclear periphery, we performed colocalization
studies with ER tracker green and complexes 2 and 3. The
merged images in Figure 10 show the exact overlap of the
fluorescence of ER tracker and the complexes. Similar
experiments with complexes 4 and 5 show only a diffused
blue fluorescence throughout the cell (Figure S26, Supporting
Information). Thus, we infer that the complexes of VB6
derivative localize in the ER of the cells and upon irradiation
generate ROS which results in ER stress. Followed by such a
cascade of events, the cells eventually undergo apoptotic death.
Again, complexes 4 and 5 do not show such specific targeting
and display a general accumulation in the cytoplasm and the
nucleus. So the pyridoxal derivative is implicated in the
preferential localization of the complexes in the ER. This
observation is both interesting and important since detouring
chemotherapeutic molecules from their conventional target, i.e.,
the nucleus, to other organelles is likely to have positive effects
on their anticancer properties.
Figure 9. Confocal microscopy images of HaCaT (panels: (i) and (ii))
and HeLa cells (panels: (iii) and (iv)) treated with propidium iodide
(
PI) as the nucleus marker and complex 3 or 5 as labeled. Panels (a)
show the bright field images of the cells, panels (b) show the
fluorescence of propidium iodide, panels (c) show the fluorescence of
either complex 3 or 5, and panels (d) show the merged images of the
PI and complex 3 or 5. The scale bar corresponds to 20 μm.
CONCLUSIONS
■
experimental conditions, which reaffirms the role of ROS in cell
death (Figure S24, Supporting Information).
Ternary iron(III) Schiff base complexes of a VB6 derivative
show specific accumulation into the cancer cells compared to
the control complexes lacking the pyridoxal moiety and exhibit
remarkable photocytotoxicity when irradiated with visible light
(400−700 nm) while remaining innocuous in the dark. The
complexes are five to six times less cytotoxic in normal lung
cells. They induce apoptotic cell death in HeLa or HaCaT cells
with the generation of ROS. An interesting observation is that
the VB6 derivative Schiff base complexes accumulate
predominantly in the ER of the cells as opposed to the control
complexes of similar make which resided in both the cytoplasm
and nucleus of the cells. Such molecules which can target
organelles other than the nucleus can dodge the NER
Cellular Localization. The localization of the complexes
−5 inside the HeLa and HaCaT cells was monitored by
2
confocal microscopy using nuclear staining dye propidium
iodide (PI). Co-localization studies indicated that complexes 2
and 3 primarily accumulate in certain portions of the cytoplasm
of the cells with no significant nuclear uptake. Though
complexes 4 and 5 showed a diffused distribution throughout
the cell, complexes 2 and 3 of VB6 derivative showed very
distinct accumulation in the periphery of the nucleus in both
cell lines as can be seen in the panels i(c) and iii(c) of Figure 10
(Figure S25, Supporting Information).
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mechanism, which often leads to intrinsic resistance of cells to
the chemotherapeutic agents. The apoptotic cell death through
ERSR thus offers a new strategic way in PDT, and the
development of such molecules can confer a higher degree of
selectivity to novel PDT agents which can target cancer cells
and cause cell death.
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AUTHOR INFORMATION
Corresponding Authors
(P.K.) E-mail: paturu@mrdg.iisc.ernet.in. Tel.: +91-80-
2932688. Fax: +91-80-23600999.
(A.R.C.) E-mail: arc@ipc.iisc.ernet.in. Tel.: +91-80-22932533.
Fax: +91-80-23600683.
■
2
(
2
(
*
2
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(
Notes
Chakravarty, A. R. Chem. Commun. 2014, 50, 5590−5592.
(
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The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
(
We thank the Department of Science and Technology (DST),
Government of India, and the Council of Scientific and
Industrial Research (CSIR), New Delhi, for financial support
(
Y. J.; Choung, P. H.; Cho, C. S.; Chung, J. H. Biomaterials 2013, 34,
3716−3728.
(
SR/S5/MBD-02/2007; CSIR/01(2559)/12/EMR-II/2012).
A.R.C. thanks the DST for J. C. Bose national fellowship and
the Alexander von Humboldt Foundation, Germany, for
donation of an electrochemical system. We are thankful to
Mr. Vashista K. and Ms. A. Kavya for the FACS analysis and Dr.
Santosh Poddar for the confocal microscopy images.
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