Iron(III) Complexes of Vitamin B6 Schiff Base with Boron-Dipyrromethene Pendants for Lysosome-Selective Photocytotoxicity

Article abstract of DOI:10.1002/ejic.201701487

Iron(III) complexes of a vitamin B₆ Schiff base and NNN-donor ligands with pendant boron-dipyrromethene (BODIPY) moieties; namely, [Fe(L^1–3)(L^4,5)](NO₃) (1–4), where L¹ is benzyl-bis[(pyridin-2-yl)methyl]methanamine (bzdpa in 1), L² is a noniodinated BODIPY-appended dipicolylamine ligand (in 2, 3), L³ is the diiodinated BODIPY analogue in 4, L⁴ is a vitamin B₆ Schiff base, namely 3-hydroxy-5(hydroxymethyl)-4-{[(2-hydroxyphenyl)imino]methyl}-2-methylpyridine (in 1, 3, and 4), and L⁵ is 2-[(2-hydroxyphenylimino)-methyl]phenol (in 2) as a nonpyridoxal Schiff base, were prepared, characterized, and their cellular localization and cytotoxic activity in light and in the dark were studied. The diiodo–BODIPY complex 4 displays remarkable photoinduced cytotoxicity in visible light (400–700 nm), with IC₅₀ values within 0.11–0.25 μm and about 200-fold lower dark toxicity. Complex 3, being fluorescent, was used for cellular imaging by confocal microscopy. Complex 4 shows supercoiled pUC19 DNA cleavage activity through the generation of singlet oxygen (¹O₂) as the reactive oxygen species (ROS). Selective uptake of the complexes is observed from competitive cellular incorporation assays in cancer and noncancer cells. The complexes also show no apparent toxicity up to 100 μm in the immortal human lung epithelial cells HPL1D in both light and the dark. Complex 3 shows preferential accumulation in lysosomes, giving a Pearson's correlation coefficient value of about 0.7.

Full text of DOI:10.1002/ejic.201701487

A Journal of
Accepted Article
Title: Iron(III) Complexes of Vitamin-B6 Schiff Base with Borondipyrro-
methene Pendants for Lysosome Selective Photocytotoxicity
Authors: Akhil Chakravarty, Somarupa Sahoo, Santosh Podder, Aditya
Garai, Shamik Majumdar, Nandini Mukherjee, Uttara Basu,
and Dipankar Nandi
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201701487
Link to VoR: http://dx.doi.org/10.1002/ejic.201701487

10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
Iron(III) Complexes of Vitamin-B6 Schiff Base with Borondipyrro-
methene Pendants for Lysosome Selective Photocytotoxicity
Somarupa Sahoo,[a] Santosh Podder,[b] Aditya Garai,[a] Shamik Majumdar,[b] Nandini Mukherjee,[a] Uttara
Basu,[a] Dipankar Nandi*[b] and Akhil R. Chakravarty*[a]
Dedication ((optional))
agents showing better activity.[3] Vitamin B6 (VB6) is one such
Abstract: Iron(III) complexes of a vitamin B6 Schiff base and NNN-
donor ligands with pendant borondipyrromethene (BODIPY) moieties,
viz. [Fe(L1-3)(L4,5)](NO3) (1-4), where L1 is benzyl-bis((pyridin-2-
yl)methyl)methanamine (bzdpa in 1), L2 is a non-iodinated BODIPY
appended dipicolylamine ligand (in 2, 3), L3 is the diiodinated-
BODIPY analogue in 4, L4 is vitamin B6 Schiff base, viz. 3-hydroxy-
5(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2-
methylpyridine (in 1, 3 and 4) and L5 is 2-[(2-hydroxyphenylimino)-
methyl]phenol (in 2) as a non-pyridoxal Schiff base, were prepared,
characterized and their cellular localization and cytotoxic activity in
molecule which plays an important role in cellular processes by
acting as a cofactor.[4] Cellular uptake of VB6 is by means of
diffusion, through specific transporting membrane carriers
known as Vitamin Transport Channels (VTC).[5] During the
uncontrolled cell proliferation in cancer, the activity of Serine
Hydroxymethyltransferase (SHMT) increases manifold as it aids
in the biosynthesis of DNA, which also leads to an increased
requirement of VB6.[6] Therefore, it is assumed that a molecule
conjugated with this moiety will have significant cellular uptake,
thus enhancing its selectivity and efficacy. We have earlier used
this molecule to target cancer cells selectively.[7,8] Iron being a
bioavailable metal present in human body in various enzymes
and receptors is chosen for our study to make covalent
conjugates with organic moieties that are selected for desired
activity. Iron reduces the metal related toxicity that can arise in
case of leaching.[9] The clinical success of iron-bleomycins as
chemotherapeutic agents showing oxidative damage of DNA by
forming cytotoxic hydroxyl radicals provides impetus in using this
metal for cancer treatment and cure.[10] Metal complexes having
ligands with a VB6 moiety are expected to show significant
enhancement in the cellular uptake compared to the control
species lacking such a moiety. BODIPY dyes are a versatile
class of photosensitizers that exhibit properties such as high
molar extinction coefficient in the absorption band, sharp
emission bands with minimal Stokes shift, high resistance to
photo-bleaching and readily amenable for structural modification.
This makes them an appropriate choice for cellular imaging and
exploiting their light-induced ROS generation ability for
cytotoxicity study.[11] Research on using these dyes to generate
singlet oxygen by photo-irradiation has received major attention
in recent years. Akkaya et al. have worked extensively in using
BODIPY dyes as photodynamic therapy (PDT) agents.[12] The
present work stems from our interest in designing new ternary
iron(III) complexes with VB6 incorporated in the Schiff base
framework that are expected to give higher cellular uptake in
cancer cells over normal cells.
light and dark were studied. The diiodo-BODIPY complex
4
displayed remarkable photo-induced cytotoxicity in visible light (400-
700 nm) with IC50 values within 0.11 to 0.25 μM and ~200-fold lower
dark toxicity. Complex 3 being fluorescent was used for cellular
imaging by confocal microscopy. Complex 4 showed supercoiled
pUC19 DNA cleavage activity via generation of singlet oxygen (1O2)
as the reactive oxygen species (ROS). Selective uptake of the
complexes was observed from competitive cellular incorporation
assays in cancer and non-cancerous cells. The complexes also
showed no apparent toxicity up to 100 μM in the immortal human
lung epithelial cells HPL1D in both light and dark. Complex 3
showed preferential accumulation in the lysosomes giving
a
Pearson’s correlation coefficient value of ~0.7.
Introduction
Two important aspects in designing an anticancer drug are: (i)
the selectivity of the drug between normal versus cancer cells
and (ii) specific localization of the drug to a particular cellular
organelle in preference to the others. To achieve the first
objective, several bioactive organic molecules are being used in
conjugation with the main drug to drive it specifically to the
cancer cells.[1] This significantly enhances the efficacy of the
drug and reduces the drug and dose related toxicity. For
instance, Frochot et al. have used glycosylated and folic acid
conjugated photosensitizers for selective cytotoxicity.[2] Low and
coworkers have developed receptor based targeted therapeutic
The other important aspect is the specific localization of
the drug to a particular cellular organelle. Cisplatin and its
analogues are known to localize in the nucleus and bind to
nuclear DNA.[13] The low efficacy of some of these
chemotherapeutic drugs is attributed to the nuclear excision
repair (NER) mechanism which is operative for nuclear DNA.[14]
Cisplatin is also known to show poor selectivity between normal
versus cancer cells. PDT has emerged as an alternative Food
and Drug Administration (FDA) approved therapeutic approach
to treat cancer.[15] This methodology is useful for selective
treatment of tumors by the drug Photofrin which as a
[a]
[b]
Somarupa Sahoo, Dr. Aditya Garai, Nandini Mukherjee, Dr. Uttara
Basu, Prof. Akhil R. Chakravarty
Department of Inorganic and Physical Chemistry
Indian Institute of Science, Bangalore 560012, Karnataka, India
E-mail: arc@iisc.ac.in; http://ipc.iisc.ac.in/~arc/index.html
Santosh Podder, Shamik Majumdar, Prof. Dipankar Nandi
Department of Biochemistry
Indian Institute of Science, Bangalore 560012, Karnataka, India
E-mail: nandi@iisc.ac.in; http://biochem.iisc.ernet.in/dpnLab/
Supporting information for this article is given via a link at the
end of the document.
This article is protected by copyright. All rights reserved.

10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
cells over the normal cells, the BODIPY-appended NNN-donor
dipicolylamine (dpa) ligand in the ternary structure in complex 4
is expected to generate singlet oxygen causing light-induced cell
death by type-II pathway that is known for Photofrin. Complex 3
contains a highly emissive BODIPY unit which can be used for
cellular imaging to identify the target organelle for these
complexes. Complex 2 contains a salicylaldehyde Schiff base
instead of vitamin B6. This complex is used to study the cancer
cell vs normal cell selectivity of the complexes as imparted by
the vitamin moiety. And finally, complex 1 which is devoid of any
photosensitizing group is used as a control to ascertain the
photosensitizing ability of the BODIPY moieties.
The significant results of this work include: (i) higher
cellular uptake in the cancer cells than in normal cells, (ii)
remarkable PDT activity in light of 400-700 nm with singlet
oxygen mediated apoptotic cell death, and (iii) significant
localization of the complex in the lysosome instead of the
anticipated mitochondria. This observation is serendipitous as
lysosome localizing PDT agents are rare in the literature.[24-28]
Tsubone et al. in a recent paper have demonstrated that
lysosome-specific photo-damage results in enhanced activity by
using new porphyrin dyes.[24] The enhanced activity is ascribed
to the lysosomal membrane damage and induction of
autophagy-associated cell death. Iron oxide based magnetic
nano particles were reported to target cancer cells via
overexpressing the epidermal growth factor receptor (EGFR)
that were found to be permeable in the lysosomal membrane
when an alternating magnetic field was applied.[25] BODIPY
based systems that target lysosomes are also being studied
recently.[26] Peng et al. have reported lysosome-targeted
BODIPY dyes showing PDT activity.[27] The present work
exemplifies the first metal-based photo-cytotoxic agent showing
light-induced cell death on localizing to this important cellular
organelle which is involved in cellular protein degradation.[28]
Herein we report the synthesis, characterization and photo-
induced anticancer activity of the iron(III) complexes 1-4.
Figure 1. Iron(III) complexes 1-4 as their nitrate salts.
photosensitizer generate singlet oxygen (1O2) as the reactive
oxygen species (ROS) on exposure to red light of 633 nm.[16]
This PDT drug shows better selectivity with its localization in the
mitochondria of the cells. The advantage of PDT over
conventional chemotherapy is based on this selectivity in which
the light-exposed cancer cells are only damaged thus leaving
the unexposed healthy cells unaffected. The current focus lies
on developing targeted drugs with minimal side effects.
Photoactive metal-based coordination complexes with versatile
structural diversity and biophysical properties are reported to
show promising results in targeting mitochondria.[17] Gasser et
al. have reported ruthenium complexes showing anticancer
activity by localizing in the lysosomes.[18] Dhar et al. have also
demonstrated enhanced activity of mitochondria targeting
platinum complexes, while Neamati et al. have shown that the
anticancer activity of chlorambucil results from its mitochondrial
localization.[19,20] Sadler et al. have suggested that preferential
localization could be an important aspect of current and future
anticancer drug design.[21]
Results and Discussion
Synthesis and Chemical Characterization
The salicylaldehyde (H2L) and pyridoxal hydrochloride
containing tridentate Schiff bases and the dipicolylamine (dpa)
ligands L2 and L3 with appended BODIPY moieties were
prepared by reported procedures.[29] Iron(III) complexes 1-4 were
prepared in good yield following general synthetic procedure in
which FeNO3.9H2O was initially reacted with the dipicolylamine
ligand in 1:1 molar equivalent in anhydrous methanol (1, bzdpa;
2 and 3, L2; and 4, L3) with a subsequent addition of the
deprotonated Schiff base ligand using triethylamine in methanol.
(Scheme S1) The product was isolated as nitrate salt and the
complexes were characterized by spectroscopic and analytical
methods. Selected physicochemical data are given in Table 1.
Complex 1, without having any BODIPY unit, was used as a
control due to its inactivity in light. Complex 2 lacking the
pyridoxal Schiff base was also used as a control to probe the
selective cellular uptake of the VB6 containing complexes.
Complexes 3 and 4 having the pendant BODIPY moieties were
used for cellular imaging and photo-induced cytotoxicity study.
Complex 3 for its fluorescent properties was used for
Combining the above mentioned strategies of targeting
cancer versus normal cells and with the knowledge that
borondipyrromethene (BODIPY) based photosensitizers prefer
mitochondrial localization[22,23]
,
we have designed and
synthesized new ternary iron(III) complexes of formulation
[Fe(L1-3)(L4,5)](NO3) (1-4), where L1 is benzyl-bis((pyridin-2-
yl)methyl)methanamine (bzdpa in 1), L2
is non-iodinated
BODIPY-appended dipicolylamine ligand (in 2, 3), L3 is
diiodinated-BODIPY analogue (in 4), L4 is vitamin B6 Schiff base,
viz.
3-hydroxy-5-(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)
methyl)-2-methylpyridine (in 1, 3 and 4) and L5 is 2-[(2-
hydroxyphenylimino)-methyl]phenol) (in 2) as a non-pyridoxal
Schiff base (Figure 1). While vitamin B6 unit is expected to
facilitate the uptake of the complex selectively into the cancer
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
Table 1. Selected physicochemical and ct-DNA binding data of
the complexes 1 - 4 as their nitrate salts.
[b]
[c]
e]
[
Com
plex
λmax[a]/nm
[ε/(M−1
λem
nm
[ΦF]
/
Epc
(V)
Kb [f] (M-1)
ΛM
/
eff
[d]
cm−1)]
(S m2
M−1
)
1
270 (46450),
386 (16025)
470 (-)
-0.63
5.86
77
9.45 x 104
2
3
4
400 (26420),
500 (42400)
560
(0.12)
5.83
5.82
5.85
-0.45
-0.49
-0.44
69
71
73
-
Figure 2. (a) Absorption spectra of the complexes 1-4 in 1:1 (v/v)
DMSO:DPBS buffer medium. (b) Emission spectra of the complexes 1-4 in
10% aqueous DMSO (λex: 300 nm for 1; 480 nm for 2 and 3; 520 nm for 4).
265 (42130),
500 (45100)
560
(0.12)
7.83 x 105
4.99 x 105
407 (25140),
532 (35520)
560
(0.01)
intense π→π* transition arising from the BODIPY and diiodo-
BODIPY units at 500 and 532 nm, respectively.[32] A ligand to
metal charge transfer (LMCT) band was seen at 400 nm for the
phenolate π orbital to the dπ* orbital of iron.[33] The dpa ligand
showed π→π* band in these complexes at ~280 nm. Complex
3 with the BODIPY unit was highly emissive in 10% aqueous
DMSO upon excitation at 480 nm (Figure 2). This complex was
found to be suitable for cellular imaging studies by confocal
imaging with its fluorescence quantum yield (ΦF) value of 0.12.
Complex 4 was found to be non-emissive with ΦF value of 0.01
(fluorescein in 0.1 M NaOH used as standard). The iodine atoms
seemed to facilitate intersystem crossing (ISC) via heavy atom
effect, hence making it suitable for photodynamic therapy (PDT).
Complexes 3 and 4 gave singlet oxygen quantum yield values of
0.11 and 0.56, respectively, when estimated using Rose Bengal
as a standard (Figure S11). The complexes were redox active
showing a quasi-reversible Fe(III)-Fe(II) couple near -0.45 V
versus SCE (saturated calomel electrode) in DMF - 0.1 M TBAP
(tetrabutylammonium perchlorate). Reduction peaks from the
BODIPY unit and the Schiff base were visible near -1.2 V. No
oxidative response was observed in the complexes in anodic
scans (Figures S6, S7). Such a low redox potential for the
Fe(III)/Fe(II) couple makes the complexes inactive as “chemical
nucleases” in the presence of cellular thiols, viz. glutathione,
thus reducing their cellular dark toxicity.[10]
[a] Visible bands within 400-700 nm in 1:1 DMF–DPBS buffer. [b] In 10%
aqueous DMSO. Quantum yield [ΦF] was measured using fluorescein as the
standard (ΦF = 0.79). [c] Epc is the cathodic peak potential of the Fe(III)/Fe(II)
redox couple vs. SCE in 5 mL DMF-0.1 M TBAP, 2.0 mmol of the complexes.
The scan rate was 100 mV s-1. [d] μeff in μB for solid samples of the complexes
at 25 °C. [e] ΛM, molar conductivity in S m2 M−1 in 10% aqueous DMF at 25 °C.
[f] Intrinsic ct- DNA binding constant (ct, calf thymus).
cellular imaging to probe any organelle selective localization of
the complex. It is known that BODIPY moieties without heavy
atoms like Br or I do not generate significant singlet oxygen (1O2)
as the ROS.[30] Complex 4 with its diiodoBODIPY unit as a
photosensitizer was hence used for the PDT activity. The
complexes gave the elemental analysis data in accordance to
the ternary complex formulation. They were soluble in methanol,
ethanol, dimethyl sulfoxide and dimethylformamide (DMF). The
BODIPY ligands alone were soluble in halogenated solvents,
however, the solubility reduced upon binding to iron(III). They
were insoluble in hydrocarbons like hexane, toluene and
benzene. The complexes were 1:1 electrolytes in DMF giving
molar conductivity values in the range of 70-80 S m2 M−1. The
mass spectra of the complexes showed single [M]+ peak in
methanol suggesting the monomeric nature of the ternary
complexes (Figures S1-S4). The IR spectra showed
characteristic bands for nitrate anion at ~1450 cm-1 and for the
C=C and C=N stretching in the range of 1500 to 1700 cm-1
(Figure S5).[31] The complexes were five-electron paramagnetic
with magnetic moment values of ~5.8 µB at room temperature
indicating high spin state of the iron(III) center.
Theoretical Studies
Photophysical and Photochemical Characterization
The two phenolic groups present on the Schiff base
ligands stabilize the metal in its high-spin +3 oxidation state due
to their strong binding affinity for the metal. This observation is
important as a stable complex reduces the possibility of metal
leaching in the biological medium. Hence, the metal-bound state
of the complexes was monitored by absorption spectroscopy in
1:1 v/v DMSO:DPBS (DPBS: Dulbecco’s phosphate buffered
saline) medium up to 36 h to observe any changes in the
spectral band intensity. The intensity remained marginally
diminished in the cell incubation window of 4 h for complexes 2-
4. The diiodo-BODIPY complex 4, however, showed decrease in
intensity after 12 h, possibly due to instability of the diiodo-
BODIPY moiety. The time dependent absorption spectra for free
ligands and complexes 2-4 are given in Figures S9, S10.
Figure 3. Geometrically optimized structures of the complexes 1, 3 and 4
obtained from DFT.
The UV-visible spectra of the complexes 2-4 in 1:1 (v/v)
DMF:PBS (PBS, phosphate buffered saline) medium showed an
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
nm were >100 μM in all the cell lines. The same for complex 3
was significantly low (~4.0 μM) due to the presence of the
photosensitizing BODIPY unit. The values reduced even further
to nanomolar range (~0.15 μM) for the diiodo-BODIPY complex
4 due to high quantum yield of singlet oxygen as the ROS.
Complexes 2 and 3 remained essentially non-toxic in HPL1D
cells (Figure S12 for cumulative depiction of the IC50 values).
This selective toxicity of the complexes towards cancer cells
over the non-cancerous cells can be attributed to their enhanced
cellular uptake in the cancer cells due to vitamin-B6 moiety and
high photosensitizing ability of the diiodo-BODIPY unit in visible
light.
The energy optimized structures of the complexes 1, 3 and
4 were obtained by density functional theory (DFT) using B3LYP
with LanL2DZ basis set for all atoms with Gaussian 09 software
package to probe the electronic nature of the complexes.[34] The
optimized structures and the HOMO-LUMO are shown in Figure
3. The frontier molecular orbital (FMO) diagram for 1 suggests
the presence of both HOMO and LUMO on the Schiff base
moiety. In complexes 3 and 4, the LUMO was based on the
BODIPY appended dipicolylamine moiety while the HOMO lies
on the Schiff base-metal unit, which gives rise to the metal to
phenolate ligand-to-metal charge transfer (LMCT) transition that
was observed near 385 nm in the UV-visible spectra. The
participation of the BODIPY core in forming the FMOs is evident
as a photosensitizing unit in the complex. The energy difference
between LUMO and HOMO for the complexes is in the range of
2.3 to 2.5 eV.
Cellular uptake
To investigate whether the VB6 moiety possessed any selective
role in facilitating the uptake of the complexes into the cancer
cells over the normal cells, flow cytometry based cellular
incorporation assay was performed using the fluorescent-VB6
containing molecule 3 and its non-vitamin analogue 2. A dose-
dependent as well as competitive uptake study along with time
dependent analysis were performed in HeLa and HPL1D cells
(Figure 4). The data demonstrated that HeLa cells have a
preferential uptake of complex 3 as compared to 2 within an
incubation time of 4 h. No such difference was observed in the
HPL1D cells. Interestingly, VB6 did not assist in quicker ingestion
of the drug moiety in HPL1D cells, which was evidenced by
varying the incubation time from 2 to 4 h in complex 3. It showed
the same amount of uptake at both time points in HPL1D cells.
However, there was a significant difference in the uptake of 3 in
HeLa cells, suggestive of higher uptake in cancer cells owing to
their increased demand for VB6 as a nutrient.[41] In addition to
that, there was an increase in the uptake of the complex into the
HeLa cells with the increase in incubation time (Figure S17).
Complete incorporation was noteworthy in HeLa cells upon 4 h
post- incubation in all the three doses used. This might be due to
the selective uptake of the complex by transporter channels
initially and subsequent internalization via simple diffusion with
the passage of time. This observation helped us to standardize
Photocytotoxicity
The antitumor activity of the complexes in light (visible light
source of 400–700 nm; Luzchem photoreactor, 10 J cm−2;
irradiation time of 1 h) and dark was studied using cell viability
assay in three cancerous cell lines, viz. HeLa, (human cervical
cancer), MCF-7 (human breast cancer) and HepG2 (human liver
carcinoma cells) and one non-cancer immortalized cell line, viz.
HPL1D (transformed human epithelial lung cells). The complexes
were administered into the cells as a 1% DMSO solution. They were
first prepared in DMSO and the final volume was adjusted to 1% by
further dilution with reconstituted DMEM medium (10% FBS) in
which the cells were cultured. The complexes were incubated for
4 h in dark and then one batch was irradiated with visible light,
while the other was kept in dark. Cytotoxicity was measured
from the IC50 values (50% inhibitory concentration) obtained
from the cell viability assay. The complexes generally showed
significant photocytotoxicity in the cancerous cell lines and were
negligibly toxic in dark conditions (Table 2, Figures S12-S16).[35-
40]
The IC50 values of the control complex 1 in light of 400-700
Table 2. IC50 (μM) values of the complexes 1-4 and other relevant compounds
Compound
HeLa
MCF-7
HEPG2
HPL1D
D
L[a]
D
L[a]
D
L[a]
D
L[a]
>50
>50
>50
>50
1
2
3
4
> 100
> 100
> 100
> 100
>100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
> 100
>100
>100
>100
>100
12.2 ± 1.1
5.5 ±1.2
12.1 ± 1.4
3.25 ± 0.08
0.20 ± 0.04
10.3 ± 1.3
3.9 ± 0.6
0.22 ± 0.01
0.11 ± 0.08
[Fe(bpyag)(pyap] (NO3)[b]
[Fe(cat)NO3][c]
> 80
>100
8.2 ± 0.6
6.2 ± 0.1
14.6 ± 0.7
[Fe(BHA)(pydpa)Cl]Cl[d]
[Fe(pybpa)(L)](NO3)[e]
Photofrin[f,g]
>100
3.3± 1.1
>41
4.3 ± 0.2
[a] Visible light of 400–700 nm (10 J cm−2). The IC50 values of the Schiff base ligands in HeLa cells were >100 μM in both light and dark. The cell viability
assay of the BODIPY appended dipicolylamine bases could not be done due to their poor solubility. [b] bpyag is N,N-bis(2-pyridylmethyl)-2-aminoethyl-β-
D-glucopyranoside and H2pyap is 3-(2-hydroxyphenylimino)-1-pyrenylbutan-1-one.[35] [c] L is 9-[(2,2’-dipicolylamino)methyl]anthracene.[36] [d] BHA is
benzhydroxamate and pydpa is (pyrenyl)dipicolylamine.[37] [e] pybpa is (pyren-1-yl)-N,N-bis((pyridin-2-yl)methyl)methanamine and (H2L is 3-hydroxy-5-
(hydroxymethyl)-4-(((2-hydroxyphenyl)imino)methyl)-2methylpyridine.[29 a] [f] The values are taken from Ref 38 and Ref 39 and converted to μM using the
approximate molecular weight of Photofrin as 600 g mol−1. At 633 nm excitation with fluence rate of 5 J cm−2. [g] Cisplatin shows an IC50 value of 13.0±1.2
in HeLa, 2.0±0.3 in MCF 7 and 7.23 ± 0.04 in HepG2 cells.[40]
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
Figure 5. 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), and late
apoptotic cells stained by both Annexin-V-FITC and PI (upper right quadrant)
in HeLa cells alone or treated with complex 3 in the dark or after exposure to
visible light for 1 h (400− 700 nm).
Figure 4. Histogram depicting cellular incorporation of complex 2, i.e. the
non-vitamin analogue in blue dotted line and complex 3, which contains the
VB6 moeity in red dotted line in (a) HPL1D and (b) HeLa cells post 4 h of
incubation at 37C. Complex concentration: 2 μM.
the incubation time of the complexes for other in-vitro studies
prior to light irradiation.
is a cellular protein which binds to the phophatidylserine, a
glycerophospholipid component of the cell membrane. Exposure
of this protein on the external surface of the plasma membrane
is considered as a hall mark feature of apoptosis and forms the
basis for the Annexin V (conjugated to a fluorescent dye FITC
emitting at 470 nm) binding assay to determine apoptotic cell
death by flow cytometer. PI is a membrane impermeant dye
which emits red at 617 nm. It is excluded from viable cells and
used as the second fluorescent marker to detect necrotic cells.
The VTC mediated uptake of the drug was also studied by
pre-saturating the cells with 4 mM VB6 (pyridoxal hydrochloride)
for 1 h prior to the addition of complex 3 so that the channels are
blocked by the externally added VB6. HeLa cells demonstrated a
noticeable difference in uptake of the complex with and without
pre-saturation as compared to that with the normal cells (Figure
S18).
Quantification of Cellular Iron by ICPMS
The cells, pretreated with 0.1 μM of complex 4, were
stained with Annexin-V FITC and PI and then studied in dark
and after photo-irradiation. Complex 4 exhibited ~19% of cell
population in the late apoptotic stage with just 30 min irradiation.
The irradiation time could not be extended any longer as the
complex (IC50 in nanomolar range) was highly phototoxic and
sufficient live cells were not available for FACS analysis on
delaying the same. The population of cells undergoing necrosis
(stained with PI) was significantly low suggesting the overall
apoptotic mode of cell death. The cells were then treated with
two concentrations of the relatively less active complex 3 (6 and
8 μM) followed by irradiation with visible light (400–700 nm) for 1
h. On performing this assay with 4 h post photo-irradiation,
about 15% cells were seen in the Q2 and Q3 (Q = quadrant)
which is indicative of early and late apoptosis respectively. With
a slightly increased concentration, this value rose up to ~55%
with a negligible necrotic population (Figure 5, Figure S19). The
data established the highly photocytotoxic properties of
complexes 3 and 4.[42]
The results obtained from the incorporation studies showed a
differential uptake of complexes 2 and 3 in the cancer cells. To
ascertain this observation, the iron content inside the cells was
measured by the ICPMS (Inductively Coupled Plasma Mass
Spectroscopy) method.
The cells were incubated with complexes 2 and 3 in dark and
the iron content was measured after an incubation period of 4 h.
Intracellular iron content was recorded to be 21 and 127 ng/L ×
105 cells, for complexes 2 and 3, respectively. Thus, the cells
treated with complex 3 showed about 6 times higher iron content
than the non-vitamin analogue 2. This observation also
correlated to the enhanced cytotoxicity seen for complex 3
despite having the same photosensitizing unit L2. This further
strengthens the argument of selective internalization of the
complex due to the appended vitamin B6 moiety.
Reactive Oxygen Species (ROS)
Annexin-V / FITC- PI Assay
ROS is one essential requirement for PDT in inducing cell death
via apoptosis. BODIPY dyes produce singlet oxygen as the ROS
upon photo-activation. Complex 4 was probed by DCFDA (2’,7’-
dichlorofluorescien diacetate) assay for the generation of any
ROS in HeLa cells on exposure to light (400-700 nm) at IC50
Apoptotic cell death was evaluated using Annexin V- fluorescein
isothiocyanate (FITC)/ Propidium Iodide (PI) method. Annexin V
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
Following this observation, a 70 μM solution of DBPF was
treated with 2 μM solution of the complexes 1, 3 and 4 and the
absorbance band intensity was monitored at 5 sec intervals of
photo-irradiation with visible light of 400-700 nm (Luzchem
photoreactor). Complex 4 caused a complete depletion of the
absorption intensity within 120 sec of photo-irradiation. The ROS
generation ability followed the sequence: 4 > 3 >> 1 (Figure 7,
Figure S20). Complex 1 having no photoactive moiety did not
show any significant singlet oxygen generation. The DPBF
absorption decay rate for complex 4 was found to be 13.5 x 10-3
sec-1 which was 4 fold higher than that for complex 3 (4 x 10-3
sec-1 ) and 12 fold higher than complex 1 (0.36 x 10-3 sec-1).
Cellular Localization
Figure 6. Flow cytometric analysis showing ROS generation by complex 4 in
the dark and upon visible light irradiation in HeLa cells. Greater positive shift in
fluorescence of DCF indicates greater extent of ROS generation in visible light
(400-700 nm).
The localization of complex 3 (fluoresces in green) was
monitored in HeLa cells by means of confocal microscopy. The
cells were treated with 5 μM of the complex and incubated for 4
h in dark. The complex showed no apparent localization in the
nucleus and accumulated primarily in some portions of the
cytoplasm. Co-localization studies with Mito-tracker red (MTR)
and Propidium Iodide (PI) showed no significant accumulation of
the complex either in mitochondria (PCC = ~0.3) or in the
nucleus (PCC= ~0.1). Co-staining with Lyso-Tracker Red gave a
Pearson’s correlation coefficient (PCC) value of ~0.7 suggesting
a positive co-localization of complex 3 with Lyso-Tracker (LTR)
(Figure 8).
concentration. Cellular ROS oxidizes DCFDA to 2’,7’-
dichlorofluorescien (DCF) which upon excitation at 488 nm emits
at 525 nm. The emission intensity of DCF was quantified by flow
cytometry giving an indirect estimate of the ROS generation.
Untreated cells did not show any DCF fluorescence, while
significant shift in the spectra was observed for complex 4 on
light exposure indicating formation of ROS. This was not
observed in dark, marking the lack of ROS in the absence of
light exposure (Figure 6). Since two kinds of ROS, i.e. singlet
oxygen via a type-II process or hydroxyl radicals via photo-redox
pathway could be generated in the PDT process, the true nature
of ROS was ascertained by DPBF (diphenylisobenzofuran)
assay and pUC19 DNA photocleavage activity.[43] DPBF forms
endoperoxides upon reaction with singlet oxygen that leads to
the quenching of its absorbance at 414 nm.[44] An earlier report
from our lab showed that the diiodinated BODIPY ligand L3 itself
has a singlet oxygen quantum yield () of 0.8.[45] An indirect
estimation of the same using Rose Bengal as the standard ( =
0.76 in DMSO)[46] gave a  of 0.56 for complex 4 and 0.11 for
complex 3.
This is an interesting observation as targeting lysosomes
can initiate the apoptotic process in cell death.[47] Lysosomes as
“suicidal bags” in the cells contain enzymes to break down the
worn out organelles and other biomolecules. Like the
mitochondria, lysosomes are the attractive targets for initiating
anticancer activity.[48] Lysosomal membrane permeabilization
(LMP) can be triggered by damaging the lysosomes leading to
proton and hydrolase leakage. Specifically, it has been
suggested that the intracellular release of cathepsins into the
cytosol by LMP can activate the intrinsic apoptotic pathway
inducing cell death via apoptosis.[49]
.
DNA Cleavage and Mechanistic Aspects
The DPBF experiments showed singlet oxygen as the
generated ROS by the complexes. To rule out the possibility of
formation of any radical based ROS, the mechanistic aspects of
the plasmid pUC19 DNA photo-cleavage reactions were studied
in the presence DABCO (1,4-diazabicyclo[2.2.2]octane), NaN3
and TEMP (2,2,6,6-tetramethylpiperidine) as singlet oxygen
quenchers, and KI and DMSO as hydroxyl radical scavengers,
catalase as a H2O2 scavenger, and superoxide dismutase (SOD)
as a superoxide radical scavenger.[37] The binding interaction of
the complexes with calf thymus (ct) DNA was studied in 5%
DMF-Tris buffer (pH = 7.2) by UV-Vis absorption method (Figure
S21). The “chemical nuclease” activity of the complexes was
studied in the presence of glutathione (GSH) as a reducing
agent and H2O2 as an oxidizing agent (Figures S22, S23).
Figure 7. (a) Absorption spectral traces of diphenylisobenzofuran (DPBF) and
complex 4 (0.1 μM) in DMSO on exposure to light (400–700 nm, 10 J.cm-2) for
exposure time of 5 sec. (b) Plot showing changes in absorbance of DPBF at
414 nm with time on light exposure with complexes 1, 3 and 4. The slope
gives an indication of the amount of singlet oxygen generated with higher
slope meaning greater singlet oxygen generation.
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
Figure 8. Confocal microscopic images of HeLa cells after 4 h incubation with complex 3: panels (a) and (e) are bright field, panels (b) and (f) are for the
fluorescence of complex 3, panels (c) and (g) are for fluorescence of Mito-Tracker Deep Red and Lyso-Tracker Deep Red respectively , panel (d) shows
the merged image of (b) and (c), and panel (h) shows the merged image of (f) and (g).
showed ∼87% cleavage of SC DNA. The control complex,
The intrinsic binding constant (Kb) values of the complexes
were determined from the absorption spectral traces of
complexes 1, 3 and 4 in 5 mM Tris-HCl buffer (pH 7.2) by
increasing the quantity of calf thymus DNA. The Kb values varied
as: 3  4 >> 1. The DNA cleavage properties were studied by
using supercoiled (SC) pUC19 DNA on photo-irradiation with a
green light source (532 nm) to excite the BODIPY band. The gel
diagram showed significant photo cleavage of DNA in green light
by both 3 and 4 (25 μM). Complexes 2 and 3 showed the
formation of ∼65% of nicked circular (NC) DNA, while complex 4
without the BODIPY unit, showed ~6% SC DNA photo cleavage.
No apparent cleavage of DNA was observed when the samples
were not photo-exposed. The DNA photo-cleavage activity of
complex 4 reduced to only ~15% from 87% in presence of
singlet oxygen quenchers, while the hydroxyl and SOD
scavengers did not show any apparent effect, suggesting
singlet oxygen as the only ROS (Figure 9, Figure S24). D2O as
solvent enhanced the cleavage activity as it increases the
lifetime of singlet oxygen.[50] The iron(III) complexes were
“chemical nuclease” inactive due to stabilization of the metal in
its trivalent oxidation state. The results indicate that PDT activity
of the diidoBODIPY complex 4 is due to singlet oxygen as the
ROS.
Conclusions
Ternary iron(III) complexes having Schiff base with cell targeting
Vitamin-B6 moiety and dipicolylamine bases with appended
photoactive BODIPY units were prepared and their cellular
activities were studied. The complexes having VB6 moiety
showed selective incorporation into the cancer cells than
immortalized transformed cells. Moreover, the complex with a
diiodo-BODIPY moiety exhibited excellent PDT activity in visible
light (400−700 nm), while remaining essentially non-toxic in dark.
The ROS generated during the PDT process was ascertained to
be singlet oxygen while the mode of cell death was apoptosis.
The present VB6 derivative Schiff base complexes were seen to
localize in the lysosomes, which makes them an effective tool to
avoid nuclear excision repair (NER) process associated with the
nuclear DNA causing drug resistance of the currently used
platinum-based chemotherapeutic drugs. The present BODIPY
complexes exemplify the metal-based PDT agents showing
apoptotic cell death by targeting lysosomes instead of the
anticipated mitochondria of the cancer cells. The diiodo-BODIPY
Figure 9. Bar diagram showing the mechanistic aspects of supercoiled (SC)
pUC19 DNA photo-cleavage activity of the diiodoBODIPY complex 4 (25 μM),
in the presence of various singlet oxygen quenchers and radical scavengers
under irradiation with diode laser light of 532 nm (100 mW, 75 min exposure,
lanes 3-12) in the form of %NC DNA (NC, nicked circular): lane 1: DNA alone;
lane 2: DNA + 4 (Dark); lane 3: DNA + 4 (Light); lane 4: DNA + 4 + TEMP (0.5
mM); lane 5: DNA + 4 + DABCO (0.5 mM); lane 6: DNA+ 4 + NaN3 (0.5 mM);
lane 7: DNA + 4 + SOD (superoxide dismutase , 4 units); lane 8: DNA + 4 +
DMSO (4 µL); lane 9: DNA + 4 + KI (0.5 mM); lane 10: DNA + 4 + catalase (4
µL); lane 11: DNA + 4 + D2O (16 µL); lane 12: DNA + 4 + Argon.
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
complex satisfies all the major criteria of PDT thus making it
Synthesis of ligands: The dipicolylamine (dpa) derivatives of
two BODIPY moieties were synthesized following procedures
mentioned in the literature.[29b] The Schiff base ligands were
synthesized by literature procedures.[29a] All the ligands were
characterized by NMR and mass spectroscopy before preparing
their metal complexes.
suitable for further studies for potential in vivo applications.
Experimental Section
Materials and Methods: The reagents and chemicals were
obtained from the commercial sources, viz. S. D. Fine
Chemicals, India, Invitrogen, USA and Sigma–Aldrich, U.S.A.
Solvents used were purified by standard methods.[51] The
complexes were prepared under nitrogen atmosphere. Plasmid
supercoiled (SC) pUC19 DNA (cesium chloride purified) was
Synthesis of [Fe(L1-3)(L4,5)](NO3) (1 - 4): The complexes were
synthesized following a general synthetic procedure where
Fe(NO3)3·9H2O (0.4 g,
1 mmol) was dissolved in 5 mL
anhydrous methanol and dipicolylamine base (0.29 g bzdpa for
1, 0.53 g L2 for 2, 3, 0.79 g L3 for 4) also dissolved in methanol
was added dropwise under nitrogen atmosphere at room
temperature. This reaction mixture was allowed to stir for 2 h.
The Schiff base ligand L4,5 (pyridoxal hydrochloride, 1 mmol,
0.26 g, for 1, 3, 4; salicylaldehyde, 1 mmol, 0.21 g for 2) was
dissolved in 10 mL methanol and then deprotonated by treating
with 2 eq. of triethylamine (0.2 g). This solution was then added
to the reaction mixture and stirred for 3 h. The solution was
filtered, concentrated and precipitated by addition of diethyl
ether. The solid was isolated and washed with cold ethanol, and
then finally dried in vacuum over P4O10.
procured
from
Bangalore
Genie
(India).
Tris(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer (pH =
7.2) was prepared using deionized and sonicated double
distilled water for DNA work. 3-(4,5-Dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT), Dulbecco’s Modified Eagle’s
medium (DMEM), 2,7-dichlorofluorescein diacetate (DCFDA),
2,2,6,6-tetramethyl-4-piperidone (TEMP), ethidium bromide (EB),
calf thymus (ct) DNA, agarose (molecular biology grade), and
Annexin-V-FITC/PI kit were purchased from Sigma–Aldrich (U. S.
A.) and used as received. The dipicolylamine (dpa), BODIPY-
appended dpa (L2) and diiodo-BODIPY-appended dpa (L3) were
synthesized
following
literature
procedures.[29]
[Fe(L1)(L4)](NO3) (1): Yield: 75%.
Anal. Calcd for
Tetrabutylammonium perchlorate (TBAP), prepared by reacting
tetrabutylammonium bromide and perchloric acid, was used for
electrochemical experiments in small quantity (caution!).
Mitotracker Deep Red (MTR), Lyso Tracker Deep Red (LTR)
and fetal bovine serum (FBS) were procured from Invitrogen
U.S.A.
C33H31FeN6O6 (MW: 663.1642): C, 59.74; H, 4.71; N, 12.67.
Found: C, 59.88; H, 4.59; N, 12.60. ESI-MS in MeOH (m/z):
601.1776 [M−NO3]+. IR data [cm−1]: 2900 (br), 2358 (w), 1575
(m), 1384 (s), 1279 (vs), 1010 (m), 818 (w), 740 (vs), 565 (w) (vs,
very strong; s, strong; m, medium; w, weak; br, broad.). UV-
visible in 1:1 DMF:DPBS [λmax/nm (ε/M−1 cm−1)]: 270 (46450),
386 sh (16025); Molar conductivity in 10% aqueous DMF at 298
K [ΛM/S m2 M−1]: 77. μeff [μB] at 298 K: 5.86.
The elemental analysis of the complexes was carried out
using a Thermo Finnigan FLASH EA 1112 CHNS analyzer. The
infrared (IR) and electronic spectra (UV-Vis, emission) were
recorded with Bruker Alpha, Perkin–Elmer spectrum one 55 and
Perkin–Elmer LS 55 fluorescence, spectrometers at room
temperature. The fluorescence quantum yields of the
compounds were obtained by a relative method as described in
the literature.[52] Molar conductivity measurements were
performed with a Control Dynamics (India) conductivity meter.
Magnetic susceptibility measurements of the samples at 298K
were carried out using a magnetic susceptibility balance of
Sherwood Scientific, Cambridge (U. K.) using Hg[Co(SCN)4] as
a standard. Cyclic voltammetric measurements were taken with
an EG&G PAR model 253 VersaStat potentiostat/galvanostat
with electrochemical analysis software 270 and a three electrode
setup consisting of glassy carbon working, platinum wire
auxiliary and a saturated calomel reference electrode (SCE) in
DMF-0.1 M TBAP as the supporting electrolyte. Electrospray
ionization (ESI) mass spectral measurements were carried out
using an Agilent Technologies 6538 Ultra High definition
accurate-mass Q-TOF LC/MS ESI model mass spectrometer.
Flow cytometric analysis were performed using the Becton
Dickinson (BD) FACS (fluorescence-activated cell sorting)
VerseTM flow cytometer. Confocal microscopy cellular images
were acquired from Leica TCS, SP5 microscope with oil
immersion lens of 63X magnification. Iron contents in complexes
2 and 3 treated HeLa cells were measured by Perkin−Elmer
Optima 7000 DV ICP-OES.
[Fe(L2)(L5)](NO3) (2): Yield: 30%.
Anal. Calcd for
C45H41BF2FeN7O5 (MW: 864.5037): C, 62.52; H, 4.78; N, 11.34.
Found: C, 62.57; H, 4.66; N, 11.53. ESI-MS in MeOH (m/z)
802.2702 [M−NO3]+. IR data [cm−1]: 1625 (vs), 1341 (vs), 1282
(vs), 1157 (s), 1024 (m), 874 (w), 749 (s), 598 (w), 523 (w). UV-
visible in 1:1 DMF:DPBS [λmax/nm (ε/M−1 cm−1)]: 270 (47185),
400 sh (26420), 500 (42400) (sh, shoulder); Molar conductivity
in 10% aqueous DMF at 298 K [ΛM/S m2 M−1]: 69; μeff [μB] at 298
K: 5.83.
[Fe(L2)(L4)](NO3) (3): Yield: 30%.
Anal. Calcd for
C46H44BF2FeN8O6 (MW: 909.2794): C, 60.74; H, 4.88; N, 12.32.
Found: C, 60.96; H, 4.85; N, 12.65. ESI-MS in MeOH (m/z):
847.2916 [M−NO3]+. IR data [cm−1]: 3000 (br), 1635 (w), 1577
(w), 1295 (vs), 1168 (m), 992 (m), 739 (m), 583(w). UV-visible in
1:1 DMF:DPBS [λmax/nm (ε/M−1 cm−1)]: 265 (42130), 386 sh
(24330), 500 (45100); Molar conductivity in 10% aqueous DMF
at 298 K [ΛM/S m2 M−1]: 71; μeff [μB] at 298 K: 5.82.
[Fe(L3)(L4)](NO3) (4): Yield: 20%.
Anal. Calcd for
C46H42BF2FeI2N8O6 (MW: 1161.3374): C, 47.57; H, 3.65; N, 9.64.
Found: C: 46.98; H, 3.62; N, 9.92. ESI-MS in MeOH (m/z):
1099.0849 [M−NO3]+. IR data [cm−1]: 3000 (br), 2354 (w), 1578
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
(m), 1389 (s), 1394 (s), 1294 (vs), 1156 (m), 992 (m), 820 (w),
751 (m), 518 (w). UV-visible in 1:1 DMF:DPBS [λmax/nm (ε/M−1
cm−1)]: 265 (37455), 407 sh (25140), 532 (35520); Molar
conductivity in 10% aqueous DMF at 298 K [ΛM/S m2 M−1]: 73;
μeff [μB] at 298 K: 5.85.
performed to observe the effect of varying concentrations of VB6
on the cellular uptake using flow cytometry.
DCFDA assay was performed for intracellular ROS estimation
using the fluorogenic dye, 2’7’-dichlorofluorescein diacetate
(DCFDA, 287810, Calbiochem®, USA). The cells were seeded
in 6 wells plate and incubated for 24 h. Subsequently, the cells
were treated with the compounds for 4 h at 37 °C in dark and
then photo-exposed for 1 h. Post exposure, the cells were
washed, trypsinized and re-suspended in DMEM medium.
Viable cell numbers were enumerated by Trypan blue exclusion
assay using a hemocytometer. About 0.25 x 106 viable cells
were stained with 10 µM of DCFDA and incubated at 37 °C for
30 min in dark. Next, the cells were washed, re-suspended in
PBS and acquired (10,000 cells in the live singlet gate) on the
BD FACS VerseTM flow cytometer (BD Biosciences, USA). The
results were analyzed and the representative histograms were
constructed using the flow cytometry analysis software, FlowJo®
Theoretical Methods: The geometries of the complexes 1, 3
and 4 were optimized by density functional theory (DFT) using
B3LYP level of theory and LanL2DZ basis set as implemented in
Gaussian 09 program.[34] The electronic transitions with their
transition probability were obtained using linear response time
dependent density functional theory (TDDFT).
Cellular experiments: The cancer cells HeLa, MCF7, and
HepG2 and the immortalized non-cancer HPL1D cells were
grown in 100 mm polymer culture dishes (SPL Life sciences,
Korea) in DMEM supplemented with 10% FBS, 100
U
(USA)[54]
.
penicillin/mL, 100 µg streptomycin/mL and incubated in a
humidified 5% CO2 incubator (Sanyo, UK) at 37 C. “MTT Assay”
was performed to understand the photocytotoxic effect of the
complexes in these cell lines. For individual cell lines, ~8 x 103
cells were seeded in two 96 well plates in 100 µL medium per
well and were grown for 24h at 37 °C in a CO2 incubator. The
complexes were dissolved in 1% DMSO and were added to the
cells at different concentrations. Incubation was continued for
another 4h. Thereafter, the medium of one plate was replaced
with DPBS and photo-irradiated for 1h in broad-band visible light
(400-700 nm) using a Luzchem Photoreactor (Model LZC-1,
Ontario, Canada; light fluence rate: 2.4 mW cm-2; light dose: 10
J cm-2). This was followed by removal of DPBS and addition of
fresh medium. The medium of the other plate was discarded and
replaced with fresh medium. Both the plates were incubated for
a further period of 20h in the dark which was followed by
addition of 25 µL MTT (4 mg mL-1) in each well and additional 3
h incubation in dark. Finally the culture medium was discarded
from each well of the plates and 200 µL DMSO was added to
each well to solubilize the purple formazan crystals. The
absorbance at 540 nm was measured using a Molecular
Devices Spectra Max M5 plate reader. The cytotoxicity of the
complexes was measured as the percentage ratio of
absorbance of treated cells to the untreated controls. The IC50
values were determined by nonlinear regression analysis (Graph
Pad Prism 6).
For quantification of cellular iron by ICPMS, ~1x105 HeLa cells
were grown in 35 mm culture dishes in DMEM supplemented
with 10% FBS. The complexes 2 and 3 were dissolved in DMSO
in their IC50 concentrations and added to the cells and incubated
for 4 h. The culture medium was then discarded, cells were
trypsinized and washed with cold DPBF. The cell pellets were
prepared by spinning them down at 1000 rpm for 10 min.
Subsequently, the pellets were digested with 70% HNO3 at 65
C for 2 h followed by a dilution to a final concentration of 2%
nitric acid. The iron content was then measured using the
spectrometer previously calibrated with standard ferric nitrate
solutions.
In Annexin V / Propidium iodide (PI) Assay, the cells were
stained with Annexin V-FITC and PI. The cells were seeded in 6-
well plates and incubated for 24 h, post which, the cells were
washed and treated with the mentioned compounds for 4 h at
37°C in dark followed by photo-exposure for 1 h. The cells were
subsequently washed, trypsinized and resuspended in DMEM
medium. The viable cells were enumerated by Trypan blue
exclusion assay using a hemocytometer and ~0.25 x 106 viable
cells were stained with Annexin V-FITC and PI, according to the
manufacturer’s instructions (APOAF, Sigma-Aldrich, USA). The
cells were acquired (10,000 events in the singlet gate) on the BD
FACS VerseTM flow cytometer (BD Biosciences, USA) and the
BD FACSDiva™ software was used to analyze, construct the
Annexin V-FITC versus PI dot-plots and quantify the results.
For cellular incorporation assay, in 6-well plates ~0.3 x 106
cells were seeded per well and were grown for 24 h.
Subsequently the cells were washed and incubated for 4 h in
dark. The cells were then washed with PBS, trypsinized, re-
suspended in PBS and acquired on the BD FACS VerseTM flow
cytometer (BD Biosciences, USA). The cells were selected on
the singlet gate (forward scatter-area versus forward scatter-
height) and live gate (forward scatter-area versus side scatter-
area) and ~10,000 events were recorded. The results were
analyzed using the flow cytometry analysis software, FlowJo®
(USA) and the histograms were constructed.[53] The experiment
was repeated with cells that were pretreated with pyridoxal
hydrochloride (2 mM) 4 h prior to treatment with the complexes.
Dose-dependent estimation of cellular uptake study was
The cellular localization pattern of the fluorescent complex
3 (10 μM) in HeLa cells was visualized using a confocal
microscope. Standard experimental procedures were adopted
for cell plating and post treatment processing.[38] To understand
the sub-cellular localization of the complex, HeLa cells were
incubated with 10 µM of complex for 4 h in dark. Post incubation,
the medium was discarded and the cells were washed thrice
with DPBS. Lyso Tracker Red (250 nM) was added to the cells
and incubated for 10 min at room temperature. The live cells
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10.1002/ejic.201701487
European Journal of Inorganic Chemistry
FULL PAPER
were then directly imaged under a confocal microscope (TCS
SP5, Leica, Germany) using 63X HCX APO L U-V-I water
immersion objective (NA 0.9).
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DNA binding and cleavage experiments: DNA binding
constants of the complexes were obtained from UV-visible
spectral studies using calf thymus (ct) DNA by the reported
procedures.[55] The DNA photocleavage activity of the
complexes was studies using supercoiled (SC) pUC19 plasmid
on light irradiation using diode lasers and by gel electrophoresis.
A continuous-wave (CW) diode laser made of Research Electro-
Optics, Colorado; (U. S. A.), model no. EXLSR-532-100-CDRH
was used as a monochromatic visible light of 532 nm (100 mW
power, 1 h exposure time, 0.32 ± 0.02 mm beam diameter).
External agents, viz., sodium azide and TEMP (2,2,6,6-
tetramethyl-4-piperidone) as singlet oxygen quenchers and
DMSO, KI and catalase as hydroxyl radical scavengers and
superoxide dismutase (SOD) as a superoxide scavenger were
used for mechanistic studies to determine the nature of the ROS
generated using procedures that are reported earlier.[37]
425–438; b) S. Pandey, P. Garg, S. Lee, H.W. Choung, Y.H.
Choung, P.H. Choung, J.H. Chung, Biomaterials 2014, 35,
9332–9342.
Supporting Information (see footnote on the first page of
this communication): General synthetic scheme of ligands and
complexes (Scheme S1), Coordinates from DFT study (Tables
S1-S4), ESI-MS spectra (Figures S1-S4), IR spectra (Figure S5),
cyclic voltammograms (Figures S6,S7), Energy optimized
geometry and HOMO–LUMO diagram of complex 2 (Figure S8),
Stability plots for ligands (Figure S9), Stability plots for
complexes (Figure S10), Singlet oxygen quantum yield plot
(Figure S11), MTT assay bar diagram (Figure S12),
Concentration vs % cell viability MTT plots (Figures S13-S16),
time dependent incorporation studies (Figure S17), effect of
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3 (Figure S20), DNA binding MVH plots (Figure S21), and gel
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Acknowledgements
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/2007,
EMR/2015/000742). A.R.C. thanks DST for J.C. Bose national
fellowship. We thankfully acknowledge the supports of the DBT-
IISc partnership program, the Bio-imaging and the flow
cytometry facility Division of Biological Sciences, IISc. We thank
Vasista Adiga from the Center for Infectious Diseases Research
(CIDR) for helping with FACS analysis. The authors also thank
the Alexander von Humboldt Foundation for donation of an
electroanalytical system.
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Keywords: Bioinorganic chemistry • Iron • Antitumor agents •
Borondipyrromethene derivatives • Vitamin B6
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Ternary iron(III) complexes
of NNN-donor ligands having
pendant BODIPY moieties
and O,N,O-donor vitamin-B6
Schiff base showed singlet
Key Topic*: Photodynamic Therapy
Somarupa Sahoo, Santosh Podder,
Aditya Garai, Shamik Majumdar,
Nandini Mukherjee, Uttara Basu,
Dipankar
Nandi*,
Akhil
R.
oxygen
induced
photo-
Chakravarty*
cytotoxicity in the cancer
cells with apoptotic cell
Page No. – Page No.
death
localization from imaging
studies. The complexes
and
lysosomal
Title:
Iron(III) Complexes of Vitamin-B6
Schiff Base with Borondipyrro-
methene Pendants for Lysosome
Selective Photocytotoxicity
displayed higher cellular
uptake in the cancer cells
compared to normal cells.
This article is protected by copyright. All rights reserved.