Mitochondria targeting photocytotoxic oxidovanadium(IV) complexes of curcumin and (acridinyl)dipyridophenazine in visible light

Article abstract of DOI:10.1002/zaac.201300569

Oxidovanadium(IV) complexes, [VO(acac)(L)Cl] (1), [VO(cur)(L)Cl] (2), and [VO(scur)(L)Cl] (3) {acac = acetylacetonate, cur = curcumin monoanion, scur = diglucosylcurcumin monoanion, L = 11-(9-acridinyl)dipyrido[3, 2-a:2',3'-c]phenazine (acdppz)}, were prepared and characterized. The complexes are non-electrolytic in DMF and 1:1 electrolytic in aqueous DMF. The one-electron paramagnetic complexes showed a d-d band near 725 nm in aqueous DMF and green emission near 520 nm in aqueous DMSO. The complexes exhibited an irreversible V^IV/V^III redox response near-0.85 V versus SCE in aqueous DMF. The complexes showed good binding strengths to calf thymus DNA (K_b: 3.1×10⁵-9.6×10⁵ M ^-1) and efficient pUC19 DNA photocleavage activity in red light of 705 and 785 nm by singlet oxygen (¹O₂) pathway. Complexes 1 and 2 exhibited significant photocytotoxicity (IC₅₀^: 0.1-1.0 μM) in visible light (400-700 nm) with low dark toxicity (IC ₅₀: >20 μM) in HeLa and HaCaT cells. Complex 3 was cytotoxic in both light and dark. DNA ladder formation experiments indicated cell death via apoptotic pathway. Confocal microscopy done with 1 and 2 revealed primarily cytosolic localization of the complexes with significant presence of the complex in the mitochondria as evidenced from the imaging data using mitotracker red. Copyright

Full text of DOI:10.1002/zaac.201300569

Job/Unit: Z13569
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Date: 12-03-14 18:51:14
Pages: 11
ARTICLE
DOI: 10.1002/zaac.201300569
Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes of
Curcumin and (Acridinyl)dipyridophenazine in Visible Light
[
a]
[a]
[b]
[a]
Samya Banerjee, Puja Prasad, Imran Khan, Akhtar Hussain,
Paturu Kondaiah,*[ and Akhil R. Chakravarty*
b]
[a]
Dedicated to Professor C. N. R. Rao on the Occasion of His 80th Birthday
Keywords: Curcumin; Vanadium; Photocytotoxicity; Apoptosis; Cytosolic localization
5
5
–1
Abstract. Oxidovanadium(IV) complexes, [VO(acac)(L)Cl] (1), to calf thymus DNA (K
b
: 3.1ϫ10 –9.6ϫ10 m ) and efficient pUC19
[
VO(cur)(L)Cl] (2), and [VO(scur)(L)Cl] (3) {acac = acetylacetonate, DNA photocleavage activity in red light of 705 and 785 nm by singlet
1
2
cur = curcumin monoanion, scur = diglucosylcurcumin monoanion, oxygen ( O ) pathway. Complexes 1 and 2 exhibited significant photo-
L = 11-(9-acridinyl)dipyrido[3,2-a:2’,3’-c]phenazine (acdppz)}, were cytotoxicity (IC50 0.1–1.0 μM) in visible light (400–700 nm) with low
prepared and characterized. The complexes are non-electrolytic in dark toxicity (IC50: Ͼ20 μM) in HeLa and HaCaT cells. Complex 3
:
DMF and 1:1 electrolytic in aqueous DMF. The one-electron paramag-
was cytotoxic in both light and dark. DNA ladder formation experi-
netic complexes showed a d-d band near 725 nm in aqueous DMF ments indicated cell death via apoptotic pathway. Confocal microscopy
and green emission near 520 nm in aqueous DMSO. The complexes done with 1 and 2 revealed primarily cytosolic localization of the com-
IV III
exhibited an irreversible V /V redox response near –0.85 V versus plexes with significant presence of the complex in the mitochondria
SCE in aqueous DMF. The complexes showed good binding strengths
as evidenced from the imaging data using mitotracker red.
Introduction
ing of this moiety to an oxophilic metal ion is reported to
increase the hydrolytic stability and bioavailability of curcu-
Curcumin, the major ingredient of turmeric, is used as tradi-
tional herbal medicine to treat infections, bite, burns, and skin
[7,8]
min.
Although several studies on the anticancer properties
of curcumin and its metal complexes are reported, their utility
as photodynamic therapy (PDT) agents remains virtually unex-
plored.[
[1]
diseases. It is known to show a wide range of biological
activities.[ It exhibits anticancer activity due to its ability to
induce apoptosis in cancer cells without any significant cyto-
toxic effects on normal cells.[ Curcumin is reported to inter-
fere with the activity of the transcription factor NF-κB which
is a protein complex involved in the transcription of DNA and
2]
3,6,9]
The motivation to explore the medicinal chemistry of curcu-
min in light has led us to design and synthesize new ternary
oxidovanadium(IV) complexes of curcumin and its glyc-
osylated derivative showing photo-induced cytotoxicity in vis-
ible light in cancer cells. Vanadium is a biocompatible metal
3]
known to enhance the activity of the tumor suppressor
protein p53.[2,4,5]
However, clinical application of curcumin
ion and its complexes have earlier been used as insulin mi-
[Hcur = 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-hepta-diene-
metics and antitumor agents.[
10,11]
Bleomycin oxidovanadi-
3,5-dione] is limited due to its poor bioavailability and phar-
2+
um(IV) and [VO(phen)(H O) ] are known to show chemical
2
2
macokinetic profile resulting from its hydrolytic instability un-
der physiological conditions.[ This is largely attributed to the
presence of a reactive β-diketone moiety in its structure. Bind-
[12,13]
6]
nuclease activity in the presence of H O .
There are few
2
2
reports on oxidovanadium(V) and peroxidovanadium(V) com-
plexes cleaving DNA on photo-activation using UV light
*
*
Prof. Dr. P. Kondaiah
source, which is undesirable in photodynamic therapy (PDT)
E-Mail: paturu@mrdg.iisc.ernet.in
Prof. Dr. A. R. Chakravarty
Fax: +91-80-23600683
[14,15]
due low tissue penetration of UV light.
In contrast, oxido-
vanadium(IV) complexes displaying a low energy d-d absorp-
E-Mail: arc@ipc.iisc.ernet.in
tion band in the red light window are suitable for PDT con-
[
[
a] Department of Inorganic and Physical Chemistry
Indian Institute of Science
[16,17]
sidering greater tissue penetration of red light.
Besides,
Bangalore-560012, India
the green emission of 11-(9-acridinyl)dipyrido[3,2-a:2Ј,3Ј-c]-
phenazine (acdppz) and curcumin (Hcur) complexes designed
in this work could be utilized to study the cellular uptake and
localization of the complexes in cancer cells by confocal
fluorescence microscopy. We have designed and prepared new
ternary oxidovanadium(IV) complexes based on our dual strat-
b] Department of Molecular Reproduction,
Development and Genetics
Indian Institute of Science
Bangalore 560012, India
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/zaac.201300569 or from the au-
thor.
Z. Anorg. Allg. Chem. 0000, ᭿,(᭿), 0–0
© 0000 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
1

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P. Kondaiah, A. R. Chakravarty et al.
ARTICLE
egy of combining imaging with therapy. Glycosylation of cur- involves the mitochondria as is known for the PDT drug Pho-
®
[22–25]
cumin is done to increase the aqueous solubility of the re- tofrin .
The mitochondria targeting PDT agents could
sulting complexes.[ In addition, appending a glucose moiety overcome any resistance mechanism that are involved for con-
has proved to be successful in augmenting the cellular uptake ventional chemotherapeutic agents targeting the nuclear DNA.
of the compounds into the cancer cells having high density of In addition, the present complexes show cellular damage in
18]
glucose receptors compared to that in the normal cells.[ We light generating singlet oxygen ( O ) as the reactive oxygen
19]
1
2
®
have chosen acdppz ligand in the ternary structure for its green species (ROS) like Photofrin as evidenced from the mechan-
emissive and strong DNA intercalating property. The increased istic data obtained from the plasmid DNA photocleavage ex-
planarity and conjugation in this ligand is expected to enhance periments. Having many functional similarities with the PDT
the photocytotoxicity of the resulting complexes.[
20]
drug Photofrin , complex 2 is unique in the chemistry of 3d
®
Herein we present the synthesis, characterization, and vis- metal-based PDT agents.[26]
ible light-induced photocytotoxicity of three new oxidovanadi-
um(IV) complexes, [VO(acac)(L)Cl] (1), [VO(cur)(L)Cl] (2)
and [VO(scur)(L)Cl] (3) {acac = acetylacetonate (in 1), Hcur Results and Discussion
=
curcumin (in 2), Hscur = diglucosylcurcumin (in 3), and
L = 11-(9-acridinyl)dipyrido[3,2-a:2Ј,3Ј-c]phenazine (acdppz) Synthesis and Characterization
in 1–3)} (Figure 1). Significant results of this study include
(
The complexes were prepared in good yield by treating van-
adyl sulfate with barium chloride in aqueous ethanol (1:5 v/v
water-ethanol) and the filtrate after removal of barium sulfate
was subsequently reacted with acdppz base in chloroform-eth-
anol (2:3 v/v) and respective acetylacetone (Hacac), curcumin
(Hcur), or glycosylated curcumin (Hscur) ligand in acetoni-
trile-ethanol mixture. The complexes were characterized from
the analytical and physicochemical data (Table 1). The ESI-
MS of the complexes showed essentially a single peak corre-
remarkable photocytotoxicity of the complexes at nanomolar
concentration in human cervical cancer cell line (HeLa) (IC :
0
5
0
.3–1.0 μM) and human skin keratinocyte cell line (HaCaT)
IC : Ͻ0.4 μM) in visible light of 400–700 nm with low dark
(
5
0
toxicity thus showing similar activity as the clinically approved
PDT drug Photofrin .
desirable cytosolic uptake of the complexes (1, 2) within 4 h
of incubation, which compares well with that of Photofrin .
Observation of mitochondrial localization of the complexes is
significant considering that the pathway of apoptosis largely
®
[21]
The cellular imaging studies showed
®
+
sponding to the [M – Cl] in MeOH. The complexes were
found to be stable in the solution phase as five-coordinate spe-
cies [VO(cur)(L)]Cl as evidenced from the mass spectroscopic
data. The complexes gave molar conductance values of approx.
2
–1
2
0 and approx. 95 S·m ·m in pure DMF and 20% aqueous
DMF, respectively, suggesting their non-electrolytic behavior
in DMF and 1:1 electrolytic nature in aqueous medium due to
dissociation of the chloride ligand.[ The room temperature
magnetic moment (μ ) values of 1.6 μ implied one-electron
27]
eff
B
1
IV
paramagnetic nature of the 3d -V complexes. The IR spectra
of the complexes showed three bands centred around 1588,
495, and 965 cm–1 for the C=O, C=C (β-diketonate), and
1
[
28]
V=O stretching vibrations, respectively.
The strong inten-
sity IR band at 1588 cm–1 (C=O stretching) indicates bidentate
coordination mode of the β-diketone (acac/cur/scur) ligand to
the central metal atom. The complexes displayed a d–d band
in the electronic spectra in the wavelength range of 717–
729 nm in 20% DMF-water (Figure 2, Table 1). Complex 2
showed an intense curcumin-based visible band at 437 nm due
to π Ǟ π* transition with a shoulder at around 454 nm. The
Figure 1. Schematic drawing of the complexes 1–3 and the ligands
used.
[
9]
Table 1. Selected physicochemical data and ct-DNA binding parameters of the complexes 1–3.
IR /cm–1
Electronic a)
:
λ
/nm (Φ
b))
μ
eff
c)
Λ
d)
/S·m ·M
2
–1
E
pc /V
e)
K
f)
/M–1
ΔT
g)
/°C
Complex
f
f
M
b
m
ν˜ (C=O)
ν˜ (V=O)
λmax /nm
3
–1
–1
(
ε /dm ·M ·cm )
1
2
3
1591
1586
1589
965
967
962
717 (47)
725 (51)
729 (54)
523(0.02)
520(0.03)
516(0.026)
1.63
1.65
1.62
111
92
81
–1.01
–0.84
–0.82
(3.1Ϯ0.2)ϫ105
5.1
6.5
7.4
5
(8.9Ϯ0.4)ϫ10
(9.6Ϯ0.1)ϫ105
a) Visible electronic spectral band in 20% DMF-water. b) Emission spectra and quantum yield of 1–3 in aqueous DMSO (1:20 v/v), λexi (nm)
390. c) Magnetic moment (μeff) in μB obtained by NMR method at 298 K. d) Λ , molar conductivity in 20% aqueous DMF at 25 °C. The
=
M
–
1
complexes are non-electrolytic in pure DMF. e) Cathodic peak potential in 20% aq. DMF-0.1 M TBAP at 50 mV·s scan rate. f) Equilibrium
ct-DNA binding constant from the UV/Vis absorption experiments. g) Change in the ct-DNA melting temperature.
2
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Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes
π Ǟ π* band for the diglucosylcurcumin complex 3 was ob- and 3 using B3LYP/LANL2DZ level of DFT.[30] The energy
served at 426 nm. The acdppz ligand in the complexes showed optimized structures of the complexes showed an oxidovanadi-
two additional bands near 362 nm and 390 nm assignable to the um(IV) moiety bonded to the chelating N,N-donor acdppz,
π–π* transition of the dppz and acridine chromophores.[
20,29]
O,O-donor monoanionic cur/scur ligand, and a chloride anion
IV
giving a V N O Cl core in a distorted octahedral arrange-
2
3
ment. The axial V=O bond length is 1.61 Å, whereas the other
V–O bond lengths are 1.97 Å. It is evident in the optimized
structure that the chloride ligand being at the trans position of
the V=O is labile with a V–Cl bond length of 2.57 Å. This
explains the dissociation of the chloride ligand in aqueous
solution phase as is evidenced from the ESI-MS and molar
conductivity data. The dihedral angle between the phenazine
ring and the acridine moiety is 68.5° and this does not allow
any effective π conjugation between two planar chromophores.
In both the structures, the HOMO is concentrated over the cur-
cumin moiety, whereas the LUMO is located over the phena-
zine ring.
Figure 2. Electronic absorption spectra of complexes 1 (___), 2 (···) Cytotoxicity
and 3 (-·-) in 20% aqueous DMF.
Photocytotoxicity of the complexes 1–3 was studied on hu-
man skin keratinocyte cell line (HaCaT) and human cervical
The complexes showed an emission band near 520 nm in
aqueous DMSO (1:20 v/v) giving fluorescence quantum yield
cancer cell line (HeLa) using an MTT [3-(4,5-dimethylthi-
(φ) values in the range of 0.02–0.03 (Figure 3, Table 1). The
[31]
azole-2-yl)2,5-diphenyltetrazolium bromide] assay.
We
curcumin ligand alone gave a φ value of 0.042 under similar
experimental conditions. The curcumin ligand-based fluores-
cence is thus retained in the oxidovanadium(IV) complexes
and this allowed us to study the cellular uptake and localization
of the complexes by confocal imaging. Cyclic voltammetry of
the complexes showed a reduction process assignable to the
have used the HaCaT cell line to know about the efficacy of
the complexes in PDT and HeLa is used as a normal cancer
cell line. The complexes were incubated for 4 h followed by
photo-irradiation in visible light (400–700 nm photoreactor,
–2
1
0 J·cm ) for 1.0 h (Figure 4). The IC50 values of complex 1
were found to be 0.34(Ϯ0.03) μM in HaCaT and
.99(Ϯ0.05) μM in HeLa cells. This complex showed no
IV III
V -V redox couple near –0.85 V versus S.C.E. in 20%
0
DMF-H O with 0.1 m [nBu N](ClO ) (TBAP) as the support-
2
4
4
dark cytotoxicity in both the cell lines. The IC50 values of
complex 2 in light were 0.18(Ϯ0.02) μM in HaCaT and
ing electrolyte. The complexes did not show any oxidative re-
2
+
sponse. The redox stability of the VO moiety over a wide
potential window could reduce the dark toxicity of the com-
plexes.
0
.71(Ϯ0.07) μM in HeLa cells. Complex 3 having a curcumin
derivative containing a sugar moiety gave IC50 values of
.13(Ϯ0.03) μM in HaCaT and 0.33(Ϯ0.02) μM in HeLa cells.
0
One reason for the higher photocytotoxicity of complex 3 may
be the presence of the sugar moiety, which possibly increases
the cellular uptake of this complex in the cancer cells. Com-
Figure 3. Emission spectra of 1–3 in aqueous DMSO (1:20 v/v)
(λexcitation = 390 nm).
Computational Study
Figure 4. Photocytotoxicity of the complexes 1–3 from the MTT assay
in HeLa and HaCaT cells on 4 h incubation in dark (black bar) and in
To rationalize the photophysical properties of the com-
plexes, computational studies were performed for complexes 2 visible light of 400–700 nm (gray bar).
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P. Kondaiah, A. R. Chakravarty et al.
ARTICLE
Table 2. The IC50 /μM values of 1–3 and selected compounds in HaCaT and HeLa cells.
Compound
HaCaT
in visible light
HeLa
in visible light
a)
a)
in dark
in dark
1
2
3
0.34(Ϯ0.03)
0.18(Ϯ0.02)
0.13(Ϯ0.03)
Ͼ10
Ͼ50
19.0(Ϯ0.2)
Ͼ30
Ͼ10
11.8(Ϯ0.6)
Ͼ10
Ͼ50
0.99(Ϯ0.05)
0.71(Ϯ0.07)
0.33(Ϯ0.02)
9.5(Ϯ0.3)
Ͼ30
Ͼ10
4.2(Ϯ0.1)
Ͼ20
Ͼ50
acdppz
Hscur
Curcumin®
Photofrin
b)
Ͼ50
8.2Ϯ(0.2)
c)
28.1(Ϯ0.4)
85.4(Ϯ0.6)
Ͼ41
d)
e)
4.3Ϯ0.2
a) Light of 400–700 nm wavelength. b) Data from Ref. [7b]. c) Data from Ref. [8]. d) Data from Ref. [21]. e) Red light of 633 nm.
plex 3, however, showed undesirable dark cytotoxicity giving by cellular ROS to generate fluorescent DCF with an emission
IC50 values of 11.8(Ϯ0.6) μM in HaCaT and 4.23(Ϯ0.08) μM maxima at 525 nm.[ The percentage of cell population gen-
in HeLa cells. This could be due to higher cellular uptake of erating ROS could be determined by flow cytometry analysis.
the complex. Cytotoxicity of the ligands, viz. curcumin, sugar- The assay data in HaCaT cell line indicate the formation of
appended curcumin, and acridine-dppz was measured and the ROS by complex 2 on exposure to visible light, while there
data are given in Table 2. The complexes showing photocyto- was no ROS formation in dark.
34]
toxicity in nanomolar concentration with minimal dark toxicity
are of importance in the chemistry of PDT.
Cellular Imaging and Uptake
The emission property of the complexes was used to study
Mechanistic Study of Cellular Damage
their cellular uptake and localization in the HaCaT and HeLa
DNA ladder assay is a convenient way to detect pro- cells by confocal microscopy. Propidium iodide (PI) was used
grammed cell death or apoptosis. Cellular apoptosis is due to as a nuclear staining dye to ascertain any nuclear uptake of the
the activation of endonuclease activity.[ This activation leads complexes. Complexes 1–3 in HaCaT and HeLa cells showed
to the degradation of genomic DNA at inter-nucleosomal significant accumulation within 2 h and were retained up to 4
linker region forming 180 to 185 base pair of DNA fragments. h. The uptake of the complexes in 4 h was found to be more
These DNA fragments can be analyzed by gel electrophoresis. than that in 2 h. On comparing the merged image with the
We analyzed the mode of cell death from the DNA laddering green fluorescence of the complex and red fluorescence of PI
experiment for complexes 1 and 2. It is evident from Figure 5 in Figure 6 (see Figure S18 for a colored image), complexes 1
that both the complexes show DNA ladder only in the presence and 2 were seen to localize mainly in the cytoplasm [panels
of visible light (400–700 nm) in HaCaT cells indicating (c), (f), (l) and (o)], whereas complex 3 localizes in the nucleus
apoptosis as the primary mode of cell death. This experiment as well as in cytoplasm [panels (i) and (r)]. The nuclear mor-
was also done for the complexes in HeLa cells, but only com- phology was found to remain intact as evident from the micro-
plex 2 gave ladder like pattern. No ladder formation was ob- scopy images indicating the nontoxic nature of the complexes
served in dark indicating that the complexes become cytotoxic in dark. The results are indeed remarkable since the complexes
only on light exposure, while remaining innocuous to cells in could serve the dual purpose of imaging the tumor for its de-
dark. We probed generation of any reactive oxygen species tection and remaining passive within the cells in absence of
32]
(
(
ROS) from 2Ј,7Ј-dichlorodihydrofluorescein diacetate light and become cytotoxic on photo-irradiation damaging the
DCFDA) assay.[ Cell permeable DCFDA could be oxidized tumor.
Further study using mitochondrial tracker deep red in
33]
HaCaT cells showed partial localization of the complexes 1
and 2 in the mitochondria as evidenced from the yellow color
in the merged image resulting from the green fluorescence of
the complex and the red fluorescence of the mitotracker red
(Figure 7, see Figure S19 for a colored image). We had to use
low concentration of the complexes 1 and 2 to avoid any light-
induced cell damage during the imaging experiments and this
resulted in low fluorescence intensity. Complex 3 could not be
used for imaging study due to its high cellular toxicity in dark.
DNA Binding Properties
With the knowledge that the complexes partially localizing
in the mitochondria and the mitochondrial DNA could be a
possible site of binding, we investigated the DNA binding
Figure 5. DNA ladder of the complexes 1 and 2 in HeLa and HaCaT
cells showing apoptosis (D = dark, L = light, M = Marker 100 bp).
4
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Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes
Figure 7. Confocal images of complexes 1 and 2 (10 μM) in HaCaT
cells after 4 h showing the cellular uptake. Panels (a) and (d) show
green fluorescence of complexes 1 and 2, respectively. Panels (b) and
e) are red fluorescence images of mitotracker red (MTR, 50 nM). Pan-
els (c) and (f) are the merged fluorescence images of the mitotracker
and the complex. Scale Bar: 10 μm (for a colored image, see Figure
S19).
(
DNA gradually dissociates to single strands on increasing the
solution temperature. The melting temperature T which is de-
m
fined as the temperature where half of the total base pairs gets
non-bonded is a valuable parameter. The DNA melting studies
in phosphate buffer (pH 7.2) showed stabilizing interaction of
all the complexes with ct-DNA (Table 1, Figure 8). The ΔTm
values for the complexes lie in the range of 5.1 to 7.4 °C and
Figure 6. Confocal images of HeLa and HaCaT cells (after 4 h) treated
it is 10.6 °C for the DNA intercalator ethidium bromide
–
1
with complexes 1–3 (10 μM) and propidium iodide (PI, 10 mg·mL ):
panels (a), (d), (g), (j), (m) and (p) show the green emission of the
complexes; panels (b), (e), (h), (k), (n), and (q) correspond to the red
36]
(
EB).[
The ct-DNA binding strengths follow the order:
3
Ͼ 2 Ͼ 1. The DNA melting data indicate intercalative mode
emission of PI; and panels (c), (f), (i), (l), (o) and (r) show the merged of binding of the complexes to the ct-DNA.
images. Scale Bar: 10 μm (for a colored image, see Figure S18).
strengths of the oxidovanadium(IV) complexes using absorp-
tion titration, DNA melting and viscosity measurements. The
equilibrium binding constants (K ) of the complexes 1–3 to
b
ct-DNA were obtained from UV/Vis absorption titrations by
monitoring the change in the absorbance of the ligand-based
band of the complexes at 267 nm (ct-DNA = calf thymus
DNA). Binding of a complex to DNA generally leads to hy-
pochromism along with a bathochromic shift of the electronic
spectral bands resulting from strong stacking interaction be-
tween the aromatic chromophore of the ligand in the complex
and the base pairs of DNA.[
35]
Figure 8. DNA melting plots of 190 μM ct-DNA alone and in the
presence of 1–3 (20 μM) in 5 mM phosphate buffer (pH 7.2).
The extent of hypochromism
thus gives an estimate of the binding strength. The observed
trend in hypochromism among the present oxidovanadium(IV)
complexes follows the order: 3 Ͼ 2 Ͼ 1. The equilibrium bind-
Viscosity measurements were carried out to examine the ef-
ing constant (K ) values for the complexes range within fect on the specific relative viscosity of ct-DNA upon addition
b
5
5
–1
(
3.1Ϯ0.2)ϫ10 to (9.6Ϯ0.1)ϫ10 m giving the same order of the complexes. DNA intercalating compounds result in a
as observed in the trend of hypochromism (Table 1). change in the relative viscosity by unwinding and elongation
Thermal behavior of DNA in the presence of the complexes of the DNA double helix. For example, EB shows significant
provides information on the conformational changes and the increase in the relative viscosity of the ct-DNA solution on
strength of the DNA complex interaction. The double-stranded intercalation due to an increase in the overall DNA contour
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P. Kondaiah, A. R. Chakravarty et al.
ARTICLE
Figure 10. Bar diagram showing the photocleavage of SC pUC19
DNA (30 μM) by the ligands and the complexes 1–3 (30 μM). The
reactions conditions are: in dark shown by black bars, in red light of
Figure 9. Plots showing the effect of increasing the concentration of
ethidium bromide (EB) (᭢), 1 (᭹), 2 (᭡), 3 (᭿), and Hoechst 33258
(᭣) on the relative viscosity of ct-DNA (150 μM) at 37.0Ϯ0.1 °C in
705 nm shown by shaded bars with stripes and in red light of 785 nm
Tris-HCl buffer (5 mM, pH 7.2).
showing dark shaded bars.
length. In contrast, groove binding or partially intercalating
molecules cause little or no effect on the relative viscosity of
centration. A 30 μM solution of 2 induced ca. 85% DNA
cleavage on exposure for 1 h. Complex 3 showed 92% cleav-
age of the SC DNA to its nicked circular (NC) form under
similar reaction conditions (Table 3). Control experiments in
dark did not show any significant DNA cleavage activity. The
curcumin and glycosylated curcumin ligands alone showed
[37]
the ct-DNA solution.
The groove binder Hoechst 33258,
used as a reference compound, showed no apparent change in
the DNA solution viscosity. The plots of relative viscosity
1
/3
(
η/η0)
vs. [complex]/[DNA] ratio for 1–3, where η and η0
are respective specific viscosity of DNA in the presence and
absence of the complexes show significant change in the rela-
tive specific viscosity (Figure 9). Comparison of the viscosity
data with EB and Hoechst dye indicates partial intercalative
binding nature of complex 1 to ct-DNA, whereas complexes 2
and 3 show significant intercalative ct-DNA binding.
1
7% cleavage of SC DNA at these wavelengths. The photoac-
tivity could be metal-assisted and involves the metal-based
d–d band resulting in an excited state that generates the cleav-
age active species. The results showing DNA cleavage activity
of the oxidovanadium(IV) complexes in near-IR light are of
significant importance in PDT considering that the vanadi-
um(V) complexes lacking any low energy electronic band are
unsuitable for PDT application.[
38]
Photoinduced DNA Cleavage
The mechanistic aspects of the DNA photocleavage reac-
The photocytotoxic effect of the complexes in the cancer tions in red light of 705 nm and 785 nm were studied using
cells and their mitochondrial and nuclear (for complex 3) up- complexes 2 and 3 in the presence of various external addi-
take prompted us to study their ability to photo-cleave su- tives. The complexes did not show any apparent DNA photo-
percoiled DNA in near-IR light. The photoactivated DNA cleavage activity in an argon atmosphere suggesting the neces-
cleavage activity of the complexes was studied using su- sity of O to generate the ROS. The ROS could be hydroxyl
2
percoiled (SC) pUC19 DNA in a medium of Tris-HCl/NaCl radical species generated from a photo-redox process or singlet
buffer on irradiation using near-IR light diode lasers of 705 nm oxygen from the energy transfer type-II pathway. The addition
and 785 nm wavelengths (Figure 10). The choice of these of singlet oxygen quenchers, viz. sodium azide or TEMP
wavelengths is due to the presence of a central metal atom d- showed significant reduction in the DNA cleavage activity in
d absorption band near 725 nm in the electronic spectra of the red light (Figure 11). Hydroxyl radical scavengers, viz. DMSO
complexes. The complexes displayed excellent DNA cleavage and KI had no apparent effect on the DNA cleavage activity
activity at these wavelengths even at very low complex con- of the complexes. Hydrogen peroxide and superoxide anion
Table 3. Selected SC pUC19 DNA (0.2 μg) cleavage data of 1–3 and the ligands in red light.
Reaction conditions a)
% NC Form
in dark)
% NC Form
(red light, 705 nm)
% NC Form
(red light, 785 nm)
(
DNA control
DNA + 1
DNA + 2
DNA + 3
DNA + curcumin (Hcur)
DNA + Hscur
2
10
9
21
11
15
5
4
69
88
94
15
18
62
84
89
16
19
a) In Tris-buffer medium (pH = 7.2). Red light from diode lasers. Photo-exposure time (t) = 1 h. Concentration of the complexes 1–3 and the
ligands used was 30 μM.
6
www.zaac.wiley-vch.de
© 0000 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 0000, 0–0

Job/Unit: Z13569
/KAP1
Date: 12-03-14 18:51:14
Pages: 11
Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes
ROS, (iii) apoptotic cell death, (iv) DNA binding and photo-
induced DNA cleavage activity in red light, (v) cytosolic local-
ization, and (vi) significant mitochondrial uptake. This work is
expected to presage designing and developing new generation
of mitochondria targeting metal-based PDT and tumor imaging
agents for their potential applications in PDT.
Experimental Section
Materials: All reagents and chemicals were procured from commer-
cial sources (s.d. Fine Chemicals, India; Aldrich, USA) and used with-
out further purification. Curcumin (95% curcuminoid content, 80%
curcumin) was purchased from Sigma-Aldrich and purified into indi-
[
41]
vidual components by following a reported procedure.
were purified by standard procedures.
Solvents
[
42]
Supercoiled (SC) pUC19
Figure 11. Bar diagram showing the extent of photocleavage of
pUC19 DNA (0.2 μg, 30 μM) by complex 3 (30 μM) forming nicked
circular (NC) form of DNA in the presence of additives in red light of
DNA (cesium chloride purified) was purchased from Bangalore Genie
India). Tris-(hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer
solution was prepared using deionized and sonicated triple distilled
water. Calf thymus (ct) DNA, agarose (molecular biology grade), dista-
mycin, catalase, superoxide dismutase (SOD), ethidium bromide (EB),
(
7
1
0
05 nm (light shade) and 785 nm (dark shade) for an exposure time of
h [D O, 16 μL; NaN , 0.5 mM; TEMP, 0.5 mM; DMSO, 4 μL; KI,
.5 mM; SOD, 4 units; catalase, 4 units].
2
3
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
and 2Ј,7Ј-dichlorofluorescein diacetate (DCFDA) were procured from
Sigma (USA). MitoTracker® Deep Red FM (Cat. no.M22426) (MTR)
radical scavengers, viz. catalase and SOD did not show any
inhibitory effect. The results suggest the involvement of singlet was purchased from Invitrogen BioServices India. 11-(9-Acridinyl)di-
1
oxygen ( O ) in the photocleavage reaction, which was further pyrido[3,2-a:2Ј,3Ј-c]phenazine (acdppz) ligand was prepared following
2
confirmed from our observation of enhanced DNA photocleav- a literature procedure using 1,10-phenanthroline-5,6-dione as a precur-
1
sor.[20] The glycosylated curcumin, viz., diglucosylcurcumin (Hscur)
age activity in D O due to longer lifetime of O in this sol-
2
2
vent.[39,40]
and 1,7-bis(3-methoxy-4-β-d-glucopyranos-1-yloxophenyl)hepta-1,6-
The DNA cleavage reaction involving molecular
oxygen ( O ) seems to proceed via a type-II process, in which
the excited electronic state of the complex through efficient
intersystem crossing generates the triplet excited state of the
photo-sensitizer that activates triplet oxygen to the reactive sin-
glet state by energy transfer.
[
18]
3
diene-3,5-dione, was prepared according to a literature method.
2
Synthesis of the complexes was carried out in a nitrogen atmosphere
using Schlenk technique. Tetrabutylammonium perchlorate (TBAP)
was prepared using tetrabutylammonium bromide and perchloric acid.
Instrumentation: The elemental analysis was done with a Thermo
Finnigan FLASH EA 1112 CHNS analyzer. The infrared and electronic
spectra were recorded with a Perkin-Elmer Lambda 35 and a Perkin-
Elmer spectrum one 55, respectively, at 25 °C. Molar conductivity
measurements were done with a Control Dynamics (India) conductiv-
Conclusions
We report remarkable visible light-induced photocytotoxi- ity meter. Electrochemical measurements were made at 25 °C with an
city of a new class of curcumin-based oxidovanadium(IV) EG&G PAR model 253 VersaStat potentiostat/galvanostat with electro-
complexes in a ternary structure having 11-(9-acridinyl)dipyr- chemical analysis software 270 using a three electrode setup consisting
of a glassy carbon working, platinum wire auxiliary and a saturated
calomel reference electrode (SCE) in 20% DMF-H O. TBAP (0.1 m)
2
ido[3,2-a:2Ј,3Ј-c]phenazine base, while being less toxic in
dark. The photocytotoxicity of the complexes is higher than
®
was used as a supporting electrolyte for the electrochemical measure-
ments. Electrospray ionization (ESI) mass spectral measurements were
made with a Bruker Daltonics make Esquire 300 Plus ESI model. The
NMR spectra were recorded with a Bruker Avance 400 (400 MHz)
NMR spectrometer. Room temperature fluorescence quantum yield
measurements were done with a Perkin-Elmer LS 55 fluorescence
that of the PDT drug Photofrin . The complexes showed DNA
photocleavage activity in near-IR light via singlet oxygen path-
way and cellular damage via apoptotic pathway. The DNA
melting and viscosity data show intercalative mode of DNA
binding of the complexes. The complexes do not show any
apparent chemical nuclease activity due to their redox stability spectrometer using coumarin-153 laser dye as a reference with a
[
43]
over a wide potential range. Confocal studies reveal mainly known Φ value of 0.56 in acetonitrile. Samples were deaerated prior
cytosolic localization of the complexes 1 and 2 in cancer cells to spectral measurements. The complexes and reference were excited
at 390 nm, maintaining nearly equal absorbance and the emission spec-
with significant mitochondrial uptake. The results showing
tra were recorded from 410 to 650 nm. The integrated emission inten-
dual effect of the complexes in cellular imaging and light-in-
sity was calculated using Origin Pro 8.1 software and the quantum
duced apoptosis by targeting mitochondria caused by singlet
S R S R S
yield was calculated using the equation: (Φ /Φ ) = (A /A )ϫ((OD) /
oxygen generation are distinctly different from the known
metal-based PDT agents.
Complex 2 is unique in the chemistry of metal-based PDT
2
2
(
OD)
yields of the sample and reference respectively, A
area under the fluorescence spectra of the sample and the reference
respectively, (OD) and (OD) are the respective optical densities of
R
)ϫ(n
S
/n
R
), where Φ
S
and Φ
R
are the fluorescence quantum
S
and A are the
R
agents having excellent functional similarities with the PDT
S
R
®
drug Photofrin , viz. (i) visible light-induced photo-cytotoxi- the sample and the reference solution at the wavelength of excitation,
1
city with low dark toxicity, (ii) singlet oxygen ( O ) as the and n
S
and n
R
are the refractive indices for the respective solvents
2
Z. Anorg. Allg. Chem. 0000, 0–0
© 0000 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.zaac.wiley-vch.de
7

Job/Unit: Z13569
/KAP1
Date: 12-03-14 18:51:14
Pages: 11
P. Kondaiah, A. R. Chakravarty et al.
ARTICLE
used for the sample and the reference.[44,45] Room temperature mag-
conductivity and electronic absorption spectroscopic data. The absorp-
netic moment of the [D ]DMSO solutions of the oxidovanadium(IV) tion spectral study using curcumin and its derivative alone showed
6
complexes containing 1% TMS (v/v) as the internal reference was
gradual degradation with time resulting to almost complete degradation
in 24 h as evidenced from the decrease in the intensity of the absorp-
tion spectral band at 435 nm with the appearance of a new band at
252 nm in DMSO-phosphate buffer (1:1 v/v, pH = 7.2 at 37 °C). The
obtained by a solution NMR method with a Bruker AMX-400 NMR
spectrometer.[
46,47]
Fluorescence microscopic investigations were car-
ried out with a Leica DM IL microscope with integrated Leica DFC400
camera and IL50 image software. Confocal microscopy was done oxidovanadium(IV) complexes of these ligands did not show any ap-
using confocal scanning electron microscope (Leica, TCS SP5
DM6000).
parent spectral change even after 48 h.
Cell Culture: HeLa (human cervical carcinoma) cancer cells and
Synthesis of [VO(acac)(L)Cl] (1), [VO(cur)(L)Cl] (2), and HaCaT (human skin keratinocyte) cells were maintained in Dulbecco’s
VO(scur)(L)Cl] (3) (L
acdppz): Vanadyl sulfate (0.16 g, Modified Eagle’s Medium (DMEM), supplemented with 10% fetal bo-
.0 mmol) and barium chloride (0.25 g, 1.0 mmol) together were dis- vine serum (FBS), 100 IU mL of penicillin, 100 μg·mL of strepto-
[
1
=
–
1
–1
solved in aqueous ethanol (24 mL, 1:5 v/v). The mixture was stirred
at 25 °C for 2 h in a nitrogen atmosphere using Schlenk technique.
The mixture was filtered using celite to remove the barium sulfate
precipitate. The blue filtrate was de-aerated and saturated with nitro-
gen. The acdppz base (0.46 g, 1.0 mmol) taken in chloroform-ethanol
mycin and 2 mM Glutamax at 37 °C in a humidified incubator at 5%
CO . The adherent cultures were grown as monolayer and passaged
2
once in 4–5 d by trypsinizing with 0.25% Trypsin-EDTA.
Cell Viability Assay: The photocytotoxicity of the complexes 1–3 was
studied using MTT assay, which is based on the ability of mito-
chondrial dehydrogenases of viable cells to cleave the tetrazolium
rings of MTT resulting formation of dark purple membrane imperme-
able crystals of formazan that could be quantified from spectral mea-
(20 mL, 2:3 v/v) was added to the filtrate. A deep greenish solution
was formed after stirring the mixture for 10 min. Ligand Hacac or
curcumin (Hcur) or glycosylated curcumin (Hscur) [Hacac, 0.10 g,
Hcur, 0.36 g, Hscur, 0.69 g (1.0 mmol)] taken in acetonitrile-ethanol
[
31]
surements in DMSO.
Approximately, 8000 cells of HeLa and
3
mixture (16 mL, 1:3 v/v), neutralized with Et N (0.10 g, 1.0 mmol),
HaCaT were plated in a 96 wells culture plate in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% fetal bovine serum (FBS)
and kept for 24 h incubation at 37 °C in a CO incubator. The stock
2
solution of the complexes, prepared in DMSO, was first diluted in the
culture medium to the desired concentration and next added to the 96
well plates keeping the quantity of DMSO constants at 1% for all
tests. Cells were incubated for 4 h in dark. The medium that was sub-
sequently replaced with PBS was photo-irradiated with visible light
was added to the previously formed deep greenish solution. The com-
plex as a precipitate was obtained after stirring the solution for 2 h in
nitrogen atmosphere. The precipitate was filtered, isolated, washed
with cold acetonitrile, ethanol, THF, chloroform, and diethyl ether and
finally dried in vacuo over P O10 (Yield: ca. 78% for 1; 81% for 2,
4
and 72% for 3).
Complex 1: (M = 660.10), C36
H
24
N
5
ClO
3
V: calcd. C 65.41; H 3.66;
OH):
(
400–700 nm) for 1 h using a Luzchem Photoreactor (Model LZC-1,
N 10.60%; found: C 65.32; H 3.61; N 10.68%. ESI-MS (CH
3
+
Ontario, Canada) fitted with Sylvania make 8 fluorescent white tubes
m/z 625.56 [M – Cl] . IR: ν˜ = 3062 w, 1591 s, 1495 vs, 1420 s, 1383
–
2
with a fluence rate of 2.4 mW·cm to provide a total dose of
m, 1277 s, 1150 m, 1121 m, 1035 w, 965 m, 841 m, 719 m, 557 w,
–
2
59 w cm–1 (vs, very strong; s, strong; m, medium; w, weak). UV/Vis
10 J·cm . After photoexposure, PBS was removed and replaced with
4
(
8
3
–1
–1
DMEM-FBS and incubation was continued for further 20 h in dark.
20% DMF-water) [λmax /nm (ε /dm ·m ·cm )]: 717 (47), 387 (19
–
1
2
–1
After the incubation period, 20 μL of 5 mg·mL of MTT was added
to each well and incubated for an additional 3 h. The culture medium
was finally discarded and 200 μL of DMSO was added to dissolve the
60), 362 (19 500), 269 (62 750). Λ
M
= 111 S·m ·m in 20% aqueous
DMF at 25 °C. μeff, μ
B
at 298 K: 1.63.
Complex 2: (M = 928.17), C52
N 7.54%; found: C 67.30; H 3.86; N 7.59%. ESI-MS (CH
36 5 7
H N ClO
V: calcd. C 67.21; H 3.90; formazan crystals and its absorbance at 595 nm was measured using a
OH): m/z
93.20 [M – Cl] . IR: ν˜ = 3069 w, 1586 s, 1492 vs, 1422 s, 1379 m,
277 s, 1153 m, 1119 m, 1030 w, 967 m, 810 m, 721 m, 560 w,
BIORAD ELISA plate reader. Cytotoxicity of 1–3 was measured as the
percentage ratio of the absorbance of the treated cells to the untreated
controls. The IC50 values were determined by nonlinear regression
analysis (GraphPad Prism).
3
+
8
1
4
–1
62 w, 436 m cm . UV/Vis (20% aqueous DMF) [λmax /nm
3
–1
–1
(ε /dm ·m ·cm )]: 725 (51), 454 sh (56 830), 437 (57 370), 389 (46
2
–1
DNA Fragmentation Analysis by Agarose Gel Electrophoresis: The
DNA fragmentation assay was performed to observe any apoptotic
mode of cell death induced by complexes 1 and 2 using HeLa and
170), 362 (34 600), 267 (77 800). Λ
M
= 92 S·m m in 20% aqueous
DMF at 25 °C. μeff, μ
B
at 298 K: 1.65.
[
48]
6
HaCaT cells.
Briefly, 0.3ϫ10 cells were taken in each 60 mm
Complex 3: (M = 1252.28), C64
H
56
N
5
ClO17V: calcd. C 61.32; H 4.50;
dish, grown for 24 h and later treated with 1 (2 μM) and 2 (1 μM)
incubated for 4 h in dark. One dish containing the complex was ex-
posed to light for 1 h and again the cells were left to grow for 4 h
along with its dark control in another dish. After 4 h, cells were tryp-
sinized, washed with DPBS and re-suspended in 0.4 mL of lysis buffer
(10 mM Tris-HCl; pH, 8.0, 20 mM EDTA, 0.2% triton-X 100) with
incubation for 20 min on ice. Lysed cells were centrifuged for 20 min
at 13000 rpm and the supernatant having soluble chromosomal DNAs
including both high molecular weight DNA and nucleosomal DNA
fragments was collected. Phenol chloroform was performed to remove
the protein present. Later supernatant was precipitated with 1/10 vol-
ume of 3 m sodium acetate (pH 5.8) and 2 volume of ethanol at –20 °C
N 5.59%; found: C 61.44; H 4.57; N 5.52%. ESI-MS (CH
1
3
OH): m/z
217.23 [M – Cl] . IR: ν˜ = 3301 br, 1589 s, 1504 vs, 1423 w, 1384
w, 1276 s, 1126 m, 1071 m, 1030 m, 962 m, 817 w, 725 w, 551 w,
+
–
1
4
(
66 w (br., broad) cm . UV/Vis (20% DMF-water) [λmax /nm
3
–1
–1
ε /dm ·m ·cm )]: 729 (54), 427 (52 760), 391 (47 510), 363 (36
2
–1
490), 266 (71 350). Λ
M
= 81 S·m ·m in 20% aqueous DMF at 25 °C.
μeff, μ
B
at 298 K: 1.62.
Solubility and Stability: The complexes showed good solubility in
DMF, DMSO, MeOH, moderate solubility in water, ethanol, acetoni-
trile, and poor solubility in hydrocarbons. The diglucosylcurcumin
complex 3 having the carbohydrate moiety showed better solubility in
water than the curcumin complex 2. The complexes were found to be for overnight. DNA pellet was washed with 70% alcohol and re-sus-
–
1
stable in solution in the monocationic form. The solution stability of pended in Tris-EDTA (pH 8) containing RNAse (100 μg·mL ) fol-
the cationic species was ascertained from their time dependent molar lowed by incubation at 37 °C for 2 h. DNA samples were resolved on
8
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© 0000 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
Z. Anorg. Allg. Chem. 0000, 0–0

Job/Unit: Z13569
/KAP1
Date: 12-03-14 18:51:14
Pages: 11
Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes
.5% agarose gel at 80 V for about 2 h and photographed under UV
1
electrophoresis using the complexes in 50 mM Tris-HCl buffer (pH
7.2) and 50 mM NaCl containing 10% DMF. The photo-induced DNA
cleavage reactions were carried out in red light using diode lasers of
light.
DCFDA Assay for Measurement of ROS Generation: DCFDA as-
say was used to detect the formation of any reactive oxygen species
7
1
1
05 nm and 785 nm wavelengths (Model: LQC705-38E and LQC785-
00C from Newport Corporation). The laser powers were 38 mW and
00 mW, respectively, measured using Spectra Physics CW laser
(ROS) in the cancer cells. Cell permeable DCFDA on oxidation by
cellular ROS generates DCF as green fluorescent compound. The per-
centage of cells generating ROS was determined by flow cytometry
analysis. To detect ROS generation, HaCaT cells were incubated with
complex 2 (1 μM) for 4 h followed by photo-irradiation (400–700 nm)
for 1 h in serum free conditions. The cells were harvested by trypsin-
ization and a single cell suspension of 1ϫ10 cells·mL was prepared.
The cells were treated with 1 μM DCFDA solution in DMSO in dark
for 20 min at room temperature. The distribution of DCFDA stained
HaCaT cells was determined by flow cytometry in the FL-1 channel.
power meter (Model 407A). Prior to light exposure, each sample was
incubated for 1.0 h at 37 °C and the photo-cleaved products were ana-
[54]
lyzed by gel electrophoresis as reported earlier.
The mechanistic
, 0.5 mM;
DMSO, 4 μL; KI, 0.5 mM; catalase, 4 units; SOD, 4 units; TEMP,
.5 mM). For the D O experiment, this solvent was used to dilute the
studies were carried out using different additives (NaN
3
6
–1
0
2
sample up to 20 μL final volume. The extent of SC DNA cleavage
was measured from the intensities of the bands using UVITEC Gel
Documentation System. Due corrections were made for the low level
of nicked circular (NC) form of DNA present in the original SC DNA
sample and for the low affinity of EB binding to SC compared to NC
Confocal Studies: Uptake and Localization: Uptake and localization
of fluorescent complexes into the cell was visualized using a confocal
[
55]
form of DNA.
was ca. 5%.
The observed error in measuring the band intensities
[
49]
scanning electron microscope (Zeiss, LSM510 apocromat).
HeLa
and HaCaT cells were grown on glass cover slips in each 12 well
plates at a seeding density of 50,000 cells in 1.5 mL of culture medium
Supporting Information (see footnote on the first page of this article):
for 24 h. Cells were treated with the complexes for 2 h and 4 h in dark, The supplementary data contain ESI-MS (Figures S1–S3), IR (Figures
fixed, and permeabilized with chilled methanol for 5 min at –20 °C.
Methanol was subsequently removed followed by washing with 1X
PBS buffer. It was later incubated with propidium iodide (1 mg·mL ) S11–S16), DCFDA assay (Figure S17), confocal images (Figure S18–
to stain the nucleus for 2 min and visualized under a confocal scanning
S20), DNA binding plots (Figure S21), DNA cleavage data (Figures
electron microscope. The cell-permeant MitoTracker® deep red having S22–S25), spectroscopic data for measuring stability (Figure S26) and
S4–S6), cyclic voltammograms (Figure S7), energy optimized geome-
try and HOMO-LUMO diagrams (Figures S8–S10), MTT plot (Figures
–
1
a mildly thiol-reactive chloromethyl moiety was used for labeling
computational data for the complexes (Tables S1 and S2).
mitochondria. A 50 nM of MitoTracker® Deep Red FM (Cat.
[
22a]
no.M22426) was used for confocal microscopy.
Acknowledgements
DNA Binding Methods: The experiments were done in Tris-HCl
buffer (5 mM, pH 7.2) using DMF solution of the complexes 1–3. Calf
thymus (ct) DNA (ca. 350 μM NP, nucleotide pair) in this buffer me-
dium gave a ratio of UV absorbance at 260 and 280 nm of ca. 1.9:1
indicating the DNA apparently free from any protein impurity. The
concentration of ct-DNA was estimated from its absorption intensity
A.R.C. thanks the Department of Science and Technology (DST, Gov-
ernment of India), and the Council of Scientific and Industrial Re-
search, New Delhi), for financial support (SR/S5/MBD-02/2007 and
CSIR/01(2559)/12/EMR-II/2012) and DST for J.C. Bose fellowship.
We are thankful to the Alexander von Humboldt Foundation for do-
nation of an electrochemical system and Mr. Sanjoy Mukherjee for
his help in computational studies. S.B. thanks the University Grants
Commission (UGC), New Delhi, for a fellowship.
at 260 nm with
a molar extinction coefficient value (ε) of
3
–1
–1 [50,51]
6600 dm ·M ·cm .
Absorption titration experiments were done
by changing the concentration of ct-DNA while maintaining constant
[
16,17]
complex concentration following a method as reported earlier.
The intrinsic equilibrium binding constant (K ) of the complexes 1–3
b
References
to ct-DNA were obtained by the McGhee-von Hippel (MvH)
method.[
52,53]
DNA thermal denaturation studies were carried out by [1] a) H. Hatcher, R. Planalp, J. Cho, F. M. Torti, S. V. Torti, Cell
Mol. Life Sci. 2008, 65, 1631–1652; b) V. S. Govindarajan, W. H.
Stahl, Crit. Rev. Food Sci. Nutr. 1980, 12, 199–301.
2] B. B. Aggarwal, C. Sundaram, N. Malani, H. Ichikawa, Adv. Exp.
Med. Biol. 2007, 595, 1–75.
monitoring the absorption intensity of ct-DNA (190 μM) at 260 nm by
varying the temperature from 40 to 90 °C in both absence and presence
of the complexes (1–3) (20 μM) using a Cary 300 bio UV/Vis spec-
trometer with a Cary temperature controller at an increase rate of
[
[
3] a) C. H. Hsu, A. L. Cheng, Adv. Exp. Med. Biol. 2007, 595, 471–
0.5 °C per min of the solution. The viscosity measurements were done
480; b) B. B. Aggarwal, A. Kumar, A. C. Bharti, Anticancer Res.
using a Schott Gerate AVS. 310 automated viscometer attached to a
constant temperature bath at 37 °C. The concentration of the ct-DNA
stock solution was 150 μM (NP) in a 5 mM Tris-HCl buffer. The com-
plex was added gradually on increasing the concentration from 0 to
2003, 23, 363–398; c) M.-H. Teiten, S. Eifes, M. Dicato, M. Died-
erich, Toxins 2010, 2, 128–162.
[4] P. Anand, S. G. Thomas, A. B. Kunnumakkara, C. Sundaram,
K. B. Harikumar, B. Sung, S. T. Tharakan, K. Misra, I. K. Priyad-
arsini, K. N. Rajasekharan, B. B. Aggarwal, Biochem. Pharmacol.
120 μM, and the viscosity was measured on each addition. The flow
2
008, 76, 1590–1611.
[5] T. Choudhuri, S. Pal, M. L. Agwarwal, T. Das, G. Sa, FEBS Lett.
002, 512, 334–340.
6] R. A. Sharma, W. P. Steward, A. J. Gescher, Adv. Exp. Med. Biol.
007, 595, 453–470.
times were monitored with an automated timer. The relative specific
1
/3
viscosity of DNA, (η/η
0
)
was plotted vs. [complex]/[DNA], where
is the
2
η is the viscosity of DNA in the presence of the complex and η
0
[
[
viscosity of ct-DNA alone in 5 mM Tris buffer medium. The viscosity
values were calculated from the observed flow time of ct-DNA con-
2
7] a) D. Pucci, T. Bellini, A. Crispini, I. DЈAgnano, P. F. Liguori, P.
Garcia-Orduña, S. Pirillo, A. Valentini, G. Zanchetta, Med. Chem.
Commun. 2012, 3, 462–468; b) A. Hussain, K. Somyajit, B.
Banik, S. Banerjee, G. Nagaraju, A. R. Chakravarty, Dalton
Trans. 2013, 42, 182–195; c) A. K. Renfrew, N. S. Bryce, T. W.
Hambley, Chem. Sci. 2013, 4, 3731–3739.
taining solutions (t), duly corrected for that of the buffer alone (t
0
),
η = (t – t )/t
0
0
.
DNA Cleavage Methods: The cleavage of supercoiled (SC) pUC19
DNA (0.2 μg, 30 μM, 2686 base-pairs) was studied by agarose gel
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Job/Unit: Z13569
/KAP1
Date: 12-03-14 18:51:14
Pages: 11
P. Kondaiah, A. R. Chakravarty et al.
ARTICLE
[
8] S. Banerjee, P. Prasad, A. Hussain, I. Khan, P. Kondaiah, A. R.
[27] a) W. J. Geary, Coord. Chem. Rev. 1971, 7, 81–122; b) I. Ali,
W. A. Wani, K. Saleem, Synthesis and Reactivity in Inorganic,
Metal-Organic, and Nano-Metal Chemistry, 2013, 43, 1162–1170.
Chakravarty, Chem. Commun. 2012, 48, 7702–7704.
[
9] a) I. K. Priyadarsini, J. Photochem. Photobiol. C: Photochem.
Rev. 2009, 10, 81–95; b) T. A. Dahl, P. Bilski, K. J. Reszka, C. F.
Chignell, Photochem. Photobiol. 1994, 59, 290–294.
[
[
[
28] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Co-
ordination Compounds, John Wiley & Sons, New York. 1997.
29] K. Toshima, R. Takano, T. Ozawa, S. Matsumura, Chem. Com-
mun. 2002, 212–213.
30] a) A. D. Becke, Phys. Rev. 1998, A38, 3098–3100; b) A. D.
Becke, J. Chem. Phys. 1993, 98, 5648–5652; c) C. Lee, W. Yang,
R. G. Parr, Phys. Rev. B 1988, 37, 785–789; d) P. J. Hay, W. R.
Wadt, J. Chem. Phys. 1985, 82, 284–298.
[
10] M. J. Clarke, F. Zhu, D. R. Frasca, Chem. Rev. 1999, 99, 2511–
2533.
[
11] a) D. C. Crans, A. S. Tracey in Vanadium Compounds: Chemistry,
Biochemistry, and Therapeutic Applications (Eds.: A. S. Tracey,
D. C. Crans), American Chemical Society, Washington, DC,
1998, vol. 711, pp 2–29; b) D. Rehder, J. C. Pessoa, C. F. G. C.
[
31] T. Mosmann, J. Immunol. Methods 1983, 65, 55–63.
Geraldes, M. M. C. A. Castro, T. Kabanos, T. Kiss, B. Meier, G. [32] D. M. Conrad, S. J. Furlong, C. D. Doucette, K. A. West, D. W.
Micera, L. Pettersson, M. Rangel, A. Salifoglou, I. Turel, D.
Hoskin, Apoptosis 2010, 15, 597–607.
[33] A. S. Keston, R. Brandt, Anal. Biochem. 1965, 11, 1–5.
Wang, J. Biol. Inorg. Chem. 2002, 7, 384–396.
[
[
[
12] J. Kuwahara, T. Suzuki, Y. Sugiura, Biochem. Biophys. Res. Com- [34] T. Takanashi, Y. Ogura, H. Taguchi, M. Hashizoe, Y. Honda, In-
vestigative Ophthalmol. Vis. Sci. 1997, 38, 2721–2728.
mun. 1985, 129, 368–374.
13] H. Sakurai, H. Tamura, K. Okatani, Biochem. Biophys. Res. Com-
mun. 1995, 206, 133–137.
14] C.-T. Chen, J.-S. Lin, J.-H. Kuo, S.-S. Weng, T.-S. Cuo, Y.-W.
Lin, C.-C. Cheng, Y.-C. Huang, J.-K. Yu, P.-T. Chou, Org. Lett.
[
[
[
35] R. B. Nair, E. S. Tang, S. L. Kirkland, C. J. Murphy, Inorg. Chem.
1998, 37, 139–141.
36] M. Cory, D. Mckee, J. Kagan, D. Henry, J. Miller, J. Am. Chem.
Soc. 1985, 107, 2528–2536.
37] a) C. V. Kumar, E. H. Asuncion, J. Am. Chem. Soc. 1993, 115,
2004, 6, 4471–4474.
8
1
1
541–8553; b) L. E. Gunther, A. S. Yong, J. Am. Chem. Soc.
968, 90, 7323–7328; c) J. M. Veal, R. L. Rill, Biochemistry
991, 30, 1132–1140; d) G. Cohen, H. Eisenberg, Biopolymers
[
15] a) D. W. J. Kwong, O. Y. Chan, R. N. S. Wong, S. M. Musser, L.
Vaca, S. I. Chan, Inorg. Chem. 1997, 36, 1276–1277; b) C. Hiort,
J. Goodisman, J. C. Dabrowiak, Biochemistry 1996, 35, 12354–
1969, 8, 45–55.
12362.
[38] Y. Sun, Y. Zheng, W.-H. Lei, Q.-X. Zhou, Y.-J. Hou, B.-W. Zhang,
[
[
[
16] P. K. Sasmal, S. Saha, R. Majumdar, S. De, R. R. Dighe, A. R.
X.-S. Wang, Dalton Trans. 2012, 41, 651–657.
[
[
39] A. U. Khan, J. Phys. Chem. 1976, 80, 2219–2227.
Chakravarty, Dalton Trans. 2010, 39, 2147–2158.
40] P. B. Merkel, D. R. Kearns, J. Am. Chem. Soc. 1972, 94, 1029–
17] S. Banerjee, A. Hussain, P. Prasad, I. Khan, B. Banik, P. Kon-
daiah, A. R. Chakravarty, Eur. J. Inorg. Chem. 2012, 3899–3908.
18] a) E. Ferrari, S. Lazzari, G. Marverti, F. Pignedoli, F. Spagnolo,
M. Saladini, Bioorg. Med. Chem. 2009, 17, 3043–3052; b) K.
Mohri, Y. Watanabe, Y. Yoshida, M. Satoh, K. Isobe, N. Sugim-
oto, Y. Tsuda, Chem. Pharm. Bull. 2003, 51, 1268–1272.
1030.
[
[
[
41] O. Vajragupta, P. Boonchoong, L. Berliner, Free Radic. Res. 2004,
8, 303–314.
3
42] D. D. Perrin, W. L. F. Armarego, D. R. Perrin in Purification of
Laboratory Chemicals, Pergamon Press, Oxford, 1980.
43] J. Guilford II, R. Jackson, C. Choi, W. R. Bergmark, J. Phys.
Chem. 1985, 89, 294–300.
[
19] a) R. S. Brown, R. L. Wahl, Cancer 1993, 72, 2979–2985; b)
S. N. Reske, K. G. Grillenberger, G. Glatting, M. Port, M. Hilde- [44] J. R. Lakowicz, Principles of Fluorescence Spectroscopy, Kluwer
brandt, F. Gansauge, H. G. Beger, J. Nucl. Med. 1997, 38, 1344–
Academic/Plenum Publishers, New York, 1999.
1348.
[45] A. T. R. Williams, S. A. Winfield, J. N. Miller, Analyst 1983, 108,
1067–1071.
[
[
[
20] M. Mariappan, M. Suenaga, A. Mukhopadhyay, P. Raghavaiah,
[
[
46] D. F. Evans, J. Chem. Soc. 1959, 2003–2005.
B. G. Maiya, Inorg. Chim. Acta 2011, 376, 340–349.
21] E. Delaey, F. V. Laar, D. De Vos, A. Kamuhabwa, P. Jacobs, P.
De Witte, J. Photochem. Photobiol. 2000, B55, 27–36.
47] D. F. Evans, T. A. James, J. Chem. Soc. Dalton Trans. 1979, 723–
726.
[
48] P. T. Daniel, I. Sturm, S. Ritschel, K. Friedrich, B. Dörken, P.
Bendzko, T. Hillebrand, Anal. Biochem. 1999, 266, 110–115.
49] T. Esteves, F. Marques, A. Paulo, J. Rino, P. Nanda, C. J. Smith,
I. Santos, J. Biol. Inorg. Chem. 2011, 16, 1141–1153.
[50] S. P. Foxon, C. Metcalfe, H. Adams, M. Webb, J. A. Thomas,
Inorg. Chem. 2007, 46, 409–416.
22] a) M. Lam, N. L. Oleinick, A.-L. Nieminen, J. Biol. Chem. 2001,
276, 47379–47386; b) R. Hilf, J. Bioenerg. Biomembr. 2007, 39,
[
85–89; c) J. S. Armstrong, Br. J. Pharmacol. 2006, 147, 239–248;
d) S. Fulda, L. Galluzzi, G. Kroemer, Nat. Rev. Drug Discovery
2010, 9, 447–467; e) C. E. Wenner, J. Cell. Physiol. 2012, 227,
4
50–456.
[51] M. E. Reichmann, S. A. Rice, C. A. Thomas, P. Doty, J. Am.
Chem. Soc. 1954, 76, 3047–3053.
[
23] J. Saczko, M. Mazurkiewicz, A. Chwilkowska, J. Kulbacka, G.
´
[52] J. D. McGhee, P. H. von Hippel, J. Mol. Biol. 1974, 86, 469–489.
Kramer, M. Ługowski, M. Snietura, T. Banas, Folia Biol. 2007,
3, 7–12.
24] a) S. Marrachea, S. Dhar, Proc. Natl. Acad. Sci. USA 2012, 109,
6288–16293; b) S. Marrachea, S. Tundup, D. A. Harn, S. Dhar,
[
[
[
53] M. T. Carter, M. Rodriguez, A. J. Bard, J. Am. Chem. Soc. 1989,
5
111, 8901–8911.
[
54] S. Saha, R. Majumdar, M. Roy, R. R. Dighe, A. R. Chakravarty,
Inorg. Chem. 2009, 48, 2652–2663.
55] J. Bernadou, G. Pratviel, F. Bennis, M. Girardet, B. Meunier, Bio-
chemistry 1989, 28, 7268–7275.
1
ACS Nano 2013, 7, 7392–7402.
[
[
25] P. Prasad, I. Khan, P. Kondaiah, A. R. Chakravarty, Chem. Eur. J.
2
013, 19, 17445–17555.
26] N. A. Smith, P. J. Sadler, Phil. Trans. R. Soc. A 2013, 371,
0120519.
Received: November 11, 2013
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Z. Anorg. Allg. Chem. 0000, 0–0

Job/Unit: Z13569
/KAP1
Date: 12-03-14 18:51:14
Pages: 11
Mitochondria targeting Photocytotoxic Oxidovanadium(IV) Complexes
S. Banerjee, P. Prasad, I. Khan, A. Hussain, P. Kondaiah,*
A. R. Chakravarty* ......................................................... 1–11
Mitochondria targeting Photocytotoxic Oxidovanadium(IV)
Complexes of Curcumin and (Acridinyl)dipyridophenazine in
Visible Light
Z. Anorg. Allg. Chem. 0000, 0–0
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