Photoactivated cytotoxicity of ferrocenyl-terpyridine oxovanadium(IV) complexes of curcuminoids

Article abstract of DOI:10.1016/j.ejmech.2014.07.098

Oxovanadium(IV) complexes, viz. [VO(Fc-tpy)(Curc)](ClO₄) (1), [VO(Fc-tpy)(bDHC)](ClO₄) (2), [VO(Fc-tpy)(bDMC)](ClO₄) (3) and [VO(Ph-tpy)(Curc)](ClO₄) (4), of 4′-ferrocenyl-2,2′: 6′,2″-terpyridine (Fc-tpy) and 4′-phenyl-2,2′:6′, 2″-terpyridine (Ph-tpy) and monoanionic curcumin (Curc), bis-dehydroxycurcmin (bDHC) and bis-demethoxycurcumin (bDMC) were prepared, characterized and their photo-induced DNA cleavage activity and photocytotoxicity in visible light studied. The ferrocenyl complexes 1-3 showed an intense metal-to-ligand charge transfer band near 585 nm in DMF and displayed Fc⁺/Fc and V(IV)/V(III) redox couples near 0.65 V and -1.05 V vs. SCE in DMF-0.1 M TBAP. The complexes as avid binders to calf thymus DNA showed significant photocleavage of plasmid DNA in red light of 647 nm forming OH radicals. The complexes showed photocytotoxicity in HeLa and Hep G2 cancer cells in visible light of 400-700 nm with low dark toxicity. ICP-MS and fluorescence microscopic studies exhibited significant cellular uptake of the complexes within 4 h of treatment with complexes. The treatment with complex 1 resulted in the formation of reactive oxygen species inside the HeLa cells which was evidenced from the DCFDA assay.

Full text of DOI:10.1016/j.ejmech.2014.07.098

European Journal of Medicinal Chemistry 85 (2014) 458e467
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Photoactivated cytotoxicity of ferrocenyl-terpyridine oxovanadium(IV)
complexes of curcuminoids
,
*
Babu Balaji a, Babita Balakrishnan b, Sravanakumar Perumalla a, Anjali A. Karande b
,
,
*
Akhil R. Chakravarty a
a Department of Inorganic and Physical Chemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560012, India
b Department of Biochemistry, Indian Institute of Science, Sir C.V. Raman Avenue, Bangalore 560 012, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Oxovanadium(IV) complexes, viz. [VO(Fc-tpy)(Curc)](ClO4) (1), [VO(Fc-tpy)(bDHC)](ClO4) (2), [VO(Fc-
tpy)(bDMC)](ClO4) (3) and [VO(Ph-tpy)(Curc)](ClO4) (4), of 40-ferrocenyl-2,20:60,200-terpyridine (Fc-tpy)
and 40-phenyl-2,20:60,200-terpyridine (Ph-tpy) and monoanionic curcumin (Curc), bis-dehydroxycurcmin
(bDHC) and bis-demethoxycurcumin (bDMC) were prepared, characterized and their photo-induced
DNA cleavage activity and photocytotoxicity in visible light studied. The ferrocenyl complexes 1e3
showed an intense metal-to-ligand charge transfer band near 585 nm in DMF and displayed Fcþ/Fc and
V(IV)/V(III) redox couples near 0.65 V and ꢀ1.05 V vs. SCE in DMFe0.1 M TBAP. The complexes as avid
binders to calf thymus DNA showed significant photocleavage of plasmid DNA in red light of 647 nm
forming ꢁOH radicals. The complexes showed photocytotoxicity in HeLa and Hep G2 cancer cells in visible
light of 400e700 nm with low dark toxicity. ICP-MS and fluorescence microscopic studies exhibited
significant cellular uptake of the complexes within 4 h of treatment with complexes. The treatment with
complex 1 resulted in the formation of reactive oxygen species inside the HeLa cells which was evi-
denced from the DCFDA assay.
Received 20 March 2014
Received in revised form
25 July 2014
Accepted 25 July 2014
Available online 28 July 2014
Keywords:
Medicinal chemistry
Vanadium
Curcuminoids
Ferrocene
Photocytotoxicity
Cellular imaging
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
mechanistic pathways are generally involved, viz. Type-I process
with the photosensitizer having a long lived triplet state reacting
Photodynamic therapy (PDT) is a promising and non-invasive
treatment modality for various cancers [1e5]. It involves the
combined action of a photosensitizer (PS), visible light and mo-
lecular oxygen to generate cytotoxic reactive oxygen species (ROS)
that causes damage of only the photoexposed cancer cells thus
leaving the unexposed healthy cells unaffected. Two common
directly with the cellular components and a Type-II process
involving formation of singlet oxygen as the ROS that triggers cell
death [6]. Photofrin®, the FDA approved PDT drug is a hema-
toporphyrinic mixture [7e9]. It generates singlet oxygen upon
photoexcitation in red light of 633 nm. It suffers from various
drawbacks like slow clearance from the body, prolonged skin
photosensitivity, and hepatotoxicity [10,11]. We are interested to
develop metal-based PDT agents which could be potential alter-
natives for the porphyrin based PDT agents. Recent reports have
shown that transition metal complexes could exhibit photo-
cytotoxicity in various cancer cells in visible light [12e17]. The
redox active transition metal complexes show photocytotoxic effect
via alternate photo-redox pathway generating hydroxyl radical or
singlet oxygen as ROS to damage the cancer cells [18,19]. We have
shown that oxovanadium(IV) complexes could be suitably designed
to achieve remarkable PDT activity [20e24].
Abbreviations: DMEM, Dulbecco's Modified Eagle's Medium; ct-DNA, calf
thymus DNA; DMF, dimethylformamide; DMSO, dimethyl sulfoxide; DNA, deoxy-
ribonucleic acid; EB, ethidium bromide; EDTA, ethylenediaminetetraacetate; FBS,
fetal bovine serum; Fc, ferrocenyl; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; MvH, McGhee-von Hippel; PBS, phosphate buff-
ered saline; PDT, photodynamic therapy; Fc-tpy, 40-ferrocenyl-2,20:60,200-terpyr-
idine; Ph-tpy, 40-phenyl-2,20:60,200-terpyridine; CurcH, curcumin; bDHCH, bis-
dehydroxycurcumin; bDMCH, bis-demethoxycurcumin; MLCT, metal-to-ligand
charge transfer; TBAP, tetrabutylammonium perchlorate; ROS, reactive oxygen
species; NAC, N-acetylcysteine; DCFDA, 20,70-dichlorofluorescein diacetate; ICP-MS,
Inductively coupled plasma mass spectrometry; EPR, Electron paramagnetic
resonance.
The present work stems from our interests to study the photo-
cytotoxic potential of new ferrocene-conjugated oxovanadium(IV)
complexes of curcuminoids. Curcumin, 1,7-bis(4-hydroxy-3-
methoxyphenyl)-1,6-heptadiene-3,5-dione, is the major bioactive
* Corresponding authors.
E-mail addresses: anjali@biochem.iisc.ernet.in (A.A. Karande), akhil_
chakravarty@yahoo.co.in, arc@ipc.iisc.ernet.in (A.R. Chakravarty).
http://dx.doi.org/10.1016/j.ejmech.2014.07.098
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
459
ingredient of turmeric. Curcumin is well known for its biological
and pharmacological properties as anticancer, antioxidant, anti-
inflammatory, antibacterial, antispasmodic, and anticoagulant
agent [25,26]. The anticancer activities of curcumin are demon-
strated in a variety of transformed cell types like T- and B-cell
lymphomas, breast, colon, gastric, ovarian, prostate, and oral
epithelial carcinoma cells [27]. In spite of having several positive
biological effects, its therapeutic potentials are restricted because of
its extremely low aqueous solubility, instability, and poor
bioavailability [28,29]. Our objective is to enhance the stability of
curcumin in a cellular medium on binding to the oxovanadium(IV)
moiety [20,30]. We have chosen ferrocene in our complex design
because of its lipophilic nature and redox activity [31]. Also ferro-
cenyl derivatives are known to exhibit antitumor, antimalarial, and
antifungal activities with the ferrocene-appended anticancer drug
of tamoxifen showing enhanced activity against both hormone-
independent (ERꢀ) and hormone-dependent (ERþ) cancer with
respect to tamoxifen [32e35]. Similarly, ferroquine which is the
ferrocene-attached version of the antimalarial drug chloroquine is
more active than chloroquine [36].
Herein, we present the synthesis, characterization and biolog-
ical activity of ferrocene-appended oxovanadium(IV) complexes,
viz. [VO(Fc-tpy)(L)](ClO4) (L ¼ Curc, 1; bDHC, 2; bDMC, 3), where
Fc-tpy is 40-ferrocenyl-2,20:60,200-terpyridine, Curc is monoanionic
curcumin, bDHC is monoanionic bis-dehydroxycurcumin, bDMC is
monoanionic bis-demethoxycurcumin (Fig. 1). To explore the role
of ferrocene we have prepared the phenyl analogue, viz. [VO(Ph-
tpy)(Curc)](ClO4) (4) as a control species, where Ph-tpy is 40-
phenyl-2,20:60,200-terpyridine. The fluorescence properties of the
curcuminoids (Curc, bDHC, bDMC) were used in cellular imaging
studies. The results show significant cellular localization of the
complexes in the HeLa cells. The photocytotoxicity of the com-
plexes 1e4 along with cellular uptake, DNA binding propensity and
DNA photocleavage activity in visible light were studied. Significant
results include observation of an enhancement in the photo-
cytotoxicity, cellular uptake, DNA photocleavage activity of the
ferroceyl complex compared to its phenyl analogue. The complexes
show significant PDT effect giving low IC50 values while remaining
essentially non-toxic in dark.
intermediate product with ammonium acetate in refluxing ethanol
[37,38]. Curcuminoids were prepared by following a literature
procedure [39]. A suspension of B2O3 and acetylacetone in DMF was
reacted for 30 min at 80 ꢂC, and then tributylborate was added.
After 30 min, the appropriate benzaldehyde was added, followed by
addition of n-butylamine. The reaction mixture was stirred for 4 h
at 80 ꢂC followed by acidification with 0.5 M HCl and cooled to get a
yellow solid. The metal precursor VOCl2 was prepared by reacting
VOSO4 with barium chloride. Oxovanadium(IV) complexes 1e4
were prepared by a general synthetic method having two steps.
First, the corresponding curcuminoid was reacted with VOCl2 in the
presence of NaOH for 30 min. Corresponding terpyridine ligand in
CHCl3eMeOH was then added. The complexes were obtained as
perchlorate salts on addition of excess aqueous NaClO4.
3. Pharmacology
Cytotoxicity of the oxovanadium(IV) complexes 1e4 in
cancerous cell lines like HeLa and Hep G2 cells and in normal
fibroblast cells (3T3) was evaluated by MTT assay in dark and visible
light (400e700 nm). The cellular localization studies were carried
out in HeLa cells by confocal fluorescence microscopy using the
green fluorescence of curcuminoids present in the complexes. The
mechanism of cell death on photo-irradiation was assessed by
Hoechst staining. The generation of reactive oxygen species (ROS)
in HeLa cells on photo-irradiation was evaluated by DCFDA assay.
The oxovanadium(IV) complexes were evaluated for their calf
thymus DNA binding strengths and plasmid DNA photocleavage
activities. The DNA binding studies were done by spectroscopic
methods, viz. electronic absorption titration, DNA melting and
viscosity measurements of the complex bound ct-DNA. The DNA
cleavage activity of the complexes was studied by determining the
ability of each complex to convert the plasmid supercoiled (SC)
form of DNA to its nicked circular (NC) form.
4. Results and discussion
4.1. Synthesis and general aspects
Complexes 1e3 were prepared from oxovanadium(IV) chloride
as a precursor. Curcumin (CurcH) or curcumin derivative (bDHCH,
bDMCH) was initially stirred for 15 min with oxovanadium(IV)
chloride and aqueous sodium hydroxide followed by addition of Fc-
tpy (in CHCl3eCH3OH) and the complexes were isolated as
perchlorate salts on addition of aqueous NaClO4. Complex 4 was
2. Chemistry
The terpyridine ligands (Fc-tpy and Ph-tpy) were prepared from
a reaction of the corresponding aldehyde with 2-acetylpyridine in
the presence of NaOH and subsequent condensation of the
Fig. 1. Schematic drawings of the complexes 1e4 having monoanionic curcumin (Curc) in 1 and 4 (R1 ¼ OMe, R2 ¼ OH), bDHC in 2 (R1 ¼ OMe, R2 ¼ H) and bDMC in 3 (R1 ¼ H,
R2 ¼ OH).

460
B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
prepared using Ph-tpy instead of Fc-tpy. Complexes 1e4 were
characterized from the analytical, mass spectral and other physi-
cochemical data (Table 1). They were 1:1 electrolytic in DMF with
molar conductance value of ~74 S m2 Mꢀ1 at 25 ꢂC. The ESI-MS
spectra of the complexes in CH3CN showed a single peak corre-
sponding to the molecular ion [Mꢀ(ClO4)]þ. The IR spectra in the
solid state showed typical C]O and C]C bands of curcuminoids
which shifted to lower wave-numbers due to binding to the metal.
ꢀ
The complexes also showed band for V]O and ClO4 within
952e962 cmꢀ1 and 1091e1096 cmꢀ1, respectively. The complexes
are one-electron paramagnetic giving meff of ~1.6 mB at 25 ꢂC. The
electronic absorption spectra showed an intense band within
414e436 nm corresponding to the pep* electronic transition of the
curcuminoids (Fig. 2) [40]. The absorption maximum (lmax) of
bDHC and bDMC complexes showed significant blue shift
compared to their curcumin analogue. Complexes 1e3 also showed
a metal-to-ligand charge transfer band (MLCT) near 585 nm
involving the Fc-tpy and oxovanadium(IV) moieties [41,42]. Inter-
estingly, this MLCT band is absent in the phenyl analogue 4. Com-
plex 4 showed a weak ded transition at 777 nm. The ded band in
1e3 was masked by the intense MLCT band. The complexes showed
fluorescence due to the presence of curcuminoids. The emission
bands in 1e4 were at 523, 468, 500 and 522 nm, respectively. The
emission of the bDHC and bDMC complexes 2 and 3 showed a
notable blue shift when compared to that of the curcumin ana-
logues 1 and 4 [43]. The fluorescence quantum yield (4) of 1e4
ranged within 0.01e0.06 in DMF (Table 1).
The complexes showed redox activity in DMFe0.1 M TBAP
(Table 1). The ferrocenyl conjugates 1e3 showed a quasi-reversible
Fcþ/Fc redox couple near 0.65 V. A significant anodic shift of
~100 mV of the Fe(III)eFe(II) couple was observed compared to that
of the Fc-tpy ligand (0.61 V) suggesting greater stabilization of the
Fe(II) centre in the complexes compared to the free Fc-tpy ligand.
The V(IV)eV(III) reduction was observed as an irreversible peak
within ꢀ0.9 V to ꢀ1.05 V vs. SCE. Complex 4 showed only the
V(IV)eV(III) reduction response near ꢀ0.98 V. The room tempera-
ture EPR study of the complexes 1e4 in DMF showed an eight line
spectra arising from the interaction of single unpaired electron
(S ¼ 1/2) with the vanadium nucleus (51V, I ¼ 7/2) indicating the
presence 3d1eVO2þ moiety in the complexes [44,45]. The DFT
calculations were performed to get the energy optimized structures
Fig. 2. Electronic absorption spectra of complexes 1e4 in DMF. The inset shows the
MLCT band.
and the time-dependent DFT (TD-DFT) calculations were done to
understand the photophysical properties of the complexes (vide
supporting information for details). The calculated absorption band
positions for the ferrocenyl complexes 1e3 at 591, 594 and 591 nm
matched with the observed ones at 585, 585 and 588 nm, respec-
tively. This was absent in the free ligand (Fc-tpy) and for the phenyl
analogue 4. The frontier orbital picture of complex 1 corresponds to
the charge transfer band from HOMO-1 to LUMO, where HOMO-1
lies on the ferrocene unit and LUMO lies on the terpyridine moi-
ety. The MO contribution analysis suggests that the HOMO is
dominated by the contributions from the ferrocenyl moiety (97%)
and the LUMO-1 is dominated by the terpyridine segment (84%)
with minor contribution coming from the V]O centre (8%). The
same observation is made for other ferrocenyl complexes 2 and 3.
Further, the calculated absorption band for curcumin at 472 nm is in
agreement with the experimental observed transition at 436 nm.
The MO picture shows the HOMO lying on the curcumin moiety
(99%). Same trend is observed for the complexes 2e4.
4.2. Stability of the complexes
Stability of the complexes was studied in 1:1 aqueous DMF using
UVeVisible spectroscopic technique. The UVevisible spectrum of
free curcumin showed a steady decrease with time up to 4 h
Table 1
Selected physicochemical data for the complexes 1e4.
1
2
3
4
IRa/cmꢀ1
yV
]
970
962
952
970
O
yClO
1097
1091
1096
1094
4
yCeO curcumin
1607
1605
1607
1610
b
lmax/nm (ε/Mꢀ1 cmꢀ1
)
436 (38 850),
585 (3140)
523 [0.02]
0.65 (181)
ꢀ0.95
414 (43 820),
585 (650)
468 [0.007]
0.62 (166)
ꢀ0.90
427 (24 730),
588 (2140)
500 [0.06]
0.64 (167)
ꢀ1.05
435 (27 860),
777 (125)
522 [0.013]
e
Emission: lem (nm)c
[
FF
Ep/mV)d
]
b
Ef/V [FcþeFc] (
D
Ep/V [V(IV)eV(III)]d
ꢀ0.98
e
meff
/
mB
1.61
76
2.01
1.64
70
2.02
1.60
80
2.02
1.62
LMf/S m2 Mꢀ1
74
2.02
g
giso
h
Kb
6.8 0.4 ꢃ 104
1.2
5.4 0.5 ꢃ 104
0.9
7.1 0.5 ꢃ 104
1
2.4 0.3 ꢃ 105
2.3
i
D
Tm /ꢂC
a
b
c
In KBr phase.
In DMF.
Excitation at their corresponding lmax of curcuminoids.
Fe(III)eFe(II) couple in DMF-0.1 M TBAP, Ef ¼ (Epa þ Epc)/2,
d
DEp ¼ (Epa ꢀ Epc), where Epa and Epc are the anodic and cathodic peak potentials, respectively. The potentials are
vs. SCE. Scan rate ¼ 50 mV sꢀ1. The cathodic peak for V(IV)eV(III).
e
In solid phase.
Molar conductance in DMF at 25 ꢂC.
From room-temperature EPR spectra in DMF.
Kb, Intrinsic DNA binding constant.
f
g
h
i
Change in the DNA melting temperature in phosphate buffer (pH ¼ 7.2).

B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
461
indicating degradation of curcumin. There was, however, no change
in the spectrum of complex 1 with time. This could be due to
binding of curcumin to the VO2þ centre arresting the hydrolytic
degradation of curcumin [30]. The same trend was observed for the
curcumin complex 4. While the ligands bDHCH and bDMCH were
more stable compared to curcumin, their oxovanadium(IV) com-
plexes showed similar stability as those of the curcumin complexes.
light (IC50) than complex 1, but it suffers from dark toxicity
(IC50 > 10 M) and solubility problem [24,50,51]. Also incorporation
of ferrocene increases the lipophilicity so that complex can easily
penetrate the cellular lipid bilayer.
m
4.4. Measurement of intracellular ROS
DCFDA was used to study generation of any intracellular reactive
oxygen species (ROS) by fluorescence-activated cell sorting (FACS)
analysis (Fig. 4). The cell-permeable DCFDA on passive diffusion
into the cells is retained in the intracellular level after cleavage by
the intracellular esterases [52]. Upon oxidation by ROS, the non-
fluorescent DCFDA gets converted to the highly fluorescent 20,70-
dichlorofluorescein (DCF). The HeLa cells on treatment with com-
4.3. Photocytotoxicity study
Cytotoxicity of the complexes and ligands was evaluated in
HeLa, Hep G2 and 3T3 cells by MTT assay. The complexes were
incubated for 4 h in dark followed by photo-exposure to visible
light of 400e700 nm for 1 h (light dose ¼ 10 J cmꢀ2). The complexes
showed dose-dependent decrease in cell viability (Fig. 3). The IC50
values are given in Table 2. The complexes showed good photo-
cytotoxicity in cancerous cell line, while being significantly less
toxic in dark. The complexes showed better photocytotoxicity in
HeLa cells than Hep G2 cells. In HeLa cells, the ferrocenyl complex 1
plex 1 (10 mM) showed a significant positive shift in the DCFDA
derived fluorescence intensity on exposure to light compared to
those kept in dark and the untreated control cells. The fluorescence
intensity was found to reduce when photo-exposure was carried
out in presence of ROS scavenger N-acetyl cysteine. The results
show that complex 1 generates ROS in the cellular medium upon
photo-irradiation but not in the dark. The positive control cells
(H2O2 treated cells) showed significant shift compared to the con-
trol cells.
showed ~4-fold increase in photocytotoxicity (IC50 ¼ 2.4
compared to its phenyl analogue 4 (IC50 ¼ 10.9 M), where as in
Hep G2 cells, it was ~3fold increase (IC50 ¼ 10.9 M for 1 and
M for 4). The IC50 values were >50 M in dark. In
mM)
m
m
m
IC50 ¼ 33.9
m
normal 3T3 cells all the complexes showed IC50 less activity
compared to the HeLa cells (Table 2). In HeLa cells Complex 1
showed better activity compared to the free ligand Fc-tpy
4.5. Cellular uptake from ICP-MS
(IC50 ¼ 34.1
m
M) and curcumin (IC50 ¼ 8.8
mM) in light. Complex
The cellular uptake of the complexes was quantitatively studied
by ICP-MS measurements. The vanadium contents associated with
1 showed comparable cytotoxicity with Photofrin® and cisplatin
[46,47]. The IC50 values of the complexes of curcuminoids in visible
light follow the order: 1 < 3 < 2 < 4. This order shows the impor-
tance ortho-methoxyphenolic groups of curcumin for its cellular
activity. A similar order of photocytotoxicity was observed for free
curcuminoids. Based on the above results the complexes are more
active in HeLa cells. When photo-irradiation was done in the
presence of N-acetyl cysteine as ROS scavenger, the activity of the
complexes was found to decrease significantly. This indicates that
the photocytotoxicity of the complexes is due to ROS generation on
photo-irradiation. Observed higher photocytotoxicity of the ferro-
cenyl complex 1 than its phenyl analogue 4 is probably due to the
involvement of the ferrocenyl terpyridine moiety in 1 in the photo-
activation process which is absent in 4. Complex 1 showed better
photocytotoxicity compared to previously reported ferrocene con-
jugated oxovanadium complexes [20,42] (Table 2). Also on
comparing the oxovanadium complexes of curcumin reported in
the literature, though those complexes showed better IC50 value in
the HeLa cells upon incubation for 4 h with complexes 1e4 (30 mM)
at 37 ꢂC were measured (Fig. 5). The DMSO (1% in DMEM media)
treated cells were used as a control. The uptake of the complexes
follows order: 1 > 2 > 3 > 4. The ferroceneyl complex 1
(3.62 0.14
to its phenyl analogue 4 (0.53 0.20
m
g/106 cells) showed better cellular uptake compared
m
g/106 cells). This could be due
to higher lipophilic nature of ferrocene. The photocytotoxicity of
the complexes, however, does not follow this trend possibly due to
different photosensitizing ability of the complexes.
4.6. Fluorescence microscopy
Incorporation of a fluorescent curcuminoids in the complexes
was primarily aimed to study the uptake and localization of the
complexes in the cellular organelles in HeLa cells. The internaliza-
tion of all the complexes was investigated using fluorescence mi-
croscopy. The images of HeLa cells treated with 1 and 4 (10 mM) for
different time intervals are shown in Fig. 6. It was observed that the
complexes after 2 h of treatment were internalized efficiently,
distributed all over the cytosol and also in the nucleus as evidenced
from the co-staining of the cells using Hoechst dye which stains the
nucleus of the HeLa cells. An increase in the fluorescence intensity
in the cells was observed on increasing the time suggesting that
higher uptake of the complexes with time. Cells treated with
complex 2 showed less cellular uptake and complex 3 showed
similar cellular uptake as complex 1 and 4. To study the PDT effect,
complexes 1 and 4 were treated with the HeLa cells that were then
exposed to visible light for 1 h and images were taken. A change in
the nuclear morphology like chromatin condensation, a morpho-
logical feature of apoptotic cell death, was observed (Fig. 7) [53,54].
The nuclei of the apoptotic cells appeared smaller and shrunken
compared to the untreated cells and cells treated with the com-
plexes but not exposed to light where no such shrinkage in the
nuclei was visible. The results show that the complexes remained
non-cytotoxic to the cells in dark and induced significant apoptosis
only on photo-exposure to visible light (400e700 nm).
Fig. 3. Cell viability plots showing the cytotoxic effect of (a) [VO(Fc-tpy)(Curc)](ClO4)
(1) and (b) [VO(Ph-tpy)(Curc)](ClO4) (4) in dark (solid symbols) and on exposure to
visible light (hollow symbols, 400e700 nm, 10 J cmꢀ2) in HeLa cancer cells.

462
B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
Table 2
A comparison of the IC50 values of complexes 1e4 with relevant compounds in different cell lines.
Compound
HeLa
IC50
Hep G2
IC50
M) Darka
3T3
IC50
(
m
M) Darka
IC50
(
m
M) Lightb
(m
IC50
(
m
M) Lightb
(
m
M) Darka
IC50 (m
M) Lightb
1
2
3
4
54.2 ( 1.1)
75.1 ( 1.4)
56.2 ( 1.3)
60.5 ( 1.2)
>41
71.3 ( 2.9)
35.5 ( 1.0)
34.7 ( 1.9)
>100
>100
>50
>10
>50
2.4 ( 0.3)
5.1 ( 0.3)
4.5 ( 0.4)
10.9 ( 1.2)
4.28
68.7 ( 3.4)
13.4 ( 1.1)
17.8 ( 1.2)
12.0
72.7 ( 1.8)
>100
66 ( 1.2)
55.6 ( 1.1)
e
e
e
e
e
e
10.9 ( 1.1)
28.4 ( 1.8)
20.5 ( 1.1)
33.9 ( 1.5)
e
e
e
e
e
e
46.4 ( 1.5)
88.3 ( 1.4)
49.4 ( 1.1)
51.3 ( 1.6)
e
e
e
e
e
e
22.5 ( 1.1)
26.3 ( 1.5)
25.8 ( 1.0)
31.6 ( 1.9)
e
e
e
e
e
e
Photofrinc
Cisplatind
[VO(Fc-tpy)(dppz)](ClO4)e
[VO(Fc-pic)(Curc)](ClO4)f
[VOCl(dppz)2]Clg
[VO(sal-argH)(dppz)]Clh
[VO(dppz)(cur)]Cli
15.4
3.3 0.4
0.71 ( 0.07)
23.1 ( 0.5)
j
[VO(cur)(acdppz)Cl]
e
e
e
e
e
[VO(napth-py-aebmz)(cur)]Clk
a
The IC50 values of the complexes in dark.
The IC50 values for the complexes and ligands correspond to 4 h incubation in the dark followed by photo-exposure to visible light (400e700 nm, 10 J cmꢀ2) for 1 h.
b
c
The Photofrin IC50 values (633 nm excitation; fluence rate: 5 J cmꢀ2) are taken from Ref. [46] (converted to
mM using the approximate molecular weight of Photofrin,
600 g Mꢀ1).
d
The IC50 values for 4 h incubation time are taken from Ref. [47].
e
f
The IC50 values are from Ref. [42].
The IC50 values are from Ref. [20].
The IC50 values are from Ref [48].
The IC50 values are from Ref. [49].
The IC50 values are from Ref. [24].
The IC50 values are from Ref. [50].
The IC50 values are from Ref. [51].
g
h
i
j
k
4.7. DNA binding and photocleavage
On intercalation the contour length of DNA is known to increase by
separation of the DNA base pairs [56]. The classical intercalator, viz.
ethidium bromide shows a significant increase in the relative
specific viscosity where as the groove binding Hoechst dye shows
less pronounced effect on the viscosity. The results indicate that
complexes 1e3 are groove binders to ct-DNA with complex 4
showing partial intercalation property.
With the knowledge that the complexes localizing
predominantly in the nucleus, we studied the DNA photocleavage
activity of the complexes using supercoiled pUC19 DNA in
TriseHCl/NaCl (50 mM, pH ¼ 7.2) buffer on irradiation with blue
and red laser light of 454 and 647 nm wavelengths. The wave-
lengths were chosen based on the visible band of curcumin and the
The DNA binding strength of the oxovanadium(IV) complexes
1e4 was studied by different spectral methods using calf-thymus
(ct) DNA (Table 1). The intrinsic equilibrium DNA binding con-
stant Kb values were obtained from the absorption spectral titration
of the complexes with ct-DNA by monitoring the band at 280 nm.
The Kb values for 1e4 were found within 5.4 ꢃ 104 to 2.4 ꢃ 105 Mꢀ1
.
The DNA binding strength follows the order: 4 > 3 > 1 > 2. A
comparison of binding constant values with some oxovandium
complexes are given in the Table S6. Thermal denaturation exper-
iments were carried out to study the DNA-binding nature of the
complexes. An increase in the DNA melting temperature indicates
an increase in the stability of the double helix [55]. A positive shift
MLCT band positions. The ferrocenyl complexes 1e3 (40 mM) in red
of 0.9e1.4 ꢂC (
observed upon addition of the complexes to the ct-DNA. The low
Tm value suggests primarily DNA surface and/or groove binding
DTm value) in the DNA melting temperature was
light showed significant cleavage of SC DNA giving ~70% nicked
circular (NC) DNA (Table 3). Complex 4 lacking the ferrocenyl
moiety is less active in red light. Control experiments like only DNA
in light and the complexes in dark did not show any significant DNA
D
nature of the complexes. Viscometric titration experiments were
done to study the effect of the complexes on the specific relative
cleavage activity. The complexes (20 mM) in blue light were found to
viscosity (
h
/
h0) of the ct-DNA (
h
and h0 are the specific viscosities of
be efficient photocleavers of DNA showing ~80% NC DNA formation.
Again, complex 4 showed less activity than its ferrocenyl analogues.
DNA in the presence and absence of the complexes, respectively).
Fig. 4. DCFDA-DCF assay used for ROS generation in HeLa cells by complex 1 (5
exposure to visible light (400e700 nm).
mM) on
Fig. 5. Cellular vanadium content as determined by ICP-MS in HeLa cells upon incu-
bation with the complexes 1e4 (30 m
M) at 37 ꢂC for 4 h.

B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
463
Fig. 6. A time-course collection of fluorescence microscopic images of untreated control cells (panels: aed), [VO(Fc-tpy)(Curc)](ClO4) (1) (10
mM) (panels: eel), [VO(Ph-tpy)(-
Curc)](ClO4) (4) (10
mM) (panels: met) in HeLa cells. Panels (e), (i), (m) and (q) show the green emission of the curcumin complex. Panels (b), (f), (j), (n) and (r) correspond to the
blue emission of Hoechst 33342 dye. Panels (c), (g), (k), (o) and (s) display the merged images of the first two panels. Panels (d), (h), (l), (p) and (t) show the corresponding bright-
field images. Scale bar ¼ 20
mm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
The DNA photocleavage activity follows the order: [VO(Fc-tpy)(-
Curc)](ClO4) (1)
> [VO(Fc-tpy)(bDMC)](ClO4) (3) > [VO(Fc-
tpy)(bDHC)](ClO4) (2) > [VO(Ph-tpy)(Curc)](ClO4) (4). Though the
ctDNA binding constant of 4 is higher than 1, the former complex
lacks MLCT band, which is responsible for better DNA cleavage
activity.
Mechanistic studies were carried out in red light of 647 nm
using complex 1 in the presence of various additives to explore any
formation of reactive oxygen species (Fig. 8). Singlet oxygen
quenchers, viz. sodium azide and TEMP did not inhibit the DNA
photocleavage activity and addition of D2O in which singlet oxygen
has longer life time, did not show any enhancement in the DNA
photocleavage activity, thus ruling out the singlet oxygen forma-
tion. The hydroxyl radical scavengers, viz. DMSO, KI and catalase
exhibited significant inhibitory effect on the DNA photo-cleavage
activity suggesting the formation of hydroxyl radical (ꢁOH) as the
ROS. Superoxide dismutase (SOD) showed only partial inhibitory
Table 3
Selected DNA (SC pUC19, 0.5
m
g) photocleavage data for complexes 1e4.
%NCb form %NCb form
Reaction conditionsa
l
¼ 454 nm ¼ 647 nm
l
DNA control
3
4
DNA þ [VO(Fc-tpy)(Curc)](ClO4) (1)
DNA þ [VO(Fc-tpy)(bDHC)](ClO4) (2)
DNA þ [VO(Fc-tpy)(bDMC)](ClO4) (3)
DNA þ [VO(Ph-tpy)(Curc)](ClO4) (4)
84
70
78
75
74
66
70
28
Fig. 7. Hoechst 33342 staining of the HeLa cells treated with complexes 1 and 4 for 4 h
in dark followed by photo-exposure to visible light (400e700 nm, 10 J cmꢀ2) for 1 h.
Panels (a), (c) and (e) are the fluorescence images of the control, complexes 1 and 4
treated cells (Hoechst stained) in dark. Panels (b), (d) and (f) are the fluorescence
images of the control and treated cells in light. The PDT effect is shown by arrows in
a
In Tris-buffer medium (pH ¼ 7.2). Concentration of complexes 1e4 was 20
M for 647 nm. Photo-exposure time (t) ¼ 2 h.
NC ¼ nicked circular form of pUC19 DNA.
mM
for 454 nm and 40
m
b
panels (d) and (f). The scale bar is 20 mm.

464
B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
procedures [60]. Supercoiled (SC) pUC19 DNA (cesium chloride
purified) and protein marker were purchased from Bangalore Genei
(India). Tris-(hydroxymethyl)aminomethaneeHCl (TriseHCl)
buffer solution was prepared using deionized and sonicated triple
distilled water. Curcumin, calf thymus (ct) DNA, agarose (molecular
biology grade), KI, catalase, NaN3,
L-histidine, SOD, 2,2,6,6-
tetramethyl-4-piperidone, bromophenol blue, xylene cyanol, Dul-
becco's Modified Eagle's Medium, propidium iodide, Hoechst
33342 and MTT were purchased from SigmaeAldrich (U.S.A.). Fc-
tpy, Ph-tpy, bDHCH and bDMCH were synthesized based on the
literature reports and the ligands were characterized from their 1H
NMR and 13C NMR spectral data [37,38].
Fig. 8. A bar diagram showing the mechanistic data for the visible light-induced SC
6.2. Instrumentation
pUC19 DNA (0.2
mg, 30 mM b.p.) cleavage activity of [VO(Fc-tpy)(Curc)](ClO4) (1, 40 mM)
in red light of 647 nm for an exposure time of 2 h.
The elemental analysis was carried out using a Thermo Finnigan
Flash EA 1112 CHN analyzer. The infrared, UVevisible and emission
spectra were recorded on PerkineElmer Lambda 35, PerkineElmer
Spectrum one 55 and PerkineElmer LS 55 spectrophotometer,
respectively. IR spectra signals were assigned as follows: br, broad;
vs, very strong; s, strong; m, medium; w, weak. Molar conductivity
measurements were performed using a Control Dynamics (India)
conductivity meter. Magnetic measurements were done in the solid
state at 25 ꢂC using Sherwood Scientific magnetic susceptibility
balance, Cambridge, UK. Cyclic voltammetric measurements were
carried out at 25 ꢂC on a EG&G PAR Model 253 VersaStat poten-
tiostat/galvanostat using a three electrode set-up with a glassy
carbon working, platinum wire auxiliary and a saturated calomel
reference electrode (SCE). Tetrabutylammonium perchlorate
(TBAP) (0.1 M) was used as a supporting electrolyte in DMF. The
electrochemical data were uncorrected for junction potentials.
Electrospray ionization mass spectral measurements were done
using Esquire 3000 plus ESI (Bruker Daltonics) Spectrometer. Room
temperature fluorescence quantum yield measurements were done
using a PerkineElmer LS 55 fluorescence spectrometer using
Fluorescein as a standard [61]. Fluorescence microscopic in-
vestigations were carried out on fluorescence microscope (Carl
Zeiss, Germany) using an oil immersion lens. FACS Calibur (Becton
Dickinson (BD) cell analyzer) at FL1 channel was used in the bio-
logical study. EPR spectral measurements were carried out using a
Bruker ER200D spectrometer. ICP-MS analysis was carried out on
Thermo scientific X-series-2-ICPMS. NMR spectral measurements
were made using Bruker NMR spectrometer.
effect, which could be due to spontaneous dismutation reaction of
superoxide radical anion to ꢁOH radicals. The complex did not show
any DNA photocleavage activity under an argon atmosphere. Its
known in the literature that on photo-irradiation, because of MLCT
band in the ferrocenyl complexes, it undergoes one electron
oxidation generating ferrocenium species (Fcþ) [57,58]. Ferroce-
nium species are known to generate hydroxyl radical via Fenton-
type reaction [59].
5. Conclusions
A new class of ferrocenyl oxovanadium(IV) complexes of cur-
cuminoids were designed, synthesized, characterized and their
DNA photocleavage and visible light-induced cytotoxic activity
studied. The ferrocenyl complexes showed significant DNA photo-
cleavage activity compared to its phenyl analogue in both blue and
red light. Mechanistic study revealed formation of hydroxyl radicals
as the reactive oxygen species. The complexes showed remarkable
PDT activity in HeLa cells compared to the normal 3T3 cells. The
effect of ferrocenyl moiety is evident from a four-fold increase in
photocytotoxicity of the ferrocenyl complex 1 compared to its
phenyl analogue 4. The IC50 value of complex 1 in light is compa-
rable to that of Photofrin®. The MTT assay results showed that the
presence of both methoxy and hydroxyl groups are essential in the
curcuminoid moiety to achieve desired cytotoxic effect. The ICP-MS
analysis of complex 1 showed a 7-fold increase in cellular uptake
compared to its phenyl analogue 4 and this could be due to lipo-
philic nature of the ferrocenyl moiety. Observed high cellular up-
take along with the presence of an MLCT band at 585 nm in 1 makes
this complex better PDT agent than its phenyl analogue 4. The
fluorescence emission property of the curcuminoids was utilized
for cellular imaging. The imaging study showed localization of the
complex in both cytosol and nucleus of the HeLa cells. Significant
photodynamic effect on nuclear morphology was observed when
HeLa cells were treated with the complex in light compared to the
non-irradiated samples. This suggests light-induced cell death by
the complexes via an apoptotic pathway. The results are of
importance toward developing the medicinal chemistry of bio-
organometallic complexes as metal-based photocytotoxic agents.
Caution! Perchlorate salts being potentially explosive, only small
quantity of the sample was used with due precautions.
6.3. Synthesis of the complexes 1e4
Vanadayl sulfate (VOSO4) (0.16 g, 1.0 mmol) was dissolved in
4 ml of ethanol and to this was added BaCl2$2H2O (0.24 g,1.0 mmol)
taken in 4 ml of ethanol and stirred for 1 h at 25 ꢂC. The mixture was
centrifuged and the clear solution was separated. To the clear so-
lution was added corresponding curcuminoids (1.0 mmol) (CurcH,
0.36 g; bDHCH, 0.33 g; bDMCH, 0.30 g) which was previously
treated with aqueous NaOH (0.04 g) and stirred for 30 min. To this
solution was added Fc-tpy (0.41 g) or Ph-tpy (0.30 g) dissolved in
(CHCl3:MeOH ¼ 1:4 v/v, 5.0 ml) and stirred for 30 min. The com-
plexes were precipitated as their perchlorate salts by adding excess
of aqueous NaClO4. The precipitate was filtered off, isolated and
washed with cold methanol and diethyl ether and dried in vacuum.
6. Experimental
6.1. Materials
Analytical or reagent grade chemicals were obtained from
commercial sources (s.d. Fine Chemicals, India; SigmaeAldrich,
U.S.A.) and used as such. Solvents were purified by reported

B. Balaji et al. / European Journal of Medicinal Chemistry 85 (2014) 458e467
465
6.3.1. [VO(Fc-tpy)(Curc)](ClO4) (1)
6.7. Cellular experiments
Yield: 70%. Anal. Calcd for C46H38N3O11ClFeV: C, 58.09; H, 4.03;
N, 4.42. Found: C, 57.78; H, 3.95; N, 4.21. ESI-MS (þ) in MeCN (m/z):
6.7.1. Cytotoxicity of complexes in HeLa cell line
851 [M-(ClO4)]þ. LM in DMF: 76 S m2
M
ꢀ1. UVeVis in DMF [lmax
,
The cytotoxicity of the complexes in two cancerous cells lines
namely, HeLa and Hep G2 and in normal 3T3 cells was assessed by
MTT assay [66]. About 1 ꢃ104 cells were seeded into 96-well plates
nm (ε, Mꢀ1 cmꢀ1)]: 436 (38 850), 585 (3140). FT-IR (cmꢀ1): 3324br,
1985w,1955w, 1607vs,1500vs,1386m,1278s, 1158m,1097s (ClO4ꢀ),
1030m, 970s (V]O), 847m, 790m, 754m, 727w, 667m, 622m,
582m, 507m, 467m, 438m, 412m. meff ¼ 1.61 mB at 298 K.
in 100 mL media per well. The cells were allowed to grow for 24 h in
the CO2 incubator at 37 ꢂC. Different concentrations of the com-
plexes dissolved in 1% DMSO were added to the cells. Incubation
was continued for further 4 h at 37 ꢂC in the CO2 incubator. After
incubation, the medium was replaced with 50 mM phosphate
buffer, pH 7.4 containing 150 mM NaCl (PBS) and photo-irradiation
was performed for 1 h in visible light of 400e700 nm using Luz-
chem Photoreactor (Model LZC-1, Ontario, Canada; light fluence
rate: 2.4 mW cmꢀ2; light dose ¼ 10 J cmꢀ2). For NAC (ROS scav-
enger) experiment, photo-irradiation was done in presence of
0.5 mM NAC containing PBS. Post irradiation, PBS was replaced
with 10% DMEM and cells were cultured for a further period of 20 h
6.3.2. [VO(Fc-tpy)(bDHC)](ClO4) (2)
Yield: 80%. Anal. Calcd for C46H38N3O9ClFeV: C, 60.12; H, 4.17; N,
4.57. Found: C, 59.89; H, 4.10; N, 4.49. ESI-MS (þ) in MeCN (m/z):
819 [M-(ClO4)]þ. LM in DMF: 70 S m2
M ,
ꢀ1. UVeVis in DMF [lmax
nm (ε, Mꢀ1 cmꢀ1)]: 414 (43 820), 439 sh (31 330), 585 (650). FT-IR
(cmꢀ1): 3080w, 1605s, 1577s, 1507s, 1479s, 1432s, 1395vs, 1280s,
1248vs, 1091vs (ClO4ꢀ), 1036s, 1008m, 962vs (V]O), 823m, 789vs,
730m, 675m, 597m, 516m, 475m, 428m. meff ¼ 1.64 mB at 298 K.
in dark followed by addition of 25
well and incubated for an additional 3 h. The culture medium was
discarded and a 200 L volume of DMSO was added to dissolve the
m
L of 4 mg mlꢀ1 of MTT to each
6.3.3. [VO(Fc-tpy)(bDMC)](ClO4) (3)
Yield: 68%. Anal. Calcd for C44H34N3O9ClFeV: C, 59.31; H, 3.85; N,
4.72. Found: C, 59.10; H, 3.79; N, 4.64. ESI-MS (þ) MeCN (m/z): 791
[M-(ClO4)]þ. LM in DMF: 80 S m2 Mꢀ1. UVeVis in DMF [lmax, nm (ε,
m
purple formazan crystals. The absorbance at 540 nm was deter-
mined using an ELISA microplate reader (BioRad, Hercules, CA,
USA). Cytotoxicity of the test compounds was measured as the
percentage ratio of the absorbance of the treated cells to the un-
treated controls. The IC50 values were determined by nonlinear
regression analysis (GraphPad Prism 5).
M
ꢀ1 cmꢀ1)]: 427 (24 730), 588 (2140). FT-IR (cmꢀ1): 3510w, 3096w,
1607vs, 1512vs, 1483vs, 1438s, 1383s, 1254s, 1166s, 1096vs (ClO4ꢀ),
1033vs, 952vs (V]O), 834s, 790s, 768m, 748m, 724m, 672m, 621s,
565m, 514m, 473m, 4362, 417w. meff ¼ 1.60 mB at 298 K.
6.3.4. [VO(Ph-tpy)(Curc)](ClO4) (4)
6.7.2. Measurement of intracellular ROSeDCFDA assay
Yield: 72%. Anal. Calcd for C42H34N3O11ClV: C, 59.83; H, 4.06; N,
4.98. Found: C, 59.54; H, 4.01; N, 4.88. ESI-MS (þ) MeCN (m/z): 743
[M-(ClO4)]þ. LM in DMF: 74 S m2 Mꢀ1. UVeVis in DMF [lmax, nm (ε,
Cell permeable DCFDA when oxidized by cellular ROS is known
to generate a fluorescent compound DCF having emission maxima
at 528 nm [52]. To determine the intracellular ROS, 0.5 ꢃ 106 cells
M
ꢀ1 cmꢀ1)]: 435 (27 860), 777 (124). FT-IR (cmꢀ1): 3441br, 3082w,
HeLa cells were incubated with complex 1 (10 mM) for 4 h in dark.
1610vs, 1506vs, 1484vs, 1420s, 1382s, 1279vs, 1158s, 1094vs (ClOꢀ4 ),
1028s, 970s (V]O), 948s, 825m, 790m, 768m, 729m, 668m, 622m,
553w, 470w, 420w. meff ¼ 1.62 mB at 298 K.
The media was replaced with PBS followed by photo-irradiation for
1 h in serum free conditions. The cells were harvested by trypsi-
nization and a single cell suspension was made. The cells were
washed with PBS to remove extracellular complex and treated with
10 mM DCFDA solution in DMSO and incubated in dark for 30 min at
6.4. Solubility and stability
room temperature and the intracellular fluorescence of formed DCF
was monitored by flow cytometry in the FL-1 channel. Three
negative control experiments were performed: (a) untreated cells,
in order to determine the contribution from auto-fluorescence of
cells; (b) curcumin treated cells, in order to determine the contri-
bution from curcumin fluorescence; and (c) cells treated with
DCFDA. Hydrogen peroxide treated cells were taken as positive
control.
All the complexes showed good solubility in DMF, acetonitrile,
DMSO and moderate solubility in CHCl3, CH2Cl2, methanol, and
ethanol. The complexes were found to be stable in the solid and
solution phases.
6.5. Computational methodology
All calculations were performed using GAUSSIAN09 program
suite [62]. Geometries of the complexes were optimized at B3LYP/
6-31g(d,p) level of theory [63,64]. Details are given as supporting
information. Time-dependent density functional theory (TD-DFT)
calculations were carried out to investigate the optical properties of
the complexes in DMF. The lowest 40 transitions, up to 400 nm,
were taken into account in the calculations of the absorption
spectra. Molecular orbital (MO) compositions were calculated using
the Multiwfn program [65].
6.7.3. Cellular uptake by ICP-MS
Uptake of oxovanadium complexes 1e4 was determined by
measuring the cellular vanadium using ICP-MS. HeLa cells
(0.5 ꢃ 106) were grown in 6 wells plate and incubated at 37 ꢂC
under 5% CO2 atmosphere for overnight. The culture medium was
removed and replaced with medium containing the complex
(30
mM). After incubation for 4 h, the medium was removed,
washed twice with 3 ml PBS to remove excess complex from
extracellular media, trypsinized and digested in concentrated nitric
acid (65% HNO3) at 70 ꢂC for 2 h. Then diluted with Milli-Q water to
the final volume of 10 ml and analyzed for ICP-MS. The instrument
was calibrated for vanadium using standard solutions containing 1,
10, 100 and 1000 ppb vanadium.
6.6. Cell culture
HeLa cells were cultured in Dulbecco's Modified Eagle's Medium
(DMEM), supplemented with 10% FBS, 100 IU mlꢀ1 of penicillin,
100 mg mlꢀ1 of streptomycin and 2 mM Glutamax and were
maintained in a humidified chamber at 37 ꢂC in 5% CO2 atmosphere.
The cells were passaged once every 4e5 days by trypsinizing with
0.25% Trypsin-EDTA.
6.7.4. Cellular uptake from fluorescence microscopy
The fluorescence of curcumin and its analogue complexes was
used to study the cellular localization using fluorescence micro-
scopy. 3 ꢃ 104 HeLa cells were grown on glass cover slips in each 6

466
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We thank the Department of Science and Technology (DST),
Government of India, and the Council of Scientific and Industrial
Research (CSIR), New Delhi, for financial support (CSIR No.
01(2559)/12/EMR-II and DST No. SR/S5/MBD-02/2007). We are
thankful to the Alexander von Humboldt Foundation, Germany, for
donation of an electroanalytical system. A.R.C. thanks DST for J.C.
Bose national fellowship. We thank Prof. S.V. Bhat and Mrs. S.K.
Bhagyashree for assistance in the EPR experiments and Dr.
Ramananda Chakrabarti for the ICP-MS facility. We thank the
Department of Biotechnology (DBT) for the FACS facility. S.P thanks
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