Curcumin "drug" Stabilized in Oxidovanadium(IV)-BODIPY Conjugates for Mitochondria-Targeted Photocytotoxicity

Article abstract of DOI:10.1021/acs.inorgchem.7b01924

Ternary oxidovanadium(IV) complexes of curcumin (Hcur), dipicolylamine (dpa) base, and its derivatives having pendant noniodinated and di-iodinated boron-dipyrromethene (BODIPY) moiety (L₁ and L₂, respectively), namely, [VO(dpa)(cur)]ClO₄ (1), [VO(L₁)(cur)]ClO₄ (2), and [VO(L₂)(cur)]ClO₄ (3) and their chloride salts (1a-3a) were prepared, characterized, and studied for anticancer activity. The chloride salts were used for biological studies due to their aqueous solubility. Complex 1 was structurally characterized by single-crystal X-ray crystallography. The complex has a VO²⁺ moiety bound to dpa ligand showing N,N,N-coordination in a facial mode, and curcumin is bound in its mono-anionic enolic form. The V-O(cur) distances are 1.950(18) and 1.977(16) ?, while the V-N bond lengths are 2.090(2), 2.130(2), and 2.290(2) ?. The bond trans to V=O is long due to trans effect. The complexes are stable in a solution phase over a long period of time of 48 h without showing any apparent degradation of the curcumin ligand. The diiodo-BODIPY ligand (L₂) or Hcur alone showed limited solution stability in dark. The emissive BODIPY (L₁) containing complex 2a showed preferential mitochondrial localization in MCF-7 cells in cellular imaging experiments. The cytotoxicity of the complexes was studied by MTT assay. The BODIPY complex 3a showed excellent photodynamic therapy effect in visible light (400-700 nm) giving IC₅₀ values of 2-6 μM in HeLa and MCF-7 cancer cells, while being less toxic in dark (～100 μM). The cell death was apoptotic in nature involving reactive oxygen species (ROS). Mechanistic data from pUC19 DNA photocleavage studies revealed photogenerated ROS as primarily ¹O₂ from the BODIPY moiety and ·OH radicals from the curcumin ligand.

Full text of DOI:10.1021/acs.inorgchem.7b01924

Article
pubs.acs.org/IC
Curcumin “Drug” Stabilized in Oxidovanadium(IV)-BODIPY
Conjugates for Mitochondria-Targeted Photocytotoxicity
†
‡
†
†
†
Utso Bhattacharyya, Brijesh Kumar, Aditya Garai, Arnab Bhattacharyya, Arun Kumar,
Samya Banerjee,† Paturu Kondaiah,* and Akhil R. Chakravarty*
,‡
,†
†
‡
Department of Inorganic and Physical Chemistry and Department of Molecular Reproduction, Development and Genetics, Indian
Institute of Science, Bangalore 560 012, India
*
S Supporting Information
ABSTRACT: Ternary oxidovanadium(IV) complexes of curcumin (Hcur),
dipicolylamine (dpa) base, and its derivatives having pendant noniodinated
and di-iodinated boron-dipyrromethene (BODIPY) moiety (L and L ,
1
2
respectively), namely, [VO(dpa)(cur)]ClO (1), [VO(L )(cur)]ClO (2),
4
1
4
and [VO(L )(cur)]ClO (3) and their chloride salts (1a−3a) were prepared,
2
4
characterized, and studied for anticancer activity. The chloride salts were used
for biological studies due to their aqueous solubility. Complex 1 was
structurally characterized by single-crystal X-ray crystallography. The complex
has a VO2 moiety bound to dpa ligand showing N,N,N-coordination in a
facial mode, and curcumin is bound in its mono-anionic enolic form. The V−
O(cur) distances are 1.950(18) and 1.977(16) Å, while the V−N bond
lengths are 2.090(2), 2.130(2), and 2.290(2) Å. The bond trans to VO is
long due to trans effect. The complexes are stable in a solution phase over a
long period of time of 48 h without showing any apparent degradation of the curcumin ligand. The diiodo-BODIPY ligand (L2)
+
or Hcur alone showed limited solution stability in dark. The emissive BODIPY (L ) containing complex 2a showed preferential
1
mitochondrial localization in MCF-7 cells in cellular imaging experiments. The cytotoxicity of the complexes was studied by
MTT assay. The BODIPY complex 3a showed excellent photodynamic therapy effect in visible light (400−700 nm) giving IC
5
0
values of 2−6 μM in HeLa and MCF-7 cancer cells, while being less toxic in dark (∼100 μM). The cell death was apoptotic in
nature involving reactive oxygen species (ROS). Mechanistic data from pUC19 DNA photocleavage studies revealed
1
photogenerated ROS as primarily O from the BODIPY moiety and ·OH radicals from the curcumin ligand.
2
INTRODUCTION
in its enolic form to an oxophilic metal ion leads to its
■
21−30
stabilization and significantly increases its bioavailability.
Transition-metal complexes of curcumin show different extent
Curcumin (Hcur) as an active ingredient of turmeric is well-
known for the treatment of a wide variety of ailments (Chart
21−24,27,28
1
−10
of labilization of the M−O (curcumin) bond.
While
1).
The anticancer activity is ascribed to its antiproliferative,
metal ions like Co(III) and Pt(II) show slow release of
curcumin from their metal complexes leading to its eventual
degradation, oxophilic metal ions like vanadium(IV) or trivalent
lanthanoids stabilize the ligand in a significant manner and
anti-metastatic, and antiangiogenic properties. In vitro studies
indicate that curcumin induces apoptosis via mitochondrial
pathways comprising caspases and the Bcl-2 family of proteins
1
1−17
and restricts the function of NF-κB.
The major
21−24,27−29
enhance its bioavailability.
predicaments are poor aqueous solubility and hydrolytic
instability in buffer of physiological pH of 7.4 that limit its
therapeutic potential. With a high dose of tolerance intake
The present work stems from our continued interests to
design curcumin based photocytotoxic agents, and we used
VO2+ moiety to stabilize the curcumin dye in its metal-bound
form. While the stabilized curcumin is expected to retain its
pendant diphenolic units (not used as metal binding site) intact
for its biological activity, dipicolylamine (dpa)-based N,N,N-
donor ancillary ligands with pendant di-iodinated boron-
(
>100 mg, i.e., ∼3 g of turmeric) per day, its delivery to a
biological target is restricted. Nelson and co-workers have made
an elaborate analysis of the published results and highlighted
the unreliability of the data leading to its classification under
pan-assay interference compounds (PAINS) and invalid
18,19
dipyrromethene (BODIPY) moiety is incorporated in the
metabolic panaceas (IMPS).
been made by other authors who have opined that vast
literature reports on curcumin and curcuminoids cannot just be
dismissed from limited observations and analysis. The real
concern of curcumin is its poor bioavailability and instability in
a buffer medium. This needs to be addressed rationally and
critically. Recent reports have shown that binding of curcumin
A rebuttal has subsequently
1
ternary structure to generate singlet oxygen ( O ) for achieving
2
light-activated cell death by a type-II pathway akin to
photodynamic therapy (PDT) of cancer by hematoporphyrin
2
0
Received: July 28, 2017
©
XXXX American Chemical Society
A
DOI: 10.1021/acs.inorgchem.7b01924
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Chart 1. Curcumin Dye (Hcur) and Its Structure in Keto and
Enolic Forms
1a−3a, L is dipicolylamine (dpa) for 1 and 1a, BODIPY
appended dpa for 2 and 2a, and diiodo BODIPY appended dpa
for 3 and 3a (Figure 1). The complexes showed aqueous
Figure 1. Ternary oxidovanadium(IV) complexes 1−3 as perchlorate
and 1a−3a as chloride salts.
solubility and stability, while the ligands alone, specifically,
curcumin (Hcur) and the BODIPY bases (L and L ) showed
poor aqueous solubility. The significant aspects of this study
include structural characterization of the curcumin complex 1
1
2
3
1−35
drug, namely, Photofrin.
The ligands in the ternary
structure [(BODIPY-dpa)VO(cur)]+ are expected to show
selectivity and targeted anticancer activity induced by light. The
advantage of PDT over conventional chemotherapy is its
selectivity causing the cellular damage of only the light-activated
cancer cells, while the unexposed healthy cells remain
by X-ray crystallography, complex 3a having diiodo BODIPY
1
unit showing remarkable singlet oxygen ( O ) mediated photo-
2
cytotoxicity in MCF-7 (human breast adenocarcinoma) and
HeLa (human cervical carcinoma) cancer cells in visible light
3
1,32
unaffected.
PDT with its three essential components,
(
400−700 nm), while being essentially nontoxic in dark, and
specifically, (i) photosensitizer (PS), (ii) light to activate the
PS, and (iii) molecular oxygen to generate reactive oxygen
species (ROS), has received current attention for developing
metal-based PDT agents that could overcome the toxicities
associated with Photofrin, namely, skin sensitivity and
the emissive complexes 1a and 2a exhibiting predominant
mitochondrial localization from the confocal microscopic
imaging studies. Mitochondrial localization is important, as it
avoids the nuclear excision repair (NER) pathway, which
reduces the efficacy of any nuclear DNA targeting anticancer
3
1,35
36,37
hepatotoxicity.
bioessential metal ion, curcumin as a nontoxic natural product
NP) for its selectivity and anticancer activity, and BODIPY
The PDT agents having vanadium as a
drug.
Stabilization of curcumin in its metal-bound form
could lead to further development of new strategies for cellular
delivery of this dye for in vivo applications without causing any
degradation or structural changes. That may enable curcumin
(
units as photosensitizers could find utility as new generation
metal-based PDT agents. We used two different BODIPY
moieties (L1 and L ). The highly emissive ligand L1 is
18,19
to list out of the classification under “PAINS”.
2
incorporated in the structure for cellular imaging to study
localization of the complex in different cellular organelle, while
EXPERIMENTAL SECTION
Materials and Methods. The reagents and chemicals were
procured from the commercial sources (s.d. Fine Chemicals, India;
Sigma-Aldrich, U.S.A., E-Merck and Alfa Aesar, U.K.). Solvents used
were purified and distilled by standard methods. Curcumin (95%
curcuminoid content, ca. 80% curcumin) was purchased from Sigma-
■
the diiodo BODIPY unit in L is used as a photosensitizer to
2
generate singlet oxygen on light activation. Additionally,
curcumin as a photosensitizer is known to generate hydroxyl
3
8
1
,2,7
radicals as the ROS on light activation.
39
Aldrich, U.S.A. and purified by following a literature method.
The ternary complexes are aimed to enhance the efficacy
compared to our earlier reported non-PDT oxidovanadium(IV)
complexes, specifically, [VO(cur)(phen/dppz)Cl], where phen
Synthesis of the complexes was performed under nitrogen atmosphere
using Schlenk technique. Supercoiled (SC) pUC19 DNA (cesium
chloride purified) was from Bangalore Genie (India). Tris-
22
and dppz are 1,10-phenanthroline and dipyridophenazine.
(
hydroxymethyl)aminomethane-HCl (Tris-HCl) buffer (pH = 7.2)
These complexes showed stabilization of curcumin on binding
to the VO2 unit. The replacement of the phenanthroline bases
by dipicolylamines with BODIPY units is to achieve enhanced
PDT activity by generating singlet oxygen on photoactivation.
While human body has enzymatic defense mechanism to
degrade the hydroxyl radicals, a similar process is absent in our
cells for the singlet oxygen. Again, the BODIPY moiety is
capable of generating ROS at low oxygen concentration that is
prevalent in the tumor cells. This is due to high yield of singlet
oxygen from the diiodo BODIPY unit. Herein, we present the
PDT activity of three ternary oxidovanadium(IV) complexes of
curcumin [VO(L)(cur)]X, where cur is the curcumin
was prepared using deionized and sonicated double distilled water.
Dulbecco’s Modified Eagle’s medium (DMEM), 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2,7-dichlorofluor-
escein diacetate (DCFDA), calf thymus (ct) DNA, agarose (molecular
biology grade), 2,2,6,6-tetramethyl-4-piperidone (TEMP), ethidium
bromide (EB), Hoechst dye, propidium iodide (PI), and annexin-V/
FITC were purchased from Sigma-Aldrich (U.S.A.) and used as
+
received. The dipicolylamine (dpa), BODIPY-appended dpa (L ), and
1
diiodo-BODIPY-appended dpa (L ) were prepared by reported
2
procedures (4,4-difluoro-1,3,5,7-tetramethyl-4-bora-3a,4a-diaza-s-inda-
40,41
cene is abbreviated as BODIPY in this work).
Tetrabutylammo-
nium perchlorate (TBAP) was prepared by reacting tetrabutylammo-
nium bromide and perchloric acid. Caution! Use in small quantities and
with care.
monoanion, X is ClO for complexes 1−3 or Cl for complexes
4
B
DOI: 10.1021/acs.inorgchem.7b01924
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
−
1
The elemental analysis of the complexes (1−3, 1a−3a) was
performed using a Thermo Finnigan FLASH EA 1112 CHNS
analyzer. The infrared and electronic spectra (UV−vis) were recorded
with Perkin−Elmer Lambda 35 and Perkin−Elmer spectrum one 55
spectrometers, respectively, at 25 °C. Molar conductivity measure-
ments were made with a Control Dynamics (India) conductivity
meter. Electrochemical measurements were made at 25 °C with an
EG&G PAR model 253 VersaStat potentiostat/galvanostat with
electrochemical analysis software 270 using a three-electrode setup
having a glassy carbon working, platinum wire auxiliary, and a
saturated calomel reference electrode (SCE) in dimethylformamide
IR data/cm : 3364 m (br), 1588 s, 1495 vs, 1421 m, 1365 w, 1271
s,1152 m, 1116 m, 1095 m, 969 m, 843 w, 756 m, 525 w, 465 w. UV−
visible in 1:1 DMF/DPBS [λ /nm (ε/M cm )]: 721 (69), 535
(30 285) 432 (27 450), 288 (65 000). Molar conductivity in 10%
aqueous DMF at 298 K [Λ /S m M ]: 84. μ , μ at 298 K: 1.61.
VO(L )(cur)]Cl (3a): Yield = 75%. Anal. Calcd for
−1
−1
max
2
−1
M
eff
B
[
2
C H BClF I N O V: C, 50.64; H, 3.93; N, 5.73. Found: C, 50.43;
53
49
2 2
5
7
+
H, 3.74; N, 5.53%. ESI-MS in CH OH: m/z 1221.0482 [M − Cl] .
Solubility. The perchlorate and chloride complexes were soluble in
ethanol, methanol, acetonitrile, DMF, and dimethyl sulfoxide
DMSO). The complexes were moderately soluble in CHCl and
3
(
3
(
DMF)−0.1 M TBAP. Electrospray ionization (ESI) mass spectral
CH Cl2 and insoluble in hydrocarbon solvents. The chloride
2
measurements were made using an Agilent Technologies 6538 UHD
Accurate-mass Q-TOF LC/MS ESI model mass spectrometer.
Fluorescence measurements at room temperature were done using a
Perkin−Elmer LS 55 fluorescence spectrometer. The fluorescence
quantum yields of the compounds were obtained by a relative method
complexes showed better aqueous solubility (∼200 μM in 99:1 v/v
DPBS/DMSO) than that of perchlorate complexes (∼100 μM in 99:1
v/v DPBS/DMSO) and were used for biological studies.
X-ray Crystallographic Procedures. The crystal structure of
[VO(dpa)(cur)]ClO4 (1) was obtained by single-crystal X-ray
diffraction method.
diffusion of diethyl ether into solution of the complex in acetonitrile. A
crystal of dimensions 0.6 × 0.3 × 0.2 mm was mounted on a crystal
mounting loop with the help of paratone oil. The intensity data were
collected using graphite-monochromated Mo Kα radiation (0.7107 Å)
at 273 K. The data quality was found to be poor. That resulted in some
high thermal parameters, higher shift/esd residue for the final cycle of
refinement, and relatively high R-indices than desired. The structure
was solved in P1 space group, although there were two similar complex
molecules in the unit cell. Our attempts to use centrosymmetric P1
space group with Z value of 2 did not yield the structure. On careful
analysis it was found that two molecules are not identical. While one
cationic complex was involved in H-bonding with a ClO4− anion, the
other was not. This was supported from the Fourier transform infrared
FT-IR) data that showed splitting of the perchlorate peak indicating
lowering of symmetry from T to C on H-bond formation. The
structure solved by direct methods using SHELXL-2014 incorporated
in WinGX (Version 1.63.04a) did not show any anomaly in the core
structure of the complex. Selected crystallographic parameters are
given in Table 1. The CCDC deposition number is 1543636.
22,42
as described in the literature.
Viscometric measurements using ct-
4
3−45
Single crystals were obtained upon vapor
DNA were performed with a Schott AVS 310 automated viscometer.
Fluorescence microscopic investigations were performed using
ApoTome.2 fluorescence microscope. Flow cytometric analysis was
performed using FACS (fluorescence-activated cell sorting) Calibur
Becton Dickinson (BD) cell analyzer at FL2 channel (595
nm).Vanadium contents in 1a−3a treated MCF-7 cells were measured
by Perkin−Elmer Optima 7000 DV ICP-OES.
Synthesis of the Complexes. Vanadyl sulfate (0.16 g, 1.0 mmol)
and calcium perchlorate (0.31 g, 1.0 mmol) for 1−3 or barium
chloride (0.24 g, 1.0 mmol) for 1a−3a were dissolved in 15 mL of
ethanol and 3 mL of water. The mixture was then stirred at room
temperature for 3 h under a nitrogen atmosphere. The mixture was
filtered to remove the white calcium sulfate or barium sulfate as the
precipitate. The blue filtrate was deaerated and saturated with
nitrogen. To this filtrate was added a deaerated solution (10 mL of
EtOH and 2 mL of MeCN) of Hcur (0.36 g, 1.0 mmol), which was
3
̅
(
d
3v
previously neutralized with Et N (0.10 g, 1.0 mmol). A deep red
3
solution thus formed after stirring the mixture for 30 min was reacted
with an ethanol solution (5 mL) of dipicolylamine (0.20 g, dpa) for 1/
1
a or a CH Cl solution (10 mL) of L or L (0.54 g, L ; 0.79g, L ,1.0
2 2 1 2 1 2
mmol) for BODIPY complexes. The product precipitated out of the
solution spontaneously after it was stirred for 2 h. The solid was
filtered, isolated, and washed with cold ethanol, CH Cl , and diethyl
Table 1. Selected Crystallographic Data for the Complex
[VO(dpa)(cur)](ClO ) (1)
2
2
ether and finally dried in vacuum over P O in a desiccator.
4
10
4
[
VO(dpa)(cur)](ClO ) (1). Yield = 75%. Anal. Calcd for
4
empirical formula
formula weight (g M−1)
C H ClN O V
33 32 3 11
C H ClN O V: C, 54.07; H, 4.40; N, 5.73. Found: C, 53.79; H,
3
3
32
3
11
+
4
.35; N, 5.44%. ESI-MS in CH OH: m/z 633.1640 [M − ClO ] . IR
data/cm : 3360 m (br), 1590 s, 1494 vs, 1373 w, 1276 s, 1150 m,
122 m, 1095 w, 968 m, 819 m, 619 m, 465 w (vs, very strong; s,
733.02
3
4
−1
crystal system
space group
a, Å
triclinic
P1
1
strong; m, medium; w, weak; br, broad.). UV−visible in 1:1
12.241(3)
12.600(3)
12.941(3)
95.769(6)
110.364(5)
114.313(5)
1634.2(7)
2
dimethylformamide/Dulbecco’s phosphate-buffered saline (DMF/
b, Å
−1
−1
DPBS) [λmax/nm (ε/M cm )]: 715 (85), 435 (45,800), 280
62,200). Molar conductivity in 10% aqueous DMF at 298 K [Λ /S
c, Å
(
M
2
−1
α, deg
m M ]: 97. μ , μ at 298 K: 1.60. [VO(dpa)(cur)]Cl (1a): Yield =
eff
B
β, deg
7
2%. Anal. Calcd for C H ClN O V: C, 59.24; H, 4.82; N, 6.28.
33 32 3 7
γ, deg
V, Å3
Found: C, 58.79; H, 4.45; N, 5.95%. ESI-MS in CH OH: m/z
3
+
6
33.1691 [M − Cl] .
[
VO(L )(cur)](ClO ) (2). Yield = 80%. Anal. Calcd for
Z
1
4
C H BClF N O V: C, 59.54; H, 4.81; N, 6.55. Found: C, 59.32;
T, K
273 (2)
1.484
53
51
2
5
11
+
ρcalc, g cm−
3
H, 4.47; N, 6.26%. ESI-MS in CH OH: m/z 969.3458 [M − ClO ] ,
3
4
−1
IR data/cm : 3363 m (br), 1589 s, 1496 vs, 1368 m, 1276 s,1154 m,
117 m, 1094 w, 965 m, 817 m, 762 m, 619 w, 465 w, 435 m. UV−
visible in 1:1 DMF/DPBS [λ /nm (ε/M cm )]: 717 (77), 501
λ, Å (Mo Kα)
μ, cm−1
0.710 73
0.452
1
−1
−1
max
data/restraints/parameters
14 768/15/733
753
(
54 680), 454sh (36 600), 434 (35 475), 285 (61 600) (sh, shoulder).
2
−1
F(000)
Molar conductivity in 10% aqueous DMF at 298 K [Λ /S m M ]:
8
M
GOF
0.931
9. μ , μ at 298 K: 1.63. [VO(L )(cur)]Cl (2a): Yield = 78%. Anal.
eff B 1
a
b
R(F0), I > 2σ(I) [R (F )]
0.1102 [0.4032]
0.1513 [0.2289]
0.407, −0.322
Calcd for C H BClF N O V: C, 63.33; H, 5.11; N, 6.97. Found: C,
w
0
53
51
2
5
7
R [R (all data)]
largest diff peak and hole (e Å−3)
6
2.82; H, 4.78; N, 6.76%. ESI-MS in CH OH: m/z 969.3288 [M −
w
3
+
Cl] .
VO(L )(cur)](ClO ) (3). Yield = 77%. Anal. Calcd for
[
2
4
a
R = ∑∥F | − |F ∥/∑|F |. R = {∑[w(F − F ) ]/∑[w(F ) ]}1/2.
b
2
o
2
c
2
2
C H BClF I N O V: C, 48.19; H, 3.74; N, 5.30. Found: C, 47.93;
5
3
49
2 2
5
11
o
c
o
w
o
+
2
2
2
−1
2
2
H, 3.54; N, 4.95%. ESI-MS in CH OH: m/z 1221.1298 [M − ClO ] ,
w = [σ F + (0.0723P) ] , where P = (F + 2F )/3.
o o c
3
4
C
DOI: 10.1021/acs.inorgchem.7b01924
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
Table 2. Selected Physicochemical Data for the Complexes 1−3 as Perchlorate Salts
IR, cm−1 VO, CO
a
λmax, nm (ε , M cm−1
b
−1
)
λ , nm [Φ ]
c
μeff
d
ΛM
e
E , V
f
K , M
b
g
−1
complex
F
F
pc
4
1
2
968, 1590
965, 1589
435(45,800), 715(85)
434(35,475),
531 [0.04]
512 [0.11]
1.60
1.63
97
89
−1.02
−0.96
(8.9 ± 0.4) × 10
(2.1 ± 0.6) × 10
5
5
7
01(54,680),
17(77)
3
969, 1588
432(27,450),
521 [0.01]
1.61
84
−1.04
(7.9 ± 0.5) × 104
5
7
35(30,285),
21(69)
a
b
c
In the solid phase. In 1:1 DMF−DPBS buffer. In 10% aqueous DMSO. Quantum yield was measured using fluorescein as the standard (Φ =
F
d
e
2
−1
f
0
.79). μ in μ for solid samples of the complexes at 25 °C. Λ , molar conductivity (S m M ) in 10% aqueous DMF at 25 °C. In 5 mL of DMF
eff
B
M
−1
with 0.1 M TBAP and 2 mmol complexes. The potentials were vs SCE at a scan rate of 50 mV s . E is the cathodic peak potential. There was no
anodic counterpart. Intrinsic ct-DNA binding constant.
pc
g
Theoretical Methods. The geometries of the complexes were
diameter) was used from a continuous-wave (CW) diode laser made of
Research Electro-Optics, Colorado (U.S.A.), model no. EXLSR-532−
100-CDRH. Different external agents, namely, sodium azide and
2,2,6,6-tetramethyl-4-piperidone (TEMP) as singlet oxygen quenchers,
DMSO, KI, and catalase as hydroxyl radical scavengers, and superoxide
dismutase (SOD) for superoxide scavenger were used for mechanistic
studies to ascertain the formation of any hydroxyl radicals (·OH) and
optimized by density functional theory (DFT) using B3LYP level of
4
6−48
theory and Lanl2DZ basis state by Gaussian 09 program.
The
coordinates were initially obtained from the crystal structure of
complex 1. They were used for optimization of the structures of the
complexes with or without having the BODIPY moiety. The optimized
coordinates are given in Tables S1−S3 along with the electronic
transitions in Figure S17 (see Supporting Information).
1
singlet oxygen ( O ) as the ROS. D O was used as a solvent in which
2
2
53
Experiments on Singlet Oxygen. Generation of singlet oxygen
singlet oxygen is known to have longer lifetime than in H O.
2
on visible light irradiation of the complexes was examined using 1,3-
49,50
diphenylisobenzofuran (DPBF) titration method.
DPBF and the
RESULTS AND DISCUSSION
■
complex in DMSO in a 50:1 molar ratio were taken and photo-
irradiated. The absorption maximum at 417 nm corresponding to
DPBF was monitored after different time intervals of the light
exposure using a broad-band light source of 400−700 nm (Luzchem
Photoreactor model LZC-1, Ontario, Canada, fitted with Sylvania-
made 8 fluorescent white tubes, having a fluorescence rate of 2.4 mW
cm , to provide a total dose of 10 J cm ). The relative rates of decay
of the DPBF-based absorption band for different complexes and the
BODIPY ligands were calculated.
Synthesis and General Aspects. Ternary oxidovanadium-
(IV) complexes of curcumin (Hcur) having dipicolylamine
dpa) bases, namely, [VO(dpa)(cur)]X (X = ClO , 1; Cl, 1a),
(
[
4
VO(L )(cur)]X (L = BOPIPY-appended dpa; X = ClO , 2;
1
1
4
Cl, 2a) and [VO(L )(cur)]X (L = diiodo BOPIPY-appended
−2
−2
2
2
dpa; X = ClO , 3; Cl, 3a), were prepared in good yield from a
4
general synthetic procedure in which vanadyl sulfate was first
reacted with calcium perchlorate or barium chloride,
respectively, for the perchlorate or chloride counteranion, in
aqueous ethanol (1:5 v/v), and the filtrate on removal of
calcium sulfate or barium sulfate was subsequently reacted with
the deprotonated solution of curcumin in ethanol−acetonitrile
mixture. Finally, a solution of the tridentate ligand (in ethanol
Cellular Measurements. Cytotoxicity studies were performed
with the complexes 1a−3a in MCF-7 (human breast adenocarcinoma
cell line) and HeLa (human epithelial cervical carcinoma cell line) by
3
-(4,5-dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT)
51
colorimetric assay following procedures that were reported earlier.
Cells were incubated with various concentrations of 1a−3a for 4 h in
dark. One set of cells was exposed to visible light (400−700 nm
for dpa or in CH Cl for L and L ) was added to the reaction
−2
2
2
1
2
Luzchem Photoreactor, 10 J cm power) for 1 h, while the other set
was kept in darkness. The stability of the cells with this light dose was
ascertained prior to the exposure of the samples containing the
complexes. Data were obtained with use of three independent sets of
experiments performed in triplicates for each concentration. Uptake of
the complexes in MCF-7 cells was studied by flow cytometry. 2′,7′-
Dichlorofluorescein diacetate (DCFDA) assay was performed with the
complexes to detect generation of any ROS in the MCF-7 cells. The
final concentration of vanadium content in 1a−3a-treated MCF-7 cells
were measured by inductively coupled plasma mass spectrometry
mixture to obtain a precipitate of the desired product. The
complexes were characterized from spectroscopic and analytical
data. Selected physicochemical data are given in Table 2. The
electrospray ionization mass spectrometry (ESI-MS) data of the
perchlorate and chloride complexes showed respective single
+
+
peak of [M − ClO ] or [M − Cl] in MeOH (Figures S1−S6,
4
Supporting Information). The complexes were 1:1 electrolytes
in 10% aqueous DMF giving molar conductivity values of ca. 90
2
−1
54
S m M at 25 °C. The magnetic moment values of ∼1.6 μB
indicate V(IV) oxidation state with one unpaired electron. The
IR spectra of the complexes showed CO and CC
stretching bands assignable to curcumin at ∼1589 and ∼1495
(
1
ICP-MS) method. The cellular localization of fluorescent complexes
a and 2a inside the MCF-7 cells was performed by confocal
microscopy experiments. Different organelle selective trackers were
used for cellular localization study using the merged images. The effect
of the complexes on cell cycle in MCF-7 was studied to analyze the
phase where the cell cycle got arrested, and annexin-V/FITC assay was
performed to confirm apoptotic cell death.
−1
cm , respectively (Figures S7−S9, Supporting Information).
−
The characteristic infrared bands of VO and ClO
anion
55
4
−1
were observed at ca. 967 and 1095 cm , respectively. The
ClO4 peak showed splitting suggesting deviation from its Td
−
DNA Binding and Cleavage Experiments. UV−vis spectral
studies were done to determine DNA binding constants of the
geometry to a lower symmetry, possibly due to hydrogen-
bonding interaction with the complex in the solid state (Figure
S10, Supporting Information). The oxidovanadium(IV) com-
plexes showed a broad and weak d−d band within 715−720 nm
in 1:1 DMF/DPBS medium (Figure S11, Supporting
5
2
complexes using calf thymus (ct) DNA by reported procedures.
Viscosity measurements were performed to ascertain the DNA binding
nature of the complexes 1a−3a compared to ethidium bromide (EB)
as a DNA intercalator and Hoechst dye as a DNA groove binder. The
supercoiled (SC) pUC19 plasmid DNA photocleavage activity of the
complexes was studied on light exposure using diode lasers followed
by gel electrophoresis. A monochromatic visible light source of 532
nm (100 mW power, 1 h exposure time, 0.32 ± 0.02 mm beam
2
1,49,56
Information).
An intense visible band at ∼434 nm with
a shoulder at ∼454 nm is assigned to curcumin π→π*
transition with the shoulder peak being prominent in complex 2
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Figure 2. (a) Absorption spectra of the complexes 1−3 in 1:1 (v/v) DMF/PBS buffer medium. (b) Emission spectra of the complexes 1−3 in 10%
aqueous DMSO (λ : 430 nm for 1 and 3; 465 nm for 2).
ex
7
(
Figure 2a). The BODIPY and diiodo BODIPY-centered
curcumin as evidenced from the absorption spectral studies of
21,22,29
intense π→π* transition was observed for complexes 2 and 3 at
01 and 535 nm, respectively.
the complexes.
The present complexes were found to be
39,40
5
This band was not visible in
stable in both solid and solution phases under dark
experimental conditions. The spectral band intensity of
curcumin alone or in its metal-bound state was monitored by
absorption spectroscopy in 1:1 DMF/DPBS medium up to 48
h. The intensity of the spectral band remained essentially
unchanged compared to that of free curcumin (Figure 3, Figure
the spectrum of complex 1, which lacks a BODIPY moiety. The
dpa ligand showed an intense band in the complexes at ∼280
nm. All the complexes were found to be emissive in 10%
aqueous DMSO (Figure 2b). Complexes 1 and 3 showed
respective emission maxima at 531 and 521 nm upon excitation
at 430 nm. The emission of complex 1 in absence of the
BODIPY moiety is due to curcumin giving a ϕ value of 0.04.
F
The emission of complex 3 with a ϕ value of 0.01 is also due
F
to curcumin as the diiodo BODIPY moiety being nonemissive
57−59
due to efficient intersystem crossing (ISC).
Complex 2 is
highly emissive due to combined green emission of curcumin
and the BODIPY unit in L giving a band at 512 nm with a
1
modest ϕ value of 0.11 upon excitation at 465 nm. The
F
emission spectral property of 2 makes it suitable for cellular
uptake and imaging studies by confocal fluorescence micros-
copy. The complexes were redox-active showing a reduction
peak assignable to the V(IV)−V(III) couple near −1.0 V versus
n
Figure 3. Absorption spectral traces of the complexes 1−3 and
curcumin (Hcur) (monitored at 435 nm for 1, 2 and curcumin; 432
nm for 3) in 1:1 (v/v) DMF/PBS buffer medium (pH = 7.2) at 37 °C
SCE in DMF with 0.1 M [Bu N](ClO ) (TBAP) as the
4
4
supporting electrolyte (Table 2, Figure S12, Supporting
Information). The curcumin-based reduction peaks in 1−3
were observed near −1.59 V. Complex 2 showed a BODIPY-
based reduction peak near −1.17 V. The complexes did not
show any oxidative response. The redox stability of the
complexes over a large potential window is expected to
decrease their chemical nuclease activity and dark cellular
toxicity in the presence of thiols, namely, glutathione (GSH).
The redox stability of the metal is also expected to reduce the
possibility of any curcumin ligand release.
2+
up to 48 h indicating the stability of the VO bound curcumin.
S13, Supporting Information). The stability follows the order: 1
> 2 > 3. The spectra of BODIPY complex 2 remained
unchanged up to 48 h, while the diiodo-BODIPY complex 3
showed reduced band intensity, possibly due to instability of
60,61
the ligand.
A similar trend was observed for the chloride
analogues (1a−3a) when their stabilities were spectroscopically
monitored in DMSO−DMEM medium (1:99 v/v, similar to
cellular medium) for 72 h (Figure S14, Supporting
Information). The spectral data revealed curcumin stability
on binding to VO2+ in buffer at pH of 7.2. Furthermore, ESI-
MS spectral studies showed that complex 2 even after 1 h of
Stability of the Complexes. Limited stability of curcumin
in the physiological buffer medium makes it unsuitable for
7
,18,19
varied medicinal applications.
Curcumin is known to
degrade via different pathways, namely, (i) reduction, (ii)
conjugation, (iii) oxidation, and (iv) cleavage to form vanillin,
ferulic acid, and feruloylmethane as the major products. One
reason for the classification of curcumin under “PAINS” and
photo-irradiation (400−700 nm) displayed ∼65% of the
+
complex remaining intact giving a peak of [M-ClO ] in
4
“
IMPS” is due to its instability. Our earlier reports have shown
MeOH with an m/z value of 969.3287 (Figure S15, Supporting
Information).
21,22
that binding of curcumin to a metal ion leads to its stability.
Cobalt(III) and platinum(II) complexes slowly release
curcumin on chemical and/or photochemical treatments.
In contrast, oxophilic metal ions do not show any release of
X-ray Crystal Structure. The crystal structure of [VO-
(dpa)(cur)]ClO4 (1) was obtained by single-crystal X-ray
diffraction method. A perspective view of the cationic complex
23,24,27
E
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Article
3,24
2
in 1 is shown in Figure 4 (Figure S16, Supporting Information).
glutathione or on photoactivation to form Co(II) species.
Selected bond distances and angles are given in Table 3. The
The redox stable VO(IV), La(III), and Gd(III) complexes with
respective M−O(cur) distances of ∼1.95, ∼2.40, and ∼2.41 Å
21,22,29,30
do not show similar release of curcumin.
Theoretical Studies. DFT studies were done to rationalize
the structural and spectral data of complexes 1−3 using B3LYP
45−47
with Lanl2DZ basis set with Gaussian programs.
The
coordinates obtained from the crystal structure of complex 1
were used for the geometrical optimization of the complexes
(
Tables S1−S3, Supporting Information). The optimized
structures along with the highest occupied molecular orbital−
lowest unoccupied molecular orbital (HOMO−LUMO) are
shown in Figure 5. From the frontier molecular orbital (FMO)
Figure 4. A perspective view of the cationic complex in [VO(dpa)-
(
cur)](ClO ) (1) showing the spatial arrangements of the ligands in
4
the ternary structure (color code: red, V; green, N; blue, O; gray, C).
The hydrogen atoms are omitted for clarity.
Table 3. Selected Bond Distances (Å) and Angles (deg) for
[
VO(dpa)(cur)](ClO ) (1)
4
V(1)−O(1)
V(1)−O(2)
V(1)−O(3)
V(1)−N(1)
V(1)−N(2)
V(1)−N(3)
O(1)−V(1)−O(2)
O(1)−V(1)−O(3)
O(2)−V(1)−O(3)
O(1)−V(1)−N(1)
O(2)−V(1)−N(1)
1.559(16)
1.950(18)
1.977(16)
2.090(2)
2.130(2)
2.290(2)
104.40(9)
107.20(8)
88.00(7)
93.20(9)
85.60(9)
O(3)−V(1)−N(1)
O(1)−V(1)−N(2)
O(2)−V(1)−N(2)
O(3)−V(1)−N(2)
O(1)−V(1)−N(3)
O(2)−V(1)−N(3)
O(3)−V(1)−N(3)
N(1)−V(1)−N(2)
N(2)−V(1)−N(3)
N(1)−V(1)−N(3)
162.40(9)
93.20(9)
157.80(8)
85.6(7)
163.70(9)
87.00(8)
83.70(8)
92.80(8)
74.50 (8)
75.96(8)
Figure 5. Optimized Structures and FMOs of the complexes 1−3.
unit-cell packing diagram showed the presence of two similar
cationic complexes and two lattice perchlorate anions (Figure
S17, Supporting Information). One perchlorate is hydrogen-
bonded to the complex involving the dpa ligand, while the
other perchlorate does not show any such interaction. The X-
ray structural observation is buttressed from the FT-IR data
showing splitting of the IR band of the perchlorate group
pictures it is evident that the HOMO for complex 1 is located
over the curcumin moiety with significant contribution of metal
and dipicolyl amine ligand, while the LUMO contains both
metal and dipicolyl amine moiety. The HOMO for complexes 2
and 3 is found to be exclusively located over the BODIPY and
diiodo-BODIPY core, respectively, whereas LUMO is predom-
inantly located over curcumin and metal for both the
complexes. The M−O(cur) distances are found to be ∼1.92,
∼2.03, and ∼2.04 Å for 1−3, respectively. Energy difference
between LUMO and HOMO for the complexes is ∼2.30 eV
(Figure S18, Supporting Information).
(
Figure S10, Supporting Information). This interaction,
however, has no impact on the core structure of the curcumin
ligand bound to the oxidovanadium(IV) moiety. The ternary
structure of the complex has a distorted octahedral VO N core
3
3
in which the tridentate N,N,N-donor dipicolylamine (dpa) base
is bonded to the oxidovanadium(IV) unit in a facial mode, and
the monoanionic curcumin (cur) ligand in its enolic form is
bonded in a O,O-bidentate chelating mode thus making the
complex as monocationic with the perchlorate as the
counteranion. The V−O(cur) distances are of 1.950(18) and
Cytotoxicity. Complexes 1a−3a showed significant cellular
uptake in MCF-7 breast cancer cells within 2 h, and the uptake
was ∼90% within 4 h (Figure S19, Supporting Information).
The vanadium metal content inside the MCF-7 cells was
measured for complexes 1a−3a by ICP-MS after 4 h of
5
incubation and found to be 125, 150, and 133 ng/1 × 10 cells,
1
.977(16) Å. The V−N bond lengths are 2.090(2), 2.130(2),
respectively. The efficacy of the complexes as anticancer agents
was tested using MCF-7 and cervical HeLa cancer cells in the
dark and upon photo-irradiation with a broad-band visible light
and 2.290(2) Å. The VO bond is of 1.559(16) Å. The bond
trans to the VO bond is the longest among V−N (2.290(2)
Å) due to trans effect. The N−H group of dpa is hydrogen-
−2
source (400−700 nm; Luzchem photoreactor, 10 J cm ;
irradiation time of 1 h) by MTT assay. The complexes were
incubated for 4 h and then irradiated with the visible light. Dark
controls were utilized to estimate the effect of light activation.
Cytotoxicity was measured from the IC50 values (50%
inhibitory concentration) obtained from the cell viability
assay. The data are given in Table 4, and the IC values are
bonded to ClO giving a distance of 2.053(2) Å. The M−
4
O(cur) bond distances in the structurally characterized
curcumin complexes are within 1.878(4) to 2.410(4) Å,
where M is a transition metal or lanthanoid (Table S4,
21−23,25,26,29
Supporting Information).
The Co(III) curcumin
complex exhibits a M−O(cur) bond length of ∼1.88 Å. This
complex releases curcumin only upon chemical reduction by
5
0
22,41,62−65
compared with related complexes.
A bar diagram
F
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Article
Table 4. IC50 (μM) Values of the Complexes 1a−3a as Chloride Salts and Relevant Compounds
MCF-7
HeLa
a
b
a
b
c
compound
IC50 (μM) dark
IC50 (μM) light
IC50 (μM) dark
IC50 (μM) light
ROS type
d
d
d
1
2
3
a
a
a
94 ± 2
87 ± 2
99 ± 3
90 ± 5
8.8 ± 0.4
6.2 ± 0.2
2.7 ± 0.4
20 ± 2
>100
15.5 ± 0.9
5.8 ± 0.5
2.5 ± 0.2
8.2 ± 0.2
3.3 ± 0.4
1.8 ± 0.6
7.9 ± 0.3
3.8 ± 0.2
4.3 ± 0.2
·OH
1
>100
O , ·OH
2
1
55 ± 2
85 ± 4
>50
O , ·OH
2
e
curcumin (Hcur)
·OH
·OH
f
[
[
[
[
VO(cur)(dppz)Cl]
g
1
VO(L )Cl ]
>50
3.4 ± 0.4
2.0 ± 0.2
>50
O2
2
2
h
h
Cu(L )(cur)]Cl
29.1 ± 0.5
32.1 ± 0.4
>41
1
Cu(L )(cur)]Cl
2
i
1O2
Photofrin
j
Cisplatin
2.0 ± 0.3
24
a
b
The sample on incubation for 4 h in the dark. MTT assay was done using maximum complex concentration of ∼100 μM. The sample on
−2
c
d
photoexposure for 1 h in visible light of 400−700 nm at 10 J cm . ROS is reactive oxygen species. The solvent medium 99:1 (v/v) DMEM
e
f
medium/DMSO. Curcumin alone under similar experimental conditions as used for the complexes 1a−3a (ref 22). dppz is dipyridophenazine. The
g
h
−2
data are from ref 22. The data from ref 48. The data are from ref 41. Light used was 400−700 nm (power: 10 J cm ). The nature of ROS was not
i
−2
j
reported. The data are from refs 62 and 63 (light source: 633 nm at 5 J cm for 2 h). The data are from refs 64 and 65 (IC values for HeLa on 4
50
h of incubation, whereas it is 96 h of incubation for MCF-7).
giving the IC50 values is shown in Figure 6 (Figures S20−S22,
Supporting Information). The complexes showed remarkable
correspond to the ROS generation ability of the ligands:
diiodoBODIPY-dpa ≫ BODIPY-dpa ≈ curcumin. The ligand
dpa is photoinactive. A comparison of the IC values of 1a and
50
2a suggest significant PDT activity of curcumin in metal-bound
form. The objective of using a BODIPY unit in 2a is to utilize
its emission property for cellular imaging, while the diiodo-
BODIPY is incorporated to generate singlet oxygen, which is of
paramount importance in PDT. The curcumin ligand, in
contrast, generates primarily hydroxyl radicals on photo-
activation. The aspect of singlet oxygen versus hydroxyl radical
generation was probed from the mechanistic studies of the
light-activated pUC19 DNA cleavage reactions (vide infra).
The photoactivity of the BODIPY ligands L and L alone
1
2
could not be studied with accuracy because of their poor
aqueous solubility. Curcumin (Hcur) alone showed significant
photocytotoxicity in cervical HeLa cells on short incubation
time of 4 h in DMEM medium giving an overall order of 3a ≫
2a > Hcur > 1a (Table 4). The dye alone showed significant
degradation on longer incubation time in the buffer medium as
evidenced from the spectral studies.
Mitochondrial Localization. Subcellular localization study
in MCF-7 cells was performed by confocal microscopy using
emissive complexes 1a (ϕF = 0.04) having photoactive
curcumin (Figure S23, Supporting Information) and 2a having
Figure 6. Bar diagram giving the IC50 values of the complexes 1a−3a
in dark and light in MCF-7 and HeLa cells (the light source is 400−
both photoactive BODIPY and curcumin moieties (ϕ = 0.11)
00 nm Luzchem photoreactor of power 10 J cm−2 and 1 h exposure
F
7
(
Figure 7). From the merged images it is evident that the
time at 37 °C). Color codes: in dark, black; in light, green. Asterisk (*)
symbol indicates up to 100 μM complex concentration for which the
corresponding complex does not reach IC .
complexes predominantly colocalized giving overlap coefficient
value of ∼0.7 with the Mito Tracker Deep Red (MTR)
indicating their selective localization in the mitochondria of the
cells. This observation is of importance, as both curcumin and
BODIPY dyes are known to target mitochondria as is observed
50
photo-cytotoxicity in both the cell lines but remained less toxic
7
,31,62,66
in the dark thus signifying their PDT effect. The IC values of
for the PDT drug Photofrin.
Anticancer agents
50
complex 1a in light of 400−700 nm were within 8.8−15.5 μM.
The values for complex 2a in light were lower within 5.8−6.1
μM due to the presence of BODIPY as an additional
photoactive moiety, while 1a had only curcumin as the
photosensitizer. The photo-cytotoxicity of 3a was found to be
excellent giving IC50 values within 2.4−2.7 μM. This is due to
higher ROS generation ability of the diiodo-BODIPY ligand.
The IC50 values of 3a are similar to those of the clinically
localizing in the mitochondria can avoid the deactivation
pathway associated with the nuclear DNA targeting drugs by
nuclear excision repair (NER) mechanism.
Reactive Oxygen Species. ROS plays very important role
to induce apoptotic cell death. The complexes were found to
induce cell cycle arrest of MCF-7 cells in G1-S phase, and
apoptotic death of the cells by 3a was confirmed by annexin-V
FITC and propidium iodide (PI) assays (Figures S24 and S25,
Supporting Information). Generation of any ROS by the
complexes 1a−3a on exposure to visible light (400−700 nm)
36,37
62,63
approved PDT drug Photofrin.
The photo-cytotoxicity of
the complexes follows the order of 3a ≫ 2a > 1a. The data
G
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triplet state, which by type-II energy transfer pathway generates
57,58
singlet oxygen.
The singlet oxygen quantum yield value
(
0
ϕ ) for the iodinated BODIPY ligand (L ) is reported as
.8. Complex 1 having only curcumin as the photoactive
Δ
2
6
8
moiety did not show any significant singlet oxygen generation.
The ROS involved for this complex could be hydroxyl radicals
2
1,22
as reported for other curcumin−metal complexes.
The
decay rate for complex 2 for DPBF absorbance (7.5 × 10− s )
4 −1
was found to be significantly lower and ∼25% of that of the
complex 3 (3.0 × 10− s ; Figure 8b).
3
−1
DNA Binding and Cleavage. Confocal microscopic
images showed that the complexes primarily located at the
mitochondria of the cells. Targeting mitochondria has several
positive aspects: (i) being the powerhouse of cell, mitochon-
drial damage arrests rapid growth of cancerous cells, (ii) NER
mechanism is absent in mitochondrial circular DNA, (iii) many
significant metabolisms of cancer cells get affected as they take
place in this important organelle, and (iv) release of
cytochrome-c from mitochondria to the cytoplasm leads to
initiate caspase-dependent apoptotic cell death. Mitochondrial
DNA being a potential target of the complexes, the interaction
of 1a−3a with ct-DNA was studied by UV−vis absorption and
Figure 7. Confocal microscopic images of MCF-7 cells after 4 h of
incubation with complex 2a: (a) bright field, (b) fluorescence of MTR,
(
c) fluorescence of 2a, and (d) merged image of (b, c). Scale bar = 10
μm.
viscosity experiments. The intrinsic binding constants (K ) of
b
5
−1
the complexes for ct-DNA were ∼1 × 10 M in 5% DMF−
Tris buffer (pH = 7.2) giving an order of 2a > 1a > 3a (Figures
S28−S30, Supporting Information). Free curcumin is known to
show weak intercalative mode of binding with herring sperm
DNA (hs-DNA), and dimethoxycurcumin (Dimc), a synthetic
analogue of native curcumin, exhibits minor groove binding
was probed in MCF-7 cells by DCFDA assay. DCFDA is a cell-
permeable fluorogenic probe. It gets oxidized to DCF in
67
presence of ROS and displays emission maxima at 528 nm.
Enhanced ROS generation leads to increase in the DCF-based
emission intensity, which could be quantified from flow
cytometry data. The assay data on MCF-7 cells showed
significant increase in DCF based emission for the complex 3a
treated cells with light (400−700 nm) for 1 h of irradiation,
while formation of DCF was insignificant in absence of light
69,70
1/3
property with ct-DNA.
From the plot of (η/η0) versus
[complex]/[DNA] using viscometric titration data of the
complexes in DMF−Tris−HCl buffer (pH = 7.2) and ct-DNA
at 37 °C it appears that the complexes are partial intercalative
ct-DNA binders considering ethidium bromide (EB) and
Hoechst dye as standard DNA intercalator and groove binder,
respectively (Figure S31, Supporting Information). DNA
binding properties of the complexes studied by EB displace-
(
Figure S26, Supporting Information). The ROS generated
could be singlet oxygen via a type-II process or hydroxyl
radicals via photoredox pathway.
30−32
DPBF experiment was
performed to ascertain formation of any singlet oxygen. DPBF
1
is known to react with singlet oxygen ( O ) to form
2
ment and emission quenching method gave K values of (3.4
app
endoperoxide, and a decrease in the DPBF based absorption
4
−1
4
−1
4
±
M
(
0.8) × 10 M , (4.4 ± 0.8) × 10 M and (3.2 ± 0.2) × 10
1
intensity at 417 nm indicates formation of O . Ligand L and
−1
2
2
for 1a−3a, respectively, giving an order of 2a > 1a ≈ 3a
complex 3 of this ligand showed similar and rapid decay of the
DPBF absorbance on visible light (400−700 nm) irradiation
with lower complex concentration (Figure 8, Figure S27,
Supporting Information). The presence of two heavy iodine
Figures S32−S34, Supporting Information).
Photoinduced DNA cleavage activity of the complexes (1a−
a) and the ligands (Hcur, L and L ) was studied (taking 10
3
1 2
μM concentration of each sample) with dark controls using SC
pUC19 DNA (30 μM, 0.2 μg) in Tris−HCl/NaCl (50 mM, pH
atoms in ligand L makes the ISC favorable thus stabilizing the
2
=
7.2) buffer (Figure 9A, Figure S34, Supporting Information).
Gel electrophoresis in agarose gel was done to assess the extent
of nicked circular (NC) DNA formation from the SC DNA in
presence of the complexes and ligands. Monochromatic visible
light source of 532 nm (100 mW power) was used from a CW
diode laser. The wavelength chosen for the DNA photocleavage
activity is based on the presence of the BODIPY-centered
electronic spectral band of the complexes 2a and 3a near 501
and 535 nm, respectively. Each sample, after treating with the
DNA solution in Tris−HCl buffer medium, was incubated for 1
h in the dark, followed by 1 h of exposure to light. The
complexes and ligands (10 μM) did not show any significant
DNA cleavage activity (≤10% NC) in the dark. However,
formation of 9, 20, and 50% of NC DNA was observed in
Figure 8. (a) Absorption spectral traces of DPBF and complex 3 (0.1
−2
μM) on exposure to light (400−700 nm, 10 J·cm ) for exposure time
of 15 s. (b) Plot showing changes in absorbance of DPBF at 417 nm
with time on light exposure with complexes 1−3. A higher slope
indicates more quantity of singlet oxygen generation.
presence of light for the ligands Hcur, L , and L alone,
1
2
respectively. Complexes 1a−3a gave, respectively, 42, 60, and
90% of NC DNA under similar experimental conditions.
H
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Article
CONCLUSIONS
■
This work presents a structurally characterized ternary
oxidovanadium(IV) complex of curcumin and dipicolylamine
(
dpa) along with its analogues having pendant photoactive
BODIPY moieties showing visible light-induced (400−700
−2
nm,10 J cm ) cytotoxicity in MCF-7 and HeLa cells. The
observed photoactivity is akin to the PDT effect reported for
the United States Food and Drug Administration-approved
hematoporphyrin drug Photofrin. Interestingly, the complexes
remained less toxic in the dark. The emissive complexes were
used for cellular imaging. They showed primarily mitochondrial
localization and cellular death via apoptotic pathway involving
ROS. This observation is of importance, as nuclear targeting
anticancer agents like cisplatin and analogues suffer from
intrinsic resistance of cells. While the complex having dpa
showed light-induced formation of hydroxyl radicals as the
ROS via photoredox pathway, the diiodo BODIPY moiety
displayed significant generation of singlet oxygen as the ROS
via type-II process as evidenced from the mechanistic data
obtained from the pUC19 DNA photocleavage studies. The
complexes were stable in the physiological buffer medium
without showing any apparent dissociation and degradation of
the curcumin ligand. Binding of curcumin to the oxophilic
VO2+ moiety has significantly enhanced its stability. The
presence of curcumin and photoactive BODIPY moieties as the
photosensitizers to the oxidovanadium(IV) moiety in the
ternary structure has resulted to a significant enhancement in its
photo-cytotoxic potential under physiological conditions.
Curcumin retaining its therapeutic potential under in vitro
cellular conditions is of importance for any in vivo applications,
as the drug in itself is categorized under “PAINS” due to its
hydrolytic instability and poor bioavailability. Further studies
are on to understand any specific role the “drug” plays with its
intact diphenolic moiety in the apoptotic cell death mechanism.
Figure 9. (A) Gel electrophoresis diagram showing the cleavage of
pUC19 DNA (0.2 μg, 30 μM base pair) by the complexes 1a−3a,
ligands Hcur, L , and L (10 μM) in light of 532 nm (100 mW, 1 h
1
2
exposure): lane 1, DNA control (L); lanes 2 and 3, Hcur (D) and
HCur (L); lanes 4 and 5, L (D) and L (L); lanes 6 and 7, L (D) and
1
1
2
L2 (L); lanes 8 and 9, 1a (D) and 1a (L); lanes 10 and 11, 2a (D) and
2
a (L); lanes 12 and 13, 3a (D) and 3a (L), where L is for light-
exposed and D is under dark condition. (B) Mechanistic data for
cleavage of pUC19 DNA (0.2 μg, 30 μM base pair) by (a) 3a (10 μM)
and (b) 1a (25 μM) in light of 532 nm (100 mW, 1 h exposure time):
lane 1, pUC19 DNA control; lane 2, complex in dark; lane 3, complex
in light; lanes 4−10 are for the complexes in the presence of various
additives: lane 4, D O (16 μL); lane 5, NaN (0.5 mM); lane 6, TEMP
2
3
(
0.5 mM); lane 7, DMSO (4 μL); lane 8, KI (0.5 mM); lane 9, catalase
(
4 μL); and lane 10, SOD (4 units).
For the mechanistic study of ROS generation, complexes 3a
10 μM) and 1a (25 μM) were treated with pUC-19 DNA and
(
photoexposed in presence of different singlet oxygen
quenchers, hydroxyl radical scavengers, and SOD as superoxide
radical scavenger (Figure 9B, Figures S35−S37, Supporting
ASSOCIATED CONTENT
■
Information). Complex 3a in D O showed higher percentage of
2
S
*
Supporting Information
NC DNA (94%), whereas in presence of singlet oxygen
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.7b01924.
quenchers like NaN and TEMP it was diminished to 30%. In
3
contrast, hydroxyl radical scavengers like KI, DMSO, and
catalase showed minor reduction giving 60% of NC DNA. No
significant decrease in percentage of NC DNA was observed in
presence of SOD. Complex 1a (25 μM), in contrast, showed no
apparent change in the percentage of NC DNA in presence of
D O or on addition of NaN or TEMP. However, a significant
Details of the experimental procedures, reaction scheme,
ESI-MS, IR spectra, d−d transitions of 1−3, cyclic
voltammograms, UV−visible stability data, ESI-MS of
complexes, crystal structure (ORTEP and unit-cell
packing diagrams showing H-bonding), electronic
transitions by DFT, time-dependent cellular incorpo-
ration assay, MTT assay plots, confocal images of 1a, cell
cycle profiles of 1a−3a, Annexin assay profile of 3a,
DCFDA assay, DPBF absorbance decay plots, DNA
binding data, DNA photocleavage bar diagrams, DFT
data for the complexes, a list of M−O(Cur) bond
distances of structurally characterized curcumin com-
plexes (PDF)
2
3
reduction in NC DNA from 85% to 20% was observed in
presence of hydroxyl radical scavengers like KI, DMSO, and
catalase. A minor reduction in %NC DNA was also observed in
presence of SOD. The mechanistic study reveals that complex
3
a having both diiodo BODIPY and curcumin produces both
singlet oxygen (∼70%) and hydroxyl radicals (∼30%) on
photoactivation. Complex 1a having only dpa produces
primarily hydroxyl radicals as the ROS. That makes complex
3
a a novel vanadium-based PDT agent with curcumin retaining
its pendant diphenolic moieties. DNA photocleavage studies
Accession Codes
reveal that the extent of total ROS generation for 1a and 2a are
∼
47% and ∼66% than that of 3a in similar experimental
CCDC 1543636 contains the supplementary crystallographic
data for this paper. These data can be obtained free of charge
via www.ccdc.cam.ac.uk/data_request/cif, or by emailing data_
request@ccdc.cam.ac.uk, or by contacting The Cambridge
Crystallographic Data Centre, 12 Union Road, Cambridge CB2
1EZ, UK; fax: +44 1223 336033.
condition in presence of light, which is in good agreement with
the DCFDA assay demonstrating that an enhancement in
DCF-based emission intensity in light is distinctly greater in
case of complex 3a in comparison to the other two complexes
(
Figure S26, Supporting Information).
I
DOI: 10.1021/acs.inorgchem.7b01924
Inorg. Chem. XXXX, XXX, XXX−XXX

Inorganic Chemistry
Article
(
13) Deng, Y.; Verron, E.; Rohanizadeh, R. Molecular Mechanisms of
AUTHOR INFORMATION
■
*
Anti-metastatic Activity of Curcumin. Anticancer Res. 2016, 36, 5639−
Corresponding Authors
5
647.
E-mail: paturu@mrdg.iisc.ernet.in. Phone: +91-80-22932688.
Fax: +91-80-23600999. (P.K.)
E-mail: arc@ipc.iisc.ernet.in. Phone: +91-80-22932533. Fax:
91-80-23600683. (A.R.C.)
(14) Pavan, A. R.; Silva, G. D. B.; Jornada, D. H.; Chiba, D. E.;
Fernandes, G. F. S.; Man Chin, C.; dos Santos, J. Unraveling the
Anticancer Effect of Curcumin and Resveratrol. Nutrients 2016, 8,
628−677.
*
+
(
15) Yoysungnoen, P.; Wirachwong, P.; Changtam, C.; Suksamram,
ORCID
A.; Patumraj, S. Anti-Cancer and Anti-Angiogenic Effects of Curcumin
and Tetrahydrocurcumin on Implanted Hepatocellular Carcinoma in
Nude Mice. World J. Gastroenterol. 2008, 14, 2003−2009.
Samya Banerjee: 0000-0003-4393-4447
Akhil R. Chakravarty: 0000-0002-6471-9167
(
16) Guo, L.; Chen, X.; Hu, Y.; Yu, Z.; Wang, D.; Liu, J. Curcumin
Notes
Inhibits Proliferation and Induces Apoptosis of Human Colorectal
Cancer Cells by Activating the Mitochondria Apoptotic Pathway.
Phytother. Res. 2013, 27, 422−430.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
(
17) Esatbeyoglu, T.; Huebbe, P.; Ernst, I. M. A.; Chin, D.; Wagner,
■
(
A. E.; Rimbach, G. Curcumin-from Molecule to Biological Function.
Angew. Chem., Int. Ed. 2012, 51, 5308−5332.
(
Walters, M. A. The Essential Medicinal Chemistry of Curcumin. J.
Med. Chem. 2017, 60, 1620−1637.
(
Product Drugs, and Pan Assay Interference Compounds (PAINS). J.
Nat. Prod. 2016, 79, 616−628.
(
The authors thank the Department of Science and Technology
DST), Government of India, for financial support (Grant Nos.
18) Nelson, K. M.; Dahlin, J. L.; Bisson, J.; Graham, J.; Pauli, G. F.;
SR/S5/MBD-02/2007 and EMR/2015/000742). The authors
also thank the Alexander von Humboldt (AvH) Foundation,
Germany, for an electrochemical system. A.R.C. thanks the
DST for a J. C. Bose national fellowship. P.K.’s research group
is funded by DBT-IISc grants. We thank Dr. Uttara, Urvashi,
and Deepika for FACS data and Saima and Santosh for confocal
microscopy images.
19) Baell, J. B. Feeling Nature’s PAINS: Natural Products, Natural
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21) Banerjee, S.; Dixit, A.; Karande, A. A.; Chakravarty, A. R.
Remarkable Selectivity and Photo-Cytotoxicity of an Oxidovanadium-
IV) Complex of Curcumin in Visible Light. Eur. J. Inorg. Chem. 2015,
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