Photorelease and Cellular Delivery of Mitocurcumin from Its Cytotoxic Cobalt(III) Complex in Visible Light

Article abstract of DOI:10.1021/acs.inorgchem.6b00554

Ternary cobalt(III) complexes of curcumin (Hcur) and mitocurcumin [Hmitocur, a dicationic bis(triphenylphosphonium) derivative of curcumin] having a tetradentate phenolate-based ligand (H₂L), namely, [Co(cur)(L)] (1) and [Co(mitocur)(L)]Cl

Full text of DOI:10.1021/acs.inorgchem.6b00554

Article
pubs.acs.org/IC
Photorelease and Cellular Delivery of Mitocurcumin from Its
Cytotoxic Cobalt(III) Complex in Visible Light
Aditya Garai,† Ila Pant,‡ Samya Banerjee,† Bhabatosh Banik,† Paturu Kondaiah,*,‡
and Akhil R. Chakravarty*,†
†Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore 560012, India
‡Department of Molecular Reproduction, Development and Genetics, Indian Institute of Science, Bangalore 560012, India
S
* Supporting Information
ABSTRACT: Ternary cobalt(III) complexes of curcumin (Hcur)
and mitocurcumin [Hmitocur,
a dicationic bis-
(triphenylphosphonium) derivative of curcumin] having a
tetradentate phenolate-based ligand (H2L), namely, [Co(cur)(L)]
(1) and [Co(mitocur)(L)]Cl2 (2), were prepared and structurally
characterized, and their photoinduced cytotoxicity was studied.
The diamagnetic cobalt(III) complexes show an irreversible
Co(III)−Co(II) redox response and a quasireversible curcumi-
noid-based reduction near −1.45 and −1.74 V SCE, respectively,
in DMF/0.1 M [nBu4N](ClO4). The complexes exhibit a
curcumin/mitocurcumin-based absorption band near 420 nm.
Complex 1 was structurally characterized by X-ray crystallog-
raphy. The structure contains the metal in a CoN2O4 distorted
octahedral coordination arrangement with curcumin binding to the metal in its enolic form. Binding to cobalt(III) increases the
hydrolytic stability of curcumin. Complex 2, having a dicationic curcuminoid, shows significant cellular uptake and photoinduced
cytotoxicity compared to its curcumin analogue 1. The dicationic cobalt(III) complex 2 has significantly better cellular uptake
and bioactivity than the neutral species 1. Complex 2 with mitochondrial localization releases the mitocurcumin dye upon
exposure to visible light (400−700 nm) in human breast cancer MCF-7 cells through photoreduction of cobalt(III) to cobalt(II).
Complex 2 displays a remarkable photodynamic therapy (PDT) effect, giving an IC50 value of ∼3.9 μM in visible light (400−700
nm) in MCF-7 cells while being much less toxic in the dark (>50 μM). The released mitocurcumin acts as a phototoxin,
generating intracellular reactive oxygen species (ROSs). The overall process leads to light-controlled delivery of a curcuminoid
(mitocur) into the tumor cells while the dye alone suffers from hydrolytic instability and poor bioavailability.
cells.10−18 Thus, curcumin as a photosensitizer with a visible
INTRODUCTION
■
band near 440 nm and as a fluorescent tag for its green
emission property is suitable for dual cellular applications: (i)
imaging cells to study the intracellular localization of the metal
complexes and (ii) killing cancer cells upon irradiation with
visible light (400−700 nm) by generating reactive oxygen
species (ROSs).10 The chemistry of curcumin in the photo-
dynamic therapy (PDT) of cancer as such or in its metal-bound
form is an emerging area because of the novel photophysical
properties and photosensitizing ability of curcumin.10−17 PDT
is a noninvasive therapeutic modality to treat cancer by
involving the activation of a photosensitizer in visible light to
generate reactive oxygen species (ROSs) that selectively kill the
light-exposed cancer cells, leaving unexposed healthy cells
unaffected.18−23 PDT reduces the drug-related toxicity on
human organs compared to that induced by the conventional
platinum-based chemotherapeutic drugs. Different types of
As the naturally occurring polyphenolic active ingredient of
turmeric, curcumin is well-known for its anticancer activity and
other medicinal values.1,2 It induces apoptosis in tumor cells by
interfering with the activity of the transcription factor NF-κB. It
enhances the activity of the major tumor suppressor protein
p53.3,4 However, the clinical use of curcumin (Hcur), 1,7-bis(4-
hydroxy-3-methoxyphenyl)-1,6-hepta-diene-3,5-dione, is se-
verely limited because of its poor bioavailability and hydrolytic
instability under physiological conditions. This is attributed to
the presence of a β-diketone moiety in its structure that is
susceptible to hydrolysis, leading to its rapid degradation.1,5
Although several modifications of the dye have been made to
improve its efficacy without much success, one strategy that has
been found to be effective is the covalent binding of curcumin
in its enolic form as a monoanionic O,O-donor chelating ligand
to an oxophilic metal ion.6−18 The coordination chemistry of
curcumin complexes is of considerable importance because of
their remarkable anticancer activity with vastly improved
hydrolytic stability of curcumin.6−10 Such complexes show
excellent photocytotoxicity in visible light in a variety of cancer
Received: March 4, 2016
© XXXX American Chemical Society
A
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metal-based PDT agents have been reported in recent
years.24−33
curcumin upon binding to the cobalt(III) metal center; (iii)
developing a mitocurcumin complex (complex 2) that shows
selective mitochondrial localization and visible-light- (400−700
nm) induced release of the dye inside cancer cells (MCF-7,
human breast cancer) with concomitant metal reduction; (iv)
obtaining a remarkable PDT effect of complex 2, comparable to
that of photofrin, giving an IC50 value of ∼3.9 μM in the light
(400−700 nm) in MCF-7 cells while being less toxic in the
dark; and (v) achieving higher cellular uptake of the dicationic
complex 2 compared to the charge-neutral curcumin complex
1, thus highlighting the importance of the structure−activity
relationship (SAR) in this class of cobalt(III) complexes.
Sadler and co-workers developed six-coordinate platinum-
(IV)-based photoactivated chemotherapeutic (PACT) agents
that, upon light activation, release two axial ligands to generate
planar platinum(II) active species.34,35 Lippard and co-workers
reported six-coordinate nontoxic platinum(IV) complexes that,
upon intracellular reduction, release their axial ligands to
generate four-coordinate active platinum(II) species.36,37 This
concept of releasing ligands from the coordination sphere of a
metal ion from its higher oxidation state to a lower one by
chemical or photochemical means could be used for the
successful delivery of curcumin inside cells, thus increasing its
bioavaiability and hydrolytic stability in a significant way.
Hambley and co-workers recently reported the release of
curcumin at the target site from its ternary cobalt(III)
complexes.18 As for the platinum(IV) complexes, the release
of curcumin is effected by intracellular reduction of cobalt(III)
to cobalt(II) with a concomitant structural change.9 Again,
similarly to the platinum(IV) prodrugs, the reduction of the
metal with ligand loss can be achieved by photoirradiation.18,38
Delivery of curcumin at the target site by photochemical means
seems to be a convenient option compared to other modes of
using nanoparticles, liposomes, and micellar systems.9,18,38 The
present work stems from our interest in probing the cobalt(III)
chemistry of curcumin. We have designed and synthesized new
ternary cobalt(III) complexes of curcumin (Hcur) and
mitocurcumin (Hmitocur) using a dianionic tetradentate
ancillary ligand (H2L) with the objective of releasing curcumin
upon photoirradiation using visible light (Figure 1). The choice
EXPERIMENTAL SECTION
■
Materials and Measurements. The reagents and chemicals were
obtained from various commercial sources (s.d. Fine Chemicals,
Mumbai, India; Sigma-Aldrich, St. Louis, MO) and used as received.
Solvents were purified according to literature procedures.42 2′,7′-
Dichlorofluoresceindiacetate (DCFDA), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), propidium iodide (PI),
Dulbecco’s modified eagle medium (DMEM), Dulbecco’s phosphate-
buffered saline (DPBS), and fetal bovine serum (FBS) were purchased
from Sigma (St. Louis, MO). MitoTracker Red was purchased from
Invitrogen (Carlsbad, CA). The bis(triphenylphosphonium) derivative
of (1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-
dione (curcumin) dichloride (the dichloro salt of dicationic
mitocurcumin, Hmitocur) and the tetradentate ligand, namely, 6,6′-
(((2-(dimethylamino)ethyl)azanediyl)bis(methylene))bis(2,4-di-tert-
butylphenol) (H2L), were synthesized by the reported procedures
(Schemes S1 and S2 in the Supporting Information).39
Elemental analyses were performed on a Thermo Finnigan Flash EA
1112 CHN analyzer. The electronic and infrared spectra were
recorded on a Perkin-Elmer Spectrum One 55 spectrophotometer
and a Perkin-Elmer Lambda 35 IR spectrometer, respectively.
Fluorescence spectra were obtained using a Perkin-Elmer LS 55
spectrophotometer. 1H NMR spectra were recorded at room
temperature on a Bruker 400 MHz NMR spectrometer. Molar
conductivity measurements were done using a Control Dynamics
(Ankleshwar, India) conductivity meter. Cyclic voltammetric measure-
ments were carried out at 25 °C on an EG&G PAR model 253
VersaStat potentiostat/galvanostat using electrochemical analysis
software 270 in a three-electrode configuration consisting of a glassy
carbon working electrode, a platinum wire auxiliary electrode, and a
saturated calomel reference electrode (SCE) in dimethylformamide
(DMF) with tetrabutylammonium perchlorate (TBAP, 0.1 M) as a
supporting electrolyte. The redox data were uncorrected for junction
potentials. Electrospray ionization (ESI) mass spectral measurements
were made using an Agilent Technologies 6538 UHD Accurate-mass
Q-TOF LC/MS and BrukerDaltonics Esquire 300 Plus ESI model
mass spectrometers. Magnetic susceptibility measurements of the
samples at 298 K were done using a magnetic susceptibility balance
from Sherwood Scientific (Cambridge, U.K.). Flow cytometric analysis
was done on a FACSCalibur Becton Dickinson (BD) cell analyzer at
the FL1 channel (595 nm). Confocal imaging studies were performed
with a Zeiss LSM 510 apochromat confocal laser scanning microscope.
Synthesis. Complexes 1 and 2 were synthesized following a
general synthetic procedure in which H2O2 (2 drops) was added to an
ethanol (25 mL) solution of Co(CH3COO)2·4H2O (12.5 mg, 0.05
mmol), followed by addition of a CH2Cl2 solution of H2L (17.3 mg,
0.05 mmol) previously neutralized with Et3N (0.05 mmol, 6.97 μL).
The solution was stirred for 30 min, and Hcur (18.4 mg, 0.05 mmol)
or Hmitocur as its dichloride salt (52.3 mg, 0.05 mmol) in ethanol (10
mL) was added. The reaction mixture was stirred for 1 h, filtered, and
kept for slow evaporation to isolate a crystalline solid in analytically
pure form. The solid was collected by filtration; washed with ice-cold
ethanol, cold methanol, and diethyl ether; and finally dried over P4O10
in a vacuum to obtain a brown red solid in ∼80% yield. The
characterization data are reported in the following subsections.
Figure 1. Molecular structures of cobalt(III) complexes 1 and 2 and
the ligands used.
of mitocurcumin with two cationic triphenylphosphonium
(TPP) moieties appended to the curcumin core was intended
to direct the complex to the mitochondria of the cells, instead
of the nucleus, for improved activity and to avoid the nuclear
excision repair (NER) mechanistic pathway.39−41 The
tetradentate phenolate-based ligand (H2L) was used to impart
stereochemical rigidity and to augment the lipophilicity of the
complexes.
Herein, we describe the synthesis, characterization, crystal
structure, and photoinduced cytotoxicity of ternary curcumin
(Hcur) and dicationic mitocurcumin (Hmitocur) cobalt(III)
complexes of a phenolate-based ligand (H2L), namely,
[Co(cur)(L)] (1) and [Co(mitocur)(L)]Cl2 (2) (Figure 1).
The significant results of this work include (i) reporting the
first crystal structure of a cobalt complex of curcumin; (ii)
observing the enhancement of the hydrolytic stability of
B
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Complex [CoIII(cur)(L)] (1). Yield = 79%. Anal. Calcd for
C55H73CoN2O8 (M = 948.5): C, 69.58; H, 7.75; N, 2.95. Found: C
69.38; H 7.61; N 3.15. Molar conductivity data in DMF (ΛM): 14 S m2
M−1. FT-IR (solid phase): 2945 s, 1585 s, 1510 s, 1460 s, 1400 s, 1370
s,1260 s, 1060 m, 1030 w, 960 w, 880 m, 830 m, 750 w, 730 w, 620 w,
540 m (s, strong; m, medium, w, weak). ESI-MS in MeOH (m/z):
Table 1. Selected Crystallographic Data for [Co(L)(cur)]·
CHCl3 (1·CHCl3)
empirical formula
formula weight (g M−1
crystal system
space group
a (Å)
C56H74Cl3CoN2O8
1068.45
)
triclinic
949.4745 [M + H]+. UV−visible in 25% DMF/Tris HCl buffer [λmax
/
P1
̅
nm (ε/M−1 cm−1)]: 424 (32900), 297 (22360). Emission in DMSO:
9.666(2)
15.854(4)
18.056(3)
84.955(12)
83.52(3)
88.33(2)
2738.1(10)
2
1
525 nm (Φ = 0.02). H NMR (400 MHz, DMSO-d6): 9.46 (s, 1H),
b (Å)
9.32 (s, 1H), 7.57−6.62 (m, 12H), 6.25 (d, J = 15 Hz, 1H), 6.11 (d, J
= 15 Hz, 1H), 5.61 (s, 1H), 3.87 (s, 6H), 3.72 (s, 2H), 3.23 (s, 2H),
3.77 (s, 4H), 2.40 (s, 6H), 1.16−1.12 (m, 36 H) ppm (s, singlet; d,
doublet; m, multiplet).
c (Å)
α (deg)
β (deg)
Complex [CoIII(mitocur)(L)]Cl2 (2). Yield = 82%. Anal. Calcd for
C97H113Cl2CoN2O8P2 (M = 1626.73): C, 71.62; H, 7.00; N, 1.72.
Found: C, 71.86; H, 6.84; N, 1.84. Molar conductivity data in DMF
(ΛM): 152 S m2 M−1. FT-IR: 2960 s, 1580 w, 1500 s, 1430 s, 1380
m,1250 s, 1020 s, 1020 m, 960 w, 870 w, 800 w, 730 m, 680 m, 500 m.
ESI-MS in MeOH (m/z): 777.9482 [M − 2Cl]2+. UV−visible in 25%
DMF/Tris HCl buffer [λmax/nm (ε/M−1 cm−1)]: 411 (33330), 275
γ (deg)
V (Å3)
Z
T (K)
ρcalc (g cm−3
100 (2)
)
1.296
λ (Å) (Mo Kα)
μ (cm−1
0.71073
)
0.513
1
(34680). Emission in DMSO: 530 nm (Φ = 0.02). H NMR (400
data/restraints/parameters
F(000)
12224/0/659
1102
MHz, DMSO-d6): δ 7.78−7.84 (m, 30H), 6.1−7.6 (m, 14H), 5.67 (s,
1H), 3.87 (s, 4H), 3.8 (s, 4H), 3.73 (m, 6H), 2.56 (m, 3H), 2.77 (s,
3H), 2.41 (m, 6H), 2.22 (m, 3H), 1.56−1.54 (m, 3H), 1.17−1.12 (m,
36 H) ppm.
goodness-of-fit
1.042
a
b
R(F0), I > 2σ(I) [Rw(F0)]
0.0945 [0.2496]
0.1777 [0.3024]
1.162, −1.509
R (all data) [Rw(all data)]
largest diff peak and hole (e Å−3
Solubility and Stability. The complexes were soluble in MeOH,
DMF, DMSO, MeCN, and aqueous mixtures of these solvents;
moderately soluble in CHCl3 and CH2Cl2; and insoluble in
hydrocarbon solvents. The complexes were stable in both solid and
solution phases under dark experimental conditions. The complexes in
solution phase were found to release curcumin or mitocurcumin in the
presence of a reducing agent, namely, ascorbic acid/glutathione
(GSH), or upon photoirradiation with broad-band visible light of
400−700 nm (Luzchem Photoreactor model LZC-1, Ontario, Canada,
having Sylvania fluorescent tubes to provide a light dose of 2.4 mW
cm−2).
Theoretical Study. The geometry of complex 2 was optimized by
density functional theory (DFT) using the B3LYP level of theory with
the LanL2DZ basis set for the cobalt center and the 6-31G basis set for
all other atoms as implemented in the Gaussian 09 program.43−45 The
initial coordinates were taken from the crystal structure of 1 and were
used for the optimization of the structure of complex 2. The electronic
transitions with their transition probabilities were obtained using
linear-response time-dependent density functional theory (TDDFT).
The coordinates of the energy-minimized structures and selected
transitions in the visible region are listed in Tables S1 and S2 (see
Supporting Information).
Crystallographic Procedures. The crystal structure of curcumin
complex 1 was determined by X-ray diffraction. Single crystals were
obtained upon slow evaporation of a methanol/chloroform solution
(2:3 v/v) of the complex. A suitable single crystal was mounted on a
crystal mounting loop with the help of paratone oil, and the intensity
data were collected using graphite-monochromated Mo Kα radiation
(0.7107 Å) at 100 K. The structure was solved by direct methods using
SHELX-2013 incorporated in WinGX.46 Empirical absorption
corrections were applied with SADABS. All non-hydrogen atoms
were refined with anisotropic displacement coefficients.47 There were
three carbon atoms in the structure, namely, C50, C51, and C52,
showing positional disorder due to two possible conformations. These
carbon atoms were treated in a positional disorder model each with
50% site occupancy. This disorder in the structure had no effect on the
chemical properties of the complex. The crystallographic parameters
are listed in Table 1. The view of the complex was obtained using
ORTEP.48 CCDC-1434561 contains the crystallographic data for this
article.
)
a
b
R = ∑||Fo| − |Fc||/∑|Fo|. Rw = ∑[w(Fo2 − Fc2)2]/∑[w(Fo)2]}1/2. w
= [σ2Fo2 + (AP)2 + BP]−1, where P = (Fo2 + 2Fc2)/3, A = 0.1933, and
B = 4.5680.
uptake of the complexes (10 μM) by flow cytometric analysis in MCF-
7 cells following reported procedures.4,51 The photorelease of
mitocurcumin in complex 2 was studied in MCF-7 cells by
fluorescence-activated cell sorting (FACS) analysis by quantifying
the intracellular curcumin-based fluorescence intensity in different
time intervals both in the dark and upon visible-light irradiation (400−
700 nm). A 10 μM solution of complex 2 was used for the
experiments. DCFDA assay was performed to examine any intra-
cellular ROS formation in MCF-7 cells treated with complex 2 (10
μM) in the light (400−700 nm) and in the dark following standard
protocols (see Supporting Information).51,52 Cellular uptake and
localization of complex 2 (10 μM) showing green fluorescence in
MCF-7 cells were visualized using a confocal laser scanning
microscope (Zeiss, LSM510 apocromat) following standard proce-
dures (see Supporting Information).4,15,27 Mitochondrial localization
was studied using cell-permeable MitoTracker Deep Red FM (catalog
no. M22426, 20 nm) to stain the mitochondria.
RESULTS AND DISCUSSION
■
Synthesis and General Aspects. Ternary curcumin
(Hcur) and mitocurcumin (Hmitocur) cobalt(III) complexes
of a dianionic tetradentate phenolate-based ligand (H2L),
namely, [Co(cur)(L)] (1) and [Co(mitocur)(L)]Cl2 (2), were
synthesized in good yields according to a general synthetic
procedure in which Co(CH3COO)2 was initially oxidized by
H2O2 in ethanol, after which H2L (previously neutralized with
Et3N) and Hcur or dicationic Hmitocur in ethanol were added
(Figure 1). The complexes were characterized using analytical
and spectroscopic data. Selected characterization data are given
in Table 2. The ESI-MS spectra of the complexes in methanol
showed a single major peak that can be assigned to [M + H]+
for complex 1 and [M − 2Cl]2+ for complex 2 (Figures S1 and
S2, Supporting Information). The IR spectra of the complexes
displayed characteristic stretching bands near 1585 and 1500
cm−1 for the CO and CC (β-diketonate) moieties,
respectively (Figures S3 and S4, Supporting Information).53
Cellular Measurements. The cytotoxicity of the complexes was
studied using MTT assay in both light and darkness using MCF-7
(breast cancer) and HeLa (cervical cancer) cells following procedures
that were reported earlier (see Supporting Information).4,12,49,50
A
cellular incorporation assay was carried out to quantify the cellular
C
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molecules in the unit cell. The complex has a discrete
mononuclear structure with the Co(III) bonded to the O,O-
donor monoanionic curcumin and the N,N,O,O-donor L2−,
giving a CoN2O4 coordination arrangement. The complex
showed minor positional disorder for three atoms in the
tetradentate ancillary ligand. The disordered atoms were refined
in two different sets of atoms labeled as A and B and refined
with a site occupancy factor of 0.5. A perspective view of the
complex having the A set of atoms is shown in Figure 3. (See
Table 2. Selected Physicochemical Data of Complexes 1 and
2
1
2
a
IR /cm−1 [υ (CO)]
1585
1580
̅
b
electronic: λmax/nm [ε/(M−1 cm−1)]
424 [32900]
525 [0.02]
14
411 [33330]
530 [0.02]
152
c
emission: λem/nm [Φ]
d
ΛM /(S m2 M−1
)
b
a
In KBr phase. Visible electronic spectral band in 25% DMF/Tris
HCl buffer. Emission data of the complexes in DMSO with λex = 415
nm. Φ is the quantum yield. ΛM, molar conductance in DMF at 25
c
d
°C.
The intense IR band at 1585 cm−1 (CO stretching) indicates
bidentate coordination mode of the β-diketone moiety of the
cur/mitocur ligand in its enolized form to the central Co(III)
center. Complex 1 is nonelectrolytic giving molar conductance
value of 14 S m2 M−1 in DMF at 25 °C. Complex 2 is dicationic
with a molar conductance value of ∼152 S m2 M−1 in DMF.54
The complexes are diamagnetic with a low-spin state of the 3d6
1
Co(III) ion. H NMR spectra of the complexes measured in
DMSO-d6 showed peaks characteristic of the Co(III)-bound
N,N,O,O-donor L2− and O,O-donor cur or mitocur ligands
(Figures S5 and S6, Supporting Information). The complexes
showed an absorption spectral band near 415 nm in 25%
DMF/Tris HCl buffer (Figure 2a). This band is assignable to
Figure 3. ORTEP view of the cationic complex of 1 with atom labeling
scheme for the metal and heteroatoms showing 50% probability
thermal ellipsoids. For clarity, hydrogen atoms are not shown.
Figures S8 and S9, Supporting Information, for unit cell
packing and the structure with the B set of atoms.) Selected
bond distances and angles are given in Table 3. The disorder in
the structure had no apparent effect on the chemistry of this
complex. At 2.018(4) Å, the Co(1)−N(1) bond is longer than
the Co(1)−N(2) bond [1.955(4) Å]. The bonds involving the
phenolate oxygen atoms, namely, Co(1)−O(3) and Co(1)−
O(4), are 1.897(4) and 1.922(4) Å, respectively. The Co(1)−
O(1) and Co(1)−O(2) bonds involving the curcumin ligand
are ∼1.88 Å. The M−O(cur) bond distances in the reported
curcumin complexes are compared in Table 4.4,6−8,10
Interestingly, the Co(III)−O(cur) bond distance was found
to be the shortest among the structurally characterized metal−
curcumin complexes. The observation of a short and strong
Co(III)−O(cur) bond is of importance, as the release of the
curcumin ligand can be achieved upon reduction of the metal
considering that the Co(II)−O(cur) bond is longer and weaker
than the Co(III)−O(cur) bond.
Theoretical Studies. Computational studies were per-
formed to rationalize the photophysical properties of the
complexes. The energy-optimized structures of the complexes
showed that the cobalt(III) center is coordinated to the
chelating cur (monoanion of curcumin in 1) or mitocur
(monocation of mitocurcumin in 2) and the N,N,O,O-donor
bisphenolate ligand (L), giving a distorted octahedral
CoIIIN2O4 coordination core as observed in the crystal structure
of complex 1 (Figure 4a). The bonding parameters are similar
to those obtained in the crystal structure. The frontier
molecular orbitals (FMOs) of complex 1 showed active
participation of the metal center and the tetradentate ligand
(L2−) in forming the highest occupied molecular orbitals
(HOMOs) and contribution of the curcumin ligand in forming
the lowest unoccupied molecular orbitals (LUMOs) (Figure
4b). The HOMO in complex 2 resides primarily on the metal
center and the tetradentate ligand (L2−), but the LUMO is
Figure 2. (a) Absorption spectra of complexes 1 (black) and 2 (blue)
in 25% DMF/Tris HCl buffer. (b) Fluorescence spectra of complexes
1 (black) and 2 (blue) in DMSO (λex = 415 nm).
the curcumin-based π → π* absorption.10−13,55 An additional
band observed near 280 nm can be assigned to the aromatic π
→ π* transition.4,10−13 Upon excitation at 415 nm, the
complexes showed curcumin-based green fluorescence near 525
nm in DMSO with quantum yield (Φ) values in the range of
0.02−0.03 (Figure 2b). The emission property of the curcumin
ligands was used to study the cellular uptake and intracellular
localization of the complexes by confocal fluorescence
microscopy. The redox-active complexes showed an irreversible
reduction process assignable to the Co(III)/Co(II) couple near
−1.45 V versus SCE in DMF/0.1 M [nBu4N](ClO4). They also
showed curcumin-based quasireversible reduction near −1.74
V. There were no oxidative responses in the cyclic voltammetric
scans. The high negative CoIII−CoII reduction potential is likely
to reduce any facile reduction of the metal by cellular reducing
agents such as thiols (GSH) and ascorbic acid. The
electrochemical data are of significance with expected reduction
of any cellular toxicity of the complexes arising from the release
of the metal-bound curcumin ligand in the dark (Figure S7,
Supporting Information).10
X-ray Crystal Structure. Curcumin complex 1·CHCl3 was
structurally characterized by single-crystal X-ray diffraction. The
complex crystallized in the triclinic space group of P1 with two
̅
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Table 3. Selected Bond Distances (Å) and Bond Angles (deg) in [Co(L)(cur)]·CHCl3 (1·CHCl3)
Co(1)−O(1)
Co(1)−O(2)
Co(1)−O(3)
Co(1)−O(4)
Co(1)−N(1)
Co(1)−N(2)
O(2)−Co(1)−O(1)
O(2)−Co(1)−O(4)
O(1)−Co(1)−O(4)
O(2)−Co(1)−O(3)
1.880(4)
1.877(4)
1.920(5)
1.897(5)
2.021(5)
1.956(5)
94.87(19)
88.6(2)
O(1)−Co(1)−O(3)
O(3)−Co(1)−O(4)
O(2)−Co(1)−N(2)
O(1)−Co(1)−N(2)
O(4)−Co(1)−N(2)
O(3)−Co(1)−N(2)
O(2)−Co(1)−N(1)
O(1)−Co(1)−N(1)
O(4)−Co(1)−N(1)
O(3)−Co(1)−N(1)
N(1)−Co(1)−N(2)
89.7(2)
177.8(2)
92.76(19)
172.34(18)
90.9(2)
91.1(2)
178.4(2)
84.86(19)
89.8(2)
88.5(2)
90.2(3)
91.4(2)
87.5(2)
+
observed to be located over the triphenylphosphonium (PPh3 )
cationic moiety.
Table 4. Comparison of the M−O(cur) Bond Distances in
Structurally Characterized Curcumin (cur) Complexes
Stability Study. Curcumin alone is hydrolytically unstable
and undergoes rapid degradation in aqueous media with a short
half-life of 20 min at pH 7.4, in addition to having poor aqueous
solubility.56 The metal complexes of curcumin are known to be
stable under physiological conditions even after 48 h in the dark
(Figure S10, Supporting Information). The cobalt(III)
complexes showed significant solution stability in the dark as
evidenced from the spectral measurements (Figure 5a,b).
Additionally, the cellular reducing agents glutathione (GSH)
and ascorbic acid, in moderate concentrations up to 10 equiv
with respect to the concentration of the complexes, did not
show any reduction of the metal to form the cobalt(II) species,
possibly because of high negative potential for the Co(III)−
Co(II) redox couple. The release of curcumin from the reduced
cobalt(II) complexes was previously reported by Hambley and
co-workers (Figure 5c).9 We have observed that an excess
concentration of the reducing agents (>10 equiv) showed only
partial release of curcumin, as evidenced by an increase in the
M−O
bond distances
(Å)
a
bond
complex
[Co(L)(cur)]
ref
Co−O
1.878(4),
1.880(4)
present
work
V−O
[VO(cur)(phen)Cl]
[Zn(phen)(cur)Cl]
1.973(5),
1.959(4)
4
Zn−O
2.016(2),
2.031(2)
6
Ru−O
La−O
[(p-cymene)Ru(cur)Cl]
2.06(1), 2.07(1)
7
[La(ph-tpy)(cur)(NO3)2]
2.400(4),
2.410(4)
12
Gd−O
[Gd(ph-tpy)(cur)(NO3)2]
2.307(4),
2.315(4)
12
a
L is the dianion of 6,6′-(((2-(dimethylamino)ethyl)azanediyl)bis-
(methylene))bis(2,4-di-tert-butylphenol) (H2L), Hcur is curcumin,
phen is 1,10-phenanthroline, and the ligand ph-tpy is 4′-(1-pyrenyl)-
2,2′:6′,2″-terpyridine.
Figure 4. Energy-optimized structures of complexes 1 and 2 along with the FMOs. Color code: C, black; N, green; O, blue; Co, red; H, yellow.
E
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The visible-light- (400−700 nm) induced release of
mitocurcumin from complex 2 in the cellular environment
was studied by FACS analysis to quantify the intracellular
curcumin-based fluorescence intensity in MCF-7 cells. A 30-
min incubation with complex 2 under darkness showed ∼27%
intracellular mitocurcumin-based fluorescence, whereas irradi-
ation with visible light (400−700 nm) for 10 min showed an
enhancement in the fluorescence intensity to ∼72%, indicating
photorelease of mitocurcumin inside the cells (Figure 6b).
Cellular Uptake and Localization. To examine the
cellular permeability and cellular uptake of the complexes and
to optimize their incubation time for in vitro cellular studies
prior to photoirradiation, a cellular uptake study was carried out
using human breast adenocarcinoma (MCF-7) cells at 2- and 4-
h time points by fluorescence-activated cell sorting (FACS)
analysis in which the intracellular fluorescence of the complex
was monitored (Figure 7a). Upon 2 h of incubation, complex 2
Figure 5. (a) Absorption spectral traces of complex 2 in the dark over
a period of 24 h in 25% DMF/Tris HCl buffer (pH 7.2, 37 °C). (b)
Emission spectral traces of 2 in the dark over a period of 24 h in 25%
DMF/Tris HCl buffer (pH 7.2, 37 °C). (c) Emission spectral traces of
2 upon gradual addition of reduced glutathione up to 10 equiv under
similar experimental conditions. (d) Change in the emission spectral
traces of 2 upon addition of an excess quantity of reduced glutathione.
fluorescence intensity of the sample over time and the
concentration of the reducing agent (Figure 5d). The curcumin
dye alone showed higher fluorescence intensity than the ligand
in cobalt(III)-bound form. Any release of curcumin thus results
in an enhancement of its emission intensity.
Figure 7. (a) Time-dependent cellular uptake of complex 1 in MCF-7
cancer cells as determined by FACS analysis. (b) Cellular uptake of
complex 2 in MCF-7 cells over periods of 2 and 4 h.
Photorelease of Curcumin. Photoinduced ligand sub-
stitution or ligand release with concomitant metal reduction is
well-known for cobalt(III) and platinum(IV) complexes.57,58
Photoactivation of the ligand-based π → π* bands is known to
effect electron transfer to the cobalt(III) center.59 To examine
such a possibility for these complexes, 25% DMSO/Tris buffer
solutions of the complexes were photoirradiated with visible
light at wavelengths of 400−700 nm, and the corresponding
changes in the fluorescence spectra were monitored in different
time intervals (Figure 6a). Photoirradiation caused a steady
increase in the emission intensity of curcumin/mitocurcumin,
indicating release of the dye from the complexes. Selective
delivery of curcumin inside the cancer cells only upon
photoirradiation with visible light is of importance in the
chemistry of PDT, as the stability of this dye in metal-bound
form in the dark is likely to enhance its in vivo bioavailability.
showed ∼66.9% cellular uptake, whereas almost complete
internalization of this complex was observed within 4 h of
incubation (Figure 7b). In contrast, the charge-neutral
curcumin complex 1 did not show any significant cellular
uptake even after 72 h of incubation (Figure 7a). Interestingly,
the curcumin complexes of cobalt(III) in dicationic forms,
reported by Hambley and co-workers, are known to show
significant cellular uptake.9 Similarly, the mitocurcumin
complex 2 with its dicationic charge showed high cellular
uptake. Further studies with additional examples are needed on
cobalt(III) curcumin complexes to draw any specific con-
clusions about the structure−activity relationship in this class of
complexes.
Localization of an anticancer drug is of importance to study
its pharmacokinetics and to enhance its efficacy.60−62 Complex
2 being green fluorescent and showing high cellular uptake was
chosen for the localization study in the MCF-7 cells by confocal
microscopy. The complex with two cationic triphenylphospho-
nium (TPP) moiety is expected to target the mitochondria of
the cells.39 Confocal images clearly showed the complex
predominantly colocalizing with the MitoTracker Deep Red
(MTR) suggesting its selective localization in the mitochondria
of the cells (Figure 8).
Photoinduced Anticancer Activity. MTT assay was
carried out to study the cytotoxicity of the complexes in the
dark and upon exposure to visible light using HeLa (human
cervical carcinoma) and MCF-7 (human breast adenocarcino-
ma) cancer cells (Figure 9a; Figures S11 and S12, Supporting
Information). Relevant data along with data for other metal−
curcumin complexes and photofrin are listed in Table 5.63 Both
of the cobalt(III) complexes were found to be less toxic in the
dark. Curcumin complex 1 did not show any toxicity in the
Figure 6. (a) Change in the emission spectral traces of 2 upon
irradiation over a period of 30 min in 25% DMSO/Tris HCl buffer
(pH 7.2, 37 °C). (b) Enhancement of the intracellular fluorescence
intensity of complex-2-treated MCF-7 cells upon photoirradiation
(400−7000 nm) for 10 min.
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MCF-7 cells. The MTT assay data show a significant reduction
of the undesired dark toxicity of the mitocurcumin dye on
binding to Co(III) while retaining its photocytotoxicity. In
addition, a significant reduction in the cytotoxicity was
observed in the presence of ROS scavenger N-acetylcysteine
(NAC), suggesting ROS-mediated anticancer activity.
Curcumin as such or in its metal-bound form is known to
generate ROSs upon light activation.10−14 Being strong
oxidizing agents, ROSs oxidize nonfluorescent cell-permeable
fluorogenic probe, namely, 2′,7′-dichlorofluorescein diacetate
(DCFDA, ) to form green fluorescent 2′,7′-dichlorofluorescein
(DCF) with an emission maximum at 525 nm that can be
monitored by FACS analysis.64,65 To probe any ROS
generation by complex 2, a DCFDA assay was performed in
MCF-7 cells using flow cytometric analysis. Untreated cells did
not show any DCF fluorescence. Upon treatment with DCFDA
a shift in the intracellular fluorescence corresponding to the
basal cellular ROSs. A similar shift in the intracellular
fluorescence was also observed when the cells were treated
with complex 2 and the dye but kept in the dark. However, cells
treated with 2 and exposed to visible light (400−700 nm) for 1
h showed a significant increase in the intracellular emission
indicating generation of fluorescent DCF through the ROS-
induced oxidation of the nonfluorescent DCFDA (Figure 9b).
The cell death event noted with complex 2 in MCF-7 cells is
likely to be due to the light-assisted generation of intracellular
ROSs.
Figure 8. Confocal microscopy images of complex 2 in MCF-7 cells
recorded after 4 h of incubation using MitoTracker Deep Red as the
staining agent for mitochondria: (a) bright-field image (differential
interference contrast), (b) Mitotracker Deep Red (MTR), (c)
fluorescence of complex 2, (d) merged image. Scale bar is 15 μm.
Figure 9. (a) Photocytotoxicity of complex 2 in HeLa and MCF-7
cells upon 4 h of incubation in the dark (black bar), followed by
photoirradiation with visible light (400−700 nm, 10 J cm−2) in the
absence (red bar) and in the presence of N-acetylcysteine (NAC, gray
bar). (b) Flow cytometric analysis (FACS) for ROS generation by
complex 2 performed using the DCFDA assay in MCF-7 cells treated
with complex 2 under different experimental conditions: (i) cells only,
(ii) cells + DCFDA, (iii) cells + DCFDA + 2 (in the dark), (iv) cells +
DCFDA + complex 2 (in the light), and (v) cells + DCFDA + H2O2.
A greater shift implies a higher amount of DCF and, thus, greater ROS
generation.
CONCLUSIONS
■
Ternary cobalt(III) complexes of curcumin and mitocurcumin
having a dianionic tetradentate phenolate-based ancillary ligand
were prepared and studied for the photoinduced release of the
dye facilitated by concomitant metal reduction. The charge-
neutral curcumin complex 1 was structurally characterized by
X-ray crystallography. The hydrolytic degradation of curumin
and mitocurcumin becomes arrested upon binding to the
cobalt(III) ion. This results in an enhanced bioavailability of the
dye. A preliminary structure−activity relationship study showed
a lower cellular uptake of the neutral curcumin complex 1,
whereas, in contrast, the dicationic mitocurcumin complex 2
displayed significant cellular uptake. This is in agreement with
the cellular uptake reported by Hambley and co-workers for
cationic cobalt(III) complexes of curcumin. The appendage of
two lipophilic cationic triphenylphosphonium (TPP) moieties
to the curcumin dye imparts not only higher cellular uptake,
but also selective delivery of the complex to the mitochondria
of the cells. Complex 2 has shown significant internalization
into the mitochondria of the MCF-7 cells with remarkable
photoinduced cytotoxicity in visible light with wavelengths of
400−700 nm with a low IC50 value (∼3 μM) while being less
toxic (>50 μM) when kept in the dark. Studies under
extracellular and cellular conditions have revealed photorelease
of the dye with metal reduction. The metal in its 3+ oxidation
state is, however, not susceptible to reduction even with
moderate concentrations of glutathione as a reducing agent.
This is due to high negative Co(III)−Co(II) reduction
potential observed in the cyclic voltammetric study. Confocal
microscopy has revealed both mitochondrial localization of this
complex and generation of ROSs which is light-dependent. In
summary, this work presents a novel methodology akin to the
one reported by Hambley and co-workers to deliver a
curcuminiod from a cobalt(III) complex in the cancer cells
upon photoactivation in visible light with remarkable PDT
Table 5. IC50 (μM) Values of the Complexes and Relevant
Compounds
IC50 (μM)
complex
in the light (400−700 nm)
in the dark
a
a
1
2
>100
>100
3.9 0.7
50.2 1.4
b
[Co(cur)(tpa)]ClO4
39
4
4
c
[Co(cur)(tpa-COOH)]
6
1
41
d
photofrin
4.3 0.2
>41
e
cisplatin
−
20.5 0.2
a
IC50 in the light is upon visible-light irradiation (400−700 nm, 10 J
cm−2) in MCF-7 cells. Complex 1 showed poor cellular uptake in
b
MCF-7 cells. IC50 values in DLD-1 colon cancer cells (from ref 9).
c
d
IC50 values in MCF-7 cells (from ref 18). Values taken from ref 63
e
for HeLa cells. Value taken from ref 6 for SH-SY5Y cells.
light possibly due to its poor cellular uptake. The mitocurcumin
complex 2 showed remarkable PDT activity. It gave an IC50
value of >100 μM in HeLa and ∼50 μM in MCF-7 cells on an
incubation of 4 h in the dark. Irradiation with a broad-band
visible light (400−700 nm) resulted in a significant enhance-
ment of its cytotoxicity, whereas for the untreated light control
cells, there was no apparent cell death, thus eliminating the
possibility of any cytotoxic effect of the light. Complex 2 gave
an IC50 value of ∼24.3 μM in HeLa and ∼3.9 μM in MCF-7
cells upon photoactivation. Mitocurcumin alone gave an IC50 of
∼3.7 μM in HeLa cells under similar experimental conditions in
visible light and ∼13.6 μM in the dark. The dye alone gave an
IC50 value of ∼1.3 μM in the light and ∼13.7 μM in the dark in
G
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Article
activity facilitated by concomitant metal reduction.18 The
results obtained from the SAR study are likely to open up new
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the therapeutically important curcumin and its derivatives from
cobalt(III) complexes.
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ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.inorg-
chem.6b00554.
All the details of the experimental procedures, reaction
schemes (Schemes S1, S2), mass spectra of the
complexes (Figures S1 and S2), IR spectra (Figures S3
and S4), NMR spectra (Figures S5 and S6), cyclic
voltammograms (Figure S7), ORTEP views and unit-cell
packing diagram (Figures S8 and S9), stability plot of
complex 1 (Figure S10), cytotoxicity data of complex 2
(Figures S11 and S12), and calculated energy levels from
the theoretical studies (Tables S1 and S2) (PDF)
CIF data for 1·CHCl3 (CIF)
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: paturu@mrdg.iisc.ernet.in. Tel.: +91-80-22932688.
Fax: +91-80-23600999.
*E-mail: arc@ipc.iisc.ernet.in. Tel.: +91-80-22932533. Fax:
+91-80-23600683.
(20) Lovell, J. F.; Liu, T. W. B.; Chen, J.; Zheng, G. Chem. Rev. 2010,
110, 2839−2857.
(21) Celli, J. P.; Spring, B. Q.; Rizvi, I.; Evans, C. L.; Samkoe, K. S.;
Verma, S.; Pogue, B. W.; Hasan, T. Chem. Rev. 2010, 110, 2795−2838.
(22) Ethirajan, M.; Chen, Y.; Joshi, P.; Pandey, R. K. Chem. Soc. Rev.
2011, 40, 340−362.
Notes
The authors declare no competing financial interest.
(23) Kamkaew, A.; Lim, S. H.; Lee, H. B.; Kiew, L. V.; Chung, L. Y.;
Burgess, K. Chem. Soc. Rev. 2013, 42, 77−88.
ACKNOWLEDGMENTS
■
(24) Mari, C.; Pierroz, V.; Ferrari, S.; Gasser, G. Chem. Sci. 2015, 6,
2660−2686.
We thank the Department of Science and Technology (DST),
Government of India, for financial support (SR/S5/MBD-02/
2007, EMR/2015/000742). A.R.C. thanks DST for a J. C. Bose
national fellowship. We are thankful to the Alexander von
Humboldt Foundation, Germany, for the donation of an
electroanalytical system. A.G. and S.B. thank IISc, Bangalore,
India, for senior research and postdoctoral fellowship,
respectively. I.P. acknowledges CSIR for a senior research
fellowship. We are thankful to IISc FACS facility and Dr.
Santosh Podder for the help extended in confocal microscopy
experiments.
(25) Knoll, J. D.; Turro, C. Coord. Chem. Rev. 2015, 282−283, 110−
126.
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DOI: 10.1021/acs.inorgchem.6b00554
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