Mitochondrial selectivity and remarkable photocytotoxicity of a ferrocenyl neodymium(iii) complex of terpyridine and curcumin in cancer cells

Article abstract of DOI:10.1039/c5dt04775g

A series of four novel neodymium(iii) complexes of the formulation [Nd(R-tpy)(O-O)(NO₃)₂] (1-4), where R-tpy is 4′-phenyl-2,2′:6′,2′′-terpyridine (Ph-tpy; 1, 2) and 4′-ferrocenyl-2,2′:6′,2′′-terpyridine (Fc-tpy; 3, 4); O-O is the conjugate base of acetylacetone (Hacac; 1, 3) or curcumin (Hcurc; 2, 4), are synthesized and characterized. The single crystal structure of 1 shows that the complex is a discrete mononuclear species with the Nd(iii) centre in a nine coordinate environment provided by a set of O₆N₃ donor atoms. Complexes 1 and 3 having the simple acac ligand are prepared as control compounds. Complex 4, possessing an appended ferrocenyl (Fc) and the curcumin moiety, is remarkably photocytotoxic to HeLa and MCF-7 cancer cells in visible light giving respective IC₅₀ values of 0.7 μM and 2.1 μM while being significantly less toxic to MCF-10A normal cells (IC₅₀ = 34 μM) and in the dark (IC₅₀ > 50 μM). The phenyl appended complex 2, lacking a ferrocenyl moiety, is significantly less toxic to both the cell lines when compared with 4. Complexes 1 and 3, lacking the photoactive curcumin moiety, do not show any apparent toxicity both in light and in the dark. The cell death is apoptotic in nature and is mediated by the light-induced formation of reactive oxygen species (ROS). Fluorescence imaging experiment with HeLa cells reveals mitochondrial accumulation of complex 4 within 4 h of incubation. The complexes bind to calf thymus (ct) DNA with moderate affinity giving K_b values in the range of 10⁴-10⁵ M^-1. The curcumin complexes 2 and 4 cleave plasmid supercoiled DNA to its nicked circular form in visible light via¹O2 and ^?OH pathways. The presence of the ferrocenyl moiety is likely to be responsible for the enhanced cellular uptake and photocytotoxicity of complex 4. Thus, the mitochondria targeting complex 4, being remarkably cytotoxic in light but non-toxic in the dark and to normal cells, is a potential candidate for photochemotherapeutic applications.

Full text of DOI:10.1039/c5dt04775g

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Journal Name
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DOI: 10.1039/C5DT04775G
Cite this: DOI: 10.1039/c0xx00000x
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ARTICLE TYPE
Mitochondrial selectivity and remarkable photocytotoxicity of a
ferrocenyl neodymium(III) complex of terpyridine and curcumin in
cancer cells†
a
b
b
a
Tukki Sarkar, Samya Banerjee,* Sanjoy Mukherjee and Akhtar Hussain*
5
Received (in XXX, XXX) Xth XXXXXXXXX 20XX, Accepted Xth XXXXXXXXX 20XX
DOI: 10.1039/b000000x
A series of four novel neodymium(III) complexes of formulation [Nd(Rꢀtpy)(OꢀO)(NO ) ] (1–4),
3
2
where Rꢀtpy is 4′ꢀphenylꢀ2,2′:6′,2′′ꢀterpyridine (Phꢀtpy; 1, 2) and 4′ꢀferrocenylꢀ2,2′:6′,2′′ꢀterpyridine
Fcꢀtpy; 3, 4); OꢀO is the conjugate base of acetylacetone (Hacac; 1, 3) or curcumin (Hcurc; 2, 4) is
(
1
1
2
2
0
5
0
5
synthesized and characterized. The single crystal structure of 1 shows that the complexes are discrete
mononuclear species with Nd(III) centre in a nine coordinate environment provided by a set of O N3
6
donor atoms. Complexes 1 and 3 having the simple acac ligand are prepared as control compounds.
Complex 4, possessing an appended ferrocenyl (Fc) and the curcumin moiety, is remarkably photocytotoxic
to HeLa and MCFꢀ7 cancer cells in visible light giving respective IC50 values of 0.7 µM and 2.1 µM while
being significantly less toxic to MCFꢀ10A normal cells (IC50 = 34 µM) and in the dark (IC50 > 50 µM).
The phenyl appended complex 2, lacking a ferrocenyl moiety, is significantly less toxic to both the cell lines
when compared with 4. Complexes 1 and 3, lacking the photoactive curcumin moiety, do not show any
apparent toxicity both in the light and dark. The cell death is apoptotic in nature and is mediated by the
lightꢀinduced formation of reactive oxygen species (ROS). Fluorescence imaging experiment with HeLa cells
reveals mitochondrial accumulation of complex 4 within 4 h of incubation. The complexes bind to calf thymus
4
5
ꢀ1
(
ct) DNA with moderate affinity giving K values in the range of 10 ꢀ10 M . The curcumin complexes 2 and 4
b
1
●
cleave plasmid supercoiled DNA to its nicked circular form in visible light via O and OH pathways.
2
The presence of the ferrocenyl moiety is likely to be responsible for the enhanced cellular uptake and
photocytotoxicity of complex 4. Thus, the mitochondria targeting complex 4, being remarkably cytotoxic in
light but nonꢀtoxic in dark and to normal cells, is a potential candidate for photochemotherapeutic applications.
1
ꢀ6
level of invasiveness and ability to overcome drug resistance.
®
Introduction
Photofrin is currently the FDA approved and clinically
frequently prescribed organic PDT drug. Unfortunately, the
limitations such as skin sensitivity and hepatotoxicity associated
with Photofrin have prompted chemists to look for its
alternatives. Consequently, the lightꢀactivatable metal complexes
7,8
Photodynamic therapy (PDT) is
a clinically attractive
5
5
6
6
0
5
0
5
chemotherapeutic modality for the treatment of cancer. In PDT, a
photoactivable drug is administered to a cancer patient and the
target tissue is irradiated with specific light in the presence of
molecular oxygen thereby resulting in the generation of cytotoxic
®
3
0
8
of nonꢀporphyrinic ligands have emerged as potential candidates
9ꢀ12
in the PDT of cancer.
Metal complexes can provide variable
1
,2
molecular species.
PDT has many advantages over
coordination geometries, versatile spectral and redox properties
and can cause cell death via photoꢀredox and/or typeꢀI pathways
besides generating cytotoxic singlet oxygen species in a typeꢀII
conventional cancer therapies such as its low systemic toxicity,
selective drug action within the photoꢀirradiated regions, low
3
5
9–12
process.
There is currently a growing interest in the coordination
a
Department of Chemistry, Handique Girls’ College, Guwahati 781001,
chemistry of lanthanides because of their promising applications
Assam, India. Eꢀmail: akhtariisc@gmail.com; Tel: +91ꢀ361ꢀ2543793
13ꢀ16
in medicinal chemistry.
The most important application of
b
4
0
Department of Inorganic and Physical Chemistry, Indian Institute of
lanthanides is the use of gadolinium(III) complexes such as
Magnevist and Dotarem as clinical MRI contrast agents.
Science, Bangalore560 012, Karnataka, India. Eꢀmail:
samya@ipc.iisc.ernet.in; Tel: +91ꢀ80ꢀ22932240.
TM
TM
15
Although several transition metalꢀbased PDT agents have been
studied, the studies on lanthanide (Ln) complexes as PDT agents
†
Electronic supplementary information (ESI†) available: Reaction
schemes, ESIꢀMS spectra, UVꢀvisible spectra, cyclic voltammograms,
packing diagrams, DFT structures and tables, stability and MTT assay
plots, DNA gel electrophoresis diagrams. See DOI: 10.1039/b000000x/
are rare apart from a few lanthanide complexes of macrocyclic
4
5
17ꢀ2
and nonꢀmacrocyclic organic dyes.
The lutetium(III)

texaphyrin (LUTRIN ) complex, for example, is known to show
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2
1
39ꢀ
promising PDT effect in the nearꢀIR light. The Ln(III)
complexes with their poor stereochemistry and high coordination
numbers can be appropriately designed by employing
photosensitizing organic ligands possessing hard O and N donor
atoms to achieve remarkable photocytotoxicity against cancer
cells. Furthermore, such complexes could be nonꢀtoxic in the
and photochemical properties and ability to induce PDT effect.
44
60
The green fluorescence emission of curcumin, moreover, offers
a convenient means to study the localization of the curcumin
41ꢀ46
complexes in cancer cells.
5
0
5
0
5
0
5
0
5
0
5
Although nucleus is the prime target of most anticancer drugs,
there is currently increasing interest in search for compounds that
absence of light since the Ln(III) ions are redox inactive thereby 65 target cell organelles other than the nucleus because of the
4
7,48
making them desirable for cellular applications in the presence of
reducing glutathione or ascorbic acid present in cells. Besides, the
photoexcited lanthanide complexes can undergo facile
intersystem crossing (ISC) due to the heavy atom effect induced
limitations associated with the nucleus targeting drugs.
The
resistance of cancer cells to cisplatin, for instance, is due to the
extensive repair of the cisplatinꢀDNA adducts by the nucleotide
1
1
2
2
3
3
4
4
5
5
49
excision repair (NER) pathway. Drugs that selectively target
by the heavy lanthanide cation thereby resulting in efficient 70 mitochondria, for instance, can serve as potential anticancer
2
2
generation of singlet oxygen.
Bioorganometallics is an emerging area of research in
medicinal chemistry.
agents because mitochondria perform key cellular functions and
any inhibition with their functions can lead to apoptosis.
Besides, mitochondria do not possess a functional nucleotide
excision repair mechanism to repair DNA adducts. Therefore,
4
7,48,50
2
3ꢀ25
Ferrocene and its conjugates have
received considerable attention because they are known to
possess many medicinal properties as antibacterial, antimalarial 75 mitochondria targeting drugs are highly desirable for successful
2
3ꢀ25
and antitumor agents.
Ferrocene conjugates have found their
chemotherapeutic applications. For example, the FDA approved
®
most promising application in the treatment of breast cancer.
Around 66% of the breast cancers are hormone dependent where
the estrogen receptor (ER+) is involved. The ferrocene moiety
clinical PDT drug Photofrin is known to target the mitochondria
51
of cancer cells and cause apoptosis in tumour cells. Although,
oxovanadium(IV) and cobalt(III) complexes showing
offers a number of advantages such as its stability in the 80 mitochondrial selectivity are known, the reports on lanthanide
4
3,44
physiological medium, lipophilicity, redox activity and nontoxic
complexes are virtually unknown .
2
6
nature in the dark. For example, the ferrocene appended
anticancer drug tamoxifen is known to show enhanced activity
against both hormone dependent (ER+) and hormone independent
Very recently, we have reported Co(III) and Fe(III)
complexes of curcumin showing photocytotoxicity against cancer
The present work results from our effort
ERꢀ) breast cancers. Similarly, ferroquine, the ferrocene 85 to study lanthanide complexes of curcumin as photocytotoxic
44,45
cells in visible light.
2
7
(
conjugated antimalarial drug chloroquine, is significantly more
agents. Herein, we present the studies on the in vitro
photodynamic potential of a series of neodymium(III) (Nd(III))
complexes. Nd(III) was chosen as the representative lathanide.
2
8
active than chloroquine. In addition to the ferrocene derivatives,
the rutheniumꢀarene and rutheniumꢀcyclopentadienyl halfꢀ
sandwich complexes are known as organometallic antitumor
5
2
Nd(III) complexes have earlier been studied as cytotoxic agents.
agents. The waterꢀsoluble ruthenium(II)ꢀareneꢀpta (RAPTA) 90 The Nd(III) complexes of acetylacetone (Hacac) have been
complexes have shown promising results as antiꢀmetastatic
prepared as controls to understand the role of curcumin (Fig. 1).
We have synthesized and characterized four new lanthanide(III)
complexes of formulation [Nd(Rꢀtpy)(OꢀO)(NO ) ] (1–4), where
2
3ꢀ25
agents.
The ferrocenium ion itself is known to show cytotoxic
23
activity. While the ferrocene appended oxovanadium(IV) and
copper(II) complexes have been studied as photocytotoxic agents,
the lanthanide complexes are virtually unexplored.
3
2
Rꢀtpy is a N,N,Nꢀdonor terpyridine ligand, viz., 4′ꢀphenylꢀ
29ꢀ31
Our 95 2,2′:6′,2′′ꢀterpyridine (Phꢀtpy; 1, 2) and 4′ꢀferrocenylꢀ2,2′:6′,2′′ꢀ
interest to design ferrocene based photocytotoxic agents is based
on the effectiveness of bioorganometallic complexes as
promising drug candidates showing enhanced selectivity and
anticancer activity.
terpyridine (Fcꢀtpy; 3, 4); OꢀO is the monoanion of acetylacetone
(Hacac; 1, 3) or curcumin (bis(4ꢀhydroxyꢀ3ꢀmethoxyphenyl)ꢀ1,6ꢀ
dieneꢀ3,5ꢀdione, Hcurc; 2, 4), and evaluated their photoꢀinduced
cytotoxicity in HeLa and estrogen ependent (ER+) MCFꢀ7 cancer
Curcumin (Hcurc) and its metal complexes have received a 100 cell lines. While HeLa represents a model of highly proiferative
great deal of current attention because curcumin shows a number cancer cell, the MCFꢀ7 cell line is particularly suitable to study
3
2,33
23ꢀ28
of of medicinal properties.
methoxyphenyl)ꢀ1,6ꢀdieneꢀ3,5ꢀdione, Hcurc) is
occurring βꢀdiketone compound present in the herb Curcuma
Curcumin (bis(4ꢀhydroxyꢀ3ꢀ
the cytotoxicity of ferrocence conjugates.
Significant results
a
naturally
of this study include: (i) the remarkable photocytotoxicity of
complex 4 in visible light with negligible toxicity in dark and to
longa L. It is known to be a potent anticancer agent and cause 105 normal cells, (ii) the enhancement in photocytotoxicity of
apoptosis selectively in cancer cells while being harmless to the
complex 4 compared to complex 2 due to the presence of the
ferrocenyl moiety, (iii) the apoptotic mode of cell death due to the
33,34
normal cells.
However, the poor bioavailability and
pharmacokinetic profile of curcumin has severely limited its
usefulness as drug despite its broad spectrum of biological
photoꢀassisted generation of intracellular ROS, (iv) the
mitochondrial localization of complex 4 thereby making the
3
4,35
activities.
Therefore, several derivatives of curcumin have 110 cytosolic organelles as the potential targets, and (v) efficint
3
6ꢀ38
been studied to circumvent its drawbacks.
One of the subtle
visible lightꢀtriggered DNA cleavage activity.
and successsful strategies takes advantage of curcumin’s metal
3
6ꢀ38
coordinating ability.
Although, there are reports on the
[Space for Fig.1]
anticancer activity of curcumin and its metal complexes, the
studies on the photodynamic potential of its metal complexes,
especially with Ln(III), are still rare despite its rich photophysical
115 Results and discussion
2
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Synthesis and general aspects
The complexes [Nd(Rꢀtpy)(OꢀO)(NO ) ] (1–4) were
of the ferrocene appended complexes 3 and 4. A significantly
higher positive shift (~ +0.2 V) of reduction potential compared
to the free ferrocene suggests stabilization of the Fc moiety
toward oxidation in the presence of the electron withdrawing
60
3
2
synthesized in ~78% yield by a general twoꢀstep procedure in
which the precursor complex, [Nd(Rꢀtpy)(NO ) ], prepared by
3
3
29b
terpyridyl (tpy) moiety.
5
0
5
0
5
0
5
0
5
0
5
reacting Nd(NO ) .6H O with either Phꢀtpy or Fcꢀtpy was reacted
3 3 2
with either Hacac or Hcurc in methanolꢀacetonitrile binary
solvent mixture (Fig. 1, Figs. S1, S2, ESI†). Because the Hacac
and Hcurc ligands usually coordinate to metal ions as
monoanions, their deprotonation was achieved by using
triethylamine as the base. The elemental analyses data on the
complexes were in good agreement with the theoretically
calculated values. The FT–IR spectra of the complexes recored in
[Space for Fig. 2]
65
X-ray crystallography
Complex 1 was structurally characterized by single crystal Xꢀ
ray diffraction method (Fig. 3(a), Table 2). It crystallized in the
triclinic space group Pī with two molecules in the unit cell. The
complex is a discrete mononuclear species with NdN O core
70 formed by the tridentate N,N,Nꢀdonor Phꢀtpy and bidentate O,Oꢀ
donor acac ligands in a nine coordinate environment. The
pyridine rings of the Phꢀtpy ligand lie in a planar conformation
and the bidentate nitrate anions are disposed trans to one another.
The ORTEP view of the complex is shown in Fig. 3(a) and the
1
1
2
2
3
3
4
4
5
5
3
6
ꢀ
1
pure solid phase displayed IR bands at 1595, 1490 and 1395 cm
corresponding to the C=O, C=C (βꢀdiketonate) and NO3
ꢀ
stretching vibrations, respectively, indicating bidentate
ꢀ
coordination mode of the βꢀdiketonate (acac or curc) and NO
3
5
3
ligands. In addition, the complexes 2 and 4, having the curc
ligand showed an additional band at ~3390 cm due to the 75 unit cell packing diagram is provided as supplementary material
ꢀ
1
phenolic hydroxyl groups of the curc ligand (Table 1). This value
suggests that the phenolic –OH group is interacting with the
(Fig. S9, ESI†). The phenyl ring of the phꢀtpy ligand makes a
dihedral angle of 25.66° with the central pyridine ring of the
terpyridine moiety due to steric encumbrance imposed by the
hydrogen atoms (Fig. S10, ESI†). The NdꢀO bond distances
neighboring –OCH group of the curcumin ligand through
3
3
5
hydrogen bonding. The molar conductivity (Λ ) measurements
M
carried out in DMF (N,Nꢀdimethyl formamide) suggested that all 80 involving the nitrate ligands are longer than those involving the
the complexes were 1:1 electrolytes corresponding to the
dissociation of one nitrate ligand from the complex in solution
curc ligand thereby making the nitrate ligand labile in the solution
phase. This lability of the nitrate ligand is reflected in ESIꢀMS
spectra of the complexes in aqueous methanol showing a
54
(
Table 1). This observation is further supported by the ESIꢀMS
ꢀ
+
measurements on the complexes in aqueous methanol showing
prominent peak corresponding to the [M–(NO )] species. The
3
ꢀ
+
the presence of a prominent [Mꢀ(NO )] peak resulting from 85 NdꢀO and NdꢀN distances are in the range 2.3391(18)ꢀ2.566(2) Å
3
release of one nitrate anion (Figs. S3–S6, ESI†). Thus, the
solution formulation of the complexes could be [Nd(Rꢀtpy)(Oꢀ
and 2.578(2)ꢀ2.635(2) Å, respectively. Selected crystallography
parameters are given in Table 2 and the bond lengths and bond
angles have been enlisted in Table 3. The structural properties
observed in the complexes are similar to those previously
55
O)(NO )]NO . The electronic absorption spectra of complexes
3
3
recorded in aqueous DMF showed a common absorption band
18b,56
around 275 nm assignable to a π – π* transition (Fig. 2(a), Figs. 90 reported for analogous lanthanide complexes.
Selected
44
S7, ESI†).
In addition, the complexes 2 and 4 showed an
crystallography parameters are given in Table 2 and chemically
significant bond lengths and bond angles have been enlisted in
intense curcuminꢀbased visible absorption band within 400˗500
4
4
nm originating from a π → π* transition. This absorption band
can be exploited to study the visibleꢀlight triggered cytotoxicity
of the complexes against cancer cells. The fluorescence emission
Table S3 (ESI ).
†
[
Space for Fig. 3]
spectra of complexes 2 and 4 measured in DMF showed a 95 Theoretical studies
curcuminꢀbased emission band centered at 525 nm with quantum
yield (φ) value of ~ 0.01 when excited at 420 nm (Fig. 2(a)). This
DFT calculations were carried out to obtain the optimized
geometries and insights into the excited state properties of the
Nd(III) complexes. The initial coordinates were obtained from
the crystal structure of complex 1 and used for optimization. The
100 frontier orbital calculations revealed that the HOMO of complex
green emission from the complexes can be exploited to study
4
1ꢀ45
their localization in cancer cells.
The solution susceptibility
measurements of the complexes gave magnetic moments (ꢁ ) in
eff
3
the range 3.56–3.62ꢁB corresponding to the [Xe]f configuration
4
is localized on the curcumin ligand and the LUMO is mainly
of Nd(III) center. The redox properties of the complexes were
studied by electrochemical cyclic voltammetry measurements
carried out in DMF in the presence of 0.1M tetrabutylammonium
perchlorate (TBAP) as the supporting electrolyte (Fig. 2(b), Fig.
S8, ESI†). The Phꢀtpy complexes 1 and 2, lacking any ferrocenyl
moiety, displayed poor voltammetric responses giving anodic
confined to the Nd(III) centre (Fig. 3(b), Figs. S11–S14, Tables
S2ꢀS5, ESI†). This observation suggests that the curcumin ligand
could act as an efficient photosensitizer and photoꢀexcitation of
05 the complex could result in electronic transitions from the
HOMO centred on the curcumin moiety to the LUMO centered
on Nd(III). This observation could also explain the remarkable
photocytotoxicity of complex 4 in visible light. In the case of
complex 2, the HOMO is not predominantly curcuminꢀcentred
10 but rather derived both from the curcumin and the Nd(III) centre,
while its LUMO has contributions from the tpy moiety.
Consequently, the electronic excitation in complex 2 is not as
facile as in complex 4 which could also explain the lower
1
peaks (E ) near ꢀ0.26V vs. SCE without any cathodic
pa
counterpart. The corresponding peaks for complexes 3 and 4 were
observed near ꢀ0.30V. The ferrocenyl moiety in complexes 3 and
1
4
is redox active and showed a quasiꢀreversible cyclic
voltammetric response near +0.61V vs. SCE. This higher
+
reduction potential value for the Fc /Fc couple compared to the
free ferrocene (+0.42 V vs. SCE) indicates better redox stability
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photocytotoxicity of the former complex. In the absence of a
Having confirmed the accumulation of complex 4 in the
photosensitizing curcumin moiety in complexes 1 and 3, facile
cytoplasm, we delved further to probe its selectivity of
electronic transition is unlikely and hence these two control 55 localization to specific cellular organelle. For this specific
4
4
compounds are not appreciably photocytotoxic in visible light.
purpose, we examined the specificity of localization of the
complex in the mitochondria (panels (d), (e), (f), Fig. 5). Dual
staining of complex 4 with the mitochondria staining dye
Mitotracker red showed that complex 4 specifically localized in
5
Solubility and stability
The synthesized complexes were found to be soluble in DMF
and DMSO (dimethylsulphoxide). They were moderately soluble 60 the mitochondria of HeLa cells as evidenced from the merged
in acetone, acetonitrile, methanol, tetrahydrofuran and
chloroform but insoluble in hydrocarbon solvents. The stability of
a complex in aqueous buffer is an important aspect for its
application as drug and so the stability of the complexes was
studied by absorption spectroscopy. The complexes were found
to be stable in 10% DMEM (Dulbecco’s Modified Eagle’s
Medium) as evidenced from the absorption study on complex 4
showing no noticeable spectral change even after 48 hours. In
contrast, the free curcumin ligand was found to undergo
hydrolytic degradation under identical experimental conditions
image (panel (h), Fig. 5). The selective localization of complex 4
in mitochondria is indeed a significant result considering the vital
1
1
2
0
5
0
role of mitochondria in mediating the intrinsic pathway of
44ꢀ49
apoptosis.
There is currently substantial interests in
65
mitochondria targeting cytotoxic drugs for applications in the
46ꢀ49
chemotherapy of cancer.
Photocytotoxicity
Having confirmed that the complexes are taken up by the
cancer cells, we were interested to evaluate their cytotoxicity in
(Fig. S15, ESI†). This observation indicates that the binding of
curcumin to Nd(III) ion arrests its hydrolytic degradation. This 70 the presence of visible light (400ꢀ700 nm). The inꢀvitro
finding is also supported by earlier reports on metal complexes of
photodynamic efficacy and hence the photoꢀinduced anticancer
activity of a compound can be reliably assessed by measuring its
cytotoxicity in terms of its IC50 value against cancer cells by
employing the well known MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
75 2,5ꢀdiphenyltetrazolium bromide) assay in the presence of light.
Thus, the photoactivated anticancer activity of the complexes was
studied by MTT assay in two human cancer cell lines, viz.,
human cervical carcinoma (HeLa) and estrogen receptor positive
(ER+) human breast adenocarcinoma (MCFꢀ7) in the presence of
1
9,43ꢀ45
curcumin.
As mentioned earlier, the hydrolytic instability
of curcumin has prevented its use as anticancer drug in clinical
settings. Thus, a metalꢀbound formulation of curcumin could be
therapeutically more effective than curcumin alone.
25
Cellular incorporation assay
This assay was performed to examine the cellular permeability
and uptake and to optimize the incubation time of the complexes
for inꢀvitro cellular assays before photoꢀirradiation. Uptake of 80 visible light (Table 3, Figs. S16–S19, ESI†). The ferrocenyl
complex 4 by HeLa cells at 2 h and 4 h time points was studied
by fluorescenceꢀactivated cell sorting (FACS) analysis in which
the intraꢀcellular fluorescence of the complex was monitored
appended complex 4, having curcumin as the photoactive ligand
moiety, was found to remarkably photocytotoxic to both the cells
giving IC50 values of 0.7±0.2 ꢁM in HeLa and 2.1±0.6 ꢁM in
MCFꢀ7 cells when irradiated with visible light (400–700 nm, 10 J
30
(Fig. 4). The number of events in the upper left quadrant of the
ꢀ
2
figure indicates the population of cells in which the complex was 85 cm ). The complex was nonꢀtoxic to both the cells in the absence
not taken up and that in upper right quadrant represents the cell
population containing the complex inside. On 2 h of incubation,
complex 4 showed almost 72 % cellular uptake whereas complete
internalization of the complex was observed within 4 h of
of light (IC50 > 50 µM). On the other hand, phenyl analogue of
complex 4, viz., complex 2, was found to be significantly less
photocytotoxic to both the cells giving IC50 values of 4.2±0.8 ꢁM
in HeLa and 9.6±1.2 ꢁM in MCFꢀ7 cells while being nonꢀtoxic to
35
incubation. Thus, we incubated the cells with the complex for 4 h 90 both the cells in the absence of light (IC50 > 50 µM). Thus, a 6ꢀ
for other cellular studies.
fold increase in photocytotoxicity against HeLa cells and a 5ꢀfold
increase against MCFꢀ7 cells were observed when the phenyl
group in complex 2 was replaced by a ferrocenyl group in
complex 4. The control complex 1 with Phꢀtpy and acac as
ligands, but lacking ferrocenyl as well as photoactive curcumin
moiety was not cytotoxic to both the cell lines neither in the
presence nor in the absence of light (Table 3). Complex 3, with
an appended ferrocenyl moiety and acac ligand, but lacking any
curcumin moiety gave IC50 values of 13.2±1.6 ꢁM in HeLa and
4
0
[Space for Fig. 4]
Cellular uptake and localization study
95
The green curcuminꢀderived emission from complex 4 was
utilized to study the cellular localization and uptake in HeLa cells
using fluorescence microscopy (Fig. 5). Complex 4 was found to
accumulate significantly within 2 h of incubation which increased
substantially during 4 h suggesting its accretion inside the cells.
Dual staining experiment with the nucleus staining dye Hoechst
45
1
00 19.9±1.8 ꢁM in MCFꢀ7 cells, while being nonꢀtoxic in dark. The
observed photocytotoxicity of complex 3 could be due to the
presence of ferrocenyl moiety. The curcumin ligand alone is
3
3342 (blue emission) showed primarily cytosolic localization of
complex 4 within the cells as revealed from the merged images
(panel (c) of Fig. 5).
known to give an IC50 value of 8.2±0.2 ꢁM in visible light and
50
41a
8
5.4±0.6 ꢁM in dark in HeLa cells. The corresponding values
[
Space for Fig. 5]
105 in MCFꢀ7 cells are 19.9 ± 1.4 ꢁM in visible light and 90.3±4.9
41a
ꢁM in dark under similar experimental conditions. Hence, the
Mitochondrial selectivity
remarkable photocytotoxicity but insignificant dark toxicity of
complex 4 is unprecedented in the chemistry of lanthanide based
4
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DOI: 10.1039/C5DT04775G
PDT agents. The presence of the lipophilic ferrcenyl moiety in
complex 4 is likely to be responsible for the enhanced cellular
uptake and photocytotoxicity of complex 4 when compared to
phosphatidylserine in healthy cells remains in the interior surface
of the lipid bilayer which is transferred to the outside portion of
the membrane at the onset of apoptosis. This change in the
complex 2. Earlier, lanthanide complexes of Phꢀtpy and 60 membrane morphology can be detected and studies by a
5
0
5
0
5
curcumin, lacking a ferroceny moiety, were reported to show
fluorophore conjugated annexinꢀV protein which is known to
bind phosphatidylserine. The assay is based on dual staining of
cells by annexin VꢀFITC (fluorescein isothiocyanate) and the
DNA binding dye propidium iodide (PI). Cells undergoing early
19
photocytotoxicty against HeLa cells in visible light. Thus, we
have achieved enhancement in photocytotoxicity of complex 4 by
appending a ferrocenyl unit in the complex. The photocytoxic
activity shown by complex 4 against the ER(+) MCFꢀ7 cells is 65 apoptosis are identified as single positive population for FITC,
1
1
2
2
indeed striking since the ferrocene conjugates are known to show
promising anticancer activity in both hormone dependent and
while necrotic cell population is stained only by PI. The double
positive population represents cells in late apoptotic stage with
compromised cell membrane. To find out whether the present
complexes are able to trigger any apoptotic cell death in the
2
9ꢀ31
independent cases.
Although, oxovanadium(IV), Fe(III),
Co(III) and Cu(II) complexes of curcumin are known to show
photocytotoxicity in visible light, the corresponding 70 presence of visible light, we carried out annexinꢀVꢀFITC/ PI
lanthanide(III) complexes are virtually unknown in the
assay using HeLa cancer cells treated with complex 4 (Fig. 7).
The results were analyzed by FACS analysis. PI emits in the red
region, while the annexin VꢀFITC dye shows green fluorescence.
This assay gave an estimate of the apoptotic cells in different
41ꢀ45
literature.
Selectivity of an anticancer drug in cancer cells
over normal cells is an important aspect for its sucessful
therapeutic application. Therefore, we studied the cytotoxicity of
the most photoactive complex 4 in breast epithelial MCFꢀ10A 75 stages. The cells were treated with complex 4 (2 ꢁM) followed by
normal cells by the MTT assay.The results showed that complex
was significantly less toxic to MCFꢀ10A cells with an IC50
1 h irradiation with visible light (400ꢀ700 nm) and the assay was
performed 18 h post photoꢀirradiation. About 21% populations of
the cells were in an early apoptotic stage and ~40% of the cells
were in a late apoptotic stage. Cells population in the necrotic
4
value of 34.1±1.8 µM in light and IC50 > 50 µM in dark (Fig.
S20, ESI†). Thus, a distinct selectivity of complex 4 for cancer
cells in comparison to the normal cells was observed. This 80 mode was found to be negligible. In the dark, the complex did not
differential activity exhibited by complex 4 could be attributed to
its preferential internalization into cancer cells compared to the
normal cells since the cancer cells are metabolically more active
induce any significant cell death at this concentration. The data
suggest an overall apoptotic mechanism of cell death photoꢀ
induced by the complex.
4
4
than the normal cells.
[
Space for Fig. 7]
DCFDA assay
85
DNA binding studies
3
3
4
4
5
0
5
0
5
0
Reactive oxygen species (ROS) playes important role in
57,58
DNA is a prime target of most anticancer drugs and so we studied
the binding propensity of the complexes towards calf thymus (ct)
DNA by using absorption spectroscopy (Fig. 8, Table 1). The
causing apoptosis by transition metal complexes.
2’,7’-
dichlorofluoresceindiacetate (DCFDA) assay is based on
fluorescence assisted cell sorting (FACS) analysis that detects the
formation of any intracellular ROS (Fig. 6). DCFDA is cellꢀ
permeable and upon its passive diffusion into the cells, it is
retained in the intracellular level after being cleaved by the
intracellular esterase enzymes. Upon oxidation by the ROS, the
nonꢀfluorescent DCFDA is converted to the highly fluorescent
equilibrium DNA binding constant (K ) values were in the range
b
4
5
ꢀ1
9
0
of 10 ꢀ10 M . The curcumin complexes 2 and 4 were found to
show stronger binding affinity and hence higher K values than
b
their acac analogues viz, 1 and 3. The binding affinity of the Phꢀ
tpy and Fcꢀtpy complexes was comparable indicating that the
curcumin moiety in both the complexes could be responsible for
interaction with the DNA. The fitting parameter (s) in the
McGheeꢀvon Hippel (MvH) equation gives an estimate of the
number of DNA bases interacting with the complex and a value
of <1.0 is typically attributed to the aggregation of hydrophobic
2
’,7’-dichlorofluorescein (DCF) thereby providing an emission
95
band centered at 529 nm. Measurements were carried out for the
fluorescence of the HeLa cells alone, cells treated with only
DCFDA and cells with DCFDA and complex 4 after 1 h
irradiation with visible light of 400ꢀ700 nm or in dark (as
controls). A large shift in the fluorescence bands was observed
for the cells treated with photoꢀirradiated complex as compared to
the cells treated with the dye alone or the dark controls. This
observation suggests that the ROS are generated by the complex
only on exposure to visible light but not in the dark and these
species could lead to the induction of cellular apoptosis via ROS
mediated cell stress.
59
molecules on the surface of DNA. The greater value of s for the
1
00 curcumin complexes 2 and 4 than the acac analogues 1 and 3 also
suggests better DNA binding propensity of the curcumin
60
complexes than the phꢀtpy analogues.
[Space for Fig. 8]
DNA thermal denaturation studies were conducted to gain
05 information on the binding of the complexes 1–4 to ctꢀDNA (Fig.
9(a), Table 1). A slightly positive shift in the DNA melting
1
[
Space for Fig. 6]
Annexin-V-FITC/PI assay
AnnexinꢀVꢀFITC is
temperature (ꢀT ) was observed on addition of the complex to
m
the DNA. This increase in the DNA melting temperature suggests
stabilization of the double helix on addition of the complex. The
a
fluorescence probe that binds
phosphatidylserine in the presence of calcium(II) ion. In the early 110 ꢀTm values of ~2.7 °C for the curcumin complexes 2 and 4
61
5
5
apoptotic stage, cells undergo significant morphological changes
suggest their groove binding nature. To delve deeper, solution
including the loss of phospholipid asymmetry. The
viscosity measurements were carried out to examine the effect of
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DOI: 10.1039/C5DT04775G
1
the added complex on the relative specific viscosity of the
solution containing the ctꢀDNA (Fig. 9(b)). The relative specific
namely, the typeꢀII process that produces singlet oxygen ( O ) or
2
•
67
a photoꢀredox process that produces hydroxyl radical ( OH).
Addition of singlet oxygen quenchers such as sodium azide,
2,2,6,6ꢀtetramethylꢀ4ꢀpiperidone (TEMP) or 4ꢀ
complex) of a DNA solution is a measure of the increase in 60 diazabicyclo[2.2.2]octan (DABCO) to DNA resulted in partial
viscosity
η/η0 (where η and η0 are the respective specific
viscosities of a DNA solution in the presence and absence of the
5
0
5
contour length of DNA as a result of the separation of base pairs
caused by intercalation. Classical intercalator such as ethidium
bromide (EB) causes significant increase in the viscosity of the
DNA solution. A partial and/or nonꢀintercalative DNA binder
inhibition of photocleavage activity of the complex. Similarly, the
addition of hydroxyl radical scavengers such as DMSO or
catalase also caused partial inhibition in the DNA photocleavage
activity. Addition of superoxide dismutase (SOD) did not inhibit
65 the photcleavage activity the complexes thereby ruling out the
involvent of superoxide radical. These observations suggest the
62
1
could result in less pronounced effect on the solution viscosity.
The groove binder Hoechst 33258 was used as a reference
compound. It did not show any appreciable change in the solution
viscosity of DNA. The viscosity changes observed for the
complexes are suggestive of a twoꢀstep binding process in which
1
•
involvement of both O and OH as the cleavage active ROS.
2
Furthermore, an enhancement in the photocleavage activity of the
1
complex was seen in D O due to longer lifetime of O in this
2
2
68
1
1
the complex first interacts with the DNA surface followed by 70 solvent than in water. Thus, the formation of O could be due to
2
6
3
groove binding.
the photoactive curcumin moiety via a typeꢀII process while the
formation of hydroxyl radicals could result from electron transfer
from the photoꢀexcited ligand to molecular oxygen generating a
[
Space for Fig. 9]
69
DNA photocleavage studies
radical cation.
The DNA photocleavage activity triggered by complexes 1−4
was investigated using supercoiled (SC) pUC19 DNA (30 ꢁM,
75
[Space for Fig. 11]
20
25
30
35
40
45
Conclusions
0
.2 ꢁg) in TrisꢀHCl/NaCl (50 mM, pH = 7.2) buffer by irradiating
the samples with visible light of 454 nm (Fig. 10). The phꢀtpy
complexes of acac ligand, viz., 1 and 3 showed only mild DNA
cleavage activity (lanes 5 and 7, Fig. 10). The curcumin
In conclusion, we report here the synthesis, characterization
and visible light induced cytotoxicity of a series of mixedꢀligand
Nd(III) complexes of functionalized terpyridine bases and
complexes 2 and 4 with their photosensitizing ability showed 80 curcumin. The curcumin complexes 2 and 4 are remarkably
efficient photocleavage (76% NC for 2 and 85% NC for 4) of SC
DNA at 10 µM concentration for an exposure time of 2 h (lanes 6
and 8, Fig. 10). Control experiments with only DNA, metal salts,
Phꢀtpy or Fcꢀtpy ligands did not show any apparent
photocytotoxic to HeLa and MCFꢀ7 cells but nonꢀtoxic in the
absence of light. The ferrocenyl appended complex 4 is
significantly more photocytotoxic than its phenyl analogueꢀ
complex 2, although both possess curcumin as the photosensitizer
photocleavage of DNA (Table S6, ESI†). The complexes were 85 ligand. The control complexes 1 and 3 having the acac ligand but
not cleavage active in dark thereby excluding the possibility of
any hydrolytic DNA damage (lane 4 in Fig.10). The curcumin
ligand alone showed ~23% cleavage in dark. This activity could
be due to generation of reactive oxygen species by curcumin in
lacking any curcumin moiety were neither active in light nor in
the dark. Thus, the presence of the ferrocenyl unit in complex 4
could result in significant enhancement in cellular uptake and
photocytotoxicity of complex 4. Complex 4 accumulates within
39a
aqueous medium. However, when photoꢀirradiated, it showed 90 the mitochondria of HeLa cells and induces apoptosis which is
~
43% photocleavage of DNA under similar conditions forming
triggered by the visible lightꢀinduced formation of ROS. The
complexes, particularly 2 and 4, are moderately strong binders
but potent cleavers of plasmid DNA. Thus, complex 4 holds
significant chemotherapeutic potential and warrants further work
3
9a,19
reactive oxygen species (ROS).
Thus, there is substantial
enhancement of DNA photocleavage activity of curcumin
possibly due to its hydrolytic stabilization and efficient
intersystem crossing (ISC) on complexation to the heavy Nd(III) 95 to design and develop ferrocene appended lanthanideꢀcurcumin
6
4
centre. Since the complexes were found to be DNA minor
groove binders, their groove binding nature was examined using
complexes with better tumour selectivity and enhanced
photocytotoxicity for potential photochemotherapeutic
applications. Further experimental efforts are in progress in our
laboratory to develop such complexes for potential applications in
6
5
the well known minor groove binder distamycinꢀA. Distamycinꢀ
A alone showed ~10% cleavage of the SC DNA in visible light.
Addition of complex 2 or 4 to the distamycinꢀA bound DNA 100 the PDT of cancer.
showed significant inhibition of photocleavage activity thereby
Experimental
suggesting the minor groove binding preference of the complexes
6
6
(lanes 9 and 10 in Fig. 10).
Materials
Unless otherwise mentioned, all the reagents and chemicals
[
Space for Fig. 10]
used in the present work were obtained from commercial
05 suppliers and used as received. Solvents were purified prior to
50
Mechanistic aspects of DNA photocleavage
1
70
To obtain mechanistic insights, we carried out DNA
photocleavage studies using the ferrocene appended curcumin
complex 4 in the presence of different additives (Fig. 11, Figs.
S21, S22, ESI†). The DNA cleavage reactions involving
their use by following standard literature methods. Curcumin
95% curcuminoid content, ca 80% curcumin content) was
obtained from SigmaꢀAldrich, U.S.A. and separated into their
(
7
1
individual components by following a literature procedure. Trisꢀ
55
molecular oxygen could occur by different two pathways, 110 (hydroxymethyl)aminomethaneꢀHCl (TrisꢀHCl) buffer solution
6
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(pH = 7.2) was prepared using deionized and sonicated double
route. An ethanol solution (15 ml) of Nd(NO ) .6H O (0.44 g, 1.0
3 3 2
distilled water. Dulbecco’s Modified Eagle’s medium (DMEM),
propidium iodide, Hoechst 33342, 3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ
mmol) was added dropwise to a dichloromethane solution (15 ml)
of the terpyridine ligand (Phꢀtpy, 0.31 g; Fcꢀtpy, 0.35 g; 1.0
2
,5ꢀdiphenyltetrazolium
bromide
(MTT)
and
2’,7’ꢀ 60 mmol) under nitrogen atmosphere. The resulting reaction mixture
5
0
5
dichlorofluorescein diacetate (DCFDA), calf thymus (ct) DNA,
agarose (molecular biology grade), distamycinꢀA, catalase,
was stirred for 1 hour at room temperature and subsequently
cooled to 0°C to obtain crystalline solids of [Nd(Phꢀtpy)(NO ) ]
3
3
2
,2,6,6ꢀtetramethylꢀ4ꢀpiperidone
diazabicyclo[2.2.2]octan (DABCO) and ethidium bromide (EB)
were procured from SigmaꢀAldrich (U.S.A) and used as received. 65 ether and finally dried in vacuum and used directly in the next
(TEMP),
1,4ꢀ
or [Nd(Fcꢀtpy)(NO ) ] as the precursor complex. The complex
3 3
was filtered, washed with iceꢀcold ethanol followed by diethyl
1
MitoꢀTracker Red was procured from Invitrogen, U.S.A.
Supercoiled (SC) pUC19 DNA (cesium chloride purified) was
purchased from Bangalore Genie (India) The terpyridine
derivatives, viz., 4′ꢀphenylꢀ2,2′:6′,2′′ꢀterpyridine (Phꢀtpy) and 4′ꢀ
ferrocenylꢀ2,2′:6′,2′′ꢀterpyridine (Fcꢀtpy) were prepared by
step without further treatment.
To a deaerated methanol/acetonitrile suspension (1:1 v/v, 20
ml) of the precursor complex [Nd(Phꢀtpy)(NO ) ] (0.32 g, 0.5
3
3
mmol) or [Nd(Fcꢀtpy)(NO ) ] (0.34 g, 0.5 mmol) was added a
3
3
7
7
8
0
5
0
deaerated methanol solution (10 ml) of the βꢀdiketone ligand
0.05 g, Hacac; 0.18 g, Hcurc, 0.5 mmol) previously neutralized
7
2
1
following literature procedures.
(
Measurements
with triethylamine (0.07 ml, 0.5 mmol). After stirring for 1 h at
room temperature, a clear solution was obtained in the case of
complexes 1 and 3 while complexes 2 and 4 precipitated as brick
red solids. Slow evaporation of the solution of complex 1
afforded single crystals suitable for diffraction studies while
complex 3 appeared as microcrystalline solid. The solids were
isolated, washed with cold methanol followed by diethyl ether
and finally dried in vacuum over P O . The characterization data
The elemental analyses were carried out on a Thermo Finnigan
Flash EA 1112 CHNS analyzer. The infrared (IR) spectra of pure
solid samples were recorded on
a Bruker ATR FTꢀIR
2
2
3
3
4
4
5
0
5
0
5
0
5
0
spectrometer. The UVꢀVisible absorption spectra were recorded
on a Perkin Elmer Lambda 650 spectrophotometer. The molar
conductivity measurements were done using a properly calibrated
digital conductivity meter (Labtronics, India). Room temperature
electrochemical measurements were carried out on a Biologic SPꢀ
50 Potentiostat/Galvenostat (Biologic Instruments, France) with a
three electrode setup consisting of a platinum working electrode,
platinum wire auxiliary electrode, and calomel reference
4
10
for the complexes are given below.
[Nd(Ph-tpy)(acac)(NO ) ] (1): Yield: ~81%. Anal. Calcd.
3
2
for C H N O Nd: C, 46.15; H, 3.28; N, 10.35; Found: C, 46.35;
26
22
5
8
H, 3.36; N, 10.43. ESI–MS in 5% aqueous methanol: m/z 614.09
ꢀ
1
ꢀ
+
ꢀ1
electrode (SCE) at a scan rate of 100 mVs . The electrochemical
[M–(NO )] . FT–IR data/ cm : 1590 s, 1531 s, 1498 vs, 1410 s,
3
measurements were made using solutions of the metal complexes 85 1383 s, 1286 s,1175 m, 1012 s, 922 w, 794 w, 760 s, 735 m, 697
prepared in HPLC grade DMF. Tetrabutylammonium perchlorate
TBAP, 0.1M) was used as a supporting electrolyte in DMF. The
m, 638 m (vs, very strong; s, strong; m, medium; w, weak; sbr,
ꢀ
1
(
strong broad). UV–visible in 5% aqueous DMF [λmax/nm (
ε
/ M
ꢀ
1
electrospray ionization mass spectra (ESIꢀMS) were recorded
using Agilent Technologies 6538 UHD Accurateꢀmass QꢀTOF
LC/MS mass spectrometer. The fluorescence quantum yields
were determined using a PerkinElmer LS 55 fluorescence
spectrometer using coumarinꢀ153 laser dye as a reference with a
cm )]: 280 (71,200). Molar conductivity in DMF at 298 K [Λ
/
M
2
ꢀ1
S cm mol ]: 85.
9
0
[Nd(Ph-tpy)(curc)(NO ) ] (2): Yield: ~78%. Anal. Calcd.
3
2
for C H N O Nd: C, 53.38; H, 3.63; N, 7.41. Found: C, 53.51;
42
34
5
12
7
3
H, 3.51; N, 7.46. ESI–MS in 5% aqueous methanol: m/z 880.11
known quantum yield (Φ) value of 0.56 in acetonitrile. The
samples for quantum yield measurements were deoxygenated
prior to experiments. The sample and the reference were excited
at 430 nm, maintaining nearly equal absorbance. The integrated
emission intensity was calculated using Origin Pro 8.1 software
and the quantum yield was calculated using the equation Φ /Φ
ꢀ
+
ꢀ1
[
M–(NO )] . FT–IR data/ cm : 3390 br, 1592 s, 1495 vs, 1390 s,
3
1
286 s, 1216 m, 1154 m, 1120 s, 1025 w, 967 m, 820 w, 760 w.
ꢀ
1
ꢀ1
9
5
UV–visible in 5% aqueous DMF [λmax/nm (ε/ M cm )]: 276
(
60,600), 430 (54,300), 455 (48,500) . Molar conductivity in
2
ꢀ1
DMF at 298 K [Λ / S cm mol ]: 71.
M
S
R
2
2
=
(A /A ) x [(OD) /(OD) ] x [(n ) /(n ) ], where, Φ and Φ are
[Nd(Fc-tpy)(acac)(NO ) ] (3): Yield: ~78%. Anal. Calcd.
S
R
R
S
S
R
S
R
3 2
the fluorescence quantum yields of the sample and reference
respectively, AS and AR are the area under the fluorescence 100 46.06; H, 3.38; N, 7.02. ESI–MS in 5% aqueous methanol: m/z
for C H N O FeNd: C, 45.92; H, 3.34; N, 7.12; Found: C,
30
26
5
8
ꢀ
+
ꢀ1
spectra of the sample and the reference respectively, (OD) and
722.08 [M–(NO )] . FT–IR data/ cm : 1596 s, 1540 m, 1505 s,
S
3
(OD)R are the respective optical densities of the sample and the
1454 vs, 1420 s, 1380 vs, 1302 m, 1273 m, 1254 m, 1160 w,
1007 s, 930 w, 826 w, 788 s, 760 w, 731 m, 655 w. UV–visible in
reference solution at the wavelength of excitation, and n and nR
S
ꢀ
1
ꢀ1
are the respective refractive indices of the solvents used for the
5% aqueous DMF [λmax/nm (ε/ M cm )]: 287 (57,200), 365 sh
7
4,75
sample and the reference.
The fluorescence microscopic 105 (2,800), 465 (1,457). Molar conductivity in DMF at 298 K [Λ
/
M
2
ꢀ1
investigations were carried out using ApoTome.2 fluorescence
microscope. The flow cytometric analysis was performed using
FACS Calibur (Becton Dickinson (BD) cell analyzer) at FL1
channel (595 nm).
S cm mol ]: 82.
[
Nd(Fc-tpy)(curc)(NO ) ] (4): Yield:~76%. Anal. Calcd. for
3
2
C
H
38
N
5
O12FeNd: C, 52.47; H, 3.64; N, 5.30. Found: C, 52.60;
46
H, 3.52; N, 5.35. ESI–MS in 5% aqueous methanol: m/z 990.14
ꢀ
+
ꢀ1
5
5
Syntheses of the complexes 1–4
110 [M–(NO )] . FT–IR data/ cm : 3385 sbr, 1602 s, 1502 s, 1423 s,
3
1
7
388 vs, 1276 vs, 1153 s, 1118 m, 1034 m, 1001 s, 970 m, 816 m,
86 m, 731 w, 668 w, 633 w. UV–visible in 5% aqueous DMF
The complexes were prepared by a general twoꢀstep synthetic
This journal is © The Royal Society of Chemistry [year]
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DOI: 10.1039/C5DT04775G
ꢀ
1
ꢀ1
[
λmax/nm (
ε
/ M cm )]: 279 (68,000), 430 (63,000), 455 55 5 min at 4° C. The supernatant was discarded and the cell pellet
2
ꢀ
ꢀ1
(
56,100). Molar conductivity in DMF at 298 K [ΛM / S cm mol
was suspended in 200 ꢁl of PBS containing 10 ꢁg ml of DNAseꢀ
1
]
: 69.
free RNAse for 12 h at 37° C for digesting the cellular RNA.
After RNAse digestion, cells were washed twice with DPBS and
the analysis was performed using FACS Calibur (Becton
X-ray crystallographic procedures
5
0
5
0
The crystal structure of 1 was obtained by the single crystal Xꢀ 60 Dickinson (BD) cell analyzer) at FL2 channel.
ray diffraction technique. Single crystals of 1 were grown by
slow evaporation of a solution of the complex in methanol.
Crystal mounting was done on glass fiber with epoxy cement. All
geometric and intensity data were collected at room temperature
using an automated Bruker SMART APEX CCD diffractometer
Fluorescence microscopy experiments
4
2
HeLa cells (~4 x 10 cells/mm ), plated on cover slips, were
incubated with complex 4 (30 µM) for 2 h and 4 h in the dark,
fixed with 4% paraformaldehyde for 10 min at room temperature
1
1
2
equipped with a fine focus 1.75 kW sealed tube MoꢀK Xꢀray 65 (RT) and washed with PBS. This was followed by staining with
α
source (λ = 0.71073 Å) with increasing ω (width of 0.3° per
frame) at a scan speed of 4 seconds per frame. Intensity data,
collected using ωꢀ2θ scan mode, were corrected for Lorentz–
Hoechst 33342 dye for 10 min at 25 °C. The cells were washed,
mounted in 90% glycerol solution containing Mowiol, an antiꢀ
fade reagent, and sealed. Images were acquired using Apotome.2
fluorescence microscope (Carl Zeiss, Germany) using an oil
7
6
polarization effects and for absorption. Patterson and Fourier
techniques were used to solve the structures and refinement was 70 immersion lens at 63 X magnification. The images were analyzed
8
1
done by full matrix least squares method using SHELX system of
using the AxioVision Rel 4.8.2 (Carl Zeiss, Germany) software.
7
7
4
2
programs. The hydrogen atoms belonging to the complexes
were in their calculated positions and refined using a riding
model. All nonꢀhydrogen atoms were refined anisotropically. The
For subꢀcellular localization study, HeLa (~4 x 10 cells/mm )
were incubated with 30 µM of complex 4 for 4 h in the dark,
following which, the cells were treated with 500 nM of
75 mitotracker red (MTR) in serumꢀfree medium for 30 min at 37
ºC. The cells were then washed with PBS, mounted on slides and
sealed with nailꢀpaint. The images were acquired using
Apotome.2 fluorescence microscope at 63X magnification and
analysed using AxioVision Rel 4.8.2.
78
perspective views of the molecules were obtained by ORTEP.
The CCDC number assigned to complex 1 is 1425834.
Computational methodology
To rationalize the photophysical properties and to obtain a
better understanding of the molecular structure and electronic
nature of the complexes, DFT computational studies were
performed using B3LYP hybrid functional and LANL2DZ
pseudoꢀpotential) basisꢀset for Fe, MWB28 (pseudoꢀpotential)
basis set for Nd and 3ꢀ21G basis set for other types of atoms,
2
5
80
Cytotoxicity
The cytotoxicity of the complexes was studied using MTT
assay in light and dark conditions in human cervical carcinoma
(HeLa) and human breast adenocarcinoma (MCFꢀ7) cells
(
82
3
0
using quadratic convergence criteria, as incorporated in Gaussian
following reported procedures. The MTT assay method is based
7
9,80
0
9 software package.
In order to ascertain stationary points, 85 on the ability of mitochondrial dehydrogenases of viable cells to
frequency tests were performed after optimisation of the groundꢀ
state structures.
cleave the tetrazolium rings of MTT, forming dark purple
membrane impermeable crystals of formazan that can be
4
6
estimated from the spectral measurements in DMSO at 540 nm.
Photoꢀirradiation was done with a broad band visible light
Cell culture
−2
35
HeLa (human cervical carcinoma), MCFꢀ7 (human breast 90 (400−700 nm, 10 J cm ) using Luzchem Photoreactor (Model
adenocarcinoma) and MCFꢀ10A (human breast epithelial cell)
cells were maintained in DMEM supplemented with 10% FBS,
LZCꢀ1, Ontario, Canada, Sylvania fluorescent white tubes with a
ꢀ
2
ꢀ2
fluence rate of 2.4 mW cm to provide a total dose of 10 J cm ).
The absorbance was measured at 540 nm using a Molecular
Devices Spectra Max M5 plate reader. Cytotoxicity of the
ꢀ
1
ꢀ1
1
00 IU mL of penicillin, 100 µg mL of streptomycin and 2
mM of Glutamax at 37 °C in a humidified incubator at 5% CO2.
40
The adherent cultures were grown as monolayer and were 95 complexes was measured as the percentage ratio of the
passaged once in 4ꢀ5 days by trypsinizing with 0.25% Trypsinꢀ
EDTA.
absorbance of the treated cells to the untreated controls. The
cytotoxicity was expressed in terms of the IC50 values which were
determined by nonlinear regression analysis using Graph Pad
Prism 5.
Cellular incorporation assay
6
For flow cytometric analysis, ~0.3x10 HeLa cells were plated
1
00 DCFDA assay for ROS generation
45
per well of a 6ꢀwell tissue culture plate in Dulbecco’s Modified
Eagle Medium (DMEM) containing 10% FBS. After 24 h of
DCFDA assay was carried out to detect any generation of
83
incubation at 37 °C in a CO incubator, complex 4 (1 ꢁM) was
intracellular ROS. Cell permeable DCFDA on oxidation by
cellular ROS generates green fluorescent 2′,7′ꢀdichlorofluorescein
2
added to the cells at 2 and 4 h incubation time intervals in the
dark, cells were trypsinized, transferred into 1.5 ml centrifuge
84
(DCF) with an emission maxima around 525 nm. To detect
50
tubes, washed once with chilled PBS and fixed by adding 800 ꢁl 105 intracellular ROS generation, HeLa cells were incubated with 2
of chilled 70% ethanol dropꢀwise with constant and gentle
vortexing to prevent cell aggregation. The cell suspension was
incubated at ꢀ20 °C for 6 h. The fixed cells were then washed
twice with 1.0 ml of chilled PBS by centrifuging at 4000 rpm for
ꢁM of complex 4 for 4 h followed by photoꢀexposure to visible
light (400ꢀ700 nm) for 1 h in PBS (50 mM phosphate buffer, pH,
7.2 containing 150 mM NaCl). After harvesting the cells by
6
ꢀ1
trypsinization, a single cell suspension of 1x10 cells ml was
8
| Journal Name, [year], [vol], 00–00
This journal is © The Royal Society of Chemistry [year]

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DOI: 10.1039/C5DT04775G
1/3
prepared. The cells were then treated with 10 ꢁM DCFDA in dark
(
η/
η0
)
vs. [complex]/[DNA], where η is the viscosity of DNA
for 15 min at room temperature. The distribution of the HeLa
cells, stained by DCFDA, was determined by FACS analysis.
in the presence of complex and η0 is that of DNA alone. The
viscosity values were calculated from the observed flow time of
DNAꢀcontaining solutions (t) corrected for that of the buffer
60
Annexin-V FITC and propidium iodide (PI) assay
alone (t0),
η = (t − t )/t and due corrections were made for the
0 0
5
5
To investigate the pathway of cell death, HeLa cells (4 x 10
viscosity of DMF solvent present in the solution.
ꢀ
1
cells ml ) were incubated with complex 4 (2 ꢁM) in 10% DMEM
for 4 h, followed by irradiation with visible light of 400ꢀ700 nm
for 1 h. The cells were then cultured for 18 h, harvested and
washed with chilled PBS at 4°C. The cells were then reꢀ
suspended in 100 µl AnnexinꢀV binding buffer (100 mM HEPES/
NaOH, pH 7.4 containing 140 mM NaCl and 2.5 mM CaCl2),
stained with AnnexinꢀV FITC and PI, and incubated for 15 min in
dark. A 400 µl of binding buffer was added to the cells after
DNA cleavage experiments
The cleavage of supercoiled (SC) pUC19 DNA (30 ꢁM, 0.2 ꢁg,
2686 baseꢀpairs) was studied by agarose gel electrophoresis
method. The photocleavage experiments on DNA were carried
out by using visible light of 454 nm with a laser source (Spectra
Physics continuousꢀwave Ar–Kr mixedꢀgas ion laser, model:
Stabilities 2018ꢀRM; laser power: 60 mW). Eppendorf vials were
used for photocleavage experiments in a dark room at room
temperature using SC DNA (1 ꢁL, 30 ꢁM) in 50 mM TrisꢀHCl
buffer (pH 7.2) containing 50 mM NaCl and the complex (2 ꢁL)
with varied concentrations. The concentration of the complex in
DMF or the additives in the buffer corresponded to the quantity
in 2 ꢁL stock solution after dilution to a final volume of 20 ꢁL
using TrisꢀHCl buffer. After light irradiation, each sample was
incubated for 1 h at 37 °C and analyzed for the photocleavage
products using gel electrophoresis. Mechanistic studies were
6
7
7
8
8
9
9
5
0
5
0
5
0
5
1
0
85
incubation, and flow cytometry was done foranalysis.
1
5
DNA binding experiments
DNA binding experiments were carried in TrisꢀHCl/NaCl
buffer (5 mM TrisꢀHCl, 5 mM NaCl, pH 7.2) using DMF solution
of the complexes 1ꢀ4. Calf thymus (ct) DNA (ca. 350 ꢁM NP) in
gave a ratio of UV absorbances at 260 and 280 nm of 1.9:1
indicating that the DNA sample was apparently free from
proteins. The concentration of ct DNA was estimated from its
absorbance at 260 nm having a known extinction coefficient
2
2
3
3
4
0
5
0
5
0
carried out using different additives (NaN , 0.5 mM; TEMP, 0.5
mM; DABCO, 0.5 mM; DMSO, 4 ꢁL; KI, 0.5 mM; catalase, 4
units) prior to the addition of the complex. For experiment in
ꢀ
1
ꢀ1 86
3
value (ε) of 6600 M cm . Absorption titration measurements
were done by varying the concentration of the ct DNA but
maintaining a constant concentration of the metal complex. Due
corrections were applied to the absorbance of ct DNA itself. Each
spectrum was recorded after incubation of the sample for 5
D O, this solvent was used instead of water for dilution of the
2
sample to a final volume of 20 ꢁL. The samples after incubation
in a dark chamber were added to the loading buffer containing
0.25% bromophenol blue, 0.25% xylene cyanol, 30% glycerol (3
minutes. The equilibrium binding constant (K ) and the binding
b
site size (s) of the complexes 1–4 were calculated by the
McGheeꢀvon Hippel (MvH) method. The expression given Bard
et al. was used and the change of the absorbances of the spectral
bands with increasing concentration of ct DNA was monitored.
The nonꢀlinear regression analysis was done by using equation (εa
ꢁL) and the solution was finally loaded on 1% agarose gel
containing 1 ꢁg/mL ethidium bromide (EB). Electrophoresis was
done in a dark room for 2 h at 50 V in TAE (Trisꢀacetate EDTA)
buffer. Bands so separated were visualized in UV light and
photographed. The extent of cleavage of SC DNA was measured
from the intensities of the bands using a Gel Documentation
System (UVITEC). Corrections were made for the low level of
nicked circular (NC) form of DNA present in the original SC
DNA sample and for the low affinity of ethidium bromide to SC
2
2
1/2
−
εf)/(εb
− εf) = (b − (b − 2K C [DNA] /s) )/2K C , where b = 1
b t t b t
+ K C + K [DNA] /2s and εa is the extinction coefficient
b
t
b
t
observed for the absorption band at a given DNA concentration,
εf is the extinction coefficient of the complex free in solution, εb
is the extinction coefficient of the complex when fully bound to
DNA, K is the equilibrium binding constant, C is the total metal
88
compared to NC and linear forms of DNA. The observed error
in measuring the band intensities was in the range 2ꢀ5%.
b
t
complex concentration, [DNA] is the DNA concentration in
t
59,87
nucleotides and s is the binding site size in base pairs.
The
Acknowledgements
nonꢀlinear leastꢀsquares analyses were carried out using Origin
Lab 8.0 software.
Work in the laboratory of A.H. is funded by the Department of
1
00 Biotechnology (DBT), Government of India (UꢀExcel grant:
BT/401/NE/UꢀExcel/2013). S.B. thanks IISc, Bangalore for a
postdoctoral fellowship. T.S. thanks the DBT for research
fellowship and S. M thanks the CSIR for SPM research
fellowship. We acknowledge the single crystal diffractometer
05 facility of SAIF, Department of Instrumentation & USIC, Gauhati
University. We thank Professor A. R. Chakravarty and Professor
A. A. Karande for their help in this work.
DNA thermal denaturation (melting) experiments were done
by monitoring the absorbance of ct DNA (200 µM) at 260 nm at
various temperatures, both in the absence and presence of the
complexes (20 µM). Measurements were carried out using a
Perkin Elmer Lambda 650 spectrophotometer with a Peltier PTPꢀ
4
5
5
5
0
5
1
1
+1 temperature controller at an increase rate of 0.5 °C/min of the
solution. Viscometric measurements were carried out with a
vibrationꢀfree Mansingh Survismeter (Borosil, Singapore Govt.
Patent No. 12609) that was thermostated at 37 ºC in a constant
temperature water bath. The concentration of ct DNA was 150
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Table 1. Selected physicochemical data for the complexes 1–4.
b
ꢀ1
IR /cm
c
ESIꢀMS
Λ
M
/
d
E
f
/ V
f
ꢀ1
g
h
K
b
/ M [s]
ꢀTm / °C
Complex
a
2
ꢀ1
Φ
ꢀ
e
µ
eff
ꢀ
Olefinic
C=C
S cm mol
(∆E / mV)
(m/z)
p
NO
3
C=O
1590
1592
1596
1602
4
1
2
3
4
614.09
880.11
722.08
990.14
1383
1390
1380
1388
1498
1495
1505
1502
85
71
82
69
ꢀ
ꢀ
3.62
3.56
3.58
3.60
3.0 (±0.1) 10 [0.1]
1.7
2.8
1.6
2.6
4
0.02
ꢀ
9.2 (±0.2) 10 [0.2]
4
0.61 (140)
0.61 (120)
2.1 (±0.2) 10 [0.1]
4
5
0
5
0
5
0.02
7.8 (±0.3) 10 [0.2]
a
ꢀ
+
b
c
3
The molecular ion peak arising from the [M–(NO )] species in aqueous methanol. Recorded on solid sample. Molar conductivity in DMF at 25
C. Fluorescence quantum yield measured in DMF solution of the complex , excitation wavelength = 420 nm.
where Epa and Epc are the respective anodic and cathodic peak potentials (vs. SCE) for the Fc /Fc couple. µeff in µ
DMSOꢀd
d
e
°
E
f
= 0.5 (Epa + Epc), ∆E
obtained by NMR method using
b
, DNA binding constant (s, binding site size). Change in the DNA melting temperature.
p
= (Epc ꢀ Epa),
+
f
B
g
h
6
solutions of the complexes at 25 °C. K
Table 2. Selected crystallographic data and structure refinement
parameters for the complex 1.
Empirical formula
Table 3: IC50 values of complexes 1-4 in HeLa and MCFꢀ7 (in the
parentheses) cell lines.
a
26 22 5 8
C H N O Nd (1)
Compound
Light / ꢁM
53.1±2.5
Dark / ꢁM
80.3± 2.1
(94.5±3.1)
>50
ꢀ
1
Formula weight (gmol )
Crystal system
Space group
676.73
1
Triclinic
(
62.6±2.8)
1
1
2
2
Pꢀ1
2
3
4
4.2±0.8
a (Å)
9.5473(5)
(
9.6±1.2)
(>50)
b (Å)
c (Å)
10.7596(6)
14.1625(8)
77.029(2)
81.295(2)
13.2 ±1.6
(19.9±1.8)
0.7± 0.2
>50
(>50)
α
β
(°)
(°)
>50
(2.1±0.6)
(>50)
b
γ
(°)
7
0.083(2)
(34.1±1.2)
8.2±0.2
(>50)
3
c
V (Å )
Z
1328.47(13)
2
Curcumin (Hcurc)
85.4± 0.6
(90.3±4.9)
>100
(19.9±1.4)
>100
d
T (K)
293(2)
1.692
Phꢀtpy
ꢀ
3
ρ
calc (g cm )
(>100)
(>100)
a
ꢀ2)
b
c
Light of 400ꢀ700 nm (10 J cm wavelength. In MCFꢀ10A cells.
λ
/Å (MoꢀK
α
)
0.71073
d
Reference 43. Reference 29. The photocytotoxicity of Fcꢀtpy ligand
could not be measured due to its insolubility in the buffer medium
ꢀ
1
ꢁ
(mm )
2.013
(Reference 29).
Theta range for data collection
Reflections collected / unique
Data/restraints/parameters
F(000)
1.48 ° to 26.00 °
21812 / 5242
5242/0/361
674
2
Goodnessꢀofꢀfit on F
1.056
a
b
R (F
R (all data)/wR (all data)
Largest diff. peak and hole (e Å )
o
) , I>2
σ(I) / wR (F
o
)
0.0236/0.0582
0.0278/0.0599
0.580, ꢀ0.348
−3
a
b
2
o
2
2
2
½
R = Σ||F
w = [
o
|−|F
c
||/Σ|F
o
|. wR = {Σ[w(F
−F
c
ꢀ1
) ]/Σ[w(F
o
) ]} ;
2
2
2
2
2
σ
(F
o
) + (0.0345 P) + 0.2390P] , where P = (F
o
+ 2F
c
)/3.
1
2
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5
Fig.1 The schematic representation of the complexes 1‒4.
Fig. 3 (a) ORTEP diagram of the complex 1 drawn at 40% probability.
The hydrogen atoms and the counter anions have been omitted for
clarity. The color codes are: metal (light brown), nitrogen (green),
oxygen (blue) and carbon (black); Nd(1)ꢀO(1) = 2.3407(19); Nd(1)ꢀO(2)
1
1
2
2
0
5
0
5
=
2.3391(18); Nd(1)ꢀN(1) = 2.578(2); Nd(1)ꢀN(2) = 2.635(2); Nd(1)ꢀ
N(3) = 2.607(2); Nd(1)ꢀO(3) = 2.528(2); Nd(1)ꢀO(5) = 2.566(2); Nd(1)ꢀ
O(6) = 2.562(2); Nd(1)ꢀO(8) = 2.566(2); (b) The DFT optimized
structure of complex 4. The structure was obtained by using B3LYP
level theory and LanLD2Z basis set. The color codes are: metal (red),
nitrogen (green), oxygen (blue) and carbon (black).
Fig. 4. Cellular uptake of complex 4 (1 ꢁM) at 2 h and 4 h of
incubation with HeLa cells as determined from the FACS analysis.
Fig. 2 (a) The electronic absorption spectrum in 5% aqueous
DMF and the emission spectrum in DMF for complex 4
(excitation wavelength, 420 nm); (b) Cyclic voltammograms of
complexes 2 (black curve) and 4 (red curve) recorded in DMF
in the presence of 0.1 M TBAP as supporting electrolyte.
Fig. 5. Fluorescence microscopic images of the HeLa cancer cells treated
ꢀ1
with 30 ꢁM of 4 on 4 h incubation and Hoechst 33342 dye (5 ꢁg mL ):
panel (a): emission from 4; panel (b): blue emission from the nucleus
staining Hoechst dye; panel (c): merged images showing cytosolic
localization of 4; panel (d): fluorescence emission from 4; panel (e): red
emission from the Mitotracker red (0.5 ꢁM) dye. The scale bar: 20 µm.
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Fig. 9. (a) Melting plots of 200 ꢁM ct DNA alone (black) and on
addition of the complex 1(green), 2 (pink), 3 (red), 4 (blue); (b) Effect
of increasing the concentration of the complex 1(▼), 2 (▲), 3 (◄),
5
0
5
0
4
(●), ethidium bromide (EB, ■) and Hoechst 33258 (♦) on the relative
viscosities of ctꢀDNA at 37.0 (± 0.1) °C in 5 mM TrisꢀHCl buffer (pH
.2) containing 2.0 – 20% DMF, [DNA] = 150 µM.
Fig. 6. DCFDA/DCF assay performed for the detection of ROS
generation by complex 4 in HeLa cells. (Light source: visible
light of 400–700 nm).
7
Fig. 10. Cleavage of SC pUC19 DNA (0.2 ꢁg, 30 ꢁM) by the complexes 1–
(10 ꢁM) in 50 mM TrisꢀHCl/NaCl buffer (pH, 7.2) on photoꢀirradiation at
54 nm (60 mW) for 2 h: lane 1, DNA control; lane 2, DNA + Hcurc (10
M, dark); lane 3, DNA + Hcurc (10 ꢁM); lane 4, DNA + 4 (10 ꢁM, dark);
lanes 5ꢀ8, DNA + complexes 1–4, respectively; lane 9, DNA + distamycinꢀ
A (50 ꢁM); lane 10, DNA + distamycinꢀA (50 ꢁM) + 4.
4
4
ꢁ
1
1
2
Fig. 7. Annexin VꢀFITC/PI coupled flow cytometry
analysis showing complex 4 (3 ꢁM) induced apoptosis
in the presence of visible light (400–700 nm).
Fig. 11. Bar diagrammatic representation of the photocleavage of SC
pUC19 DNA (0.2 ꢁg, 30 ꢁM) induced by complex 2 (light gray) and
complex 4 (dark gray) in the presence of various additives in TrisꢀHCl
buffer. The additive concentrations/quantities are: sodium azide, 0.5
Fig. 8. (a) Plots showing the absorption spectral decrease of
complex 4 in 5 mM TrisꢀHCl buffer (pH 7.2) on increasing the
concentration of ct DNA; (b) The leastꢀsquares fit of ꢀ
ε
af/ꢀ
ε
bf vs.
[
DNA] for complexes 1 (▲), 2 (●), 3 (■) and 4 (▼) to calculate K
b
mM; KI, 0.5 mM; TEMP, 0.5 mM; DABCO, 0.5 mM; D O, 16 ꢁL;
2
using MvH equation.
DMSO, 4 ꢁL; catalase, 4 units; SOD, 4 units. Light source: visible
light of 454 nm (60 mW).
1
4
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DOI: 10.1039/C5DT04775G
Mitochondrial selectivity and remarkable photocytotoxicity of a
ferrocenyl neodymium(III) complex of terpyridine and curcumin in
cancer cells
a
b
b
a
Tukki Sarkar, Samya Banerjee,* Sanjoy Mukherjee and Akhtar Hussain*
5
Synopsis: Mixedꢀligand neodymium(III) complex of ferrocene appended terpyridine and curcumin shows remarkable visibleꢀlight
induced cytotoxicity in HeLa and MCFꢀ7 cancer cells. The cell death is apoptotic in nature and triggered by the formation of ROS on
photoirradiation. Fluorescence imaging studies reveal mitochondrial localization of the complex.
Keywords: Neodymium(III), Curcumin, Crystal Structure, Mitochondria, Photodynamic Therapy, Apoptosis
Running title. “Mitochondrial selectivity and remarkable photocytotoxicity ...”
Pictogram:
1
1
2
0
5
0
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Journal Name, [year], [vol], 00–00 | 15