Tri- and tetranuclear RuII-Gd III 2 and RuII-Gd III 3 d-f heterometallic complexes as potential bimodal imaging probes for MRI and optical imaging

Article abstract of DOI:10.1039/c5nj03393d

The synthesis of tri- and tetranuclear Ru^II-GdIII2 and Ru^II-GdIII3 d-f heterometallic complexes of 4-aminopyridine-appended DO3A (DOTA-AMpy, 4) with 1,10-phenanthroline and 4′-(p-tolyl)-2,2′:6′,2′′-terpyridine ancillary ligands and their relaxometry and in vitro cytotoxicity studies are reported. Complexes [Ru(phen)₂{Gd(DOTA-AMpy)(H₂O)}₂]Cl₂ (7) and [Ru(ttpy){Gd(DOTA-AMpy)(H₂O)}₃]Cl₂ (9) exhibit the "per Gd" longitudinal relaxivity of 6.19 and 7.47 mM^-1 s^-1 and 15.37 and 18.61 mM^-1 s^-1, respectively, in aqueous solution and in the presence of HSA (20 MHz, pH = 7.4, phosphate buffer saline, 37 °C). Complex 7 exhibits an emission band at 590 nm. The cell viability and cytotoxicity studies of complexes 7 and 9 against the HeLa cell lines by MTT assay demonstrate their cytotoxic activity with the IC₅₀ values of 52.1 and 27.9 μM, respectively. Morphological assessment of apoptosis by acridine orange and ethidium bromide staining and by Hoechst-33342 assay shows marked morphological signs of apoptosis in a dose-dependent manner. Flow cytometric analysis by propidium iodide staining indicates the inhibition of HeLa cell proliferation and DNA ladder assay shows apoptotic cell death, lending support to the antitumor activity of 7 and 9. A molecular docking study reveals that these complexes intercalate with DNA and bind to HSA. Relaxometry and cytotoxicity studies indicate that complexes 7 and 9 could be used as potential bimodal imaging probes and as anticancer agents.

Full text of DOI:10.1039/c5nj03393d

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III
III
A tri- (RuII-Gd2 ) and tetranuclear (RuII-Gd3 ) d-f Heterometallic
Complexes as Potential Bimodal Imaging Probes for MRI and
Optical Imaging
A. Nithyakumar and V. Alexander*
Synthesis of a tri- and a tetranuclear RuII-Gd2III and RuII-Gd3III d-f heterometallic complexes of 4-aminopyridine appended DO3A
(DOTA-AMpy, 4) with 1,10-phenanthroline and 4′-(p-tolyl)-2,2′:6′,2′′-terpyridine ancillary ligands and their relaxometry and in
vitro cytotoxicity studies are reported. The complexes [Ru(phen)2{Gd(DOTA-AMpy)(H2O)}2]Cl2 (7) and [Ru(ttpy){Gd(DOTA-
AMpy)(H2O)}3]Cl2 (9) exhibit the “per Gd” longitudinal relaxivity of 6.19 and 7.47 mM-1s-1 and 15.37 and 18.61 mM-1s-1,
respectively, in aqueous solution and in the presence of HSA (20 MHz, pH = 7.4, Phosphate buffer saline, 37 °C). The complex 7
exhibits an emission band at 590 nm. The cell viability and cytotoxicity study of the complexes 7 and 9 against the HeLa cell
lines, studied by MTT assay, demonstrates their cytotoxic activity with the IC50 values of 52.1 and 27.9 μM, respectively.
Morphological assessment of apoptosis by acridine orange and ethidium bromide staining and by Hoechst-33342 assay show
marked morphologic signs of apoptosis in a dose-dependent manner. Flow cytometric analysis by propidium iodide staining
indicates inhibition of HeLa cell proliferation and DNA ladder assay show apoptotic cell death lending support for the
antitumor activity of 7 and 9. Molecular docking study reveals that the complexes intercalate with DNA and bind with HSA.
The relaxometry and and cytotoxicity studies indicate that the complexes 7 and 9 could be used as potential bimodal imaging
probes and as anticancer agents.
Received 00th 2015,
Accepted 00th 2015
DOI: 10.1039/x0xx00000x
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Introduction
images which reveal more details than when the two techniques
are used separately. In the search for the development of new,
Gadolinium(III) complexes are widely utilized as contrast agents
(CAs) for magnetic resonance imaging (MRI) in clinical diagnosis.1
Among the multiple diagnostic imaging techniques, MRI is
especially advantageous as a noninvasive modality: it can image
deep into tissues, there is no ionizing radiation, it provides 3D
images with submillimeter spatial resolution, excellent soft tissue
contrast, there are multiple types of contrast available, the imaging
is not operator dependent, and it can provide whole body images
with excellent spatial and anatomical resolution.1,2 Ever since the
introduction of MRI as a diagnostic tool, the interest in the
development of efficient, responsive, and tissue-specific CAs has
grown tremendously.3 Despite the high spatial resolution and tissue
penetration of MRI, this technique suffers from a low sensitivity.
Further, the efficiency of commercial MRI contrast agents is too low
for the application of MRI to molecular imaging.
promising contrast agents, multimodal imaging agents are gaining
nowadays great attention in the field of clinical and preclinical
imaging applications to investigate samples in exquisite detail.5
Thus, bimodal optical/MRI reporters are of high interest because
they combine the high resolution of MRI with the high sensitivity of
optical imaging.6 The possibility of using bimodal imaging probes
which permit simultaneous microscopy and whole body imaging is
explored and the recent developments in the field of combined
MRI/optical probes is reviewed.7 The relatively long lived
luminescence from lanthanide and transition metal complexes
makes them ideal as probes for time-gated luminescence based
assay and imaging.8
The use of Gd(III)-containing metallostars as MRI CAs is gaining
attention because of their improved relaxometric properties.9
Furthermore, it has been shown that heteropolymetallic complexes
combining gadolinium and luminescent transition metal ions can
potentially be used as CAs for bimodal imaging.10 Ru(II) polypyridine
complexes display emission in the visible region with lifetimes in the
microsecond range that allows their use in time-resolved applications,
large Stokes shift (> 5000 cm−1) that minimizes self-quenching, and
high photostability that permits continuous monitoring of biological
Luminescence-based imaging, on the other hand, can provide high
resolution images, but this technique is only suitable for thin tissue
samples because of the low optical transparency of biological
tissues.4 As the optical imaging technique is much more sensitive
than MRI, the combination of these two techniques may result in
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Scheme 1. Synthesis of the ligand DOTA-AMpy (4
) and the RuII-GdIII tri- and tetranuclear heterometallic complexes.
events by fluorescence spectroscopy and microscopy.11 Thus, Ru-Gd
d-f heterometallic assemblies have the potential to function as
bimodal imaging probes combining the good spatial resolution of
the MRI technique with the high resolution images of thin tissue
samples or cells with luminescence based imaging,4,10a leading to
complementary information.6b,10a Other d-f heterometallic bimodal
imaging probes reported are: Ru-Gd6 metallostar by Bunzli et al.,12
Ru-Gd heterobimetallic complex by Parac-Vogt et al.,13 Re-Gd single
molecule dual imaging agent by Faulkner et al.,10a heterobimetallic
Ru-Gd3 metallostars by Parac-Vogt et al.,14 and by Picard et al.,15
and a tripodal Ru-Gd3 metallostar by De Borggraeve et al.,16
Heterometallic f-f assemblies such as Eu-Gd dinuclear complex17
and Eu-Gd3 heteropolymetallic complex18 containing both Gd and
luminescent lanthanide metal ions are also reported as bimodal
imaging probes.
Results and discussion
Synthesis of ligand and characterization. 4′-(p-Tolyl)-2,2′:6′,2′′-
terpyridine (ttpy) is synthesized by the one pot synthetic protocol25
based on Hantzsch synthesis (pyridine ring fusion). The pyridine-
amide linker molecule
3 is synthesized by the reaction of
4-aminopyridine with chloroacetyl chloride in dry acetonitrile at -5
°C in the presence of potassium carbonate as the proton scavenger.
The pyridine-amide linker appended DO3A (DOTA-AMpy, 4) is
synthesized by the reaction of DO3A with 3 in a 1:1 mole ratio in
water in the presence of potassium carbonate at 70 °C for 24 h.
Synthesis of complexes and characterization. cis-[Ru(phen)2Cl2]26
(6) and [Ru(ttpy)Cl3]27 (8) are synthesized by the literature
procedure. The gadolinium(III) complex [Gd(DOTA-AMpy)(H2O)] (5)
is synthesized by the reaction of gadolinium(III) chloride hydrate
with the preformed ligand DOTA-AMpy (4) in a 1:1 mole ratio in
water at 70 °C for 24 h. The ESI-MS of 5 shows peaks at m/z 633 and
679 corresponding to the species [M-H]+ and [M-H+2Na]+,
respectively (Fig. S12 in the ESI). The heterometallic trinuclear
complex [Ru(phen)2{Gd(DOTA-AMpy)(H2O)}2]Cl2 (7) is synthesized
by the reaction of cis-[Ru(phen)2Cl2] (6) with 5 in a 1:2 mole ratio in
water/ethanol (1:1, v/v) under reflux in an argon atmosphere for 8
h (Scheme 1) followed by purification by preparative reversed-
phase high-performance liquid chromatography. The complex is
isolated as a dark yellow solid and its purity is checked by analytical
HPLC (Fig. S13 in the ESI). The ESI-MS of the complex 7 shows peaks
at m/z 556 and 615 corresponding to the species [M-2Cl-H2O+6H]3+
Ru(II) polypyridyl complexes are used as potent anticancer agents,19
DNA probes,20 DNA intercalators,21 in cellular imaging,22 and in
protein monitoring.23 Although multimodal imaging agents are
developing at a growing pace, a challenge that needs to be
overcome in the development of d-f heterometallic systems
suitable for medical applications is achieving good solubility in
water. We have recently reported a RuII-GdIII d-f heterometallic
2
assembly as a potential bimodal imaging probe for MR and
fluorescence imaging.24 In this paper we report the synthesis,
photophysical, relaxometric, and in vitro fluorescence imaging
study of a trinuclear RuII-GdIII and a tetranuclear RuII-GdIII d-f
2
3
heterometallic complexes incorporating the Gd(III) chelates of 4-
aminopyridine-appended DO3A (Scheme 1) as bimodal MRI/optical
imaging probes and their cytotoxicity towards the HeLa cell lines.
and [M-H+4Na]3+
, respectively (Fig. S14 in the ESI). The
heterometallic tetranuclear complex [Ru(ttpy){Gd(DOTA-AMpy)-
2 | New J. Chem., 2015, 00, 1-10
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(H2O)}3]Cl2 (9) is synthesized by reaction of [Ru(ttpy)Cl3] (8) with 5 in Relaxometric studies. The efficiency of an MRI contrast agent is
a 1:3 mole ratio in water/ethanol (1:1, v/v) in the presence of usually measured by its water proton relaxivity (r1p), defined as the
N-methylmorpholine under reflux in an argon atmosphere (Scheme paramagnetic longitudinal relaxation rates induced by a 1 mM
1) followed by purification by preparative reversed-phase high- solution of the contrast agent. The complexes 7 and 9 exhibit the
performance liquid chromatography. The complex is isolated as a “per Gd” longitudinal relaxivity of 6.19 and 7.47 mM-1s-1 (pH = 7.4,
red solid and its purity is checked by analytical HPLC (Fig. S15 in the PBS, 20 MHz, and 37 °C), respectively, which is significantly higher
ESI). The ESI-MS of the complex 9 shows peaks at m/z 1209 and 374 than that of [Gd(DOTA)(H2O)]− (3.38 mM-1 s-1, 37 °C)29 and
corresponding to the species [M-2Cl-H2O+3H+3Na]2+ and [M-2Cl- [Gd(DO3A)(H2O)2] (4.80 mM-1 s-1, 40 °C).30 The DOTA-amide
5H2O+H]6+, respectively (Fig. S16 in the ESI). When the complex derivative DOTA-AMpy (4) coordinates with the Gd(III) ion through
[Gd(DOTA-AMpy)(H2O)] (5) is reacted with the preformed Ru(II) the four ring nitrogen, three carboxylate oxygen, and the amide
complexes, the pyridine pendant arm of the Gd-bound DOTA-AMpy oxygen donors with one coordinated water molecule (q = 1).17,31
ligand coordinates with the Ru(II) metal ion and forms the The higher “per Gd” longitudinal relaxivity of the complexes 7 and 9
heterometallic RuII-GdIII complexes by self-assembly with phen or compared to that of [Gd(DOTA)(H2O)]− and [Gd(DO3A)(H2O)2] is
ttpy ancillary ligands.
attributed to an increase in their molecular weight. As the
molecular weight and dimension of complex increases its
a
Photophysical studies. Photophysical studies. The electronic molecular tumbling rate decreases leading to high proton relaxivity.
absorption spectra of the complexes [Ru(phen)2{Gd(DOTA- However, the “per Gd” relaxivity of 7 and 9 are lower than that of
AMpy)(H2O)}2]Cl2 (7) and [Ru(ttpy){Gd(DOTA-AMpy)(H2O)}3]Cl2 (9) in the other heterometallic assemblies such as [Fe{Gd2bpy(DTTA)2-
0.1 M Tris-HCl buffer at 298 K contain an absorption band at 473 (H2O)4}3]4− (r1p = 9 mM-1s-1),9b [{Gd(DTPA-ph-phen)}3(H2O)3Ru]− (r1p
=
and 493 nm due to the spin-allowed 1MLCT transition and an 12 mM-1s-1),14 [Ru{Gd2bpy(DTTA)2(H2O)4}3]4− (r1p = 12.5 mM-1s-1),12
absorption band at 265 and 273 nm assignable to the ligand- and [Ru(Gd-PMNTA)3(H2O)6]− (r1p = 17.0 mM-1s-1).15
centered π→π* transiꢀon, respecꢀvely (Fig. S17 in the ESI). The
excitation spectrum of the complex 7 contains an excitation maxima
at 447 nm. Upon excitation it exhibits an emission band at 590 nm,
characteristics of the Ru(II) polypyridine complex originating from
Interaction of the Complexes with HSA. The “per Gd” longitudinal
proton relaxivity of the complexes 7 and 9 in PBS containing 4.5%
HSA are 15.37 and 18.61 mM-1 s-1, respectively, which is about 3.0
and 2.5 times higher than their relaxivities in the absence of HSA.
The increase in the longitudinal relaxivity of the complexes in the
presence of HSA confirms their binding with the HSA.
3
the MLCT excited state, at room temperature (298 K). At 77 K, the
emission maxima is slightly blue-shifted to 586 nm. The complex 9
does not emit at room temperature due to the thermal quenching
of the lowest lying 3MLCT (Ru-tpy) excited state by the nonradiative
decay of the low-lying 3MC state. At 77 K, it exhibits an emission
band at 644 nm. The photophysical properties of the complexes are
comparable to that of other luminescent Ru(II) polypyridine
complexes.10a,16,28 In these RuII-GdIII heterometallic complexes the
Ru(II) luminescence is not quenched by the Gd(III) ion since its
excited states energies are much higher (> 31,000 cm-1) than that of
the 3MLCT state of Ru(II) chromophore, preventing any Ru→Gd
energy transfer process.15 The normalized emission spectra of the
complexes in 0.1 M Tris-HCl buffer are presented in Fig. 1.
Fig 2. Plot of the concentration of the complexes 7 and 9 versus
1/T1 in PBS and in the presence of HSA (pH = 7.4, 37 °C).
The interaction between the complexes and HSA is confirmed by
ultrafiltration method and the estimated association constants are
Ka = 6980 ± 748 and 7379 ± 658 M-1, respectively, obtained by fitting
the data in eq 1.13 The number of equivalent and independent sites
is set to be 1.
(1)
-1
=1000{(r1fs0) + 0.5(r1c-r1f)[Np0 + s0 + Ka-1] - √[(Np0 + s0 + Ka
)
2 - 4Ns0p0]}
p,obs
R1
Fig. 1 Normalized emission spectra of the complexes 7 and 9 in 0.1
where, p0 is the protein concentration, s0 is the concentration of the
paramagnetic complex, r1f is the relaxivity of the free complex, r1c is
the relaxivity of the noncovalently bound complex, and N is the
number of independent and identical interaction sites. A decrease
M Tris-HCl buffer (pH = 7.4) at 25 °C. Inset: excitation spectra (λex
=
447 and 454 nm).
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in the mobility of the adduct formed by the complexes with HSA The HeLa cell lines incubated with 25 μM solution of the complexes
leads to an increase in their proton relaxation rates. The plot of the display a weak fluorescence with significant cell reduction, whereas
concentration of the complexes 7 and 9 versus 1/T1 in PBS and in at 50 μM concentration of the complexes the cells display the
the presence of HSA are depicted in Fig. 2.
fluorescence of the apoptotic cells with marked morphological
signs of apoptosis such as cell reduction, cell shrinkage, rounding of
the cells, increase in the detached cells, decrease in the adherent
cells, and the appearance of a large number of suspended cells
accompanied by cell debris, apoptotic bodies, and other
characteristics. While apoptotic bodies displaying different size and
irregular morphology are observed upon incubation of the HeLa cell
lines with the complexes, no such changes are observed in the
control group. Fig. 3 clearly indicates that the density of the cells
gradually decreases while increasing the concentration of the
complexes. These results indicate that the complexes 7 and 9
induce apoptosis in a dose-dependent manner on the HeLa cell lines
and demonstrates the utility of the complexes as potential
anticancer agents.
Cell viability and cytotoxicity study by MTT assay. The MTT (MTT =
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay
is a rapid quantitative colorimetric assay developed by Mosmann32
for mammalian cell survival and proliferation. It is based on the
ability of live cells to convert the pale yellow MTT into a dark blue
formazan product by the cleavage of the tetrazolium ring in active
mitochondria. This method detects living, but not dead cells and the
absorbance of the color generated (optical density) is dependent on
the degree of activation of the cells and can, therefore, be used to
measure cytotoxicity, proliferation, or activation.33 The MTT assay is
performed to evaluate the cytotoxic effects of the RuII-GdIII
complexes against the HeLa cell lines. The HeLa cell lines treated
with 1 and 10 μM solution of the complexes for 48 h at 37 °C in PBS
display moderate cytotoxic activity, whereas at 25 μM
concentration of the complexes the cell viability is 49.2 and 38.6%,
respectively. When the HeLa cell lines are treated with 50 μM
solution of the complexes the cell viability is 19.5 and 16.5%,
respectively (Fig. S18 in the ESI). The IC50 values of the complexes 7
and 9 are 52.1 and 27.9 μM, respectively. These results indicate
that the complexes exhibit good cytotoxic activity against the HeLa
cell lines and have the potential to be explored as anticancer
agents.
Morphological assessment of apoptosis by acridine orange (AO)
and ethidium bromide (EB) dual staining. When cells are exposed
to cytotoxic agents, there are various patterns of cell death that
they may undergo. The two major types of cell death are apoptosis
and necrosis. Apoptosis or programmed cell death can be initiated
by two central mechanisms, the extrinsic (death receptor) and
intrinsic (mitochondrial) pathways. Apoptosis is characterized by
cell shrinkage, chromatin condensation, and blebbing of the plasma
membrane.35 The HeLa cell lines incubated with 25 μM solution of
the complexes 7 and 9 for 24 h at 37 °C and stained with AO and EB
display the green fluorescence of early apoptotic cells and weak red
fluorescence of necrotic cells with condensed structure. When the
cell lines are incubated with 50 μM solution of the complexes they
display the bright green fluorescence (blue arrow) of apoptotic cells
and the red fluorescence of necrotic cells (white arrows) with cell
reduction and chromatin condensation as shown in Fig. 4. As the
concentration of the complexes increases there is an increase in the
appearance of the apoptotic nuclear bodies in the HeLa cell lines,
wherein the nuclei have been condensed. The results indicate that
the complexes 7 and 9 exhibit the dose-dependent inhibition effect
on the cancer cell lines, whereas the control HeLa cell lines do not
show any change in their nuclear morphology.
Cell viability assay by Hoechst-33342 staining. Hoechst stains are a
family of blue fluorescent dyes used to stain DNA,34 developed by
Hoechst AG. This protocol describes the use of Hoechst 33342
(2'-(4-ethoxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5'-bi-1H-benzimi-
dazole trihydrochloride trihydrate) to label nuclear DNA of cells
grown in cultures. It is a cell permeable fluorescent compound and,
thus, stains live cells by binding with high affinity to the adenine-
thymine (A-T) rich region of DNA in the minor groove. On binding
with DNA it emits blue fluorescent emission upon excitation in the
UV region. The HeLa cell lines are incubated with the complexes 7
and 9 at two different concentrations (25 and 50 µM) for 24 h at 37
°C and stained with Hoechst-33342 and examined by fluorescent
microscopy.
Fig 4. Fluorescence images of the HeLa cells incubated with the
Fig 3. Morphological changes observed for the HeLa cell lines
incubated with the complexes 7 (top row) and 9 (bottom row) for complexes 7 (top row) and 9 (bottom row) for 24 h, 37 °C at 25 and
24 h, 37 °C at 25 and 50 µM concentrations and stained with
50 µM concentrations and stained with acridine orange and
Hoechst-33342.
ethidium bromide (AO/EB).
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Flow cytometric analysis of cell cycle by propidium iodide staining. complex 9) in DMEM culture medium at 37 oC for 24 h. The DNA
Cell cycle analysis employs flow cytometry to distinguish cells in fragments from both the samples are isolated, subjected to 1.2%
different phases of the cell cycle. The flow cytometric analysis of agarose gel electrophoresis, and photographed in
a
gel
cell cycle by propidium iodide (PI) staining has been widely used for documentation system. The images of both the test samples exhibit
the rapid and quantitative evaluation of cell apoptosis.36 Incubation ladder formation as shown in Fig. 6, indicating cell apoptosis,
of the HeLa cell lines with the complexes 7 and 9 followed by whereas the DNA isolated from the control HeLa cell lines does not
staining with PI reveals nuclear staining of the chromosomal DNA. exhibit any ladder formation. The results indicate that the
Fig. 5 shows the cell cycle profile of the HeLa cell lines incubated complexes promote apoptosis in the HeLa cell lines lending support
with 25 μM solutions of the complexes for 24 h at 37 °C. Upon for their antitumor activity.
incubation of the HeLa cell lines with the complexes there is an
increase in the proportion of the cells in the G2/M phase from DNA intercalation. Intercalation is defined as the non-covalent
18.89% for the control to 41.50% in the presence of the complex 7 stacking interaction occurring due to the insertion of planar
and 82.33% in the presence of the complex 9. Fig. 5 indicates that heterocyclic aromatic rings between base pairs of the DNA double
the complexes inhibit the HeLa cell proliferation by inducing the cell helix.38 To elucidate the mode of interaction and binding affinity,
cycle arrest in the G2/M phase and shows a dose-dependent molecular docking studies have been performed on DNA (PDB ID:
inhibition effect on the cell proliferation.
1BNA) with the complexes 7 and 9. The planarity of the 1,10-
phenanthroline and tolylterpyridine ligands coordinated to the
Ru(II) centre are compatible for strong π-π stacking interactions.
Our docking results show that the complex 7 binds with the major
groove with a better match inside the DNA strands which facilitates
hydrogen bonding with the G-C and A-T rich region of the
d(CGCGAATTCGCG) sequence, while the complex 9 intercalates with
the DNA and does not form hydrogen bonding. The overall
geometric shape and complementary score value of the complexes
7 and 9 are 4870 and 6250, respectively. Graphical representation
of the intercalated complexes with the DNA is given in Fig. 7.
Fig 5. The cell cycle profiles of the HeLa cell lines incubated with 25
µM solutions of complexes 7 and 9 for 24 h, 37 °C and stained with
propidium iodide after fixation.
Apoptotic DNA ladder assay. DNA laddering is a distinctive feature
of DNA degraded by caspase-activated DNase (CAD), which is a key
event during apoptosis. CAD cleaves genomic DNA at
internucleosomal linker regions, resulting in DNA fragments that
are multiples of 180-185 base-pairs in length. Separation of the
fragments by agarose gel electrophoresis results in a characteristic
“ladder” pattern.37 The dose-dependent inhibition effects of the
complexes 7 and 9 on the HeLa cell lines is confirmed by DNA
fragmentation assay.
Fig 7. Graphical representation of the intercalated complexes 7 (top
row) and 9 (bottom row) with DNA (PDB ID: 1BDNA).
Molecular docking with HSA. Human serum albumin (HSA) is the
most abundant protein in the blood plasma which accounts for
about 60% of the plasma’s total protein contents. The molecular
docking results reveal that the complexes 7 and 9 have good
binding affinity towards the protein receptor with a binding energy
of -466.84 and -476.85 kcal mol-1, respectively. The amino acids
involved in binding with the protein receptor are ALA 194, ARG 145,
ARG 197, ASP 108, GLN 459, GLU 425, HIS 146, LYS 190, PHE 149,
PRO 147, TRY 148, and SER 193. Structural studies have shown that
HSA consists of three homologous domains: I (residues 1-195), II
(196-383), and III (384-585). The complexes are bound to HSA by
electrostatic and hydrogen bonding interactions. The distance of
separation between the hydrogen bonded protons of the protein
Fig 6. DNA ladder assay: HeLa cell lines incubated with the
complexes 7 and 9 at 25 and 50 μM concentrations in DMEM
medium.
The HeLa cells are incubated with different concentrations of the
complexes (M, base pair; control, lane 1; 25 µM, lane 2; 50 μM, lane
3 for the complex 7; 25 μM, lane 4 and 50 μM, lane 5 for the
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and the nitrogen and oxygen atoms of the bound complexes, received. HPLC grade water was used for relaxivity studies. Thin
computed using MM software HEX 5.0, are 3.12 and 3.47 Å for the layer chromatography was performed on silica gel 60F-254 glass
complex 7 and 3.45, 2.96, and 3.51 Å for the complex 9, indicating plates. Reaction products were purified by flash chromatography
that the complex 7 is in close proximity to the THE 161 and PHE 134 using silica gel (100-200 mesh).
amino acids, while the complex 9 is in close proximity to the SER
Physical Measurements. Infrared spectra were recorded on a
517, ARG 186, and HIS 146 amino acids. The possible hydrogen
Perkin-Elmer Spectrum RX-I FT IR spectrometer in the range of
4000-400 cm-1 using KBr pellets. Electrospray ionization (ESI) mass
bonding interactions between the complexes with the receptor are
presented in Fig. 8.
spectra were recorded on a Quattro-II Triple Quadrupole Mass
Spectrometer coupled with waters 2795 HPLC. MALDI-TOF mass
spectra were recorded on a Micro Mass TOF Spec 2E Mass
Spectrometer using a nitrogen laser (wavelength 337 nm, 4 ns
pulse) and α-cyanohydroxy cinnamic acid was used as the matrix. 1H
and 13C NMR spectra were recorded on a Bruker Avance 400
Spectrometer operating at 400 MHz for 1H and 100 MHz for 13C
NMR. Preparative HPLC analyses were carried out using Varian
PrepStar 218 (Varian Instruments Inc., USA) binary gradient solvent
delivery module equipped with a UV-Vis detector (Model 345)
operating in the range 190-1100 nm. Analytical HPLC analyses were
performed using 1220 infinity LC system (Agilent Technologies,
USA) equipped with a UV-Visible detector operating in the range
190-600 nm (SB-C18 column, 4.6 mm x 250 mm; particle size 5 μm).
ICP-OES analyses were performed on a Perkin-Elmer Optima 2000
DV model.
Fig 8. Molecular docked model of the complexes 7 (top row) and 9
(bottom row) with HSA (PDB ID: 1h9z).
The electronic absorption spectra were recorded on a Shimadzu
UV-2450 UV-Vis spectrophotometer, controlled by the UV probe
version 2.33 software in the range 190-900 nm at 25 °C using a
matched pair of Teflon stoppered quartz cell of path length 1 cm.
Fluorescence spectra were recorded on a Fluorolog-3 FL3-221
Spectrofluorometer. The excitation source was a 450 W CW Xenon
lamp. The band pass for the excitation and double-grating emission
monochromator was set at 2 nm. A matched pair of quartz cell of
path length 10 mm was used. All emission spectra were corrected
for the instrumental function. All photophysical studies and
measurements were made in deoxygenated 0.1 M Tris-HCl buffer
(pH = 7.4) using HPLC grade water by purging argon at 25 °C. CHN
microanalyses were carried out using Perkin-Elmer 2400 Series II
CHNS/O Elemental Analyzer interfaced with the Perkin-Elmer AD6
Autobalance. Helium (99.999% purity) was used as the carrier gas.
Conclusions
III
III
The tri- and tetranuclear RuII-Gd2 and RuII-Gd3 d-f
heterometallic complexes function as contrast enhancing
agents for MRI and as optical probes for fluorescence imaging.
Our study demonstrates that the complexes have good cell
membrane permeability which promises their use for further
in vitro and in vivo studies. In vitro cytotoxicity studies using
cultured HeLa cell lines show that the complexes
7 and 9
exhibit good anticancer activity and the results may be useful
in the development of new anticancer agents. The DOTA-
AMpy type ligand framework is suitable for the development
of a variety of d-f heterometallic assemblies by replacing the
pyridine pendant arm by other polypyridine ligands. The
fluorescence properties and the binding affinity of such
complexes could be finetuned by changing the ancillary ligands
coordinated to the Ru(II) center. The synthetic strategies
developed in the present work could be exploited to develop a
variety of d-f heterometallic assemblies.
Synthesis of the Ligand
1,4,7-Tris(tert-butoxymethane)-1,4,7,10 tetraazacyclododecane (t-Bu-
DO3A) (1). Compound
1 was synthesized by the literature
procedure.39 To a mixture of cyclen (5 g, 29.02 mmol) and sodium
bicarbonate (5.80 g, 69.64 mmol) in dry acetonitrile (350 mL)
maintained at -5 °C was added tert-butyl bromoacetate (13.58 g,
69.64 mmol) dropwise over 30 min under stirring. The reaction
mixture was further stirred for 48 h at room temperature, filtered,
and flash evaporated to leave a beige solid. The solid product was
dissolved in chloroform, filtered to remove the undissolved
particles, the filtrate was concentrated, and the resulting solid
product was recrystallized from hexane/toluene mixture (2:1, v/v)
to afford a white crystalline compound: yield (10.32 g, 69%); m.p.
190 ˚C. 1H NMR (400 MHz, CDCl3, 25 °C): δ 3.37 (s, 4 H, (CH2)2
acetates), 3.29 (s, 2 H, CH2 unique acetate), 3.10 (m, 4 H, (CH2)2 cyclen ring),
2.88-2.93 (m, 12 H, (CH2)2 cyclen ring), 1.55 (s, 27 H, C(CH3)3 t-butyl); 13C
NMR (100 MHz, CDCl3, 25 °C): δ 28.1 (-C-(CH3)), 47.5 (-CH2CH2 cyclic
Experimental
Materials. All reagents and solvents were of analytical reagent
grade and used without further purification. 1,4,7,10-
Tetraazacyclododecane (cyclen) (Strem Chemicals); ruthenium(III)
chloride (99.98%), 4-aminopyridine (98%), gadolinium(III) chloride
hexahydrate (99.999%), potassium bromide (99.99%), human
serum albumin (≥ 97%), Hoechst-33342 (≥ 97%), acridine orange
(90%), ethidium bromide (∼ 95%), propidium iodide (≥ 94%) (Sigma-
Aldrich); tert-butyl bromoacetate (98%) (Alfa Aesar); 1,10-
phenanthroline monohydrate (99.9%), chloroacetyl chloride,
potassium carbonate, trifluoroacetic acid (98%), xylenol orange,
lithium chloride, sodium acetate (Merck, India) were used as
6 | New J. Chem., 2015, 00, 1-10
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ARTICLE
ring, asymmetric), 49.1 (-CH2CH2 cyclic ring, asymmetric), 51.3 (-CH2CH2 cyclic ring, 1716 (C=O acid), 2977 (CH2), 3437 (O-H acid), 3451 (N-H amide) cm-1.
symmetric), 58.1 (-CH2COO− acetate), 81.6 (-C-(CH3)3 t-butyl), 169.6 (- MALDI-MS (m/z): 480 [M]+, 524 [M-2H+2Na]+. Elemental analysis
CH2COO− unique acetate), 170.5 (-CH2COO− acetate). FT-IR (KBr): 1135 (C-O calcd (%) for [C21H32N6O7]: C, 52.49; H, 6.71; N, 17.49; found: C,
ester), 1744 (-C=O ester), 2983, 2879 (C-H), 3429 (N-H amine) cm-1. ESI- 52.09; H, 6.45; N, 17.11.
MS (m/z): 515 [M+H]+. Elemental analysis calcd (%) for [C26H51N4O6]: Synthesis of Complexes
C, 60.58; H, 9.70; N, 10.87; found: C, 60.34; H, 9.42; N, 10.58.
[Gd(DOTA-AMpy)(H2O)] (5). To a solution of 4 (2 g, 4.16 mmol) in
1,4,7-Tris(carboxymethyl)-1,4,7,10-tetraazacyclododecane (DO3A) water (100 mL) was added GdCl3.6H2O (1.54 g, 4.16 mmol) under
(2). A solution of t-Bu-DO3A (7 g, 13.58 mmol) in trifluoroacetic stirring and the reaction mixture was heated at 65 °C for 8 h by
acid/dichloromethane (1:1, v/v; 100 mL) was stirred at room maintaining the pH of the solution at 4.0-6.5 using 0.1 M aqueous
temperature for 24 h. The solvent was flash evaporated, the HCl. The reaction mixture was cooled to room temperature,
resulting solid product was dissolved in 50 mL methanol, and filtered, and the filtrate was flash evaporated to yield a hygroscopic
diethyl ether (250 mL) was added under vigorous stirring. The solid solid. Free Gd(III) ions were removed by chelex 100 resin and their
product that precipitated out was filtered, washed with diethyl absence checked by testing with the arsenazo indicator solution.
ether and acetone, and dried in vacuum to get a white crystalline The gadolinium(III) concentration was determined by ICP-OES: yield
solid: yield (2.58 g, 49%); m.p. 240 °C. FT-IR (KBr): 1724 (C=O acid), (1.72 g, 63%). FT-IR (KBr): 1400 (COO− acid) 1550 (N-H amide), 1598
2466 (CH2 cyclen), 3323 (N-H amine), 3453 (O-H acid) cm-1. ESI-MS (m/z): (COO– acid), cm-1. ESI-MS (m/z): 633 [M-H]+, 679 [M-H+2Na]+.
347 [M]+. Elemental analysis calcd (%) for [C14H26N4O6]: C, 48.54; H, Elemental analysis calcd (%) for [C21H29GdN6O7]: C, 38.64; H, 4.79;
7.57; N, 16.17; found: C, 48.26; H, 7.31; N, 15.88.
N, 12.87; found: C, 38.56; H, 4.86; N, 12.98.
2-Chloro-N-(pyridine-4-yl)acetamide (3). To
a
solution of 4- [Ru(phen)2{Gd(DOTA-AMpy)(H2O)}2]Cl2 (7). To a solution of cis-[Ru
aminopyridine (6 g, 63.64 mmol) in dry acetonitrile (300 mL), taken (phen)2Cl2] (6) (0.5 g, 0.93 mmol) in water/ethanol (1:1, v/v) (60 mL)
in a double wall RB flask connected to cryogenic circulator water under reflux in an argon atmosphere was added a solution of
bath, was added potassium carbonate (8.84 g, 63.64 mmol) at -5 °C [Gd(DOTA-AMpy)(H2O)] (5) (1.34 g, 2.05 mmol) in water (25 mL)
under stirring over 30 min. A solution of chloroacetyl chloride (7.18 through a dropping funnel for 15 min and refluxed overnight. The
g, 63.64 mmol) in acetonitrile (75 mL) was added slowly to the solution was concentrated in a rotavapor and the resulting dark
reaction mixture and stirred for 24 h at room temperature by yellow solid was purified by preparative HPLC (acetonitrile:
monitoring the progress of the reaction by TLC (dichloromethane: water:TFA, 6:4:0.1 v/v). The fraction was concentrated and dried in
methanol:aq.NH3, 9:1:0.1 v/v). The reaction mixture was filtered vacuum. The purity of the compound was checked by analytical
and flash evaporated to dryness. The resulting crude compound HPLC with acetonitrile/water and 0.1% trifluoroacetic acid as the
was purified by column chromatography (dichloromethane: eluent, resulting in a single peak (tR = 4.53 min): yield (0.89 mg,
methanol:aq.NH3, 9:1:0.1 v/v) to give a white solid: yield (4.92 g, 52%). FT-IR (KBr): 1594 (COO− acid), 1544 (N-H amide), 1412 (COO−
)
acid
38%); m.p. 265 °C (dec). 1H NMR (400 MHz, D2O, 25 °C): δ 7.89-7.87 cm-1. UV-Vis λmax(Tris-HCl/nm): 265 (ε/mol-1dm3cm-1 1,46,166), 473
(d, 2H, J = 8 Hz, CH aromatic), 7.23 (1H, N-H amide), 6.85-6.83 (d, 2H, J = (18,333). ESI-MS (m/z): 556 [M-2Cl-H2O+6H]3+, 615 [M-H+4Na]3+.
8 Hz, CH aromatic), 3.33 (s, 2H, -CH2 ethyl); 13C NMR (100 MHz, D2O, 25 Elemental analysis calcd (%) for [C66H78N16O16RuGd2Cl2]: C, 44.86; H,
°C): δ 173.2, 158.7, 143.5, 109.6, 60.1. FT-IR (KBr): 1657 (C=O amide), 4.45; N, 12.68; found: C, 44.78; H, 4.58; N, 12.79.
2992 (CH2), 3457 (N-H amide) cm-1. ESI-MS (m/z): 175 [M-Cl+K]+, 153
[Ru(ttpy){Gd(DOTA-AMpy)(H2O)}3]Cl2 (9). To
a
solution of
[M-OH]+. Elemental analysis calcd (%) for [C7H7N2ClO]: C, 49.28; H,
4.14; N, 16.42; found: C, 49.06; H, 4.08; N, 15.97.
[Ru(ttpy)Cl3] (8) (0.5 g, 0.94 mmol) in water/ethanol (1:1, v/v) (60
mL) and a few drops of N-methyl morpholine under reflux was
added a solution of [Gd(DOTA- AMpy)(H2O)] (5) (2.02 g, 3.10 mmol)
1-(N-(4-Pyridinyl)acetamido)-1,4,7,10-tetraazacyclododecane-,4,7,10-
triacetic acid (DOTA-AMpy) (4). A solution of DO3A (3 g, 8.65 mmol) in water (25 mL) through a dropping funnel for 25 min and refluxed
and potassium carbonate (1.19 g, 8.65 mmol) in water (200 mL) was overnight in an argon atmosphere. The solution was concentrated
stirred for 20 min and
a solution of 2-chloro-N-(pyridine-4- in a rotavapor and the resulting red solid was purified by
yl)acetamide (1.48 g, 8.65 mmol) in water (50 mL) was added preparative HPLC (acetonitrile:water:TFA, 6:4:0.1 v/v). The fraction
dropwise for 30 min. The reaction mixture was heated at 70 °C for was concentrated and dried in vacuum. The purity of the compound
24
h by monitoring the progress of the reaction by TLC was checked by analytical HPLC with acetonitrile/water and 0.1%
(dichloromethane/methanol/aq.NH3, 9:1:0.1 v/v), cooled to room trifluoroacetic acid as the eluent, resulting in a single peak (tR = 5.93
temperature, filtered, and flash evaporated to give a white solid. min): yield (0.98 g, 43%). FT-IR (KBr): 1602 (COO−), 1547 (N-H), 1402
The compound was purified by column chromatography (silica gel, (COO−) cm-1. UV-Vis λmax(Tris-HCl/nm): 272 (ε/mol-1dm3cm-1 98,333),
5% methanol in dichloromethane/aq.NH3, 9:1 v/v) to give 4 as a 317 (22,166), 493 (4333). ESI-MS (m/z): 1209 [M-2Cl-
white crystalline solid. The purity of the compound was checked by H2O+3H+3Na]2+, 374 [M-2Cl-5H2O+H]6+. Elemental analysis calcd (%)
HPLC analysis using acetonitrile/methanol (6:4 v/v) and 0.1% for [C85H110N21O24RuGd3Cl2]: C, 42.85; H, 4.65; N, 12.34; found: C,
trifluoroacetic acid as the eluent resulting in a single peak (tR = 3.31 43.01; H, 4.73; N, 12.44.
min): yield (2.65 g, 63%); m.p. 290 °C (dec). 1H NMR (400 MHz, D2O,
HPLC analysis and assessment of the purity of the complexes 7 and
ppm): δ 7.81-7.79 (d, 2H, J = 8 Hz, CH aromatic), 7.20 (1H, N-H amide),
9. The purity of the tri- and tetranuclear complexes 7 and 9 were
6.77-6.75 (d, 2H, J = 8 Hz, CH aromatic), 3.60 (m, 8H, CH2 acetate), 3.19
(m, 16H, -CH2 cyclen); 13C NMR (100 MHz, D2O, ppm): δ 177.6, 173.0,
checked by analytical HPLC on a SB-C18 column (Fig. S13 and S15 in
the ESI). The complexes were eluted with acetonitrile/ water (6:4,
158.7, 143.4, 109.5, 59.9, 55.3, 49.6. FT-IR (KBr): 1664 (C=O amide),
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v/v) containing 0.1% trifluoroacetic acid. The presence of the culture clusters (Costar) at a volume of 100 μL per well, and
complex in the eluate was monitored using the variable wavelength incubated for 24 h at 37 °C. The cells were then treated with
detector. The shorter wavelength of 265 nm for 7 and 273 nm for 9 different concentrations of the complexes 7 and 9 (5-50 μg mL-1)
were chosen due to their high sensitivity toward the impurities. The and incubated for 24 h. A solution of MTT (100 μL, 5 mg mL-1) was
presence of a single peak suggests that the complexes exist in added to each well, incubated for a further period of 4 h (37 °C, 5%
solution as
a
single or “averaged” species under the CO2), the cell lysate (100 μL) was added to each well, maintained for
chromatographic conditions employed in this study. The HPLC 12 h at 37 °C, and the plates were analyzed on a Microplate Reader
retention times of complexes 7 and 9 are tR = 4.53 and 5.93, at 570 nm (Biorad 680). The medium and the complex free control
respectively.
samples were prepared simultaneously. The percentage of growth
inhibitory rate of the treated cells was calculated using the
relationship (Acontrol - Acompound/Acontrol - Acell-free) x 100 (A is the mean
value calculated by using the data from the triplicate tests). The IC50
values were determined by plotting the percentage viability versus
concentration on a logarithmic graph and reading the concentration
at which 50% of cells were viable relative to the control.
Longitudinal relaxivity measurements. The longitudinal relaxivity of
the complexes were determined from the spin-lattice relaxation
time (T1). The T1 measurements were carried out on a Bruker
minispec mq 20 NMR Analyzer operating at a frequency of 20 MHz
using the standard inversion recovery pulse sequence (180°-τ-90°)
with a phase sensitive detection and τ values ranging from 50 μs to
6 s for each concentration of the complex. The temperature was Cell viability assay by Hoechst-33342 staining. The HeLa cell lines
maintained using temperature console at 37±0.1 °C. The were seeded into 24 well plates (corning) and grown to
a
instrument parameters were optimized for each T1 measurement. approximately 40% confluence. The growth medium was removed
Solutions of 0.05, 0.1, 0.15, 0.2, 0.4, 0.6, and 1.2 mM concentration and the cells in each well were exposed to 500 μL of the culture
were prepared in phosphate buffer saline (PBS) (pH = 7.4) using medium containing the complexes 7 and 9 (25 and 50 μM) for 24 h.
HPLC grade water (Merck, India) in 5 mL standard measuring flask The cells were washed with cold PBS, fixed with 4%
(vensil, class “A”). The presence of free gadolinium(III) ion in the paraformaldehyde at room temperature (298 K) for 10 min, washed
solution was checked by xylenol orange test. The T1 values for each with cold PBS, labeled with Hoechst-33342 (5 μg mL-1 in PBS) for 5
concentration were measured by taking the solution in 180 x 10 min, and analyzed immediately with a Fluorescence Microscope
mm stoppered glass tube. The computer program “winfit” was used (Olympus, America).
to plot the time versus signal intensity to get a monoexponential
Morphological assessment of apoptosis by acridine orange (AO)
plot and T1 was calculated from the plot. The T1 curves for all
and ethidium bromide (EB) dual staining. The HeLa cell lines were
concentrations have a monoexponential decay character. The
seeded into a 24-well plate (50,000 cells per well) and cultured in a
longitudinal relaxivity was calculated from the slope of the
RPMI 1640 medium supplemented with heat inactivated FBS (10%),
penicillin (100 μg mL-1), and streptomycin (100 μg mL-1). Cells were
regression line, obtained by the plot of the concentration of the
complex versus 1/T1, by least square fitting method. The instrument
maintained at 37 °C in a CO2 incubator, washed three times with
was calibrated by measuring the relaxivity of [Gd(DO3A)(H2O)2] in
aqueous solution (r1p = 4.80 mM-1s-1, 20 MHz, 40 °C).30
PBS solution, and treated with a PBS solution of the complexes 7
and 9 (25 and 50 μM) and incubated for 24 h at 37 °C. The cells
Preparation of 4.5% (w/v) HSA and GdL/HSA samples. HSA was were harvested, washed with PBS, and 40 µL of AO/EB solution (1
dissolved in 10 mM sodium phosphate and 150 mM sodium part of 100 µg mL-1 of AO in PBS; 1 part of 100 µg mL-1 of EB in PBS)
chloride (pH = 7.4) solution. Two HSA solutions (4.5% w/v) were was added. After staining, the cells were washed with PBS twice,
made: one containing the complex with a concentration of 2 mM suspended in 200 µL of PBS, and the cell morphology examined
and the other without the complex.
under
a Fluorescence Microscope (Olympus, America) at 20x
Ultrafiltration study of binding of Ru-Gd complexes with HSA. RuII-
resolution.
GdIII/HSA solutions were prepared by combining appropriate Flow cytometric analysis of cell cycle by propidium iodide staining.
amounts of HSA (4.5%, w/v) with solutions of the complexes 7 and The HeLa cell lines were incubated with the complexes 7 and 9 (25
9 of concentrations 0.05, 0.1, 0.15, 0.2, 0.25, 0.4, 0.6, and 1.2 mM. μM) for 24 h. The cells were harvested and centrifuged (5 min at
For each study, 1 mL solution was incubated at 37.0±0.1 °C for 20 800 g), fixed in 2 mL of 70% aqueous ethanol (v/v), incubated for a
°
min, placed in
a
30 kDa Ultrafiltration Unit (Amicon), and period of at least 12 h at -20 C, and centrifuged (15 min at 800 g)
centrifuged at 3500 g for 10 min. The filtrates were used to and washed twice with ice cold PBS. The cells were resuspended in
measure the concentration of the free Gd(III) complex. Experiments 200 μL staining solution containing PI (10 μg mL-1) and DNase free
were performed in duplicate. The concentration of gadolinium(III) RNase (100 μg mL-1) and analyzed by a BD FACSCaliburTM Cytometer
present in the RuII-GdIII/HSA solutions and in the ultrafiltrates were (Becton Dickinson, Heidelberg, Germany). The number of cells
determined by ICP-OES.
analyzed for each sample was 10,000 and the experiments were
repeated at least three times under identical conditions. Data were
collected by BD CellQuest™ Pro software and analyzed by ModFit LT
2.0 software.
Cell viability and cytotoxicity study by MTT assay. The HeLa cell
lines were grown in a RPMI 1640 medium supplemented with fetal
bovine serum (FBS) (10%), penicillin (100 μg mL-1), and streptomycin
(100 μg mL-1) and incubated at 37 °C in a humidified incubator with Apoptotic DNA ladder assay. The HeLa cell lines were seeded into
5% CO2 and 95% air. Cells at the exponential growth phase were 24 well plates (triplicate wells of 105 cells per well) and incubated
diluted to 2.5 x 103 cells/well with RPMI 1640, seeded in 96 well with different concentrations of the complexes 7 and 9 in fresh
8 | New J. Chem., 2015, 00, 1-10
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Eppendorf tube, spinned down, resuspended with 0.5 mL PBS in 1.5
mL Eppendorf tubes, and 55 μL of lysis buffer (40 mL of 0.5 M EDTA,
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phenol:chloroform (gentle agitation for
a
1:1 mixture of
min followed by
5
4
centrifugation), and precipitated in two equivalent of cold ethanol
and one-tenth equivalent of sodium acetate. The contents in the
Eppendorf tubes were spinned down, decanted, and resuspended
the precipitates in 30 μL of deionized water-RNase solution (0.4 mL
water and 5 μL RNase) and 5 μL of loading buffer for 30 min at 37
°C. DNA ladder (marker) (2 μL) was inserted on the outer lanes and
1.2% gel was run at 5 V for 5 min before increasing to 100 V. After
the dye front reached 75% of the gel, the image of DNA shearing
illuminator was monitored at 312 nm using a UV lamp.
5
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Molecular docking study. Molecular docking studies of the
complexes 7 and 9 with DNA and HSA were performed using HEX
5.0 software and Q-site finder which is an interactive molecular
graphics program for the interaction, docking calculations, and to
identify possible binding site of the biomolecules. The coordinates
of metal complexes were taken from their optimized structure as a
.mol file and were converted to .pdb format using PYMOL software.
The crystal structure of B-DNA (PDB ID: 1BNA) and human serum
albumin (PDB ID: 1h9z) were retrieved from the protein data bank
(http://www.rcsb.org/.pdb). Visualization of the docked systems
has been performed using PYMOL Tool. Default parameters were
used for the docking calculations with correlation type shape and
FFT mode at 3D level, grid dimension of 6 with receptor range 180,
ligand range 180 with twist range 360, and distance range 40.
9
Acknowledgements
We thankfully acknowledge the financial support from the
Department of Science and Technology (DST) and the
Department of Biotechnology (DBT), Government of India. ESI-
MS and MALDI-MS were recorded in SAIF, Punjab University
1
and Jawaharlal Nehru University. The H and 13C NMR spectra
were recorded in SAIF, IIT-Madras. We thank the Pondicherry
Centre for Biological Sciences, Pondicherry, India, for their help
in carrying out the cell culture and fluorescence imaging
studies.
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Table of contents
III
III
A tri- (RuII-Gd2 ) and tetranuclear (RuII-Gd3 ) d-f heterometallic complexes which function as
contrast agents for MRI and as optical probes for fluorescence imaging are reported. In vitro
studies with the HeLa cell lines show that the complexes exhibit anticancer activity.