Luminescent EuIII and TbIII Complexes Containing Dopamine Neurotransmitter: Biological Interactions, Antioxidant Activity and Cellular-Imaging Studies

Article abstract of DOI:10.1002/ejic.201800732

Two luminescent water-soluble lanthanide(III) complexes, [Ln(DTPA-DOPA)(H₂O)] (1–2) where Ln = Eu^III (1), Tb^III (2), formed by reaction with diethylenetriaminepentaacetic acid (DTPA) carrying the neurotransmitter dopamine

Full text of DOI:10.1002/ejic.201800732

A Journal of
Accepted Article
Title: Luminescent Eu(III) and Tb(III) Complexes Containing Dopamine
Neurotransmitter: Biological Interactions, Antioxidant Activity and
Cellular Imaging Studies
Authors: Khushbu Singh, Anshika Goenka, Subramaniam Ganesh, and
Ashis K. Patra
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To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201800732
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European Journal of Inorganic Chemistry
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FULL PAPER
Luminescent Eu(III) and Tb(III) Complexes Containing Dopamine
Neurotransmitter: Biological Interactions, Antioxidant Activity
and Cellular Imaging Studies
[a]
[b]
[b]
*[a]
Khushbu Singh , Anshika Goenka , Subramaniam Ganesh and Ashis K. Patra
Abstract: Two luminescent water soluble lanthanide(III) complexes,
Ln(DTPA-DOPA)(H O)] (1-2) where Ln = Eu(III) (1), Tb(III)(2) formed
for time-resolved live cell or in-vivo imaging application. Such
time-gated techniques will eliminate interfering background
autofluorescence, scattering in biological milieu and
enhancing signal-to-noise ratios. Chelation of lanthanides to
multidentate chelating ligands forms stable coordinatively
saturated Ln(III) complexes which protects it from quenching
through non-radiative vibrational energy dissipation for
application in luminescence imaging or sensing. Thus,
design of the Ln(III) complexes with polydentate ligands like
[
2
by reaction with diethylenetriaminepentaacetic acid (DTPA) carrying
neurotransmitter dopamine with catechol functional groups i.e. DTPA-
bis(3-hydroxytyramide) are characterized and their photophysical
properties were studied. The strong characteristic red and green
luminescence in the visible region was observed for 1 and 2 upon
photo-excitation, followed by efficient energy transfer from dopamine
to Ln(III) leading to intraconfigurational f-f transitions. They show
distinguishable sharp emission bands due to intraconfigurational
5
7
5
7
DTPA, DOTA appended with a sensitizing antenna
D
0
 F
J
transitions in Eu(III) and D
4
J
 F transitions in Tb(III). They
established as an important approach for designing highly
emissive lanthanide based bioprobes for cellular imaging
and sensing.[2,3] Recently, K.-L. Wong et al. strategically
designed unique lanthanide probes for selective
photodynamic therapeutic (PDT) applications,[4] photo-
triggered controlled delivery and tracking of anticancer
exhibit long excited-state lifetimes: = 0.52 ms (1) and 0.33 ms (2)
and calculated quantum yields (Фoverall) = 0.022 (1) and 0.038 (2). The
luminescent complexes were studied for their antioxidant activity,
ability to interact with calf thymus-DNA (CT-DNA) and bovine serum
albumin (BSA), hydrolytic cleavage of pUC19 supercoiled plasmid
DNA and as cellular imaging probes for both cancer cells and
neuroblastoma cells. Both Eu(III) and Tb(III) complexes exhibit potent
antioxidant activity. These complexes showed moderate binding
[
5]
drugs , imaging probe for early prognosis of disease
biomarkers. [6]
4
-1
3
-1
b
affinity towards CT–DNA (K ~10 M ) and BSA (KBSA =10 M ).
Dopamine
is
an
important
catecholamine
Complexes at 200 µM exhibit good hydrolytic cleavage of supercoiled
pUC19 DNA. The non-cytotoxic nature of these complexes makes
them efficient candidates for cellular imaging probes. Cytosolic
localization is observed for both the complexes in HeLa cells and
mouse neuroblastoma (Neuro-2a) cells. The present systems with
advantageous antioxidant activity may have potential application in
tracking and delivery of dopamine, which is important for dopamine
replacement therapy in various neurodegenerative disorders.
neurotransmitter and regulates cognitive abilities and various
motor functions in humans.[7-10] Several pathobiological
studies reveal an intricate relationship between the
imbalance of dopamine and several neurodegenerative
diseases: viz. Perkinson’s disease (PD), Alzheimer disease,
Huntington’s disease, multiple sclerorosis etc. Dopamine
mediates its action through binding with dopamine receptors,
thus expression, distribution and mapping of various
dopamine receptor subtypes by bioimaging intended to be
an effective tool towards diagnosis and early prognosis of
various neurodegenerative diseases. Moreover, metabolism
of various catecholamine-based neurotransmitters like
dopamine, norepinephrine, serotonin provides an antioxidant
defense in brain against oxidative stress damage induced by
Introduction
Luminescent lanthanide probes have certain distinct
incentives in designing and development of traceable
delivery vehicles for therapeutic or diagnostic loads. This is
due to their unique photophysical properties like sharp
distinguishable emission bands, long excited state lifetimes
and large ligand-induced pseudo–Stokes shift.[1-3] The f-f
transitions are Laporte and spin-forbidden leading to long
excited state lifetimes in the ms - µs range. These favourable
optical properties of lanthanides leads to many opportunities
free radicals or iron independent or dependent lipid
peroxidation.[11-13] Certain biological characteristics such as
water solubility, efficient cellular internalization and non-
toxicity at working concentration are important criteria for
lanthanide complexes to be used as a cellular probe. This
work stems from our current interest in developing
luminescent Ln(III) complexes for cellular imaging and
delivering therapeutic loads.[14]
Here we have conjugated dopamine as an antenna and
as potential antioxidant to diethylenetriaminepentaacetic
acid (DTPA) and prepared respective luminescent probes:
[
a]
Department of Chemistry, Indian Institute of Technology Kanpur,
Kanpur 208016, Uttar Pradesh, India. E-mail: akpatra@iitk.ac.in
https://www.iitk.ac.in/new/ashis-k-patra
[
b]
Department of Biological Sciences and Bioengineering, Indian
Institute of Technology Kanpur, Kanpur 208016, Uttar Pradesh,
India.
[Ln(DTPA-DOPA)(H
2
O)] [Ln(III) = Eu(III) (1), Tb(III) (2)]
(Scheme 1). Eu(III) and Tb(III) were used for their longer
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European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
excited state lifetimes (

) in µs-ms range and emission in
Physiochemical
Data
[Eu(DTPA-DOPA)(H
2
O)]
2
[Tb(DTPA-DOPA)(H O)]
visible region. The photophysical properties, interactions with
CT-DNA and BSA, anti-oxidant activity, DNA hydrolysis, and
potential cellular imaging applications were studied.
(1)
(2)
ESI-MS in H
2
O
814.17
820.18
(
m/z) [M−H
2
O+H]+
FT-IR[a] (KBr
1631 (s, νasym CO
1527(s, νCO of CONH)
384 (s, νsymCO
−)
−)
1587 (s, νasym CO
1528(s, νCO of CONH)
−)
2
2
pellet, νmax, cm−1
)
1
2
1384 (s, νsymCO
2
−)
λ
max[b]/ nm
282 (8325)
282 (7134)
(
ε/M−1cm−1
)
[
c]

H2O and D2O
Eu and qTb[d]
Eu and ϕTb[e]
0.52 and 1.43
1.2
0.33 and 0.36
1.1
q
ϕ
0.038
0.022
[a]In KBr phase. [b]UV-visible spectra in MQ water. [c] Lifetime measured in H
and D
O (τ ± 15%). [d]Hydration number (q =10%).[e]Overall quantum yield (ϕ ±
0%) using quinine sulphate as standard.
2
O
2
1
This strong coordinative property led the compounds 1 and 2 to
be stable in aqueous medium for prolonged time at room
temperature. The structural integrity of the complexes were also
+
evidenced by ESI-MS which showed [M-H O+H] peak and
matching isotopic distribution pattern in mass spectral profile
2
2
Scheme 1. Simplified scheme for [Ln(DTPA-DOPA)(H O)] (Ln= Eu(III) (1) and
Tb(III) (2)) complexes showing the energy transfer to Ln(III) through dopamine
clearly showing the formation of [Ln(DTPA-DOPA)(H
stoichiometry.
2
O)] in 1:1
moiety and multiple biological importance of the systems.
Results and Discussion
Synthesis and Characterization
The H
reaction of DTPA bis(anhydride) with dopamine in dry DMF
at 70 C with 90% yield. The luminescent complexes and
are synthesized by reacting DTPA-DOPA with
Eu(NO ·6H O and TbCl ·6H O in 1:1 mole ratio in water at
80% yield and characterized by physicochemical methods,
3
DTPA-DOPA ligand was synthesized from acylation

1
2
H
3
3
)
3
2
3
2
+
~
Figure 1. ESI-MS of (a) complex 1 with m/z [1 H
2
O + H] (C32
H
38EuN
5
O
12) and
+
(
b) complex 2 with m/z [2 H
2
O + H] (C32
H38TbN
5
O
12) in aqueous medium. Inset
ESI-MS, FTIR and UV-visible spectroscopy. The selected
physicochemical data are given in Table 1. The ligand based
absorption peaks were observed at 282 nm due to π→π*
figure shows the theoretical isotopic distribution pattern.
Photophysical Properties
transition. The IR data shows strong absorption ~1600 cm-1
Complexes 1 and 2 exhibit characteristic absorption bands at 282
nm which can be assigned to the ligand based π→π* transition.
-
ascribed to
C=O of CO
2
which shows shifts in the stretching
frequency by 20-60 cm-1 upon complexation suggesting
oxygen coordination to the Ln(III). These results suggests
that DTPA-bisamide coordinates to the Ln(III) by three
nitrogens, three carboxylate oxygen atoms, two amide
oxygen and the ninth site is occupied by water molecule to
form water-soluble thermodynamically stable complexes.[17]
The Gd(III) complexes of analogous ligands were previously
At
ex
λ = 282 nm, Eu(III) and Tb(III) complexes showed
5
7
characteristic sharp emission peaks due to D
0
→ F
(J = 6-3) transitions respectively (Figure 2). The excited
O and D O at RT and
J
(J = 0-4) and
5D
→7F
4
J
state lifetimes were measured in H
2
2
luminescence decay profile has been fitted by single exponential
fitting to obtain H2O = 0.52 ms; D2O = 1.43 ms for complex 1
and H2O = 0.33 ms; D2O = 0.36 ms for complex 2 confirming the
presence of single luminescent lanthanide species in solution.
The decay profiles and their mono exponential fittings were shown
reported as potential receptor-specific MRI contrast
agents.[15] Moreover, the redox stability of these trivalent
2
in Figure 2 (c, d). In D O, the emissive lifetime increases due to
Ln(III) complexes is crucial towards intracellular applications
non-radiative deactivation generated by O-D vibration is lower in
comparison to O-H vibration.[ The hydration numbers, qEu =1.2
and qTb = 1.2 denotes the number of coordinated water
molecule(s) in the coordination sites calculated through modified
Horrock's equation.[19] The q values are in consistent with eight
coordination sites that are bonded to DTPA-bisamide derivative
and the ninth site is occupied by one water molecule.
in presence of various reducing agents such as thiols, GSH
and ascorbates.[16]
18]
Table 1. Selected physiochemical data for complexes
1 and 2
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European Journal of Inorganic Chemistry
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The luminescence quantum yields measured for the complexes 1
and 2 were 0.038 and 0.022 respectively which is indicative of the
observed in the absorption spectral titration of complexes
with CT-DNA attributed to the possible perturbation of π -
orbitals of DNA base pairs by the complexes. The intrinsic
3
fact that the triplet state ( T) of dopamine transfers sufficient
5
5
energy to the emissive D
0
or D
4
excited state of Eu(III) and Tb(III)
b
binding constants (K ) of complexes towards CT-DNA: 0.78
respectively.
4
-1
4
-1
x 10 M (1) and 1.5 x 10 M (2) respectively, suggesting
The luminescence intensity of the probes were sensitive to
changes in pH. We observed significant decrease in emission
intensity of complex 1 with lowering pH due to protonation of
carboxyl moieties of DTPA-DOPA ligand and possible breaking
down of the complex (Figure 2 (e, f) . Loss of structural integrity
and exposure of Ln3+ core of the complexes leads to significant
moderately strong binding propensity towards CT-DNA.
Ethidium bromide (EthB) is a well-defined emissive probe
that shows intense emission upon binding to CT-DNA due to
its strong intercalation between the adjacent DNA base pairs.
The complexes exhibit competitive binding to DNA and
displaces EthB and thereby decreasing emission intensity at
quenching by vibrational energy transfer to solvent molecules.
605 nm (Table 2). The displacement of EthB by these
complexes suggestive towards an intercalative or minor
groove binding.[21] The apparent binding constant (Kapp
)
)
4
-1
4
-1
calculated are 9.8 x 10 M for (1) and 7.2 x 10 M for (2
suggesting intercalative or minor grove binding mode for
complexes with moderate affinity.[22]
Table 2. Binding parameters for complexes with CT-DNA and BSA.
Physiochemical
Data
[Eu(DTPA-DOPA)(H
2
O)]
[Tb(DTPA-DOPA)(H
2
O)]
(1)
(2)
K
K
K
b
[a]/ M-1
app [b]/ M-1
ITC[c]/ M-1
0.78 (±0.2) x 104
9.8 x 104
1.5 (±0.2) x 104
7.2 x 104
K
1
2
= 1.81 x 104
= 1.10 x 103
K
1
2
= 9.94 x 104
= 8.47 x 103
K
K
[
d]
9.8 (±0.3) x 103
9.8 x 1011 [1.1]
8.7 (±0.1) x 103
8.7 x 1011 [0.98]
K
K
BSA /M-1
[e]
/M-1 [n] [f]
q
[a]K
constant. [c]
, intrinsic equilibrium DNA binding constant. [b]
K
app, apparent DNA binding
b
[d]
KITC, Affinity constant from ITC titration.
KBSA , Stern-Volmer
quenching constant for BSA fluorescence. [e]
K
q
, quenching rate constant. [f] n,
number of binding sites.
Figure 2. (a) UV – vis spectra of ligand H
100 µM) in aqueous medium at 298 K. (b) Excitation and time-resolved
luminescence spectra for complexes 1 and 2 in aqueous medium at 298 K.
Lifetime measurement of (c) complex 1 and (d) 2 (100 µM) in H O and D
medium and fitted by single-exponential decay curves. (e) The emission
3
DTPA-DOPA, complexes 1 and 2
(
2
2
O
intensity changes of complex 1 in aqueous medium with variation of pH at ex
=
82 nm. (f) The changes in emission intensity of 5
D
0
 F
7
transition at 616 nm
2
2
as a function of pH.
DNA Binding Studies
DNA is a critical pharmacological target for metallodrugs like
cisplatin, carboplatin or oxaliplatin or Pt(IV) prodrugs where
they cross-link with nitrogen of nucleobases of DNA, thereby
arresting transcription or replication and trigger cell-death
pathways.[20] Considering potential biological activity of
Figure 3. (a) Electronic spectral traces of 1 with increasing [CT-DNA] in saline
Tris–HCl buffer (5 mM, pH 7.2) at 25 C. Inset: plot of Δεaf/Δεbf vs. [DNA] and
[
DNA] / εaf vs. [DNA] for 1. (b) Quenching of emission intensity of EthB bound
CT-DNA adduct with increasing [1] in 5 mM saline Tris–HCl buffer (pH 7.2) at
5 C; ex = 546 nm, em= 605 nm, [EthB] =3.0 μM, [CT-DNA] = 280 μM. Inset:
plot of relative fluorescence intensity (I/I ) at λem = 605 nm vs. concentration of
complexes 1 and 2. (c) ITC plot showing the interaction of CT-DNA (0.1 mM)
[
2
Ln(DTPA-DOPA)(H O)] (1, 2), the mode of interaction of the
complexes with CT-DNA at physiological condition were
studied using different spectroscopic techniques. The
bathocromic shift (2-4 nm) and hypochromism (72-75%)
2
0
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European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
with complex 1 (1 mM) in 5 mM Tris–HCl/NaCl buffer (pH 7.2) at 25C. The top
plot shows the raw ITC data for 20 injections of 2 μL each. The bottom panel
represents extent of evolved heat vs. mole ratio of [1]/[CT-DNA].
in structural conformation of BSA. The quenching constants
(
K
SV) for the complexes was determined from Stern-Volmer
equation[27] The KSV values obtained for the complexes are:
3
-1
3
-1
9
.8 x 10 M
binding constants (K) and number of binding sites (n) were
obtained from curve fitting of Scatchard equation: log(I -I)/I =
logK + nlog[Q].[28] The k
values for the complexes and
1011 M s suggesting existence of static quenching
(1) and 8.7 x 10 M (2) respectively. The
Isothermal titration calorimetry (ITC) experiments, designed
to investigate the DNA-binding thermodynamics of the
complexes with CT-DNA were performed in Tris-HCl/NaCl
0
q
1
2
buffer (5 mM, pH 7.2) at 30 C using MicroCal iTC200 system.
−1
−1
are
∼
The binding data were fitted by two mode of binding
interaction with CT-DNA. The ITC fitting reveals that
interaction of these complexes with DNA is exothermic and
biphasic at two binding sites with varying affinities attributed
for such hairpin shaped complexes.[23] The calculated
mechanism (Table 2).[29]
Hydrolytic Cleavage of DNA
The high Lewis acidity and higher effective charge
associated with the trivalent lanthanide complexes makes
4
-1
binding constant values are K
1
= 1.81 x 10 M , K
2
= 1.10 x
4
-1
3
-1
1
03 M-1
(1
) and K
1
= 9.94 x 10 M , K
2
= 8.47 x 10 M (2).
them effective catalysts for phosphodiester bonds of DNA or
RNA under physiological conditions.[30,31] The ability of Ln-
The ITC plot and best-fitting model are shown in Figure 3 (c).
The affinity constants obtained are consistent with the K and
app values measured through UV-vis and emission titration.
b
complexes to readily catalyze the DNA hydrolysis is highly
desirable to develop lanthanide-based artificial nucleases
K
important for various clinical applications including DNA-
finger printing, sequence-specific DNA lesions etc.[31] The
Interaction with Serum Protein
BSA is the most extensively studied serum transport protein
due to its structural homology with human serum albumin
complexes
supercoiled DNA (SC, form I) to nicked circular (NC, form II)
Both the complexes at 100 µM
concentrations effectively promoted cleavage of SC-DNA to
0% NC form when incubated for 5 h (Figure 5 a). The
1 and 2 displayed efficient cleavage of plasmid
(
HSA) and serve important roles in drug transport, storage
form in dark.[32]
and metabolism.[24,25] The fluorescence of BSA originates
from the intrinsic fluorescence of the tryptophan residues,
Trp-134 and Trp-212 located at the surface and within a
hydrophobic binding pocket respectively. The interaction of
the tested complexes and BSA was investigated using
tryptophan fluorescence quenching. The intrinsic BSA
fluorescence at 342 nm gets quenched in presence of these
complexes, indicating their effective binding interaction with
BSA (Figure 4). The fluorescence quenching possibly
induced from a variety of molecular interactions like
collisional encounters between the fluorophores and
quenchers or ground-state complex formation.
8
gradual disappearance of SC-DNA form and the appearance
of NC form of DNA with increasing time is observed. The
linear curve fitting of ln(%SC-DNA) vs. time plot suggesting
pseudo-first-order reaction profile. The rate constant (k) for
this conversion obtained was 5.9 x 10-3 s-1 and 6.0 x 10-3 s-1
in presence of 100 M complexes 1 and 2 respectively. To
investigate the mechanism of DNA phosphodiester cleavage
by the complexes, hydroxyl radical scavengers (DMSO and
glycerol), singlet oxygen scavengers (NaN
prior to addition of complex and it was observed that DNA
cleavage was not inhibited. The results showed that DNA
3
) were added
1
cleavage by complex
1
is not inhibited by any of the classical

1
).[31]
radical scavengers ( OH, O
2
Figure 4. (a) Fluorescence spectral traces of BSA showing the quenching effect
upon addition of complex 1 in saline Tris-buffer (5 mM, pH 7.2) at 25 C. Inset:
the plot of I
0 0
/I vs. [Complex] for 1 and 2. (b) Scatchard plots between log (I -I)/I
vs. log[complex] ex=295 nm, [BSA]=5 M.
The other possible factors include perturbation of secondary
structure of BSA, subunit association, conformational
changes induced by these quenchers.[26] The observed small
blue shift in em (~15 nm) possibly resulted from the changes
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European Journal of Inorganic Chemistry
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Figure 5. (a) Agarose gel electrophoresis of pUC19 DNA (0.2 μg, 30 μM) in
saline Tris–buffer (50 mM, pH 7.2) with complex 1 (100 µM) on increasing the
incubation time showing the disappearance of SC DNA and appearance of NC
DNA. Inset: Gel-electrophoresis picture snapped for both complex 1 and 2 with
increasing time. (b) Plot of ln(%SCDNA) vs. time for complex 1 (100 µM). (c)
Gel electrophoresis of pUC19 DNA incubated for 5 h at 37 °C and pH 7.2
(
2
50 mM Tris–HCl) with increasing [complex]: L1, DNA control; L2, Eu(NO
00 μM; L3–L7, complex 1 of 10, 20, 40, 100, 200 μM concentrations
respectively. (d) gel electrophoresis of pUC19 DNA of 1 with different additive:
L1, DNA control; L2, DNA + 1; L3, DMSO (0.4 M); L4, glycerol (0.4 M); L5, NaN
2 mM); L6, H (50 μM) after incubation at 37 C for 5 h.
3 3
) ,
Figure 6. (a) DPPH radical scavenging ability of tested compounds expressed
as percentage reduction of absorbance of DPPH measured at max = 517 nm in
aqueous medium of pH 7.2 at 298 K. (b) ABTS cation radical scavenging ability
of tested compounds measured as percentage inhibition of ABTS absorbance
at λmax = 734 nm in aqueous medium of pH 7.2 at 298 K.
3
(
2 2
O
2 2
When oxidant (50 µM H O ) was added to the reacting
mixture, it could not enhance the DNA cleavage induced by
the complexes. Therefore, the DNA cleavage promoted by
the complexes might not occur via an oxidative pathway. The
hydrolytic cleavage of phosphodiester bonds possibly
involve occurred via readily oxidizable appended catechol
group forming dopamine semiquinone or quinone radicals.
Antioxidant activity
The antioxidant activity of the complexes
1 and 2 was further
studied by scavenging of cationic ABTS+ radicals by
measuring percentage inhibition of the initial ABTS+
absorbance at λmax= 734 nm. The Eu(III) and Tb(III)
complexes showed 53.3% and 59.1% scavenging ability
compared to Trolox (89.1%) used as reference antioxidant


(Fig. 6b). Both the tested Ln-compounds are more active
Overproduction of free radicals due to imbalance of defence
or immune mechanism eventually leads to a wide range of
diseases such as chronic inflammation, cancer, rheumatoid
arthritis, cardiovascular disease, diabetes, aging and
especially neurodegenerative diseases.[33,34] The role of
antioxidants are to be antagonistic towards the free radicals
thereby protecting us from such diseases. The oxidative
stress originated from ROS and free radicals contributes to
the progression of several neurodegenerative disorders like
Alzheimer’s disease, Parkinson’s disease etc.[34,35]
Therefore, considerable attention has been directed for the
development of anti-oxidant based therapies for limiting
neuronal cell damage.[36,37] Dopamine and related
catecholamine based neurotransmitters (viz. L-DOPA,
epinephrine, norepinephrine) or phenolic-OH containing
than free H DTPA-DOPA ligand (41%) and in consistent with
3
the DPPH scavenging assay. Enhanced radical scavenging
by Ln-complexes compared to the free ligand clearly show
significant role of Ln(III) centre.
Intracellular localization studies
In order to understand the potential ability of the emissive Eu(III)
and Tb(III) compounds as visible bioimaging probes, the cellular
imaging capabilities of the complexes were studied using HeLa
cells and mouse neuroblastoma cells (Neuro-2a) by fluorescence
microscopy (Figure 7). DAPI was used as nucleus stain to
ascertain the intracellular localization of the complexes. The
conjugation of dopamine with DTPA is a possible promising
strategy in visualizing delivery or understanding mechanism of the
dopamine action pathways for neurodegenerative diseases by
utilizing the intrinsic luminescence of Eu(III) and Tb(III) in visible
region avoiding background autofluorescence.[
38]
tyramine, -tocopherol, tyrosine reported to show antioxidant
activity due to presence of catechol or phenolic-OH
moiety.[11-13]
The complexes showed intrinsic lanthanide luminescence
upon direct excitation of Ln(III) within HeLa cells and mouse
neuroblastoma cells depicting their cellular localization within 24
h of incubation. The Eu(III) and Tb(III) complexes showed their
characteristic red and green luminescence upon internalization.
The cells remained healthy and viable during complete
experiment tenure suggesting their non-cytotoxic nature (Figure
The presence of appended dopamine moieties in
[
Ln(DTPA-DOPA)(H
ability to scavenge free radicals to evaluate promising
antioxidant capacity. The ability of the complexes and to
2
O)] (1, 2) prompted us to study their
1
2
+

scavenge DPPH and ABTS (ABTS ) radicals was assessed
and compared with the well-established antioxidant agents
7
a). Counterstaining with DAPI indicated cytoplasmic localization
of the complexes inside the HeLa and mouse neuroblastoma cells
Figure 7b,c) suggesting towards effective application of these
complexes as cellular imaging agents.
(e.g. butylatedhydroxytoluene (BHT), 6-hydroxy-2,5,7,8-
(
tetramethylchromane-2-carboxylic acid (Trolox)) used as
reference standard to evaluate potential antioxidant activity.
DPPH free radical exhibit a strong absorption peak at
 =
max
517 nm which on gaining protons from antioxidants
decreases its intensity. The quantification was done after 20
and 60 minutes, which did not show any notable change
demonstrates that the scavenging action is time
independent. The effectiveness of the complexes to
scavenge DPPH radical is quite promising compared to BHT
as reference (
60% for complexes, 30.4% for
3
H DTPA-
DOPA and 73.6% for BHT) (Figure 6a).
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European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
thus promising candidates for cellular imaging studies. The
fluorescence imaging study with HeLa and neuroblastoma cell
lines revealed that these complexes showed cytosolic localization
inside the cell convincing towards their good candidature for cell
imaging application. This strategy developed here may have
potential application towards delivery and in situ tracking of
dopamine, other neurotransmitters or therapeutic loads important
for the treatment of neurodegenerative diseases.
Experimental
Materials: The A.R. grade chemicals used were purchased
commercially and used with no further purifications. The solvents
were of spectroscopic grade or were purified by standard
procedures.[ Eu(NO
39]
3 3 2 3 2
) ·6H O and TbCl ·6H O were purchased
from Sigma-Aldrich. 2,2-diphenyl-1-picrylhydrazyl (DPPH) and
,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)
ABTS) were purchased from Sigma (U.S.A). 3-(4,5-
Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide (MTT),
2
(
trypsin–EDTA, Dulbecco’s modified eagle’s medium (DMEM,
Gibco® Life Technologies, Bengaluru, India), penicillin–
streptomycin antibiotic, bisBenzimide H33258 and gelatin (from
cold water fish skin) were purchased from Sigma-Aldrich
(Bengaluru, India) and used as received. Tris-(hydroxymethyl)-
aminomethane-HCl (Tris-HCl) buffer solution was prepared using
Milli-Q water (18.2 MΩ). Calf thymus DNA (CT-DNA), bovine
serum albumin (BSA), ethidium bromide (EthB) were purchased
from Sigma (USA).
Measurements: The elemental microanalyses for C, H and N
were done using a Perkin-Elmer 2400 Series-II elemental
analyzer instrument. Infrared spectra were taken in Perkin-Elmer
model 1320 FT-IR spectrometer in KBr pellets in the 4000–400
cm range. Electrospray ionization mass spectral (ESI-MS)
measurements were achieved using a WATERS Q-TOF Premier
mass spectrometer. Electronic spectra were recorded at room
Figure 7. (a) Cytotoxicity profile of the complexes 1 and 2 with HeLa cells on 24
h incubation at 37 C as evaluated by MTT assay. (b) Fluorescent microscopic
ᴏ
images of HeLa cells on treatment with complexes 1 and 2 (100 μM) for 24 h at
−1
ᴏ
−1
3
7 C and DAPI (5 μg mL ): λex (1) = 405 nm, λex (2)= 488 nm; scale bar = 10
μM. (c) Fluorescence microscopic images of mouse neuroblastoma cells
ᴏ
(
Neuro-2a) on treatment with complex 1 (100 μM) for 24 h at 37 C and DAPI
−1
temperature using
a
Perkin-Elmer Lambda 25 UV-vis
dye (5 μg mL ): λex = 405 nm; scale bar = 10 μm for all the panels.
spectrophotometer and fluorescence spectra by Agilent Cary
eclipse fluorescence spectrophotometer at 298 K. ITC binding
study were measured using MicroCal iTC200. Lifetime
measurements for complex 1 and 2 was conducted by using
pulsed xenon lamp at λex =282 nm and λem = 616 nm and 543 nm
for Eu(III) and Tb(III) complexes respectively with a delay time and
gate time of 0.1 ms under ambient condition. Decay curve fitting
was done through non-linear least square method. The
luminescence quantum yield measurement was performed in
Conclusions
In conclusion, this work focusses on the detailed investigation on
nine-coordinated luminescent DTPA-bisamide dopamine
complexes viz. [Ln(DTPA-DOPA)(H
2
O)] of Eu(III) (1) and Tb(III)
(2). The complexes were water soluble and thermodynamically
ᴏ
aqueous medium at pH 7.2 at 25 C by taking quinine sulphate as
stable under experimental condition. Eu(III) complex exhibited red
and Tb(III) showed green luminescence on excitation at the ligand
centered band at 282 nm which is indicative of efficient energy
transfer from the triplet excited state of the ligand (chromophore)
reference with the help of reported procedure using the following
equation[
40]
퐴ref퐼푛2
5
7
5
7
∅overall = ∅ref
2
0 j 4 J
to the Eu(III) ( D → F) and Tb(III) ( D → F ). DNA binding assay
퐴퐼ref
푛
ref
of the complexes 1 and 2 were performed by UV–Vis, emission
spectroscopy and isothermal titration (ITC) and the results
obtained proves that the complexes interacts to CT-DNA by
groove binding or partial intercalation. They are also effective
towards the cleavage of pUC19 DNA through the hydrolytic
pathway. The antioxidant activity demonstrates their promising
free radical scavenging activity than free ligands and reference
standards. The complexes were non-cytotoxic upto 100 µM and
where A, I and n represents the respective absorbance at the
excitation wavelength, area under the emission spectral curve
and
refractive
index
of
the
solvent
respectively.
The ϕref represents the quantum yield of the standard quinine
sulfate solution. The lifetime measurements in excited state in
H
2
O and D
2
O allowed the determination of the coordinated water
molecules (q) directly attached to the respective lanthanide center
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
−
1
using the following modified Horrocks' equations for Eu(III) and
Tb(III)
822.18 (27.5%). FT-IR (KBr pellet, νmax, cm ): 3402 (w), 1587 (s,
−), 1528 (s, νC=O of CONH), 1440 (m), 1384 (s, νsymCO −
),
1328(m), 1115 (m), 930 (m), 817 (m), 715 (m). UV-vis (H O, 298
respectively.[19]
ν
asym CO
2
2
1
1
1
1
2
qEu=1.ꢀ (
-
- 0.ꢀ5) , qTb=5.0 (
-
- 0.06)
−1
−1
-1
-1
τHꢀO τDꢀO
τHꢀO τDꢀO
K), λmax, nm (ε, M cm ): 282 (7135 M cm ).
Fluorescence images were captured using an epifluorescence
microscope (Axiovision) attached with the ApoTome module (Carl
Zeiss, Bangalore, India).
DNA Binding Methods
Interaction of Eu(III) and Tb(III) complexes were carried out
by UV-visible absorption titration, ethidium bromide (EthB)
displacement assay, and isothermal titration calorimetry
Synthesis of N,N’-bis(3-hydroxytyramide)diethylenetriamine
(ITC) study.
3
N, N’, N’’-triacetic acid (H DTPA-DOPA)
The ligand was prepared following a modified literature
procedure and characterization data shows close resemblance
The binding study of the complexes
1 and 2 with CT-DNA
were conducted in saline-Tris buffer (5 mM, pH 7.2). The
A260/A280 ratio of 1.9 for CT-DNA in Tris buffer showed DNA
[
15]
with reported values. 3-hydroxytyramine hydrochloride (0.2 g,
.05 mmol) was added to DMF solution of
diethylenetriaminepentaacetic acid dianhydride (0.189 g, 0.52
mmol) and then stirred at 80 C under N atmosphere for 8 h. The
1
is free from proteins. The DNA concentration is calculated
-1 [41]
from the A260 value and known 260 = 6600 M . In this
assay, the complex concentration was kept constant while
varying the concentration of DNA during titration. The
2
solvent was removed by evaporation, 5 mL of distilled water was
added to the resulting compound, and pH was adjusted to 8.0 with
intrinsic interaction constant (K
 ) = [DNA]/(
where [DNA] is the CT-DNA concentration, ε
extinction coefficient of complexes, ε and ε
b
) was derived from the
 ) + 1/ K 
is the apparent
represent the
0.1 M NaOH. The solvent was further evaporated under reduced
equation [41]: [DNA]/(

a
f

b
f
b
(
b
f
)
pressure, washed with ethanol, and dried in vacuo overnight.
a
+
(
C
Yield: 0.36 g, 92%). ESI-MS in H
2
O (m/z): [M + H] calcd for
1
f
b
30
H
41
N
5
O12: 664.27; Found: 664.28. H-NMR (D
2
O), δ (ppm):
extinction coefficient of complex in free form and fully bound
form to CT-DNA. The intrinsic binding constant is calculated
2.64 (t, 4 H); 2.98 (t, 4 H); 3.10 (m, 4 H); 3.39 (m, 4 H,); 3.50 (s, 2
H); 3.56 (s, 4 H) ; 3.70 (s, 4 H); 6.56–7.12 (m, 6 H). FT-IR (KBr
-
1
1
by ratio of slope and intercept of [DNA]/(

a
f
 ) vs. [DNA] plots.
pellet, νmax, cm ): 3420 (br), 1651 (vs, νC=O of CONH ), 1529 (s,
ν
2
For DNA binding study through emission spectroscopy, ethidium
bromide (EthB) was used as emissive spectral probe in saline Tris
buffer (5 mM, pH 7.2) to calculate the apparent binding constant
of the complexes with CT-DNA. The fluorescence spectra were
recorded on excitation at 546 nm with an emission at 605 nm with
increasing concentration of the complexes. The apparent binding
C=O of CONH ), 1440 (m), 1404 (s), 1286 (m), 1120 (s), 1050 (m),
9
20 (m), 817 (m), 707 (m) (vs, very strong; s, strong; m, medium;
-
1
-1
2
br, broad). UV-vis (H O, 298 K), λmax, nm (ε, M cm ): 282 (5150
M cm ).
Synthesis of [Ln(DTPA-DOPA)(H
-
1
-1
O)] (Ln = EuIII (1), TbIII
2
(
2)): The complexes 1 and 2 were prepared by following general
procedure. H DTPA-DOPA ( 0.2 g, 0.3 mmol, pH 8) dissolved in
mL of distilled water was added dropwise to the 5 mL aqueous
solution of respective lanthanide salts, viz. Eu(NO •6H O (0.101
g, 0.3 mmol) and TbCl3•6H O (0.079 g, 0.3 mmol) at 50 C. The
constant (Kapp) was calculated from the equation:21
Kapp [C]50 =
3
K
EthB  [EthB], where [C]50 is the complex concentration when
5
emission intensity reaches half of the CT-DNA bounded ethidium
bromide emission.
)
3 3
2
2
The isothermal calorimetric titration (ITC) was performed to
study the CT-DNA interaction with Eu (III) and Tb (III) complexes
by MicroCal iTC200 system. The titration was done in
homogeneous solvent medium for both CT-DNA and complexes
reaction was continued for 6 h to obtain light yellow coloured
solution. The solution was evaporated and the desired product
was washed with diethyl ether and finally dried in a vacuum over
P
4
O10 [yield: ~80%].
ᴏ
at 30 C in saline tris buffer (5 mM, pH 7.2). The samples were
[
Eu(DTPA-DOPA)(H O)] (1): Yield: 0.213 g (81%). Anal. calcd for
2
thoroughly degassed before titrating. The sample cell was feed
with 0.02 mM CT-DNA and the complex was loaded in 40 µL
syringe at stirring rate of 1000 rpm. Titration initiated first by
C
30
H
40EuN
5
O
13: C, 43.38; H, 4.85; N, 8.43. Found: C, 43.62; H,
+
4.59; N, 8.55. ESI-MS in H
2
O (m/z): [M − H
2
O + H] calcd for
C
30
H
38EuN
5
O12 (relative abundance): 814.17 (100.0%), 812.17
baseline stability.
A titration experiment consisted of 20
(55%), 813.17 (29%), 815.17 (54%), 816.17 (19.0%). Found:
consecutive injections of 2 μL volume and 4 sec duration each,
with a filter period of 5 sec. The reference power was set at 5
μcal/sec with an initial delay of 60 sec. The resulting data were
fitted by sequential binding site model (number of sites= 2) using
8
14.17 (100.0%), 812.17 (89%), 813.17 (41%), 815.17 (54%),
−
1
816.18 (14 %). FT-IR (KBr pellet, νmax, cm ): 3410 (w), 1631
−
(s,νasym CO
2
), 1527(s, νC=O of CONH), 1441 (m), 1384
−
(s, νsymCO
2
), 1328(m), 1285 (m), 1116 (m), 930 (m), 816 (m). UV-
®
O, 298 K), λmax, nm (ε, M− cm ): 282 (8325 M cm ).
1
−1
-1
-1
MicroCal ORIGIN software supplied with the instrument.
vis (H
2
Protein Binding Assay: The interaction of proteins with complex
[
C
4
C
Tb(DTPA-DOPA)(H O)] (2): Yield: 0.185 g (78%). Anal. calcd for
2
1
and 2 was studied by quenching of fluorescence of tryptophan
30
H
40TbN
5
O
13: C, 43.02; H, 4.81; N, 8.36. Found: C, 43.41; H,
+
in saline tris buffer (5 mM, pH 7.2). Buffer containing BSA was
excited at λmax 295 to get an emission spectra at 344 nm. The
emission peak was monitored by adding the complex 1 and 2
which showed significant quenching on increasing concentration
.71; N, 8.65. ESI-MS in H
2
O (m/z): [M − H
2
O + H] calcd for
30
H
38TbN
5
O12 (relative abundance): 820.18 (100.0%), 821.18
(33.4%), 822.18 (8.5%) Found: 820.18 (100.0%), 821.18 (52.4%),
This article is protected by copyright. All rights reserved.

European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
of the complexes. The quenching constants (KSV or KBSA) for the
Cellular Viability Assay
complexes was determined from Stern-Volmer equation:[27]
I
0
/I= 1
and I denotes the fluorescence
intensities in the absence and presence of quencher of
concentration [Q], τ the average lifetime of the fluorophore in the
absence of quencher (10−8 s), k
is quenching rate constant, and
SV is the Stern–Volmer quenching constant. For static quenching
Cellular Viability assay was done using HeLa cell line
obtained from the national repository, the National Centre of
Cell Science, Pune, India. The cells were grown in
Dulbecco's modified Eagle's medium (Sigma Aldrich
Chemicals Pvt. Ltd., New Delhi, India) supplemented with
+
q 0 0
k  [Q] = 1 + KSV[Q]. Here I
0
q
K
10% (v/v) fetal calf serum, 100 units/ml penicillin and 100
interaction, both the binding constant (K) and the number of
mg/ml Streptomycin. Cells grown on gelatin coated dishes
were incubated with increasing concentrations of Eu(III) and
Tb(III) complexes for a period of 24 hours.
binding sites (n) can be determined according to the Scatchard
equation: log (I
calculated by the slope and the intercept of the double logarithm
regression curve of log(I − I)/ I versus log[Q].
0
− I)/ I = log K + n log[Q]. The n and K can be
0
Concentrations of 1, 10, 25, 50 and 100 µM were used for
each of the two compound and these were harvested along
with their experimental controls. Cell survival was assessed
Hydrolytic Cleavage of DNA: DNA cleavage assay was
performed with SC pUC19 DNA of (30 µM, 0.2 µg) by observing
its conversion from supercoiled (SC) form to nicked circular (NC)
and linear DNA forms. The experiment was executed in 50 mM
saline Tris buffer at pH 7.2 by the treatment of SC pUC19 DNA
with varying concentration of Eu(III) and Tb(III) complexes
performed by further dilution upto 20 µL by agarose gel
electrophoresis technique. The treated DNA and complex solution
using the MTT method for measuring cellular viability. Cells
were incubated with 0.5 mgml-1 3-(4,5-dimethylthiazol-2-yl)-
2
,5-diphenyltetrazolium bromide (MTT) for 2 h at 37 °C prior
to harvesting and the absorbance of the metabolic product
was measured at 570 nm using a spectrophotometer. The
absorbance was thus plotted as a measure of percentage of
cell survival.
ᴏ
was kept for incubation for varying time at 37 C and then analysis
of the cleaved DNA was done by gel electrophoresis. The
mechanistic study was performed by adding different additives
Intracellular localization Studies
HeLa cells and rat neuroblastoma cells were cultured at 37
3 2 2
DMSO (0.4 M), NaN (2 mM), glycerol (0.4 M), H O (50 µM) prior
to the addition of complexes.
°
C and incubated with complexes 1 and 2 (100 µM) for 24 h.
After 24 h, cells were fixed with 4% paraformaldehyde. For
post fixation, the nuclei were stained with 4',6-diamidino-2-
phenylindole (DAPI) reagent. The cells were then mounted
in 1,4-diazabicyclo[2.2.2]octane (DABCO) mounting medium
and processed for imaging. Fluorescence images were
captured using an epifluorescence microscope (Axiovision)
attached with the ApoTome module (Carl Zeiss, Bangalore,
India) and the images were assembled using the Adobe
Photoshop.
Antioxidant Assay
DPPH Radical Scavenging Activity: To examine the free
radical scavenging activity of the Eu(III) (1) and Tb(III) (2)
complexes, DPPH assay was done using absorption
spectroscopy. DPPH being free radical, give strong absorption
peak at 517 nm at room temperature. On interaction of DPPH with
H
3
DTPA-DOPA (0.1 mM), complexes 1 and 2 in aqueous medium,
change in absorption spectra is observed and depicted as %
reduction of the absorbance of DPPH. H
O was used as a control Acknowledgements
2
medium. The spectra were recorded after 20 and 60 minutes for
each tested compounds.BHT is used as a reference compound.
We thank Science and Engineering Research Board (SERB),
Government of India (EMR/2016/00521) and IIT Kanpur for
financial support. K. S. thank the Council of Scientific and
Industrial Research (CSIR) for research fellowship.
ABTS Radical Scavenging Activity: Stock solution of aqueous
ABTS (2 mM) on reaction with potassium persulfate (0.17 mM)
•+
produced ABTS .The complete mixture was kept in dark for 12-
4 h before being used. The reaction between potassium
1
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the compound to be tested is added and spectra was measured
after 2 minutes of mixing. The ability of the compounds for
scavenging ABTS radicals was plotted as the percentage
inhibition of the absorbance with respect to the initial ABTS
solution (ABTS %).
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European Journal of Inorganic Chemistry
10.1002/ejic.201800732
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Luminescent Ln(III)
Probe: Luminescent
lanthanide(III) complexes
of DTPA bisamide
Key Topic*
Khushbu Singh, Anshika
Goenka, Subramaniam
Ganesh and Ashis K. Patra*
Page No. – Page No.
Luminescent Eu(III) and
Tb(III) Complexes
dopamine: [Ln(DTPA-
DOPA)(H
2
O)] (Ln= Eu(III)
(
1), Tb(III) (2)) were
explored for their
Containing Dopamine
Neurotransmitter:
Biological Interactions,
Antioxidant Activity and
Cellular Imaging Studies
photophysical properties,
interactions with DNA and
BSA, DNA hydrolysis,
anti-oxidant and neuronal
cell imaging properties.
The complexes exhibit
efficient antioxidant activity
and intracellular
luminescence via via f-f
transitions in HeLa and
neuroblastoma cells
showing potential for
bioimaging applications.
This article is protected by copyright. All rights reserved.