A luminescent europium(III)-platinum(II) heterometallic complex as a theranostic agent: A proof-of-concept study

Article abstract of DOI:10.1039/c5dt04470g

A luminescent heterometallic multifunctional theranostic Eu-Pt₂ complex [{cis-PtCl₂(DMSO)}₂Eu(L)(H₂O)] has been synthesized, possessing two therapeutic Pt-centers as covalent DNA binders and one emissive Eu³⁺-center which is sensitized by platinum-based metal-to-ligand charge-transfer excited states.

Full text of DOI:10.1039/c5dt04470g

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Luminescent europium(III)-platinum(II) heterometallic complex as
theranostic agent: a proof-of-concept study†
Anirban Chandra,a, ‡ Khushbu Singh,a, ‡ Swati Singh,b Sri Sivakumarb and Ashis K. Patra*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
A luminescent heterometallic multifunctional theranostic Eu-Pt2 bioassays since they offer
complex, [{cis-PtCl2(DMSO)}2Eu(L)(H2O)] has been synthesized,
O
possessing two therapeutic Pt-centers as covalent DNA binders
and one emissive Eu3+-center which is sensitized by platinum-
S
Cl
Cl
DNA-binding site
Pt
T
based metal-to-ligand charge-transfer excited states.
E
O
N
N
N
Multifunctional cancer theranostic agents having multiple
therapeutic and diagnostic centers in a single platform gains
popularity in recent years.1-3 Recently, there are few reports
on heterometallic hairpin-shaped lanthanide-Pt2 complexes for
DNA recognition and magnetic resonance imaging (MRI) based
theranostic agents or selectively delivering gadolinium to
tumor cell nuclei.4 In this regard, theranostic Gd3+-based
platinum complexes reported4c in which Gd3+ and conjugated
cis-[Pt(NH3)2Cl]+-moieties act as MRI contrast and therapeutic
centers, respectively. These complexes showed therapeutic
efficiency along with improved MR imaging capability.
However, their DNA-binding ability is limited because of only
two available DNA-cross linking sites. In addition, the clinical
use of platinum drugs is severely affected by drug resistance
mediated by inadequate levels of platinum reaching to critical
target DNA.5 Notwithstanding with this progress, it is highly
desirable to increase the DNA binding sites that significantly
enhances platinum content at target site along with tagging of
bright luminescent center as diagnostic probe. To this end, we
report the design of multimodal targeted theranostic Eu-Pt2
conjugate possessing four DNA binding sites which can
effectively target nuclear DNA along with highly luminescent
Eu3+ center to enable interference-free live-tracking of the
drug through fluorescence microscopy. Luminescent
lanthanide complexes were widely exploited in various
O
ν
h
ν
H
f
O
-
f
E
H
H
m
i
s
s
i
o
n
N
O
Eu
MLCT Excitation
O
O
H
N
O
N
h
ν
N
O
Cl
Cl
E
T
Pt
O
DNA-binding site
S
O
Scheme 1 Molecular structure of Eu-Pt2 complex 1 showing Pt→ Eu energy transfer
pathway upon MLCT photo-excitation leading to f-f emission from Eu3+ and dissociable
Pt-Cl bonds as DNA-cross linking sites.
unique photophysical properties like narrow emission bands,
large Stokes’ shift and long-lived excited state lifetimes.6,7
Since direct excitation of the Ln3+ f-f transition is very
inefficient, chemists have designed a variety of chelating
agents conjugated to a sensitizing organic chromophore called
antenna which can transfer its excited state energy efficiently
to the emissive Ln3+ ion leading to bright luminescence
compared to the direct excitation of Ln3+ ions.8,9 Our approach
exploit the advantages of transition metal complexes as they
exhibit many desirable properties as sensitizer than organic
chromophores such as tunable absorption bands, long-lived
excited states to maximize the ET to Ln3+, excellent
photochemical stability and kinetic inertness.10,11
Herein, we demonstrate a multifunctional Eu-Pt2 complex,
[{cis-PtCl2(DMSO)}2Eu(L)(H2O)]
(1) (Scheme 1) which has
cytotoxic cis-[PtCl2(DMSO)] moieties enabling the DNA binding
whereas, EuL unit acts as luminescent reporter. Thus, a
combination of a luminescent imaging probe and a conjugated
therapeutic agent in a single hybrid 5d-4f complex can provide
real-time feedback on drug delivery, distribution and target
site localization in a non-invasive manner through fluorescence
microscopy. Other key design feature of this complex is having
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four potential DNA cross-linking sites through labile Pt-Cl fluorescence (Kapp = 4.9 x 105 M-1) , circular dichroism (CD) and
bonds, thus higher level of activated platinum reaching DNA, a isothermal titration calorimetry (ITC) (Fig. S6-S8, ESI†). ITC
possible way to lower the drug resistance. Eu-Pt2 complex
1
titration plot
was prepared in a sequential manner (Scheme S1, ESI†)
starting with a multidentate DTPA-bisamide ligand, H3L = N,N’’-
Bis(3-amidoquinolyl)diethylenetriamine-N,N’,N’’-triacetic acid
derived from acylation of 3-aminoquinoline by DTPA-
bis(anhydride). [Eu(L)(H2O)] was synthesized by reacting 1:1
molar ratio of deprotonated ligand and Eu(NO3)3⋅6H2O in
water. The [{cis-PtCl2(DMSO)}2Eu(L)(H2O)] ( ) was isolated on
1
reaction of EuL with freshly prepared cis-[Pt(DMSO)2Cl2] in 1:2
molar ratio. Detailed ESI-MS studies of Eu-Pt2 complex reveal
m/z 714.52 corresponding to {M-2Cl}2+ with matching isotopic
distribution profile unequivocally allows attribution of Eu-Pt2
formulation alongwith other physicochemical data (Fig. S1,
ESI†). We observed dissociable chloride ligands in aqueous
solution which is crucial towards forming cross-link with base
pairs in nuclear DNA.
The UV-vis spectra of
1
exhibits high energy band at 273
* transitions and a broad
* MLCT
nm are due to ligand centered
π
→π
Fig. 1 (a) UV-vis and (b) emission spectrum (λex = 330 nm) of Eu-Pt2 complex 1 in Tris-
HCl buffer. (c) Evolution of emission spectra with increasing [EuL] to a solution of
[PtCl2(DMSO)2] in DMF (λex = 330 nm), Inset: the plot of relative emission intensity at
615 nm vs. [Eu]/[Pt] ratio. (d) Luminescence decay profile from 5D0 state of Eu3+ in
complex 1 at λem = 616 nm (λex = 330 nm) (black) and in presence of CT-DNA (blue) in
Tris-HCl buffer (pH=7.2). [1] = 90 μM, [DNA] = 450 μM, delay time and gate time = 0.1
ms. The solid lines are the best fits considering single-exponential behaviour of the
decay.
band ranging from 332-350 nm corresponding to
π→π
transitions of quinoline bound Pt2+ moiety (Fig. 1a). Here we
3
have judiciously utilized this MLCT excited state as means to
populate 5D0 emissive states of europium through efficient
energy transfer. The 3MLCT
→f energy transfer through such
photosensitization is shown in few Eu-Pt complexes.12 Addition
of cis-[Pt(DMSO)2Cl2] to EuL resulted in appearance of new
suggests a biphasic sequential binding interaction13 of Eu-Pt2
with CT-DNA with an initial favorable exothermic binding event
band at 350 nm with isobestic point at ∼270 nm indicative of
formation of Eu-Pt2 complex during titration (Fig. S2 in ESI†).
1
(K1 =1.7x105 M-1,
ꢀ
H1 = -26.0 kcalmol-1), followed by a second
H2 = -77.6 kcalmol-1)
Upon excitation at MLCT band, the Eu-Pt2 complex under time-
gated mode displayed narrow emission bands spanning from
exothermic event (K2 = 9.1x105 M-1,
ꢀ
presumably due to successive sequential covalent cross-link
575-700 nm characteristic of the 5D0
→
7FJ (J = 0-4) f-f
transitions of Eu3+ (Fig. 1b). The luminescence spectra
demonstrates efficient photosensitized energy transfer from
MLCT excited state of quinoline bound Pt2+ moiety to emissive
formation with base pairs of duplex-DNA (Fig. 2a). The intrinsic
binding constant (Kb=1.5(± a
0.3) x 105 M-1) along with
hypochromism and bathochromic shift of the electronic
5D0 excited state of Eu3+ with a overall quantum yield (φoverall
)
spectral bands suggests favorable binding interaction of the
of 0.04. Spectrophotometric titration of cis-[Pt(DMSO)2Cl2]
complex
1 with DNA due to EuPt2-DNA adduct formation,
strong electrostatic interaction with activated {Eu-Pt2}n+ and
favorable stacking interactions of the two planar –Pt(L)
chromophores with planar base pairs of ds-DNA as observed in
similar hairpin shaped Eu-Pt2 complexes.4a,4b Formation of such
bis(bifunctional) platinum-DNA cross-links should induce major
unrepairable structural distortion of DNA double-helix. The
significant decrease in ellipticity in CD spectra of CT-DNA in the
with [Eu(L)(H2O)] at λex = 330 nm showed the formation of
[{cis-PtCl2(DMSO)}2Eu(L)(H2O)] (1) with gradual increase in
europium centered emission until it reached a plateau at Eu/Pt
5
=0.5 (Fig. 1c). The decay rate of emissive D0 excited state was
measured at 616 nm results in monoexponential decay curve
with a lifetime (τobs) of 0.65(±10%) ms in aqueous buffer
medium, indicating the presence of single chemical
environment. The enhancement of τobs in presence of DNA
presence of
1 (Fig. 2b) also suggests unwinding of DNA helix
(
τobs = 0.89 (±10%) ms) indicate minimization of nonradiative
and major structural deformation of DNA induced by Pt-DNA
cross-links.14 This structural distortion of DNA could be beyond
cellular DNA repair machinery and thereby inhibit transcription
and replication, triggering cell-death pathways.5 The decrease
in DNA migration rate in presence of Eu-Pt2 compared to EuL in
gel electrophoretic mobility assay using SC pUC19 DNA also
relaxation pathways in DNA bound form for Eu-Pt2 complex
(Fig. 1d).
Since DNA is the most important target for therapeutic
platinum drugs, we attempted detailed binding studies of
1
with DNA. The hairpin-shaped complex 1 gets activated by
suggest unwinding of supercoiled DNA helix by
We have observed significant enhancement of Eu-based
1
(Fig. 2 c).15
aquation through substitution of chlorides by water generating
potent cation, [Eu(L)(H2O){cis-Pt(OH2)n/2(DMSO)}2]n+ (n = 1-4),
which can readily cross-link with nucleobases of ds-DNA
(Scheme S2, ESI†). The binding interaction of Eu-Pt2 complex
with calf-thymus DNA (CT-DNA) were studied using UV-vis
titration, competitive displacement of ethidium bromide by
5
luminescence intensity originating from D0→
7FJ transitions of
Eu3+ upon addition of DNA due to efficient energy transfer to
Eu3+ and enhanced excited state lifetime in a hydrophobic
environment created due to binding of Eu-Pt2 complex with
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DNA (Fig. 2d). Serum albumin proteins constitutes a major binding propensity (KBSA = 1.50
COMMUNICATION
±
component in blood plasma proteins and plays important roles efficient transport to the pathological site (Fig. S9, ESI†).
0.03 x 105 M-1) desirable for
in drug transport and metabolism. The interaction of the Eu-
Pt2
To test our original ‘theranostic’ design, we performed in
vitro cytotoxicity assay by MTT on a human cervical carcinoma
HeLa and lung carcinoma H460 cell lines. The IC50 values for
Eu-Pt2 complex 1 are 51.0±1.05 ꢁM in HeLa cells and
30.0 1.27 M in H460 cells (Fig. 2d). It exerts anticancer
±
ꢁ
activity via extensive DNA-adduct formation through
conjugated cis-[PtCl2(DMSO)] moieties in a similar mechanism
to cisplatin.
The cellular internalization of the Eu-Pt2 complex was
investigated to probe diagnostic aspect of [Eu(L)(H2O)] utilizing
long luminescence lifetime and intrinsic luminescence from
Eu3+ by confocal fluorescence microscopy (panel A, Fig. 3).
Theranostic Eu-Pt2 complex showed significant cellular uptake
within 4 h of incubation with HeLa cells. Staining with nuclear
staining dye Hoechst 33258 demonstrate both nucleus and
cytosolic distribution of the complex (panel C, Fig. 3). The red
spots observed in some nuclei originate from luminescence
from the Eu3+ reporter tag in Eu-Pt2 theranostic conjugate (Fig.
S11, ESI†).
In conclusion, we have developed a luminescent multimodal
heterometallic Eu-Pt2 theranostic system using sensitization
and energy transfer from
a
conjugated Pt2+ based
chromophore to Eu3+. The complex shows strong binding
propensity to DNA via formation of Pt-DNA cross-links through
four potential DNA binding sites. The complex exhibit
cytotoxicity through DNA damage and nuclear localization
observed due to Eu-based f-f transition by fluorescence
imaging. Thus, such systems will have great potential towards
designing theranostic agents and delivery vehicles for cancer
chemotherapy. Further studies are on towards designing
potent lanthanide based theranostic agents as efficient drug
delivery platform and understanding mechanism of their
action.
Fig. 2 (a) ITC plot profile for the interaction of Eu-Pt2 (0.1 mM) with CT-DNA (0.01 mM)
in 5 mM Tris-HCl/NaCl buffer (pH 8.5) at 30 0C. The top panel represents the raw data
for 20 injections of 2 μL each. The bottom panel shows the amounts of heat evolved on
interaction of the Eu-Pt2 complex with CT-DNA against molar ratio of Eu-Pt2 complex to
CT-DNA. Data were fitted by sequential binding site model. (b) CD spectra of CT-DNA in
the presence of EuL and Eu-Pt2 in Tris-HCl buffer medium. (c) Agarose gel
electrophoresis of SC pUC19 DNA after incubation with EuL and Eu-Pt2 in 50 mM Tris-
HCl buffer at 37 °C for 16 h. Lane 1: DNA control, lanes 2-8 (10, 15, 20, 40, 60, 80, 100
μM complexes respectively) (d) Luminescence spectra of Eu-Pt2 (λex = 330 nm) with
addition of CT-DNA in 5 mM Tris-HCl buffer. Inset: the relative emission intensity (I/I0)
of 1 at 615 nm vs. [DNA]/[Eu-Pt2] ratio. (e) Cell viability plots showing the cytotoxicity
of Eu-Pt2 complex with HeLa and H460 cells on 16 h incubation by MTT assay.
A.K.P. acknowledges SERB, Govt. of India and IIT Kanpur for
funding. A.C. and K.S. acknowledge UGC and CSIR their
research
fellowships.
complex with bovine serum albumin (BSA) studied from
tryptophan emission quenching experiment showing high
Fig. 3 Confocal laser scanning fluorescence microscopic images of the HeLa cells treated with Eu-Pt2 complex (25 μM) on 4 h incubation and Hoechst 33258 dye (5 μg mL-1): (A)
cells incubated with the Eu-Pt2 complex; (B) incubated with the Hoechst 33258 dye for staining nucleus showing blue emission; (C) merged images showing nuclear and cytosolic
localization
of
the
complex;
(D)
DIC
images.
Scale
bar
=
20
µm.
1
(a) H. Koo, M. S. Huh, I.-C. Sun, S. H. Yuk, K. Choi, K. Kim and
I. C. Kwon, Acc. Chem. Res., 2011, 44, 1018-1028; (b) T.
Lammers, S. Aime, W. E. Hennink, G. Storm and F. Kiessling,
Acc. Chem. Res., 2011, 44, 1029-1038.
Notes and references
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2
(a) M. K. Yu, J. Park and S. Jon, Theranostics, 2012, 2, 3-44;
(b) J. D. Rocca, D. Liu and W. Lin, Acc. Chem. Res., 2011, 44
,
957-968; (c) M. Liong, J. Lu, M. Kovochich, T. Xia, S. G.
Ruehm, A. E. Nel, F. Tamanoi and J. I. Zink, ACS Nano, 2008,
2, 889-896.
3
4
A. S. Paraskar, S. Soni, K. T. Chin, P. Chaudhuri, K. W. Muto, J.
Berkowitz, M. W. Handlogten, N. J. Alves, B. Bilgicer, D. M.
Dinulescu, R. A. Mashelkar and S. Sengupta, Proc. Natl. Acad.
Sci. USA, 2010, 107, 12335-12340.
(a) P. B. Glover, P. R. Ashton, L. J. Childs, A. Rodger, M.
Kercher, R. M. Williams, L. D. Cola and Z. Pikramenou, J. Am.
Chem. Soc., 2003, 125, 9918-9919; (b) E. L. Crossley, J. B.
Aitken, S. Vogt, H. H. Harris and L. M. Rendina, Angew. Chem.
Int. Ed., 2010, 49, 1231-1233; (c) Z. Zhu, X. Wang, T. Li, S.
Aime, P. J. Sadler and Z. Guo, Angew. Chem. Int. Ed., 2014,
53, 13225-13228; (d) H. Li, R. Lan, C.-F. Chan, L. Jiang, L. Dai,
D. W. J. Kwong, M. H.-W. Lam and K.-L. Wong, Chem.
Commun., 2015, 51, 14022-14025.
5
6
7
(a) D. Wang and S. J. Lippard, Nat. Rev. Drug Discovery, 2005,
4, 307-320; (b) L. Kelland, Nat. Rev. Cancer, 2007, 7, 573-584.
S. Cotton, Lanthanides and actinides, McMillan Physical
Science Series, McMillan Education, London, 1991.
(a) J.-C. G. Bünzli, Chem. Rev., 2010, 110, 2729-2755; (b) J.-C.
G. Bünzli, and S. V. Eliseeva, Chem. Sci., 2013,
(c) E. J. New, A. Congreve and D. Parker, Chem. Sci., 2010,
4, 1939-1949;
1,
111–118; (d) E. J New, D. Parker, D. G. Smith and J. W.
Walton, Curr. Opin. Chem. Biol., 2010, 14, 238-246.
8
(a) M. C. Hefferin, L. M. Matosziuk and T. J. Meade, Chem.
Rev., 2014, 114, 4496; (b) A. J. Amoroso and S. J. A. Pope,
Chem. Soc. Rev., 2015, 44, 4723-4772; (c) S. J. Butler, M.
Delbianco, L. Lamarque, B. K. McMahon, E. R. Neil, R. Pal, D.
Parker, J. W. Walto and J. M. Zwier, Dalton Trans., 2015, 44
4791-4803.
,
9
X. Wang, H. Chang, J. Xie, B. Zhaoa, B. Liu, S. Xu, W. Pei, Na
Ren, L. Huang and W. Huang, Coord. Chem. Rev., 2014,
273/274, 201-212.
10 (a) S. Faulkner, L. S. Natarajan, W. S. Perry and D. Sykes,
Dalton Trans., 2009, 3890-3899; (b) M. D. Ward, Coord.
Chem. Rev. 2010, 254, 2634-2642; (c) F.-F Chen, Z.-Q. Chen,
Z.-Q. Bian and C.-H. Huang, Coord. Chem. Rev., 2010, 254
972-990.
,
11 (a) S. I. Klink, H. Keizer and F. C. J. M. van Veggel, Angew.
Chem., Int. Ed. 2000, 39, 4319-4321; (b) S. J. A. Pope, B. J.
Coe, S. Faulkner, E. V. Bichenkova, X. Yu, and K. D. Douglas, J.
Am. Chem. Soc., 2004, 126, 9490-9491; (c) E. Baggaley, D.-K.
Cao, D. Sykes, S. W. Botchway, J. A. Weinstein and M. D.
Ward, Chem. Eur. J. 2014, 20, 8898-8903.
12 (a) P. Kadjane, C. P.-Iglesias, R. Ziessel and L. J. Charbonnière,
Dalton Trans., 2009, 5688-5700; (b) R. Ziessel, S. Diring, P.
Kadjane, L. Charbonnière, P. Retailleau and C. Philouze,
Chem.–Asian J., 2007,
2, 975-982; (c) T. K. Ronson, T.
Lazarides, H. Adams, S. J. A. Pope, D. Sykes, S. Faulkner, S. J.
Coles, M. B. Hursthouse, W. Clegg, R. W. Harrington and M.
J. Ward, Chem. Eur. J., 2006, 12, 9299-9313.
13 (a) N. Inukai, T. Kawai and J. Yuasa, Chem.-Eur. J. 2013, 19
,
5938-5947; (b) N. Inukai, T. Kawai and J. Yuasa, Chem.
Commun. 2011, 47, 9128–9130; (c) J. Yuasa, T. Ohno, H.
Tsumatori, R. Shiba, H. Kamikubo, M. Kataoka, Y. Hasegawa,
and T. Kawai, Chem. Commun., 2013, 49, 4604–4606; (d) C.
P. Montgomery, E. J. New, D. Parker and R. D. Peacock,
Chem. Commun., 2008, 4261–4263.
14 W. C. Johnson, Circular Dichroism: Principles and
Applications, Eds. K. Nakanishi, N. Berova, R. W. Woody,
VCH, New York, 1994, pp. 523-540.
15 M. V. Keck and S. J. Lippard, J. Am. Chem. Soc., 1992, 114
3386-3390.
,
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TOC Graphics
Synopsis: In this communication, we present a luminescent heterometallic multifunctional theranostic Eu-Pt2 complex, [{cis-
PtCl2(DMSO)}2Eu(L)(H2O)], possessing two cytotoxic Pt-centers having four DNA-binding sites showing intracellular Eu-based red
luminescence sensitized by platinum based MLCT excited states.
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