Selective cytotoxicity and luminescence imaging of cancer cells with a dipicolinato-based EuIII complex

Article abstract of DOI:10.1039/c7cc06753d

Four new species [Ln(dipicNH₂)₃]^3- (Ln = La^III, Eu^III, Gd^III, Tb^III), with the ligand dipicNH₂^2- (dipic = dipicolinato), were synthesized. Incubation of the Eu^III complex with glioma NG97 and pancreatic cancer PANC1 cells showed that it penetrates the cell membrane and can be used to image the cells, while also being moderately cytotoxic.

Full text of DOI:10.1039/c7cc06753d

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Selective cytotoxicity and luminescence imaging of
cancer cells with a dipicolinato-based EuIII complex†
Cite this:DOI: 10.1039/c7cc06753d
J. H. S. K. Monteiro,ab D. Machado,c L. M. de Hollanda,c M. Lancellotti,c F. A. Sigoli*a
b
Received 28th August 2017,
Accepted 9th October 2017
and A. de Bettencourt-Dias
*
DOI: 10.1039/c7cc06753d
rsc.li/chemcomm
Four new species [Ln(dipicNH2)3]3À (Ln = LaIII, EuIII, GdIII, TbIII), with transfers it to the LnIII ion, which then emits light.19–21 For use
2À
the ligand dipicNH2
(dipic = dipicolinato), were synthesized. as labels the compounds should be water-soluble, and should
Incubation of the EuIII complex with glioma NG97 and pancreatic show high emission efficiencies.22 A wide variety of ligands23–30
cancer PANC1 cells showed that it penetrates the cell membrane has been shown to efficiently sensitize LnIII emission. Among
and can be used to image the cells, while also being moderately these are ligands based on dipicolinato; they are well known to
cytotoxic.
form water-soluble LnIII complexes with high emission efficien-
cies along with long emission lifetimes.31,32 In addition, they are
Cell luminescence imaging is of interest to aid in the character- very easy to functionalize at the para position of the pyridine
ization, in situ and in vivo, of cellular processes,1,2 or in disease ring, for example with groups that allow interaction with bio-
diagnosis.3,4 Luminescent labels, such as organic dyes,3–5 tran- molecules (such as –NCS or –NH2, as used in this study).18,33,34
sition metal complexes6 and nanoparticles,7–11 are known, yet At low concentrations most LnIII complexes show no or low
photobleaching in the case of the organic dyes, as well as short cytotoxicity, but, in some cases, at high concentrations cytotoxi-
emission lifetimes, and narrow Stokes shifts, limit their appli- city has been observed,35 and has enabled their use as cytotoxic
cation. In the case of some types of cancer, such as glioblastoma agents against OE33 (esophageal carcinoma),36 HeLa (cervical
and pancreatic cancer, both common and aggressive, early cancer)37 and B16 (melanoma).38,39 LnIII complexes which can be
detection and subsequent treatment are challenging, as many used as photoluminescent labels, but also display cytotoxicity,
luminescent labels and drugs cannot cross the blood–brain are not widely known5,10,40 and thus of interest for both therapy
barrier (BBB) and location of the tumour impedes surgical and diagnostics, and their development contributes to the
removal or there is a lack of effective drugs.12–15 Thus, the growing field of theranostics.
search for new cancer-specific imaging agents, which can, for
example, aid in cell identification pre- and post-surgery and/or complexes for use as imaging agents, as well as potential
drugs capable of crossing the BBB, is ongoing.16,17
therapeutics for glioblastomas should they display cytotoxicity,
With the goal of increasing our knowledge of luminescent
Lanthanide(III) [LnIII] ions are very attractive for application we isolated LnIII (LnIII = LaIII, EuIII, GdIII, TbIII) complexes of an
as photoluminescent labels18 due to long emission lifetimes, NH2-functionalized dipicolinato, dipicNH22À. H2dipicNH2 was
which enable time-gated detection and thus increased signal- synthesized from chelidamic acid in 33% yield,41,42 (Scheme S1,
to-noise ratio, and narrow emission bands. As the emission is ESI†) and characterized using standard techniques (Fig. S4, S6(c)
due to parity-forbidden f–f transitions, a chromophore bound and S7, ESI†). Its 3 : 1 (L : Ln) metal complexes were obtained by
to the metal ion is used as sensitizer; it absorbs energy and mixing the LnIII (Ln = LaIII, EuIII, GdIII, TbIII) oxide with H2dipicNH2
in water, adjusting the pH with M2CO3 (M = Na+, K+ or Cs+), and
purifying by re-crystallization.43 The complexes were characterized
by standard techniques (Fig. S5 and S8–S14, ESI†).
a
˜
Institute of Chemistry, University of Campinas, Sao Paulo, Brazil.
E-mail: fsigoli@iqm.unicamp.br
b Department of Chemistry, University of Nevada, Reno, NV 89557.
X-ray quality crystals of [CsK2(H2O)5(MeOH)2][Gd(dipicNH2)3]
(A) and [CsNa2(H2O)5(MeOH)][Eu(dipicNH2)3] (B) were obtained
through vapour diffusion. Both complexes crystallize in the mono-
clinic space group P21/n with cell parameters a = 9.5125(2) Å, b =
24.0522(6) Å, c = 14.1809(3) Å, b = 104.6492(15)1, V = 3139.07(12) Å3
and a = 9.4903(1) Å, b = 24.0900(3) Å, c = 14.2494(2) Å, b =
104.2850(7)1, and V = 3156.99(7) Å3, respectively. Except for
E-mail: abd@unr.edu
c
˜
Institute of Biology, University of Campinas, Sao Paulo, Brazil
† Electronic supplementary information (ESI) available: Experimental proce-
dures, NMR and FT-IR spectroscopy, mass spectrometry of ligand and complexes,
cell viability studies, confocal microscopy images and emission spectra of cell.
CCDC 1534740 and 1534741. For ESI and crystallographic data in CIF or other
electronic format see DOI: 10.1039/c7cc06753d
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log b3 = 22.4 Æ 0.3 determined for [Eu(dpa)3]3À (dpa =
dipicolinato).45 The latter species is known to dissociate upon
dilution under physiological conditions (water and pH B 7.4).45
The concentration chosen by us to obtain the spectroscopy data
and cell imaging was, at 1 Â 10À4 M and 12.5–200 mg mLÀ1
(ESI†), respectively, high enough to ensure the presence of
almost exclusively the 1 : 3 (Ln : L) species, as seen by determin-
ing the speciation from complex emission lifetimes as a func-
tion of the concentration (Fig. S17, ESI†).
The emission efficiencies f and excited state lifetimes t of the
complexes in aqueous TRIS/HCl buffered solutions are summar-
ized in Table 1 and Table S3 (ESI†). Efficiencies of B33% for
Fig. 1 (a) Plot of [Gd(dipicNH2)3]3À with thermal ellipsoids at 50% prob-
ability. Hydrogen atoms, solvated counter-cations and solvent molecules
of crystallization were omitted for clarity. (b) Tricapped trigonal prism EuIII and 24% for TbIII, respectively, were observed. These data
coordination polyhedron around the GdIII ion.
are similar to related dipicolinato-based complexes46 and com-
pare favourably with EuIII and TbIII emission in other aqueous
systems.46 The lifetimes in buffered water and D2O (Table 1)
solvent molecules of crystallization and solvated counter-cations,
confirm the absence of water molecules (q) coordinated to the
the complexes are isostructural with similar coordination environ-
metal ion and therefore that the ligand effectively prevents
ments around the LnIII. A will be discussed here representatively
vibrational quenching of the luminescence through the high
(plots of B are shown in Fig. S19, ESI†). As shown in Fig. 1a, the
energy O–H oscillators,47 resulting in high intrinsic quantum
metal ion is bound to six oxygen and three nitrogen atoms of
yields (fLLnn) and emission lifetimes of 1.2 and 1.5 ms, respec-
three dipicNH22À ligands for a coordination number of 9 with a
tively. The emission lifetimes of B1.48 ms of the TbIII complexes
tricapped trigonal prism geometry with pseudo-D3 symmetry
are slightly lower than the B1.58 ms reported by Lamture and
(Fig. 1b), similar to analogous dipicolinato-based complexes.
co-workers,48 yet these authors used a different experimental
Eu–O distances, in the range 2.437–2.462 Å, Eu–N distances, in
method for the measurement and did not report the experi-
the range 2.482–2.523 Å, and Gd–O and Gd–N distances, in the
mental error. The favourable position of the singlet and triplet
ranges 2.423–2.451 and 2.473–2.516 Å, respectively, are similar
states of the ligand and short donor–acceptor distance (RL,
to analogous reported complexes.43,44
Table S3, ESI†),44 results in high sensitization efficiencies Zsens
Aqueous solutions of the potassium salts of the EuIII and
for the EuIII complexes (Table 1 and Table S3, ESI†).
TbIII complexes display the characteristic metal-centred emis-
Cancerous cells and fibroblasts were incubated for 48 h hours
sion spectra (Fig. 2 right) with transitions 5D0 - 7FJ (J = 0–4) for
with aqueous solutions of H2dipicNH2 and K3[Ln(dipicNH2)3]
5
7
EuIII (Fig. 2(a)) and D4 - FJ (J = 6–0) for TbIII (Fig. 2(b)). The
excitation spectra (Fig. 2, left) closely resemble the absorption
spectra (Fig. S16, ESI†), indicating that the metal-centred emis-
sion is sensitized by the ligand. The stability constants (log b)
determined for the 1 : 1, 1 : 2 and 1 : 3 species (Eu : dipicNH2) are
7.2 Æ 0.1, 15.0 Æ 0.1, and 22.4 Æ 0.3, respectively (Fig. S18,
ESI†). The value for the 3 : 1 species is comparable with
(Ln = LaIII, EuIII, TbIII) (ESI†). In vitro cytotoxicity studies show
that H2dipicNH2, and the LnIII triflate and LnIII chloride salts do
not affect the viability of NG97 (brain) and PANC-1 (pancreatic)
cancer cells. However, the LnIII complexes greatly reduce their
viability from 100 to B50% (Fig. S20–S23, ESI†). As a proof of
concept, NIH/3T3 fibroblasts were used to represent a generic
non-cancerous cell.49–51 For these cells, none of the species,
metal salts, ligands or the metal complexes, was cytotoxic.
The cancer cells incubated with K3[Eu(dipicNH2)3] showed red
emission (Fig. 3 and Fig. S24 for NG97 and Fig. S25 (ESI†) for
PANC-1), while NIH/3T3 fibroblasts do not show red lumines-
cence (Fig. S26, ESI†), which we attribute to lack of penetration of
the complexes into the latter, non-cancerous cells. The analogous
TbIII complex was not used for imaging due to overlap of the
emission with cell auto-fluorescence.
High resolution emission spectra of the cells incubated with
the complexes could only be obtained by confocal Raman
spectroscopy, due to the small amount of cells available, and
show the characteristic peaks of the f–f transitions (Fig. 4 for
NG97 and Fig. S28 (ESI†) for PANC-1), confirming that the
emission is from EuIII. For the fibroblasts only very low inten-
sity metal-centred emission peaks are seen, confirming that the
complex does not penetrate the cell membrane (Fig. S27, ESI†).
The difference in peak splitting between the emission spectra
of the complex in the cells and in solution (Fig. 2 and 4) is
Fig. 2 Excitation (left) and emission spectra (right) in aqueous TRIS/HCl
buffer (pH B 7.4) of [Ln(dipicNH2)3]3À. (a) Ln = EuIII, (b) Ln = TbIII. (lexc
300 nm and lem = 615 nm, for EuIII, or 524 nm, for TbIII).
=
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Table 1 Singlet (1S) and triplet (3T) state energies of the ligand in the GdIII complex, quantum yield (f), intrinsic emission efficiency (fLLnn), sensitization
efficiency (Zsens), lifetime (t) indicated with standard deviations and water molecules (q) in the first coordination sphere obtained for the complexes in
aqueous TRIS/HCl or TRIS/DCl buffered solutions (pH B 7.4)
Ln
Ln
Complexes
1S [cmÀ1
]
3T [cmÀ1
]
f [%]
f
[%]
Zsens [%]
tH O [ms]
tD O [ms]
q
2
2
K3[Eu(dipicNH2)3]
K3[Tb(dipicNH2)3]
26 200 Æ 150
22 080 Æ 130
33 Æ 2
24 Æ 1
55
—
60
—
1.210 Æ 0.001
1.477 Æ 0.001
2.282 Æ 0.002
1.771 Æ 0.005
0.1
0.5
while solutions of the LnIII salts and of the ligand are not. Further,
as evidenced by unchanged emission lifetimes and mono-
exponential decays, the 3 : 1 species do not undergo decom-
plexation in the presence of the biologically relevant cations
CaII, ZnII and FeIII (Table S2, ESI†).53
To evaluate the K3[Eu(dipicNH2)3] complex as possible
therapeutic and luminescent dye for glioblastomas, we tested
its ability to cross the BBB.54 The barrier was simulated according
to a previously described procedure.54,55 After 24 hours of simula-
tion (Fig. S29, ESI†), the NG97 cells exhibit EuIII-centred lumines-
cence, indicating that the complex crosses the simulated barrier.
In summary, an easily accessible ligand derived from
dipicolinic acid with the functional group –NH2, H2dipicNH2,
was synthesized and its 3 : 1 (L : Ln) LnIII ion complexes isolated
and characterized. The EuIII and TbIII complexes showed efficient
metal-centred luminescence in aqueous solution at physiological
pH and stability of the 3: 1 species at the concentrations used.
The complexes showed elevated cytotoxicity against NG97 and
PANC-1 cancer cells, but not the healthy NIH/3T3 cells. Owing to
its high emission efficiency in the easily accessible red region of
the spectrum and ability to transfect the cell membrane of the
cancerous cells chosen, K3[Eu(dipicNH2)3] was used as lumines-
cent dye for the NG97 and PANC-1 cells. Finally, this complex was
able to cross a simulated BBB. The results shown here indicate
the promise of this type of LnIII complexes for use as theranostic
agents; they show the characteristic metal-centred luminescence
within cancer cells, and, albeit moderate, selective cytotoxic
towards those cells.
Fig. 3 Confocal imaging of the NG97 cells after incubation with
K3[Eu(dipicNH2)3] in (a) absence of complex and after an incubation time
of (b) 12 and (c) 24 h. The first column is the bright field image, the second
is the luminescence and the third is the overlay between first and second
columns (K3[Eu(dipicNH2)3] = 200 mg mLÀ1, lexc = 405 Æ 10 nm).
Financial support from NSF-CHE 1363325 (AdBD), FAPESP
08/53868-0 (FAS), CNPq 303433/2014-0 (FAS and AdBD), FAPESP
2012/15064-3 (ML) is gratefully acknowledged. The authors
thank Prof. Carlos Lenz Cesar, for access to equipment and
assistance provided by INFABIC at the University of Campinas.
Conflicts of interest
There are no conflicts to declare.
Notes and references
Fig. 4 Emission spectra of the NG97 cells in (a) absence of complex and
(b) after incubation for 24 h. (K3[Eu(dipicNH2)3] = 200 mg mLÀ1, lexc = 488 nm).
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