Journal of Inorganic Biochemistry 178 (2018) 32–42

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Journal of Inorganic Biochemistry

journal homepage: www.elsevier.com/locate/jinorgbio

Synthesis, photophysical and cellular characterisation of folate and T

methotrexate labelled luminescent lanthanide complexes
Zhangli Dub,1, Jing Sund,1, Christie A. Baderb, Doug A. Brooksb, Minqi Lid, Xun Lia,⁎,
Sally E. Plushb,c,⁎⁎
a
Department of Medicinal Chemistry, Key Laboratory of Chemistry and Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences, Shandong
University, Ji'nan, Shandong, P.R. China
b
Mechanisms in Cell Biology and Diseases Research Group, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research, University of South Australia,
Adelaide, Australia
c
Future Industries Institute, University of South Australia, Mawson Lakes, SA 5095, Australia
d
Department of Bone Metabolism, School of Stomatology, Shandong University, Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Ji'nan, P.R. China

A B S T R A C T

In this work we have developed a series of highly emissive europium(III) and terbium(III) complexes tethered to
either folic acid (FA) or methotrexate (MTX), with the aim of developing visual probes that enable the imaging of
folate receptors in cancer cells. The synthesis, photophysical properties and cellular behaviour are reported for
four new lanthanide Ln(III) complexes, where either FA or MTX are tethered to 1,4,7-tris(carbonylmethyl)-10-
(4′-quinolineacetic acid, (7′-acetamido)-1′,2′-dihydro-2′-oxo)-1,4,7,10-tetraazacyclododecane Ln(III) complex,
and Ln(III) = Eu(III) or Tb(III); herein referred to as Eu-FA, Eu-MTX, Tb-FA or Tb-MTX. All four complexes
were found to be sensitive to the presence of the folate receptor in a range of cell lines. The MTX conjugates
showed different cellular specificity in an oral adenosquamous carcinoma cell line (CAL-27) compared with the
analogous FA conjugates. This suggests that it is viable to explore differences in folate receptors using folate vs.
anti-folate probes, with labels that have different emissive properties (e.g. Eu-FA vs. Tb-MTX). The MTX
complexes were found to be the most cytotoxic, with Eu-MTX showing greater cytotoxicity than free MTX or the
isostructural Tb-MTX. This suggested that there could be a synergistic effect on toxicity for the Eu(III) chelate
and the MTX components of the complex.

1. Introduction
Folates (also called vitamin B9, folacin, pteroyl-L-glutamic acid, or
pteroyl-L-glutamate) are essential for the maintenance of the human
genome and cell health, due to their central role in key metabolic
functions, such as RNA and DNA biosynthesis [1,2]. The critical role of
folates in DNA synthesis, repair and methylation [3] has been mainly
attributed to the reduced tetrahydrofolate form [4], which has an im-
portant function in rapid cell division and growth. Reduced amounts of
folate have been associated with the onset of cancer, including color-
ectal [5], breast [6] and lung cancer, presumably due to increased DNA
damage and an enhanced mutation rate [7]. Folate can be internalised
into cells via at least three transport mechanisms: the reduced folate
carrier (RFC) [8], which is an anion exchanger and mainly transports
reduced folate; the proton-coupled folate transporter (PCFT) [9], which
can transport folate in an acidic environment; and the folate receptors
(FRs) [10], which have high affinity and can transport folic acid (FA)
into cells via endocytosis [11,12].
In contrast to the RFC and PCFT, which are ubiquitously expressed
in both tumour and normal tissues [13], the FRs usually have minimal
expression in normal tissue, but increased expression in malignant
cells/tissues, including, colorectal, ovarian, breast, lung, cervical, renal,
kidney, brain and nasopharyngeal carcinomas; and this increased ex-
pression has been linked with tumour progression [14–16]. This, makes
the FR a potential target for cancer diagnosis/therapy; [17,18] where
FA (small and stable over broad range of temperatures and pH) can be

⁎
Correspondence to: Dr. Xun Li, Department of Medicinal Chemistry, Key Laboratory of Chemistry and Chemical Biology (Ministry of Education), School of Pharmaceutical Sciences,
Shandong University, Ji'nan, Shandong, P.R. China.
⁎⁎
Correspondence to: Dr. Sally E. Plush, Mechanisms in Cell Biology and Diseases Research Group, School of Pharmacy and Medical Sciences, Sansom Institute for Health Research,
University of South Australia, Adelaide, Australia.
E-mail addresses: tjulx2004@sdu.edu.cn (X. Li), sally.plush@unisa.edu.au (S.E. Plush).
1
These two authors contributed equally to this work.

http://dx.doi.org/10.1016/j.jinorgbio.2017.10.003
Received 11 August 2017; Received in revised form 19 September 2017; Accepted 8 October 2017
Available online 10 October 2017
0162-0134/ Crown Copyright © 2017 Published by Elsevier Inc. All rights reserved.
Z. Du et al.
used as a biocompatible and non-immunogenic targeting motif to
covalently conjugate with an optical imaging agent or a therapeutic
[19]. Anti-folates, such as methotrexate (MTX) [20], have been directly
exploited as therapeutic agents [21]. MTX was first synthesised in the
1940's [22] and is still extensively used in the treatment of tumours,
including acute lymphocytic leukaemia [23], breast [24], head and
neck cancers [25]. It is also used in certain autoimmune diseases [26].
MTX competitively inhibits dihydrofolate reductase, a key step in the
reduction of folate to its tetrahydrofolate form, inhibiting DNA and
RNA synthesis to cause cell apoptosis [27]. Folates and anti-folates can
also be used as targeting agents and this has been successfully employed
in a wide variety of imaging applications, with clinical trials in humans
underway for magnetic resonance imaging (MRI), computed tomo-
graphy (CT) and positron emission tomography (PET) imaging of solid
tumours [28,29]. Fluorescent agents tethered to FA have been in-
vestigated for intra-operative identification of malignant disease [30].
In contrast to the conventional modalities of MRI, CT and PET, optical
imaging agents offer sub-micrometre spatial resolution, allowing for
rapid results at the sub-cellular level [31]; where early stage biological
changes can be more easily detected using low cost and more accessible
instrumentation. This opens opportunities for the non-invasive diag-
nosis of cutaneous and subcutaneous cancers and for example endo-
scopic diagnosis of oesophageal malignancies, where the issues of tissue
transparency are usually negated [28]. Furthermore, the use of imaging
agents for microscopy will allow cell biologists working in the field of
cancer to investigate and monitor changes at a cellular level prior to,
during and post intervention, in real time. To achieve these important
imaging outcomes, there is a need to be able to monitor the optical
signal for long periods of time, without photo-bleaching and to be able
to differentiate the signal from endogenous fluorescence. Consequently,
new imaging technology is required to address this unmet need.
Luminescent lanthanide (Ln) complexes are particularly attractive
as imaging agents, due to their distinctive photo-physical properties
[32]. They offer extended resistance to photobleaching, have large
Stokes shifts (large separation between absorption and emission wa-
velengths) that avoid concentration-dependant self-absorption pro-
blems, and exhibit long luminescence lifetimes (range 1 μs to 5 ms) that
enable time gated measurement of luminescence to avoid either non-
specific background or specific auto-fluorescence. Furthermore, com-
pared with fluorescent bands (> 200 nm bandwidths), lanthanides
have sharp emission bands (10–20 nm bandwidths) that do not overlap
with one another, resulting in optimal signal to noise ratios [33].
Lanthanides have an advantage over other luminescent metal ions, as
they are considered to be isostructural, which means that the emission
range can be tuned by ‘swapping’ the metal, using the same synthetic
strategy to deliver complexes with theoretically similar physiochemical
and biological targeting properties, but with different wavelength
emissions [34]. One key consideration with lanthanide ions is
their intrinsically low extinction coefficients (in the order of
0.5–3 M− 1 cm− 1), which necessitates incorporating an antenna group
into the lanthanide complex, to effectively populate the lanthanide's
excited state [35]. Although a wide range of lanthanide complexes have
been evaluated for the optical imaging of cells, most notably by Parker
and co-workers [36–39], further development is required to establish a
true structure activity relationship (SAR) and to determine the critical
parameters for the design and optimisation of these imaging agents.
This design process may be further complicated in the case of folates as
targeting groups, as there are differences in cellular uptake efficiency
and receptor binding affinities for substituted analogues of folic acid
[40–43].
We and others have recently explored the relationship between the
folate moiety (FA consists of three moieties; pterin head group, p-
aminobenzoate and glutamate residue), the length of the linker be-
tween the folate and lanthanide complex, and the site of conjugation of
the lanthanide complex; and the effects upon cellular uptake and lu-
minescent emission for folate targeted Eu(III) conjugates [44,45]. This
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Journal of Inorganic Biochemistry 178 (2018) 32–42
has shown that each portion of FA is critical and that when a short
linker is used to tether a luminescent Eu(III) complex using the γ-car-
boxylic acid of FA, higher cellular uptake is achieved [44]. However, to
be optimal for live cell imaging, these complexes need to be highly
emissive and excitable within biologically compatible wavelengths.
In this study, we report the synthesis, photophysical and cellular
properties of four new imaging agents, Eu-FA, Eu-MTX, Tb-FA and Tb-
MTX, which each consists of a highly luminescent antenna complex,
covalently conjugated using a short linker, to one of the carboxylic acid
groups present in either FA or MTX (Fig. 1). Both Eu(III) and Tb(III)
complexes were explored to evaluate any differences in emission and to
compare the uptake/targeting for these two isostructural ions. It is
important to ascertain how changes in the core metal may influence the
potential to develop near infrared emitting complexes or even to use Gd
(III) as an effective MRI contrast agent. We also aimed to explore how
uptake, cytotoxicity and emission varied in a range of cells when folate
(in the form of FA) was substituted for the anti-folate, MTX. Differences
in the uptake and cytotoxicity of FA and MTX have implications for the
targeting and potential treatment of cancer, as FA is often given as an
antagonist drug to lessen and limit side effect toxicity (e.g. non-tumour
toxicity) during MTX treatment. Therefore, it is important and mean-
ingful that cell biologists are equipped with tools that have the potential
to clearly visualise how these two analogues behave in cells, to develop
better strategies for cancer treatment.
2. Experimental
2.1. General materials and methods
1,4,7,10-tetraazacyclododecane (cyclen) was obtained commer-
cially from Strem, USA. All other solvents and chemicals used were
obtained from Sigma-Aldrich or Merck, Australia. Sephadex G10 resins
were purchased from Sigma-Aldrich, Australia. Thin layer chromato-
graphy (TLC) was performed on silica gel 60 F254 plates obtained from
Merck, Australia. The infrared spectroscopy (IR) was recorded on a
Shimadzu FTIR-8400S. Electrospray ionization mass spectrometry (ESI-
MS) was recorded using a Perkin-Elmer® Scoex API 3000. 1H NMR and
13
C NMR were recorded on a Bruker 500 MHz NMR spectrometer. All
chemical shifts are given in ppm with coupling constants in Hz. All pH
measurements were conducted on an Orion Ross pH meter. Optical
spectroscopy experiments were recorded in 100% water at constant
ionic strength (I = 0.01 (NaCl)) using a Varian CARY 50 UV–Vis
spectrophotometer or a Varian Cary Eclipse spectrophotometer at room
temperature. High pressure liquid chromatography (HPLC) was per-
formed on a Shimadzu LC-20AD with manual injection fitted with a
60 Å, 4 μM Nova-Pak phenyl analytical column (3.9 × 150 mm) with a
flow rate of 0.8 mL/min and was analysed using a Shimadzu SPD-20A
detector at 280 nm. Linear elution was used with mobile phases A (H2O
+ 0.1% TFA) and B (CH3CN + 0.1% TFA); 15%–60% over 10 min.
Elemental analysis was conducted in Campbell Microanalytical
Laboratory, University of Otago.
2.2. Synthesis
2.2.1. 2,2′,2″-(10-(2-(4-(2-(2-(tert-butoxycarbonylamino)ethylamino)-2-
oxoethyl)-2-oxo-1,2-dihydroquinolin-7-ylamino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid.Eu (Eu.3)
Compound Eu.1 [46] (131 mg, 0.17 mmol) was dissolved in DMSO
using sonication (170 W, 30 min), after which DIPEA (45 mg,
0.35 mmol) was added. The reaction mixture was then stirred for
10 min. Then HOBt (27 mg, 0.20 mmol) and BOP (159 mg, 0.36 mmol)
were added. Following this, a solution of tert-butyl 2-ami-
noethylcarbamate (60 mg, 0.37 mmol), DIPEA (23 mg, 0.18 mmol) in
DMSO (2 mL) was added. The reaction mixture was then left to stir at
room temperature for 24 h. The crude product was then precipitated
out by the addition of diethyl ether/acetone (10 mL, 7/3 (v/v)). The
Z. Du et al.
Fig. 1. The chemical structures of folic acid (FA), methotrexate (MTX) and four lanthanide complexes; Eu-FA, Tb-FA, Eu-MTX and Tb-MTX (all lanthanide complexes displayed as γ-
isomer).
precipitate was collected by centrifugation at 8300g for 5 min and used
in the next step without any further purification. Yield: 127 mg (83%).
IR (KBr) v = 3433 (br), 2978, 2920, 2870, 1616 (br), 1389, 1319,
1281, 1246, 1165, 1084, 1018 cm− 1. 1H NMR (D2O, 500 MHz): δ
33.92, 30.87, 29.66 (d), 11.87 (br), 10.74 (br), 9.02 (br), 7.81 to 0.32
(m), −1.07, − 3.08, −3.56, − 3.89, −4.99, − 6.68, 8.06 (d),
− 10.42, −12.12 (d), − 13.52 (d), − 14.63, − 15.94 (d), − 17.24. ESI-
MS+: m/z = 897.4 (M + 1)+.
2.2.2. 2,2′,2″-(10-(2-(4-(2-(2-aminoethylamino)-2-oxoethyl)-2-oxo-1,2-
dihydroquinolin-7-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic acid.Eu (Eu.5)
Compound Eu.3 (170 mg, 0.19 mmol) was dissolved in a DCM/TFA
(5 mL, 1/1 (v/v)) solution and stirred at room temperature overnight.
The solvent was then removed under reduced pressure to yield a brown
residue. The residue was then redissolved in water (8 mL) and the pH of
the solution adjusted to 7 using 1 M NaOH. The solvent was removed by
reduced pressure to yield the crude product. The crude product was
purified by a Sephadex G10 column in CH3OH/H2O (1/1) to yield a
yellow crystal. Yield: 133 mg (88%). M.p. > 300 °C. IR (KBr):
v = 3414 (br), 3063, 2986, 2916, 2870, 1597 (br), 1404, 1323, 1281,
1242, 1204, 1177, 1130, 1084, 1018 cm− 1. 1H NMR (D2O, 500 MHz): δ
34.08 (d), 30.84 (br), 29.46 (br), 11.90 (br), 7.80–0 (m), − 0.36,
− 1.02, − 3.03 (br), − 3.97, −4.93, − 8.10 (m), − 10.34, −11.73,
− 12.13, −12.95, −13.44, − 14.33, − 14.66, − 15.91 (d), −17.26.
ESI-MS+: m/z = 797.5 (M + 1)+.
2.2.3. Eu-FA
Folic acid (48 mg, 0.11 mmol) was dissolved in DMSO (4 mL).
DIPEA (28 mg, 0.21 mmol) was added, followed by the sequentially
addition of HOBt (15 mg, 0.11 mmol) and BOP (53 mg, 0.13 mmol).
The reaction mixture was stirred for 5 min, then was mixed with Eu.5
(88 mg, 0.11 mmol) together with DIPEA (14 mg, 0.11 mmol) in DMSO
(1 mL). The mixture was stirred for 24 h at room temperature and
34
Journal of Inorganic Biochemistry 178 (2018) 32–42
protected from light. The product was then precipitated by adding
diethyl ether/acetone (10 mL, 7/3 (v/v)) which was collected after
centrifugation of the suspension at 8300g for 5 min. The residue was
dried, then rinsed with CH3OH (3 mL). The product was collected as a
yellow powder. Yield: 110 mg (82%). M.p. > 300 °C. TLC: silica iso-
propanol/ammonia (5 M) (7/3), Rf 0.1. IR (KBr): v = 3410 (br), 2986,
2920, 2866, 1605 (br), 1389, 1323, 1277, 1242, 1184, 1084,
1018 cm− 1. Calculated for C48H56EuN15O14 ⋅3H2O ⋅2CH3SOCH3: C,
43.70; H, 5.22; N, 14.70. Found: C, 43.61; H, 5.36; N, 14.55. 1H NMR
(DMSO-d6, 500 MHz): δ 41.66, 38.83, 33.59, 32.11, 30.63, 18.92 (br),
15.35 (br), 13.01, 12.27, 10.76, 10.60, 9.26 to 1.23 (m), −1.32, − 3.59
(d), − 4.54, − 6.57, − 7.59 (d), − 10.46, −13.86 (d), −15.78,
− 17.39 (m), − 19.72 (br), −23.48. ESI-MS−: m/z = 1218.8 (M-1)−.
HPLC: t = 4.1 min, 4.4 min.
2.2.4. 2,2′,2″-(10-(2-(4-(2-(2-(tert-butoxycarbonylamino)ethylamino)-2-
oxoethyl)-2-oxo-1,2-dihydroquinolin-7-ylamino)-2-oxoethyl)-1,4,7,10-
tetraazacyclododecane-1,4,7-triyl)triacetic acid.Tb (Tb.4)
Compound Tb.4 was prepared using the protocol described to
synthesise Eu.3, except that Tb.2 (see ESI for synthesis) (193 mg,
0.25 mmol) was used to react with tert-butyl 2-aminoethylcarbamate
(85 mg, 0.53 mmol) instead of Eu.1. Yield: 187 mg (83%).
M.p. > 300 °C. IR (KBr) v = 3433 (br), 2982, 2920, 2874, 1609 (br),
1389, 1323, 1281, 1246, 1165, 1084, 1018 cm− 1. 1H NMR (D2O,
500 MHz): δ 260.40, 252.39, 226.19, 136.21, 127.27, 117.41, 82.59 50
to − 50 (br), − 52.08, − 63.48, −66.56, − 73.65, − 95.84, −99.23,
− 102.00, − 113.40, −128.81, −144.22, −351.92, − 369.80,
− 400.00, − 409.86. ESI-MS+: m/z = 903.5 (M + 1)+.
2.2.5. 2,2′,2″-(10-(2-(4-(2-(2-aminoethylamino)-2-oxoethyl)-2-oxo-1,2-
dihydroquinolin-7-ylamino)-2-oxoethyl)-1,4,7,10-tetraazacyclododecane-
1,4,7-triyl)triacetic acid.Tb (Tb.6)
Compound Tb.6 was prepared using the protocol described for the
synthesis of Eu.5 except that Tb.4 (207 mg, 0.23 mmol) was stirred in a
Z. Du et al.
solution of DCM/TFA (5 mL, 1/1 (v/v)) overnight instead of Eu.3. The
product was isolated as a yellow crystals. Yield: 133 mg (71%).
M.p. > 300 °C. IR (KBr): v = 3433 (br), 2990, 2916, 2878, 1620 (br),
1400, 1319, 1281, 1242, 1169, 1084, 1022 cm− 1. 1H NMR (D2O,
500 MHz): δ 259.78, 249.3, 233.28, 223.11, 135.90, 126.04, 116.79,
82.90, 50 to − 50 (br), − 52.39, − 63.48, − 67.49, − 73.04, − 95.53,
− 99.54, − 101.39, −113.40, − 128.20, − 143.30, −351.61,
− 369.49, − 398.46, − 408.62. ESI-MS+: m/z = 803.4 (M + 1)+.
2.2.6. Tb-FA
Compound Tb-FA was prepared using the procedure described for
Eu-FA except that Tb.6 (95 mg, 0.12 mmol) was used instead of Eu.5 to
react with folic acid (52 mg, 0.12 mmol), HOBt (17 mg, 0.13 mmol),
BOP (57 mg, 0.13 mol) and DIPEA (46 mg, 0.36 mmol) in DMSO
(5 mL). The final product was collected as a yellow powder. Yield:
95 mg (65%). M.p. > 300 °C. TLC: silica isopropanol/ammonia (5 M)
(7/3), Rf 0.1. IR (KBr): v = 3422 (br), 2990, 2920, 2874, 1605 (br),
1400, 1319, 1242, 1184, 1126, 1084, 1003 cm− 1. Calculated for
C48H56TbN15O14 ⋅ 3H2O⋅ 2CH3SOCH3: C, 43.48; H, 5.19; N, 14.63.
Found: C, 43.68; H, 5.31; N, 14.64. 1H NMR (DMSO-d6, 500 MHz): δ
284.74, 232.97, 155.62, 145.76, 50 to − 50 (br), −73.04, −102.62,
− 117.10, −156.86, − 183.67, − 345.45, −363.63. ESI-MS−: m/
z = 1224.7 (M-1)−. HPLC: t = 4.1 min, 4.4 min.
2.2.7. Eu-MTX
Methotrexate (54 mg, 0.12 mmol) was dissolved in DMSO (4 mL) and
DIPEA (31 mg, 0.26 mmol) added, followed by the sequentially addition
of HOBt (18 mg, 0.13 mmol) and BOP (56 mg, 0.13 mmol). The reaction
mixture was stirred for about 5 min, then was mixed with Eu.5 (96 mg,
0.3 mmol) together with DIPEA (16 mg, 0.12 mmol) in DMSO (5 mL).
The mixture was stirred for 24 h at room temperature in the dark. The
product was then precipitated by the addition of ethyl ether/acetone
(10 mL, 7/3 (v/v)) which was collected by centrifugation (8300 g,
5 min). The residue was dried and washed by CH3OH (3 mL). The pro-
duct was collected as a yellow powder. Yield: 95 mg (64%).
M.p. > 300 °C. TLC: silica isopropanol/ammonia (5 M) (7/3), Rf 0.1. IR
(KBr): v = 3426 (br), 2990, 2920, 1605 (br), 1385, 1319, 1242, 1200,
1084, 1022 cm− 1. Calculated for C49H59EuN16O13 ⋅H2O⋅5CH3SOCH3: C,
43.19; H, 5.59; N, 13.66. Found: C, 43.26; H, 5.59; N, 13.89. 1H NMR
(DMSO-d6, 500 MHz): δ 41.54, 38.95, 33.65, 32.30, 30.63, 18.98, 15.53,
12.33, 10.79, 8.60-0 (m), −1.17, −3.45 (d), −4.47, −6.56, −7.55,
−10.42, −13.84, −17.29 (m), −19.82. ESMS−: m/z 1231.8 (M-1)−.
HPLC: t = 4.3 min, 4.6 min.
2.2.8. Tb-MTX
Tb-MTX was prepared through the same method that was used to
prepare Eu-MTX except Tb.6 (70 mg, 0.087 mmol) was used to react
with methotrexate (40 mg, 0.087 mmol), HOBt (14 mg, 0.10 mmol), BOP
(44 mg, 0.10 mmol) in DMSO (5 mL). Yield: 72 mg (67%). TLC: silica
isopropanol/ammonia (5 M) (7/3), Rf 0.1. M.p. > 300 °C. IR (KBr):
v = 3422 (br), 2990, 2916, 2882, 1605 (br), 1385, 1319, 1242, 1200,
1084, 1022 cm− 1. Calculated for C49H59Tb.N16O13 ⋅H2O⋅4CH3SOCH3:
C, 43.62; H, 5.46; N, 14.28. Found: C, 43.57; H, 5.50; N, 14.09. 1H NMR
(DMSO-d6, 500 MHz): δ 295.84, 208.32, 167.64, 153.16, 143.60, 93.07,
50 to −50 (br), −72.73, −102.31, −118.03, −156.55, −183.36,
−347.30, −365.79, −429.27. ESMS−: m/z 1238.0 (M-1)−. HPLC:
t = 4.3 min, 4.6 min.
2.3. Spectroscopic methods
All pH measurements were conducted using an Orion Ross pH
meter. Deionised water; that had been purified with the MiliQ-Reagent
system to produce water with a specific resistance of > 18.2 MΩ cm− 1,
then boiled for 30 min to remove CO2 and cooled under a drying tube
filled with soda lime; was used to prepare all aqueous solutions. All
solutions were prepared freshly prior to measurement. Samples of the
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Journal of Inorganic Biochemistry 178 (2018) 32–42
complexes were first dissolved in NaH2PO4.2H2O (20 mM) solution and
then further diluted with water (I = 0.01 (NaCl)). Optical spectroscopy
experiments were recorded in 100% water at constant ionic strength
(I = 0.01 (NaCl)) using a Varian CARY 50 UV–Vis spectrophotometer
or a Varian Cary Eclipse spectrophotometer at room temperature. The
UV–Visible absorbance spectra were recorded over the wavelength
range of 250–450 nm with a scan rate of 600 nm/min. Baseline cor-
rection measurements were used for all spectra with a blank containing
0.01 M NaCl. For the fluorescence emission spectra data was obtained
between approximately 400–600 nm with both excitation slit and
emission slit widths at 10 nm. The concentrations of the samples were
the same as that used for the UV–Vis absorbance measurements. The
excitation wavelength was 355 nm. The Ln(III) emission spectra were
recorded on a Varian Cary Eclipse spectrophotometer. The data was
obtained between approximately 550–750 nm for Eu(III) complexes
and 450–650 nm for Tb(III) complexes with both the excitation and
emission slit widths at 10 nm. The concentrations of the samples were
the same as that used for the UV–Vis absorbance measurements. The
excitation wavelength was 355 nm. Luminescent lifetime measure-
ments were performed on a Varian Cary Eclipse spectrophotometer
with a sample dissolved in a minimal amount of NaH2PO4·2H2O
(20 mM) or of NaD2PO4 (20 mM) solution and then further diluted to a
concentration of 10− 5 M (Eu(III) complexes) or 0.5 × 10− 5 (Tb(III)
complexes) in either H2O (3 mL) or D2O (3 mL). The measurement of
emission was at 615 nm for Eu(III) complexes and 545 nm for Tb(III)
complexes. The PMT voltage was set at 550 V.
2.4. Epi-fluorescence imaging
2.4.1. Cell culture
Cells used in the assay, including HeLa (human cervical cancer
cells), 293 T (human embryonic kidney cells), MDA-MB-231 (human
breast cancer cells), U251 (human glioma cells), RAW264.7 (murine
macrophage cells), A549 (human lung epithelial cells), PC-3 (human
prostate cancer cells), U-2 OS (human osteosarcoma cells), Cal-27
(human tongue squamous carcinoma cells), MLO-Y4 (human normal
osteocyte cells), ST2 (mouse mesenchymal stem cells), MC3T3-E1
(mouse pre-osteoblast cells), HL7702 (human normal liver cells) were
commercially available from the Shanghai Cell Center. The passage
numbers for each cell line used for the experiments ranged from 3 to 10.
Cells were seeded at the density of 4 × 105 cells/well in a six-well
polystyrene plate. Cells were cultured in the Dulbecco's Modified Eagle
Medium (DMEM; Hyclone, Logan, UT, USA) with 10% fetal bovine
serum (FBS; Gibco, Thermo Fisher Scientific Inc., Waltham, MA, USA)
and 1% penicillin/streptomycin. Cultures were maintained at 37 °C in a
fully humidified atmosphere of 5% CO2 in air. The culture medium was
changed every 3 days.
2.4.2. Cell fluorescence detection
For microscopy, cells seeded on coverslips were incubated with the
complex (Eu-FA, Tb-FA, Eu-MTX, Tb-MTX; 50 μM) dissolved in fresh
medium at 37 or 4 °C in a 12-well plate for 24 h or 48 h. Then the cells
were washed with PBS at least three times and immediately fixed by
pre-cooled methanol (− 20 °C) for 10 min, washed with PBS for 5 min
twice, and mounted onto slides with a drop of glycerinum for detection.
Cells were examined by microscopy using the appropriate excitation
and emission filters, which include the narrow band UV excitation
(exciter filter BP360-370, dichroic beamsplitter DM400, barrier filter
BA 420-460), narrow band interference filter blue excitation (exciter
filter BP470-495, dichroic beamsplitter DM505 and barrier filter
BA510-550), narrow band interference filter green excitation (exciter
filter BP540-550, dichroic beamsplitter DM570 and barrier filter
BA575-725). Images were captured by fluorescence microscopy
(Olympus BX53, Tokyo, Japan) with an image size of
1600 × 1200 pixels (200 × magnification at 1.40 NA).
Z. Du et al.
2.5. Anti-proliferative methods
The tested chemicals were dissolved in DMSO or PBS and diluted to
the desired concentration with culture medium before using. The cell
lines were maintained in RPMI-1640 medium supplemented with 10%
(v/v) heat-inactivated fetal bovine serum (FBS) and incubated at 37 °C
in a humidified incubator with 5% CO2. Cell proliferation was de-
termined by the MTT (3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-
tetrazolium bromide) or MTS methods. Briefly, cells were seeded in a
96-well plate (104 cells per well). After 4 h incubation, chemicals were
subsequently added to wells to achieve final concentration of 200, 150,
100, 50, and 10 μM. Control wells were prepared by addition of culture
medium. Treated cells were then incubated for 48 h. Once completion
of incubation, 1% of 0.5 mg/mL MTT solution was added to each well
and incubated for an additional 4 h. Formazan formed from MTT was
extracted by adding 100 μL of DMSO and mixed for another 15 min.
The optical density was measured using an enzyme-linked im-
munosorbent assay (ELISA) reader (Model 680, BIO-RAD) at 570 nm.
For MTS method HeLa cells were seeded at 1 × 105 cells/mL in 96
well plates. Cells were then incubated with FA, MTX, Eu-FA, Eu-MTX
or Eu.1 at 200, 150, 100, 50 or 10 μM in cell culture media containing
1% DSMO. Control cells were also incubated with cell culture media
with 1% DMSO. Following a 48 h incubation, cells were wash 2 times
for 5 min with PBS before cell culture media was replaced. Cells were
then incubated with 10% v/v MTS reagents (BioVision) for 2 h to assess
cell viability. Absorbance was read on an EnSpire® multimode plate
reader (Perkin Elmer®). Results were calculated as the relative absor-
bance of control cells incubated in cell culture containing 1% DMSO.
The growth inhibition rate was calculated as [(ODc − ODt) /
(ODc − ODz)] × 100%. In this formula, ODc represents the OD values
of the control group, ODt represents the OD values of the treating
groups, and ODz represents the OD values of the zero-setting groups.
Three independent experiments with triplicated samples were per-
formed to achieve the cytotoxic results, and the IC50 values were cal-
culated according to inhibition ratios.
3. Results and discussion
3.1. Synthesis of the probes
The synthesis of the final complexes was a multistep procedure,
involving the initial formation of Eu.1 or Tb.2. Eu.1 and Tb.2 were
formed using a ring closing reaction to generate the carbostyril an-
tenna, attachment to the cyclen based ligand and Ln(III) complexation
(synthesis of Eu.1 is described elsewhere [46], see ESI for synthesis of
Tb.1). This was followed by the attachment of a reactive linker inter-
mediate to generate Eu.3 or Tb.4, then removal of linker protecting
group and finally coupling to either FA or MTX to yield Eu-FA, Tb-FA,
Eu-MTX or Tb-MTX, respectively, Scheme 1. To ensure sufficient
flexibility in the targeting moiety for optimal cellular uptake and to
take account of our previous findings [44], a small ethane linker group
was included in the synthesis to provide a ‘spacer’ unit between the
rigid carbostyril antenna and the FA or MTX residue. The carbostyril
antenna was chosen as it is known to be very effective at populating the
excited states of both Eu(III) and Tb(III) ions [47,48]. Furthermore, it
has two reactive sites, which allows it to act as conduit between the
ligand and the targeting group. In addition, it is relatively small in size
which ensures that the Ln complexes will have a suitable molecular
weight/volume, which is very important for biological applications
[49].
Conjugation of the linker, tert-butyl 2-aminoethylcarbamate, to ei-
ther Eu.1 or Tb.1 was achieved via an amide coupling reaction with 1-
hydroxybenzotriazole (HOBt), (benzotriazol-1-yloxy)tris(dimethyla-
mino)phosphonium hexafluorphosphate (BOP), N,N-diisopropylethyla-
mine (DIPEA) in dimethyl sulfoxide (DMSO). No change in the broad IR
υC = O peak for either complex was observed following conjugation,
36
Journal of Inorganic Biochemistry 178 (2018) 32–42
confirming that the attachment of the linker did not affect the Ln(III)
coordination. To generate the free amine site for consequent FA or MTX
coupling, the tert-butyl group on the ethyl linker was removed by stir-
ring Eu.3 or Tb.4 in trifluoroacetic acid (TFA)/dichloromethane (DCM)
(1/1, v/v), followed by purification using a Sephadex column yielding
either Eu.5 or Tb.6, respectively. The synthesis of the four complexes,
Eu-FA, Tb-FA, Eu-MTX and Tb-MTX was achieved by coupling the free
amine site of Eu.5 or Tb.6 to folic acid or MTX in the presence of HOBt,
BOP and DIPEA in DMSO; yields for this final step ranged from 60% to
80% (see ESI Figs. S4–5 for representative for 1H NMR spectra of Eu-FA
and Eu-MTX). No bis-coupling of FA or MTX to either Eu.5 or Tb.6 was
detected by MS analysis for any of the final complexes. Elemental
analysis of Eu-FA, Tb-FA, Eu-MTX and Tb-MTX were in close ap-
proximation with the calculated values and indicated that all complexes
existed in the presence of small number of solvent molecules. The
presence of the solvent molecules are not unexpected in lanthanide
complexes and have often been reported [50,51]. The HPLC analysis of
all four targeted complexes showed two peaks of relatively equal area
(see ESI Figs. S6–9 for HPLC traces of Eu-FA, Tb-FA, Eu-MTX and Tb-
MTX). This suggested that coupling occurred at equal rates at the two
carboxylate groups (α or γ) for both FA/MTX (Fig. 2). This was ex-
pected based on literature precedence [52,53] and our own previous
work [44], where the reduced steric hindrance at the γ-COOH would
result in the formation of the γ-isomer as the major product. The rela-
tively equal formation rates of the two isomers suggests that the in-
clusion of the carbostyril antenna may alter the steric hindrance within
the molecule or act in a synergistic manner, and hence, the formation of
a predominant isomer was less affected by the space limitation. There is
a significant number of conflicting reports within the literature with
regards to isomer selectivity, with a large proportion of work on FA
conjugates involving mixed isomeric solutions [10,40–43], hence for
this work we used the Eu(III) and Tb(III) labelled FA and MTX deri-
vatives as mixed isomeric solutions for all further characterisation.
3.2. Photophysical characterisation
3.2.1. Lifetimes and q values
The emission lifetimes of the Eu-FA, Tb-FA, Eu-MTX and Tb-MTX
complexes were determined to provide information that was relevant
for time-gated measurements and cell imaging. The lifetimes also allow
the q value (the number of metal bound waters) to be quantified for
each complex, which helped to confirm the coordination environment
around the lanthanide ion. The inner sphere hydration state (q) (i.e. the
number of metal bound water molecules) was defined by measuring the
excited state lifetimes of the Eu-FA, Tb-FA, Eu-MTX and Tb-MTX
complexes, in H2O and D2O, respectively. The q values were calculated
using q = 1.11 (kH2O-kD2O-0.31) [54] for Eu(III) complexes and q = 4.2
(kH2O-kD2O) [55] for Tb(III) complexes (Table 1). The tau (τ) values
found in Table 1 are the reciprocal of k used in the two equations and
were determined using the Cary Eclipse Win FLR lifetime program. A
q ~ 1 was found for all of the imaging agents, indicating that both
complexes possessed one water molecule in the inner coordination
sphere, supporting the formation of an 8 coordination complex with the
ligand.
3.2.2. UV–Visible, fluorescence and lanthanide ion emission spectroscopy
Under UV light excitation all four complexes were found to be
highly emissive, with Eu-FA and Eu-MTX appearing as bright red due
to the sensitised emission of europium while as anticipated Tb-FA and
Tb-MTX both appeared as bright green due to the sensitised terbium
emission. The two Tb(III) complexes were significantly more emissive
than the corresponding Eu(III) complexes, and this is attributed to the
more closely aligned triplet state energies between carbostyril and Tb
(III) ion compared to the Eu(III) ion [56]. These differences in emission
can be easily observed using a benchtop UV light (see ESI Fig. S10 for a
comparative image of Eu-FA vs. Tb-FA). As a result of this difference in
Z. Du et al. Journal of Inorganic Biochemistry 178 (2018) 32–42

Scheme 1. Synthesis of the four Ln(III) complexes; Eu-FA, Tb-FA, Eu-MTX and Tb-MTX.

emission, a higher concentration of the europium complexes was Table 1

therefore used to determine the absorbance and emission properties, Luminescence lifetimes and inner sphere hydration numbers (q).
when compared to the terbium complexes.
Probe τ (H2O)/ms τ (D2O)/ms q
The photo-physical behaviour of each of the four Eu-FA, Tb-FA, Eu-
MTX and Tb-MTX complexes was investigated as a function of pH. As Eu-FA 0.722 ± 0.0 1.611 ± 0.1 0.8
cellular pH varies significantly (from pH 7 in the cytoplasm to pH < 4 Tb-FA 0.936 ± 0.0 1.169 ± 0.0 0.9
in the lysosome), it was important to understand the photo-physical Eu-MTX 0.643 ± 0.0 1.943 ± 0.0 1.1
Tb-MTX 0.921 ± 0.1 1.116 ± 0.1 0.8
properties of each of the complexes prior to imaging in cells.
Understanding the influence of pH may also give insight into the in-
fluence that the metal ion has on the overall functionality of the com- observed. The molar absorptivity of the n-pi* band at ~385 nm in-
plex. The absorption spectra of Tb-FA and Eu-MTX is shown as a creased upon the addition of base to varying degrees for each complex.
function of pH in Fig. 3 (see ESI Fig. S11 for Eu-FA and Tb-MTX). The Absorbance versus pH plots (inset Fig. 3a and b for Tb-FA and Eu-MTX,
UV–Visible spectrum of each complex at acidic pH exhibits an intensive respectively; see ESI Fig. S11 for Eu-FA and Tb-MTX) show that the
band at around 290 nm. This was attributed to the pi-pi* transitions molar absorptivity for each complex underwent small changes between
within the structure of each complex and agrees with previously re- pH 3 and pH 7. The pKa values of FA are: 2.4 (N1H+), 3.4 (α-COOH),
ported absorptions of the p-aminobenzoate structure of FA [57]. A 4.8 (γ-COOH) and 8.0 (N3H/CO) [60]. From HPLC studies conducted, it
number of smaller emission bands were also observed between 330 and is known that all the complexes exist as a roughly 1:1 ratio of α to γ
350 nm, which are typical absorption bands from the carbostyril con- isomers. Therefore, the change in molar absorptivity below pH 7 may
jugate structure [48,58,59]. A less intensive n-pi* band was also ob- be associated with deprotonation of either the α-COOH or γ-COOH
served at ~385 nm. On increasing pH, the pi-pi* absorption bands at group, the N3H/CO group (pKa reduced due to presence of Ln(III) ion)
~ 290 nm increased in molar absorptivity and underwent a bath- or a combination of these groups.
ochromic shift, the extent of which varied for each complex, indicating The fluorescence spectra for the titration of each of the complexes
a small increase in electron delocalisation. The two characteristic bands was recorded upon incremental addition of base (NaOH) at an excita-
from the carbostyril antenna, however, only underwent an increase in tion wavelength of 355 nm (see Fig. 3c and d for Tb-FA and Eu-MTX,
molar absorptivity upon increasing pH; and no wavelength shift was respectively; see ESI Fig. S12 for Eu-FA and Tb-MTX). A large structure-

Fig. 2. Structures of the two isomeric forms of Eu-FA or Tb-FA

complexes.

37
Z. Du et al.
Fig. 3. Changes in absorbance and fluorescence emission for complexes Tb-FA (0.5 × 10− 5 M) and Eu-MTX (5 × 10− 5 M) as a function of pH; a) UV–Visible spectra of compound Tb-FA
(inset shows changes at 280 nm), b) UV–Visible spectra of compound Eu-MTX (inset shows changes at 280 nm), c) fluorescene spectra of compound Tb-FA (inset shows changes at
455 nm, λex 355 nm), d) fluorescene spectra of compound Eu-MTX (inset shows changes at 455 nm, λex 355 nm).
less band was observed at ~450 nm for each complex. A slight shoulder
at 490 nm can be observed in the spectra of Tb-FA, Eu-MTX and Tb-
MTX, but not in Eu-FA. The fluorescence emission of both Eu-FA and
Tb-FA was only slightly altered upon increasing pH. A slight increase in
emission intensity was observed as the pH increased from pH 3 to pH 6,
where the emissions plateaued, and the Tb-FA complex showed the
largest change in emission. The slight emission enhancement at acidic
pH may be assigned to the protonation effect of either or a combination
of the α-COOH or γ-COOH from the FA (the pKa values of the two
carboxylates are 3.4 (α-COOH) and 4.8 (γ-COOH)) [60]. In contrast to
Eu-FA and Tb-FA, the MTX complexes Eu-MTX and Tb-MTX showed a
significant increase in fluorescence emission intensity at higher pH
(Fig. 3c and d). This difference in fluorescence emission response be-
tween the FA and MTX compounds may be attributed to the deproto-
nation of the amine group at the 4-position of the MTX pteridine ring
upon the addition of base. This effect was not significantly noticeable in
the UV–Visible spectra for either of the MTX compounds and suggests
that the changes in the fluorescence emission may be due to a singlet
state effect in either the carbostyril antenna or the aromatic structures
of MTX in both Eu-MTX and Tb-MTX.
The Eu(III) emission spectra of Eu-FA and Eu-MTX recorded at
acidic pH displayed four characteristic emission bands from the deac-
tivation 5D0 level to 7FJ (J = 1, 2, 3 and 4) (Fig. 4a and c). A plot of the
hypersensitive ΔJ = 2 band as function of pH (Fig. 4a(inset) and c
(inset)) showed that the Eu(III) emission intensity between pH 3 and
pH 6 remained relatively constant for both the Eu-FA and Eu-MTX
complexes, respectively. A slight reduction in Eu(III) emission intensity
can be observed between pH 6–8 for Eu-FA that is not observed for Eu-
MTX. Above pH 8 a significant reduction in Eu(III) emission is observed
for both Eu(III) complexes, which is most likely caused by the depro-
tonation of the metal bound water (Eu-FA and Eu-MTX were defined to
have one metal bound water molecule, see Table 1). However, a com-
binatorial effect from deportation of the pteridine ring N3H/CO and the
amide adjacent to the cyclen [61] can not be ruled out. The emission
spectra from the Tb(III) ions of Tb-FA and Tb-MTX demonstrated four
bands from the deactivation 5D4 level to 7FJ (J = 6, 5, 4 and 3) with a
ΔJ = 5 band showing the highest emission intensity (Fig. 4b and d).
While the emission of Eu-FA and Eu-MTX was essentially unchanged
between pH 3 and pH 8 (N.B. Eu-FA had a slight decrease between pH 6
38
Journal of Inorganic Biochemistry 178 (2018) 32–42
and pH 8), the emission from Tb-FA and Tb-MTX decreased con-
tinuously upon increasing pH, which was most notable above pH 9
(Fig. 4b and d). The different emission response observed between
pH 3–8 is presumably an effect of the ligand energy levels versus the Tb
(III) energy levels undergoing a change. The quenching of the Tb(III)
emission after pH 8 was attributed to the deprotonation of the metal
bound water (both Tb-FA and Tb-MTX were calculated to have one
metal bound water molecule, see Table 1). Importantly, high emission
was recorded over biologically relevant pH ranges of 4–8 for each of the
complexes in water, which suggests that these complexes are suitable
for cell imaging.
3.3. Evaluation of cellular uptake and cytotoxicity
To assess the influence of the lanthanide ion and also to explore the
differences between FA and MTX targeting, all four complexes were
evaluated for cellular uptake in a panel of: FR-negative non-malignant
cells (MLO-Y4osteocyte cells, ST2 human mesenchymal stem cells,
MC3T3 pre-osteoblasts cells, and HL7702 hepatocytes); and malignant
cancer cell lines which were either FR-positive (HeLa cervical cancer
cells, 293 t embryonic kidney cancer cells, MDA-MB-231 breast cancer
cells, U251 glioma cells, U2OS osteosarcoma cells and RAW264.7 au-
toimmune leukaemia cells) or FR-negative (A549 lung cancer cells, PC-
3 prostate cancer cells, and CAL-27 human tongue squamous cell car-
cinoma cells). The emission from each complex was easily recorded in
cells using either a blue (360–370), green (470–495) or red (540–550)
laser filter. This choice of wavelength is important for cell biologists as
it ensures that these complexes are able to match commonly available
lasers; a key parameter in the design of optical imaging agents. Fig. 5
shows representative images taken from control (MLO-Y4), FR-negative
malignant (PC-3 and CAL-27) and FR positive malignant (HeLa) cell
lines following 24 h incubation with each of the complexes (see ESI for
comprehensive range of images in all cell lines evaluated, Figs.
S13–S25).
For non-malignant cell lines, no obvious fluorescence enhancement
was observed following 24 h incubation at all wavelengths (360–370,
470–495 and 540–550 nm) using any of the four Ln(III) complexes
when compared with untreated cells. Furthermore, for two of the FR-
negative cell lines evaluated, A549 (lung cancer) and PC-3 (prostate
Z. Du et al. Journal of Inorganic Biochemistry 178 (2018) 32–42

Fig. 4. Changes in lanthanide emission for each complex as a function of pH; λex 355 nm; a) Eu(III) emission from Eu-FA (5 × 10− 5 M) with inset shows changes at 616 nm, b) Tb(III)
emission from Tb-FA (0.5 × 10− 5 M) with inset shows changes at 545 nm, c) Eu(III) emission from Eu-MTX (5 × 10− 5 M) with inset shows changes at 616 nm, d) Tb(III) emission from
Tb-MTX (0.5 × 10− 5 M) with inset shows changes at 545 nm.

Fig. 5. Fluorescence images of cells fol-

lowing 24 h incubation either in the absence
(labelled control) or in the presence of one
of Eu-FA, Tb-FA, Eu-MTX or Tb-MTX taken
from control (MLO-Y4), FR-negative malig-
nant (PC-3), FR positive malignant (HeLa)
and FR-negative cell lines CAL-27 (human
tongue squamous cell carcinoma) cell lines.

39
Z. Du et al.
cancer), no emission increase was observed when incubated with any of
the complexes as compared with untreated cells. However, all four Ln
(III) complexes showed strong fluorescence emission in malignant FR-
positive cells following 24 h incubation at all wavelengths as compared
with untreated cells. This indicates that all four of the complexes were
taken up in the six FR positive cell lines evaluated. These findings de-
monstrate that, as expected, all four Ln(III) complexes showed selective
cellular uptake for FR-positive tumour cells, compared with either the
non-malignant cells or the A549 or PC-3 (FR-negative) tumour cells.
This data suggests that all four complexes enter the cell via the FR and
thus validated our strategy for designing potent FA/MTX-tethered lu-
minescent Ln(III) complexes as tumour cell-oriented imaging agents. No
difference in the use of FA or MTX as targeting group or Eu(III) vs. Tb
(III) as emissive metal, could be observed in emission intensity fol-
lowing incubation in the malignant FR-positive cells, non-malignant
controls or the malignant FR-negative A549 or PC-3 cell lines.
Interestingly, we did observe a difference in uptake between the FA
and MTX tethered complexes, as assessed by changes in fluorescence
emission, in the FR-negative cell lines CAL-27 (Fig. 5 last column).
Specifically, more emission was detected from CAL-27 cells incubated
with the MTX-tethered complexes, Eu-MTX and Tb-MTX, compared
with either untreated cells or the FA-tethered counterparts, Eu-FA or
Tb-FA. Negligible difference in fluorescence emission was detected
between untreated cells and those incubated with either Eu-FA or Tb-
FA. As CAL-27 cells express only very low quantities of FR, this result
suggests that the antifolate analogues (Eu-MTX and Tb-MTX) may
enter the CAL-27 cells via an alternate uptake mechanism. A noticeable
difference in cell counts can also be observed in Fig. 5 in CAL-27 cells
treated with either Eu-FA or Tb-FA analogues versus those treated with
the therapeutic MTX analogues, Eu-MTX and Tb-MTX. This suggests
that MTX even when tethered to the lanthanide chelate may have re-
tained a portion of its toxic therapeutic activity.
Given the anti-folate MTX has been widely used in clinical treat-
ments, albeit with severe side effects due to its high toxicity in healthy
cells, we anticipated that the MTX-tethered Ln complexes, Eu-MTX and
Tb-MTX, might exert anti-neoplastic efficacy when they enter tumour
cells, in addition to the above-mentioned tumour-oriented imaging ef-
fects. We therefore initially observed the cellular morphology after
extended incubation times with all four Ln(III) complexes in the FR-
positive HeLa cell line, as shown in Fig. 6. Importantly, significant cell
death was observed for cells incubated with either of the MTX-tethered
complexes, Eu-MTX or Tb-MTX, following a 48 h incubation. Cell death
was also observed for the FA-modified complexes, Eu-FA and Tb-FA,
but to a reduced degree compared with that of MTX-modified com-
plexes. This may be attributable to the Ln(III) complexes having some
Fig. 6. Transmitted light images of HeLa cells following 24 and 48 h incubations with Eu-FA, Tb-FA, Eu-MTX and Tb-MTX.
40
Journal of Inorganic Biochemistry 178 (2018) 32–42
cytotoxicity in their own right. The increased cytotoxicity of MTX-
modified complexes might be due to a synergistic effect of Ln(III) ion/
chelate in close proximity to the MTX moiety, or the release of MTX
following degradation of the amide bond after a long incubation time.
To further understand the cytotoxic effects of these lanthanide
complexes we investigated a panel of tumour cells (MDA-MB-231,
HeLa, PC-3, CAL-27) and non-malignant cell (MLO-Y4) lines. The data
in Table 2, illustrated that both the MTX-tethered Ln complexes had a
cytotoxic effect when compared to the FA-derived compounds. This was
consistent with a role for the MTX moiety in these cytotoxic effects. The
cytotoxic effect of the four complexes was most noticeable on the two
FR + ve cell lines, MDA-MB-231 and HeLa, whereas the overall effect
on proliferation for either PC-3 or MLO-Y4 cells as compared with the
effect of MTX alone was relatively limited. The MTX analogues showed
a greater cytotoxic effect in the CAL-27 cells as compared with the FA
analogues. This correlates with the quantity of fluorescence detected
from each of the cell lines following 24 h incubation. Interestingly, both
Eu-FA and Eu-MTX were found to have greater cytotoxicity than the Tb
(III) analogues in the FR + ve cell lines, suggesting that the presence of
the Eu(III) ion has had an influence. Moreover, Eu-MTX was found to
have a greater effect on cytotoxicity in both the FR + ve cell lines than
MTX alone. The differences in cytotoxicity between the ‘isostructural’
Eu(III) and Tb(III) complexes was not expected and it is note-worthy
that this difference is only observable in the FR + ve cell lines. The
cause of this difference in cytotoxicity is not clear, however the var-
iance in pH sensitivity of the Eu(III) vs. Tb(III) analogues may hold a
clue. These results are of course preliminary and additional con-
firmatory studies will be required to explore this difference in influence
on cell survival. It is important to note that all four complexes were
found to be highly stable in acidic and basic aqueous environments with
no loss of metal ion from the chelate observed.
To further explore the relationship between the Eu(III) ion and the
targeting moiety (FA or MTX) on activity, an MTS cell proliferation
assay was performed on HeLa cells using Eu.1, Eu-FA and Eu-MTX, free
FA and free MTX (see ESI Fig. S26). This data shows that Eu.1 and Eu-
FA have effects comparable to free FA. Eu-MTX was found to be more
cytotoxic than free MTX in solution, in line with the anti-proliferative
assay, suggesting that there may be a synergistic effect between the Eu
(III) ion/chelate and MTX.
4. Conclusion
Recent efforts to develop selective and sensitive methods for cancer
imaging have focused on targeted imaging agents to detect cell surface
receptors that have increased expression in cancer cells. This strategy
Z. Du et al. Journal of Inorganic Biochemistry 178 (2018) 32–42

Table 2
The cytotoxic effects of Ln-tethered complexes.

MDA-MB-231a (IC50, μM) HeLaa (IC50, μM) PC-3a (IC50, μM) CAL-27a (IC50, μM) MLO-Y4a (IC50, μM)

Eu-FA 3.59 ± 1.33⁎ 1.67 ± 0.52⁎ 9.60 ± 2.52⁎ 8.97 ± 3.23⁎ 12.86 ± 4.61⁎
Tb-FA 5.24 ± 1.27⁎ 2.25 ± 0.86⁎ 10.04 ± 3.35⁎ 9.19 ± 4.09⁎ 12.79 ± 3.56⁎
Eu-MTX 0.26 ± 0.06⁎ 0.58 ± 0.79⁎ 7.33 ± 2.49⁎ 3.61 ± 1.45⁎ 9.07 ± 3.25⁎
Tb-MTX 1.90 ± 0.32⁎ 1.64 ± 0.79⁎ 7.85 ± 3.03⁎ 3.88 ± 1.21⁎ 9.22 ± 4.17⁎
MTX 1.60 ± 0.22 1.91 ± 0.17⁎ 2.82 ± 0.31 2.42 ± 0.14 2.91 ± 0.25

a
Results are presented as the mean ± SD for at least three experiments for each group.
⁎
p < 0.05 compared to the control (MTX).

depends on binding affinity, receptor specificity and cancer specific Technology Major Projects of Shandong Province (No 2016GSF201175
receptor expression and the development of highly emissive targeted to X. Li; Major Key Technology, No. 2015ZDJS04001 to F.S. Wang), and
imaging agents. We have successfully generated four targeted lumi- ZD was supported by a University of South Australia President's post-
nescent lanthanide complexes that are highly stable under a range of graduate scholarship.
conditions. We have also shown that folate and MTX tethered com-
plexes are not efficiently internalised into non-malignant cells. Appendix A. Supplementary data
However, all four complexes were found to be internalised by FR + ve
cancer cell lines, confirming that both MTX and FA can be used as Supplementary data to this article can be found online at https://
targeting groups for the folate receptor. As expected the MTX tethered doi.org/10.1016/j.jinorgbio.2017.10.003.
complexes were found to be more toxic than the FA, with the MTX
complexes offering the potential to image the site of therapeutic ac- References
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