Effect of lanthanide complex structure on cell viability and association

Article abstract of DOI:10.1021/ic500282n

A systematic study of the effect of hydrophobicity and charge on the cell viability and cell association of lanthanide metal complexes is presented. The terbium luminescent probes feature a macrocyclic polyaminocarboxylate ligand (DOTA) in which the hydrophobicity of the antenna and that of the carboxyamide pendant arms are independently varied. Three sensitizing antennas were investigated in terms of their function in vitro: 2-methoxyisophthalamide (IAM(OMe)), 2-hydroxyisophthalamide (IAM), and 6-methylphenanthridine (Phen). Of these complexes, Tb-DOTA-IAM exhibited the highest quantum yield, although the higher cell viability and more facile synthesis of the structurally related Tb-DOTA-IAM(OMe) platform renders it more attractive. Further modification of this latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic amino acid based moieties generated a family of complexes that exhibit high cell viability (ED ₅₀ > 300 μM) regardless of the lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of 100 μM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM(OMe)) disfavors the cellular association of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of extracellular species.

Full text of DOI:10.1021/ic500282n

Article
pubs.acs.org/IC
Effect of Lanthanide Complex Structure on Cell Viability and
Association
Katie L. Peterson, Jonathan V. Dang, Evan A. Weitz, Cutler Lewandowski, and Valerie C. Pierre*
́
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455, United States
S
* Supporting Information
ABSTRACT: A systematic study of the effect of hydro-
phobicity and charge on the cell viability and cell association of
lanthanide metal complexes is presented. The terbium
luminescent probes feature a macrocyclic polyaminocarbox-
ylate ligand (DOTA) in which the hydrophobicity of the
antenna and that of the carboxyamide pendant arms are
independently varied. Three sensitizing antennas were
investigated in terms of their function in vitro: 2-methoxy-
isophthalamide (IAM(OMe)), 2-hydroxyisophthalamide
(IAM), and 6-methylphenanthridine (Phen). Of these
complexes, Tb-DOTA-IAM exhibited the highest quantum
yield, although the higher cell viability and more facile
synthesis of the structurally related Tb-DOTA-IAM(OMe) platform renders it more attractive. Further modification of this
latter core structure with carboxyamide arms featuring hydrophobic benzyl, hexyl, and trifluoro groups as well as hydrophilic
amino acid based moieties generated a family of complexes that exhibit high cell viability (ED50 > 300 μM) regardless of the
lipophilicity or the overall complex charge. Only the hexyl-substituted complex reduced cell viability to 60% in the presence of
100 μM complex. Additionally, cellular association was investigated by ICP-MS and fluorescence microscopy. Surprisingly, the
hydrophobic moieties did not increase cell association in comparison to the hydrophilic amino acid derivatives. It is thus
postulated that the hydrophilic nature of the 2-methoxyisophthalamide antenna (IAM(OMe)) disfavors the cellular association
of these complexes. As such, responsive luminescent probes based on this scaffold would be appropriate for the detection of
extracellular species.
permeability.7 Although cell-penetrating peptides have been
INTRODUCTION
■
applied to increase the cell uptake of lanthanide complexes,8 the
Luminescent metal complexes are increasingly employed as
goal of this project is to modify the intrinsic properties of the
biomolecular and cellular probes due to their unique photo-
metal complex to control cellular association. Presented here is
physical properties that make them particularly well suited to
monitor biological processes.1 Examples include not only
a systematic study to evaluate the effects of structural variations
on a core structure common to most luminescent lanthanide
complexes of transition metals such as Pt, Ir, and Ru but also
probes and an investigation of the practicality of such probes
those that incorporate lanthanides, most commonly Eu and
Tb.1d,2 In particular, the long luminescence lifetime of the
for cellular and tissue applications requiring extracellular
imaging agents.
emitting metal mitigates the interference of background
fluorescence originating from the biological sample. Moreover,
since the large energy difference between the absorbing and
emissive states of these complexes removes self-absorption
issues, their luminescence signal intensity is proportional to
their concentration over a wide range of values. With this in
The most common platform of luminescent lanthanide
complexes features a chelating polyaminocarboxylate ligand
substituted with one or more carboxamide pendant arms from
which additional moieties are attached, notably the probe’s
antenna. The Laporte-forbidden nature of the f−f transition
dictates that, for practical applications in biologically relevant
mind, lanthanide-based molecular probes have been applied to
the detection of reactive oxygen species (ROS),3 redox-active
settings, the probe must incorporate a sensitizing antenna. In
metals,4 pH,5 and intracellular analytes, such as ATP.6 The
terms of the design of a responsive probe, the luminescence
intensity of the lanthanide complex can then be modulated by
continued applications of such metal-based luminescent probes,
altering, among other parameters, the excited triplet state
however, further rely on the ability to limit their cellular
energy level of the antenna and the antenna−lanthanide
association altogether for certain tissue imaging experiments or,
alternatively, to direct them to specific cellular regions. Prior
studies indicate that structural properties of metal complexes,
such as their hydrophobicity and size, influence their cell
viability and cellular association, including their membrane
distance.2a For biological applications, it is thus important to
Received: February 4, 2014
Published: June 5, 2014
© 2014 American Chemical Society
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select an antenna with suitable energetic properties for
lanthanide sensitization that is also resistant to quenching by
the medium. Work by Parker and co-workers identified that the
nature of the antenna, specifically its point of attachment to the
cyclen backbone of the ligand, affects the subcellular local-
ization of the complexes.7d This work focused primarily on
azaxanthone and other extended aromatic antenna.7c−e,9 Given
the increasing use of phenanthridine and isophthalamide
antennas for the design of luminescent lanthanide probes,6,10
a deeper understanding of the structural parameters influencing
their effect on cellular viability and cell association is necessary
to further optimize their structures for biological application
both extra- and intracellularly. Herein, two parameters were
evaluated for their effect on cell viability and association: the
nature of the antenna and that of the pendant amide arms.
Each complex features a luminescent Tb3+ ion chelated by a
DOTA(m)-type ligand. The macrocyclic polyaminocarboxylate
ligand provides kinetic inertness and thus predicted lower
toxicity on cell viability.11 Furthermore, the overall positively
charged lanthanide complexes of DOTA tetraamide (DOTAm)
ligands are kinetically more inert than the DOTA analogues as
a result of the decreased basicity of the nitrogen atoms.12
Moreover, the macrocyclic framework is less susceptible to
enzymatic degradation.12,13 The high kinetic inertness of
macrocyclic lanthanide complexes and their ability to resist
degradation is critical for their use in vivo. Three different
antennas were investigated: the hydrophilic 2-hydroxyiso-
phthalamide (IAM), a reported sensitizer of both Tb and
Eu,10c,d the closely related and synthetically more facile 2-
methoxyisophthalamide (IAM(OMe)), and the extended
aromatic 6-methylphenanthridine (Phen) (Chart 1). For each
Chart 2. Chemical Structures of Derivatives of [Tb-1] with
Varying Charged and Hydrophobicities
a
a
All modifications were performed on the pendant arm.
1. Briefly, the oxidation of bis(hydroxymethyl)-p-cresol with
MnO2 and KOH generated the diacid 11. Subsequent
treatment with methyl iodide generated the methoxy-protected
diester 12, which was then converted to the diamide 13 with
methylamine. The aldehyde 14 was generated via a Riley
oxidation of the aryl methyl group in naphthalene at 215 °C.
Periodinane oxidation methods (Dess−Martin and 2-iodox-
ybenzoic acid), permananganates, and chromates (Jones’
reagent and PCC) were unsuccessful mediators of this
transformation. Reaction of the aldehyde 14 with hydroxyl-
amine hydrochloride generated the oxime 15, which was
crystallized by addition of diethyl ether to an acidic solution of
the crude reaction mixture in ethyl acetate and methanol and
then reduced to the amine form of the IAM antenna 16. The
tris-BOC-protected cyclen 17 was synthesized as reported14
and further coupled to the amine 16 using HATU as the
activating agent. BOC deprotection in a hydrochloric acid/
methanol mixture afforded the IAM(OMe) cyclen conjugate
19, which served as a common intermediate in the synthesis of
complexes [Tb-1], [Tb-2]−, and [Tb-4]3+−[Tb-10]3−.
Chart 1. Chemical Structures of Tb-DOTA-IAM(OMe)
([Tb-1]), Tb-DOTA-IAM ([Tb-2]−), and Tb-DOTA-Phen
([Tb-3])
Alkylation of the cyclen derivative 19 with tert-butyl
bromoacetate yielded the protected ligand 20 (Scheme 1).
Deprotection in a hydrochloric acid/methanol mixture yielded
the free ligand DOTA-IAM(OMe) (1). Harsher deprotection
conditions with boron tribromide removed both the tert-butyl
groups and the aryl methyl ether to yield DOTA-IAM (2). The
final complexes [Tb-1] and [Tb-2]− were obtained from their
respective ligands, 1 and 2, by heating with TbCl3 in aqueous
solutions at neutral to slightly basic pH for 2 days or more.15
The phenathridine complex [Tb-3] was synthesized according
to the published procedure.6,10a
In order to evaluate the role of charge and hydrophobicity of
the lanthanide complex in its cellular compatibility, seven
derivatives of Tb-DOTA-IAM(OMe) ([Tb-1]) were synthe-
sized (Chart 2 and Scheme 1). The synthesis was designed to
take advantage of the common intermediate 19, described
above. Each bromoacetamide arm was synthesized using a
complex, the quantum yield, cellular viability, and cell
association were evaluated. The effect of charge and hydro-
phobicity on cell viability and association was subsequently
evaluated with complexes comprising the IAM(OMe) antenna
functionalized with varying carboxamide arms (Chart 2).
RESULTS AND DISCUSSION
■
Synthesis of Lanthanide Complexes. The isophthala-
mide complexes Tb-DOTA-IAM(OMe) ([Tb-1]) and Tb-
DOTA-IAM ([Tb-2]−) were synthesized according to Scheme
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Scheme 1. Synthesis of Tb-DOTA-IAM(OMe) ([Tb-1]), Tb-DOTA-IAM ([Tb-2]−), and Tb-DOTA-IAM(OMe) Complexes
a
with Varying Charge and Hydrophobicity ([Tb-4]3+−[Tb-10]3−)
a
Reagents and conditions: (a) MnO2, CH3Cl, 60 °C, 20 h; (b) KOH (s), 230 °C, 1 h; (c) CH3I, K2CO3, acetone, 56 °C, 16 h; (d) NH2CH3,
CH3OH, 65 °C, 18 h; (e) SeO2, naphthalene, 215 °C, 2.5 h; (f) hydroxylamine hydrochloride, pyridine, 22 °C, 24 h; (g) Pd/C (10%), HCl, ethanol/
H2O, 5 bar, 16 h; (h) HATU, DIPEA, DMF, 22 °C, 40 h; (i) HCl, CH3OH, 22 °C, 18 h; (j) Cs2CO3, CH3CN, 40 °C, 18 h; (k) HCl, CH3OH, 22
°C, 18 h; (l) BBr3, CH2Cl2, 25 °C, 18 h; (m) CH3OH, 65 °C, 16 h; (n) TbCl3, H2O/CH3OH, pH 7, 45 °C, 48 h; (o) 0.2 M KOH, H2O, 22 °C, 20
h.
biphasic method in which bromoacetyl bromide in dichloro-
methane and aqueous potassium carbonate were simulta-
neously added dropwise to a solution of the corresponding
amine in dichloromethane at 0 °C. This method resulted in
moderate to high yields (65−85%) of the desired products in
high purity. The ligands were obtained upon reaction of the
bromoacetamide arms with the intermediate 19. Metalation
with TbCl3 at neutral pH yielded [Tb-4]3+−[Tb-8]3+. The
carboxylic acid derivatives [Tb-9] and [Tb-10]3− were obtained
by saponification of their corresponding esters [Tb-7]3+ and
[Tb-8]3+, respectively. Synthetic details and characterizations
are given in the Supporting Information.
Quantum Yield and Quenching in Cell Lysate. The
nature of the sensitizing antenna of a lanthanide complex can
affect its function in a biological environment. Here, we
compare [Tb-1], [Tb-2]−, and [Tb-3], three complexes that
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feature either an isophthalamide- or phenanthridine-based
antenna for sensitizing the lanthanide emissions (Chart 1).
Note that each complex has the same acetate pendant arms. All
three complexes luminesce in the green region of the visible
spectrum, with maximum emission at 545 nm upon excitation
at 350 nm (Figure S1 (Supporting Information)). These three
antenna platforms have different properties in terms of
hydrophobicity and interactions with biomolecules, including
nucleic acids, reducing agents, and proteins. The hydrophilic
isophthalamide and methoxyisophthalamide are composed of a
single aromatic ring and have no known interactions with
biomolecules, while the phenanthridine is an extended aromatic
system capable of base stacking with nucleotides and DNA
intercalation.6,10a
of protein/mL, the luminescence intensity of [Tb-1] is
quenched by 22
2.8%; at 0.5 mg of protein/mL the
luminescence intensity is reduced by 30 2.4% (Table 1 and
Table 1. Luminescence Quenching of [Tb-1] by Whole Cell
a
Lysate
b
concn, mg of protein/mL
quenching (%)
0.25
0.50
22 2.8
30 2.4
a
Experimental conditions: [Tb-1] = 0−15 μM, PBS, pH 7.8, time
delay 0.1 ms, excitation wavelength 345 nm, slit widths (excitation and
emission) 5 nm, integrated emission intensity from 470 to 635 nm, T
= 20 °C. Results are mean
SD (n = 3). Whole cell lysate was
generated from L6 myoblasts; the protein concentration was
The quantum yield, Φ, of the three Tb complexes in PBS was
investigated by comparing the luminescence intensity of each
complex to that of a quinine sulfate standard in 0.1 M sulfuric
acid (Φr = 0.577)16 according to the optically dilute method.10d
At pH 7.8 and 22 °C, the quantum yields of [Tb-1] and [Tb-3]
b
determined by a BCA assay. Percent quenching was calculated
using the percent change in the slope of the integrated luminescence
intensity versus the [Tb-1] (μM) plots (Figure S2 (Supporting
Information)).
are statistically identical (4.6
1.2% vs 4.7
2.2%,
respectively). The quantum yield of the phenathridine
derivative [Tb-3] is comparable to that of similar complexes
featuring either triamide- or triphosphinate-substituted DOTA
ligands.17 However, [Tb-2]− is a more efficient emitter with an
Figure S2 (Supporting Information)). Advantageously, the
isophthalamide antenna is less susceptible to quenching than
complexes containing electron-poor sensitizing moieties. For
instance, the metal-centered emission of terbium azaxanthone
complexes is quenched by 30−60% in the presence of 0.2 mM
human serum albumin.22 This highlights the potential of the
IAM(OMe) antenna for biological applications.
average quantum yield of 9.8
1.5%, indicating that
substituting the methoxy for the hydroxyl group on the
isophthalamide antenna substantially affects the quantum yield
of the complex. Note that quantum yields of ca. 50% have been
reported by Raymond and co-workers for Tb complexes with
tetra-IAM-substituted ligands.10d The substantial difference
between these complexes results from their structures. In
contrast with the case for [Tb-2]−, in the tetra-IAM complex
H(2,2)-IAM the isophthalamide directly coordinates the
lanthanide, thereby enabling a Dexter-type energy transfer
from the antenna to the Tb that is much more efficient than the
Influence of the Nature of the Antenna on Cellular
Compatibility. The effect of [Tb-1], [Tb-2]−, and [Tb-3] on
the viability of L6 rat muscle myoblast cells was investigated
using an MTT assay. It was hypothesized that the 2-
methoxyisophthalamide would have a reduced effect on cell
viability due to the protecting group on the phenol that could
block nonspecific interactions with the cell proteins and
coordination of cellular metals, such as iron and copper.
Additionally, the phenathridine complex [Tb-3] was expected
to have a greater effect on cell viability due to its ability to
intercalate DNA and RNA.10a,23 Following a 24 h incubation
with 0−300 μM Tb complex, the phenanthridine derivative
[Tb-3] decreased cell viability to 74% at 100 μM and to 63% at
300 μM. On the other hand, the cellular viability was
maintained at or above 80% for both isophthalamide complexes
[Tb-1] and [Tb-2]− (Table 2 and Figure 1). The similar
profiles indicate that the methoxy protecting group on the
isophthalamide does not significantly improve cell viability at
the doses studied here. This is in contrast to a pair of Rh probes
chelated by chrysenequinone diimine and N-functionalized
dipyridylamine ligands, for which small structural changes were
shown to have a substantial impact on cell viability. In that case,
altering the ethanol substituent to a propyl reduces the viability
of HCT116N and HCT116O cells from 80% to 60% after a 24
h incubation with 40 μM complex.7b
Overall, the complexes in this study influence cell viability
slightly less than complexes containing tetraazatriphenylene or
azaxanthone antennas with phenyl or ethyl ester substituted
carboxamide arms, which have ED50 values in the 100 μM to
>240 μM range.7d,e,24 They affect cell viability similarly to
Ru(III) polypyridine complexes, which also have ED50 (72 h)
values ≥300 μM in MCF-7 and HT-29 cell lines.25 It is
interesting to note that the europium compound of 1,4,7,10-
tetraazacyclododecane-1,4,7,10-tetraacetic acid containing no
antenna moiety, [Eu-DOTA]−, has a negligible effect on cell
viability from 0 to 500 μM with a 72 h incubation.26 Thus, with
Forster-type, through-space mechanism likely taking place in
̈
[Tb-2]−. On the other hand, [Tb-2]− has substantially higher
water solubility (M range) than the cryptand H(2,2)-IAM (μM
range) and enables the design of responsive probes.
Nonetheless, the quantum yields of [Tb-1], [Tb-2]−, and
[Tb-3] are comparable to those of luminescent d-block
compounds used for cellular imaging. For example, the
quantum yields of Ir complexes featuring 2-phenylpyridine
ligands are between 4 and 8% in PBS buffer.18 Not surprisingly,
the quantum yields of Tb-centered luminescence are lower than
those of commonly used fluorescence dyes such as Cy3 (4% in
PBS), Cy5 (30% in PBS), Alexa750 (12% in phosphate buffer),
and Texas Red (30% in water).19 However, in comparison with
phosphorescent metal complexes, organic dyes suffer from
short fluorescence lifetimes and small Stokes shifts.20
As for any luminescent or fluorescent probe, emission is
susceptible to quenching by reducing agents, proteins, and
other components of biological media. In the case of
luminescent Tb and Eu complexes, the quenching pathways
can involve electron or charge transfer from the excited states
to intracellular reducing agents such as urate, ascorbate, and
glutathione that are present in mM concentrations in cells.21 In
addition, luminescence quenching by proteins, such as human
serum albumin, has been reported.22 The degree of
luminescence quenching was evaluated by measuring the
time-delayed luminescence intensity of [Tb-1] with respect to
its concentration in PBS and whole cell lysate at two different
protein concentrations. In whole cell lysate containing 0.25 mg
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quantitative nature of measuring cellular association of metal
complexes by ICP-MS is balanced by the inability of the
technique to distinguish between internalized or membrane-
bound complexes.27 It was anticipated that the extended
aromatic antenna of [Tb-3] would facilitate membrane
permeability, as suggested by the increased cellular uptake of
more lipophilic Ru complexes and phenanthridine-substituted
cisplatin.7a,27,28 However, the cell association values of the
hydrophilic Tb-DOTA-IAM(OMe) ([Tb-1]) and Tb-DOTA-
IAM ([Tb-2]−) are statistically not different from the values for
the hydrophobic Tb-DOTA-Phen ([Tb-3]) (Table 2).
Small structural changes to the ligands of metal complexes
have been reported to exert varied effects on the intracellular
accumulation and distribution. Using a group of lanthanide
complexes with tetraazatriphenylene- or azaxanthone-based
antennas, Parker identified that how the antenna was linked to
the cyclen ring affected membrane permeability and localization
more than small structural modifications on the antenna itself.7d
For the isophthalamide complexes, however, conversion from
the methoxy group in [Tb-1] to the phenolate of [Tb-2]− does
affect cellular association. This observation is more in concert
with the findings of Barton, who measured substantial
differences in cell association between an alkyl-substituted
dipyridyl Rh complex (705 ng of Rh/mg of protein) in
comparison to a complex that introduced a terminal alcohol on
the alkyl group (165 ng of Rh/mg of protein).7b The cellular
association values measured for the hydroxyisophthalamide
[Tb-2]− are comparable to the values reported for a Tb
complex with a tetraazatriphenylene antenna and phenyl-
substituted amide arms, which had a maximum accumulation of
10 μmol/g of protein into NIH 3T3 cells after a 4 h incubation
at 50 μM.7d Unfortunately, further comparisons with cellular
accumulation values reported in the literature are limited due to
differences in the method of data collection (ICP-MS vs flow
cytometry7a,21a,29), and data presentation of metal per amount
of protein or per cell.7e,24,25,28,30
Table 2. Cellular Viability and Association of Macrocyclic Tb
Complexes
a
b
cell viability
(%)
cell association (μmol of Tb/g of
protein)
[Tb-1]
96
80
74
81
63
90
78
90
81
6
3
9
2
4
8
8
6
4
6.0
12
3
5
3
[Tb-2]−
[Tb-3]
5.3
[Tb-4]3+
[Tb-5]3+
[Tb-6]3+
[Tb-7]3+
[Tb-8]3+
[Tb-9]
1.4 0.6
22 10
8.3
3
2.2 0.5
0.32 0.07
1.3 0.4
[Tb-10]3−
88 10
100
0.47 0.2
0.07 0.04
control (no Tb
complex)
6
a
Obtained by an MTT assay after a 24 h incubation with 100 μM
b
complex. Results are mean SD (n = 3). Determined by ICP-MS
after a 4 h incubation with 50 μM complex. Results are mean SD (n
= 3).
Figure 1. Viability (24 h) of L6 myoblasts treated with 0−300 μM
[Tb-1] (solid squares), [Tb-2]− (open circles), and [Tb-3] (solid
triangles) as determined with an MTT assay. Results are expressed as
mean SD (n = 3).
Notable differences among the three metal complexes
include the increased effect of [Tb-3] on cell viability above
100 μM in spite of its only modest cell association. The
synthesis of [Tb-2]− requires a synthetically challenging boron
tribromide deprotection to produce the 2-hydroxyisophthala-
mide antenna, and the resulting complex has a cellular
association only slightly higher than that of its analogue [Tb-
1]. The more facile synthesis of the 2-methoxyisophthalamide
of [Tb-1] in comparison to [Tb-2]− thus became a significant
respect to cell viability, [Tb-1] and [Tb-2]− are preferred for
cellular imaging applications compared to [Tb-3].
The nature of the antenna was also expected to affect the
cellular association of the complexes. Following a 4 h
incubation with 50 μM of Tb complex, the cellular
accumulation of the metal was determined by ICP-MS and
expressed relative to the protein concentration as determined
by a BCA assay. Importantly, it should be noted that the
Figure 2. Viability (24 h) of L6 myoblasts treated with 0−300 μM Tb complexes as determined with an MTT assay: (a) [Tb-1] (solid squares),
[Tb-4]3+ (solid triangles), [Tb-5]3+ (open circles), and [Tb-6]3+ (open inverted triangles); (b) [Tb-7]3+ (solid squares), [Tb-8]3+ (solid triangles),
[Tb-9] (open circles), and [Tb-10]3− (open inverted triangles). Results are expressed as mean SD (n = 3).
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factor in selecting an antenna for further studies evaluating the
effects of variable arms on the cellular compatibility of
lanthanide complexes.
complex alone cannot be used to predict the cellular association
of metal complexes.
Of the amino acid derivatized complexes, the alanine-based
[Tb-7]3+ and [Tb-9] have higher cell association values than
the glutamine-based compounds [Tb-8]3+ and [Tb-10]3−.
Positively charged complexes containing esters are reported
to have enhanced cell association in comparison to carboxylic
acid derivatives,7a and this is observed with the slightly higher
cell association values of the ethyl ester complex of alanine [Tb-
7]3+ in comparison to the neutral acid complex [Tb-9]. This
reflects the finding that the cell association of [Eu-DOTAm]3+
is 5 times that of [Eu-DOTA]−, which showed negligible
cellular accumulation.26 Interestingly, this trend is not repeated
for the glutamine ethyl ester and acid complexes, [Tb-8]3+ and
[Tb-10]3−, respectively, which both have low cell association
values despite their large differences in overall complex charge.
This is in contrast with to lanthanide complexes studied by
Parker in which the intracellular concentration of a 3− charged
glutamate-functionalized DOTA with a tetraazatriphenylene
antenna was approximately 3 times that of the neutral Ln-
DOTA-tetraazatriphenylene.21a An important conclusion is that
the parameters influencing cellular association of lanthanide
complexes are multifaceted and include both structural features
(lipophilicity, nature of the antenna, terminal functional
groups) and, to a lesser extent, overall complex charge.
This study investigated the effect of structural modifications
of the ligand on cell association in the absence of targeting
moieties; however, conjugating Ln complexes to cell-penetrat-
ing peptides32 or receptor targeting groups has successfully
increased the cellular uptake. Progress in the field of cell-
penetrating, lanthanide-based contrast agents has been
reviewed8,33 and includes the use of polyarginine34 and Tat-
based peptides.35 This technique has also been applied to
luminescent metal complexes36 and dual imaging agents.37
Additional approaches to increase cellular accumulation include
coupling complexes to moieties that facilitate interactions with
cellular membrane receptors,38 transporters,39 or special groups
such as exofacial thiols.40 The translation of these approaches to
the luminescent macrocyclic polyaminocarboxylate complexes
described herein is being further investigated by our group.
Fluorescence Microscopy. The cellular association of the
Tb complexes was also investigated with epifluorescence
microscopy. L6 myoblasts were treated with Tb complex
(200 μM for 4 h), washed with PBS at room temperature, and
fixed with formaldehyde prior to imaging. Results of
representative cells indicate the presence of weak cellular
association for the trifluoro-substituted [Tb-6]3+, while the
fluorescence intensity of phenanthridine-containing [Tb-3] is
comparable to that of the control cells (Figure 3). The
remaining Tb complexes exhibit similar weak cellular
fluorescence (Figure S3 (Supporting Information)). Due to
reports that different staining patterns can be observed in
experiments that differ only in whether cells were imaged in a
live or fixed state,41 the cellular association of [Tb-6]3+ was also
examined in live cells via fluorescence microscopy (Figure S4
(Supporting Information)). In these images, an overall decrease
in the fluorescence to levels comparable to those for the
untreated control cells was observed.
Effect of Structural and Electronic Variations in
Pendant Arms on Cell Viability and Association. The
role of charge and hydrophobicity of the polyaminocarboxylate
arms on the cell viability and association of the lanthanide
probes was evaluated with a library of derivatives of [Tb-1].
Complexes [Tb-4]3+−[Tb-10]3− share a common cyclen
backbone with a 2-methoxyisophthalamide antenna but differ
in the nature of the remaining substituents (Chart 2). The alkyl,
benzyl, and trifluoro groups impart increasing hydrophobicity
to the complexes, whereas alanine and glutamine amino acid
based substituents in either an ethyl ester or carboxylic acid
form render greater hydrophilicity. The resulting complexes
also differ in overall complex charge; neutral carboxamide arms
generate the 3+ charged complexes [Tb-4]3+−[Tb-8]3+, the
addition of monoanionic arms forms the zwitterionic complex
[Tb-9], and the dianionic glutamate arm creates [Tb-10]3−
bearing a net 3− charge.
As with the previous complexes that differ with respect to the
antenna, the cell viability (24 h) was measured for the library of
complexes featuring pendant arms with structural and
electronic variations. Of the hydrophobic complexes, only
[Tb-5]3+ decreases cell viability significantly to 60%, while the
values for [Tb-4]3+ and [Tb-6]3+ are comparable to that of the
parent complex [Tb-1], maintaining a viability above 80% in
the concentration range measured (Figure 2a and Table 2).
Each of the amino acid derivatives [Tb-7]3+, [Tb-8]3+, [Tb-9],
and [Tb-10]3− also sustain viability above 75% even in the
presence of 300 μM Tb complex (Figure 2b and Table 2).
Interestingly, the notable reduction in cell viability upon
addition of medium-length alkyl chains matches the results of a
previous study comparing a family of Ir(III) cyclometalated
probes. In that study, the Ir complexes featuring the shortest
(C2) and longest (C18) alkyl chains had comparably minimal
effects on HeLa cell viability (ED50 ≈ 15 μM), while the
median length alkyl chain (C10) was significantly more
cytotoxic (ED50 = 2 μM).29b The hexyl alkyl derivative [Tb-
5]3+ excluded, the relatively low effect on cell viability (ED50
values >300 μM) of this entire class of Tb complexes suggests
they have only limited interactions with cellular components
and bodes well for their application.
The role of the pendant arms on the cellular association of
the Tb complexes was also investigated by ICP-MS. It was
hypothesized, on the basis of the results of Barton,7a,27 that
[Tb-4]3+, [Tb-5]3+, and [Tb-6]3+ would penetrate cells more
efficiently due to their lipophilic substituents and cationic
character that would assist transport across the membrane.31
Indeed, higher Tb accumulation is observed with the
hydrophobic hexyl-substituted [Tb-5]3+ and trifluoro-contain-
ing [Tb-6]3+ than with the more hydrophilic amino acid
derivatives (Table 2). However, this conclusion cannot be
universally applied to all hydrophobic arms, since the cellular
association of the benzyl-substituted [Tb-4]3+ is less than that
of the parent complex [Tb-1]. The alkyl-substituted Ir
complexes discussed previously also do not illustrate a direct
relationship between hydrophobicity and cellular association. In
that study, each complex investigated displayed measurable
cellular association by ICP-MS, but the C10 version afforded the
greatest association, followed by the C2 and then the C18
derivative.29b Thus, it is clear that the lipophilicity of the
The reduced punctate staining in live cells in comparison to
fixed cells has also been observed by Belitsky.41 However,
unlike our 2-hydroxyisophthalamide complexes, lanthanide
complexes featuring tetraazatriphenylene antennas containing
phenyl, ester, or carboxylate substituents are taken up by CHO
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were diluted appropriately and analyzed in the presence of a 20 ppb of
In internal standard using the He/H2 collision-reaction mode. UV−vis
spectra were measured with a Varian Cary 100 Bio spectrophotometer.
Data were collected between 220 and 800 nm using a quartz cell with a
path length of 10 mm. Luminescence data were recorded on a Varian
Eclipse Fluorescence spectrophotometer using a quartz cell with a path
length of 10 mm.
Determination of Quantum Yield (Φ). The absorbance and
integrated luminescence intensities of [Tb-1]−[Tb-3] in PBS (pH 7.8,
n = 1.3345) were compared to the reference fluorescence of quinine
sulfate (Φr = 0.577)16 in 0.1 M sulfuric acid (n = 1.333). Absorption
spectra were collected on a Cary 100 Bio UV−visible spectropho-
tometer; emission spectra were recorded on a Cary Eclipse
fluorescence spectrophotometer. Quantum yields (Φ) were calculated
according to the optically dilute method using the equation
2
⎛
⎞
⎟
⎠
⎛
⎞
⎛
⎞
Ar nx
Ix
Φ = Φ
⎜
⎜
⎝
⎟
⎜
⎝
⎟
⎠
x
r
⎠ n 2
A
I
⎝
x
r
r
Figure 3. Fluorescence microscopy images of representative L6
myoblasts treated with 200 μM [Tb-3] and [Tb-6] for 4 h at 37 °C:
(a) fluorescence images, 60× objective, 325−375 nm excitation filter,
470−750 nm emission filter, 0.4 s exposure time; (b) bright field
images, 0.04 s exposure time. Cells were rinsed with PBS and fixed
with formaldehyde prior to imaging. Scale bars represent 10 μm.
where A is the absorbance at the excitation wavelength (λ), n is the
refractive index, and I is the integrated intensity.10c,d The subscripts r
and x refer to the reference and samples, respectively. All data were
collected using the same spectrophotometer on the same day, resulting
in a constant intensity of the excitation light (λex = 350 nm).
Measurements were obtained at 22 °C in a quartz cell of 1 cm path
length in degassed solutions using an excitation wavelength of 350 nm
and excitation and emission slit widths of 10 nm for both complexes.
The fluorescence response of quinine sulfate was reported as the
integrated emission intensity from 365 to 665 nm; the Tb
luminescence of each complex was collected with a time delay of 0.1
ms and reported as the integrated emission intensity from 470 to 688
nm. The experiment was performed in triplicate (n = 3).
Cell Culture. Rat muscle L6 myoblast cell line was obtained from
ATCC (Manassas, VA). Cells were cultured in 25 or 75 cm2 vented
culture flasks in Dulbecco’s modified eagle medium (DMEM)
supplemented with 10% (v/v) bovine serum (BS) and 10 μg of
gentamicin mL−1 at 37 °C and 5% CO2. The cells were maintained by
splitting every 3−4 days before reaching confluence; cells were rinsed
with PBS, lifted using 0.25 g L−1 typsin for 5 min, and diluted in fresh
medium. All cells were propagated at low passage numbers (<20) to
maintain their myoblastic character.
Preparation of Whole Cell Lysate. L6 cells were harvested from
two 75 cm2 culture flasks grown to 85% confluence and centrifuged at
400g for 10 min at 4 °C. The resulting pellet was rinsed with ice-cold
PBS (2 mL) and centrifuged. The cells were then suspended in PBS (1
mL) and transferred to a tight-fitting homogenizer (B clearance
0.0008−0.0022 in.). Approximately 90% of the cells were ruptured
after 120 strokes, as determined by cell counting with trypan blue and
a hemocytometer. The total protein concentration of the whole cell
lysate was determined with a bicinchoninic acid (BCA) assay (BCA
Protein Assay Kit, ThermoScientific) using bovine serum albumin
(BSA) protein standard solutions. The whole cell lysate was then
diluted to a protein concentration of 1.0 mg/mL, and aliquots (450
μL) were stored at −20 °C for later use.
Luminescence Intensity in Whole Cell Lysate. An aqueous
suspension of [Tb-1] (100 μM) in whole cell lysate diluted in PBS
(0.25 or 0.5 mg of protein/mL) was titrated into an aqueous solution
of whole cell lysate in PBS (0.25 or 0.5 mg of protein/mL). The time-
delayed emission profile was recorded in the presence of 0−15 μM of
Tb complex. The titration was repeated in pure PBS buffer (pH 7.8)
without cell lysate. Measurements were recorded with an excitation
wavelength of 345 nm, a time delay of 0.1 ms, and excitation and
emission slit widths of 5 nm at a temperature of 20 °C. The
luminescence response was reported as the integrated emission
intensity from 470 to 640 nm. Each experiment was repeated in
triplicate (n = 3). The percent quenching of the luminescence was
calculated from the slopes of the integrated luminescence intensity
versus the concentration of Tb complex (μM) plots in PBS and whole
cell lysate at the two protein concentrations.
or NIH 3T3 cells and can be visualized with fluorescence
microscopy regardless of the nature of their pendent arms.7d
Likewise, Bunzli observed lanthanide helicates ranging in
̈
overall charge from 6− to 6+ localized to endosomes and
lysosomes, consistent with endocytotic uptake mechanisms
irrespective of the polarity or overall charge of the complex.7c
Regardless of the reasons, the low cell associations exhibited by
this entire class of complexes render them particularly useful for
monitoring extracellular species.
CONCLUSIONS
■
A family of luminescent lanthanide complexes featuring
different sensitizing antennas varying in charge and hydro-
phobicity and pendant arms of variable structural and electronic
properties were synthesized. The cell viability and association of
this family of complexes have been evaluated. With respect to
the antenna, the 2-methoxyisophthalamide of [Tb-1] was
selected as the ideal candidate for further studies due to its
synthetic ease (in comparison to [Tb-2]−), suitable quantum
yield, little effect on cell viability, and modest cell association.
Surprisingly, the addition of hydrophobic moieties (benzyl,
hexyl, and trifluoro) did not increase cell association. All of the
complexes investigated minimally affect cell viability and exhibit
low cellular association, regardless of the overall complex
charge or relative hydrophobicity; thus, it is presumed that it is
the hydrophilic nature of the 2-methoxyisophthalamide antenna
that confers the low membrane permeability of this class of
compounds. Thus, the probes based on the structural
framework presented here are well-suited for monitoring
extracellular analytes such as group I ions, polysaccharides,
hormones, or other signaling molecules.
EXPERIMENTAL SECTION
■
General Considerations. Unless otherwise noted, starting
materials were obtained from commercial suppliers and used without
further purification. Water was distilled and further purified by a
Millipore cartridge system (resistivity 18 MΩ). Elemental analyses
were performed by inductively coupled plasma mass spectrometry on a
Thermo Scientific XSERIES 2 ICP-MS fitted with an ESI PC3 Peltier
cooled spray chamber, SC-FAST injection loop, and SC-4 autosampler
at the Department of Geology at the University of Minnesota. Samples
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Cell Viability. L6 myoblasts (225 μL) were plated at ∼4.0 × 104
cells mL−1 in a 48-well cell culture plate. Cells were allowed to recover
24 h, after which Tb complexes in PBS (25 μL) were added to the
culture media (final concentrations of 0, 1, 3, 10, 30, 100, and 300
μM). For the negative control, PBS (without Tb complex) was used.
The MTT assay was performed during the last 3 h of the compound
exposure time as follows. MTT (3-(4,5-dimethylthiazolyl-2)-2,5-
diphenyltetrazolium bromide, 25 μL of 5 mg/mL stock solution, 0.5
mg/mL final concentration) was added to the media, and the plate was
returned to the cell culture incubator for 3 h. When a purple
precipitate was clearly visible, 250 μL of 0.04 M HCl in isopropyl
alcohol was added to each well. After an incubation of 4 h at room
temperature in the dark, the samples were centrifuged at 10000g for 5
min to pellet cellular debris. For each sample, a portion of the
supernatant (200 μL) was transferred to a corresponding well of a 96-
well plate, and the absorbance at 570 and 690 nm was recorded using a
microtiter plate reader. The cell viability was calculated according to
the following equation using the adjusted absorbance of the cells
treated with Tb complex in comparison to the control cells.9a
collected to determine the position of cells (exposure time 0.04 s).
The contrast of bright field images was adjusted to a contrast range of
0−100 (normal range 0−255) using HCImage software.
ASSOCIATED CONTENT
* Supporting Information
■
S
Text and figures giving synthetic details and characterization
data for [Tb-1]−[Tb-10]3−, time-delayed excitation and
emission luminescence profiles, fluorescence emission profiles,
and absorption spectra of [Tb-1]−[Tb-3], and supplementary
fluorescence microscopy images of Tb complexes incubated
with L6 myoblasts. This material is available free of charge via
the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*E-mail for V.C.P.: pierre@umn.edu.
■
Notes
(A570 − A690 Tb complex
)
viabilityMTT (%) =
The authors declare no competing financial interest.
(A570 − A690 control
)
ACKNOWLEDGMENTS
Results are expressed as mean SD (n = 3).
■
Cellular Association by ICP-MS. The cellular association of [Tb-
1]−[Tb-10] was investigated by seeding in triplicate L6 myoblasts
(225 μL) at 4.5 × 104 cells mL−1 in a 48-well plate (∼10000 cells
mL−1). Cells were allowed to recover for 48 h and had grown to 80%
confluence, at which time a concentrated solution of Tb complex in
PBS (25 μL) was added to achieve a final concentration of 50 μM in
the cellular media. Cells grown in Tb complex free media (addition of
only PBS) were included as a negative control. Following 4 h of
incubation, the media were removed, and the wells were washed twice
at room temperature by adding PBS and rocking the culture plate back
and forth several times. The addition of a lysis buffer (75 μL of 10 mM
Tris, pH 7.5, 100 mM NaCl, 1 mM Na2EDTA, and 1% Triton X-100)
followed by a 20 min incubation at 4 °C ruptured the cell
membranes.24 Aliquots (3 × 8 μL) from each well were removed
for a BCA assay to determine the protein concentration. Samples (50
μL) were combined with concentrated nitric acid (50 μL) and heated
to 95 °C for 18 h in flame-sealed glass ampules prior to ICP-MS
analysis. The Tb content (ppb) was determined by ICP-MS, and the
cellular association of each Tb complex (CTb), expressed as μmol of
Tb/g of protein, was calculated according to the equation
We thank Edgar Arriaga, Michelle Henderson, and Greg
Wolken for helpful discussions and assistance with cell culture
and fluorescence microscopy and Rick Knurr for ICP-MS
sample analysis. This work was supported by the National
Science Foundation Grant CAREER 1151665. Support to
K.L.P. from the NIH-CBITG (GM 08700), J.V.D. from the
Heisig/Gleysteen Chemistry Summer Research Program from
the Department of Chemistry at the University of Minnesota,
E.A.W. through a Wayland E. Noland Fellowship from the
Department of Chemistry at the University of Minnesota, and
C.L. from a UROP fellowship from the University of Minnesota
are gratefully acknowledged.
ABBREVIATIONS
■
ATP, adenosine triphosphate; BOC, tert-butoxycarbonyl;
DIPEA, N,N-diisopropylethylamine; DMEM, Dulbecoo’s modi-
fied eagle medium; DMF, dimethylformamide; DNA, DNA;
DOTA, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic
acid; DOTAm, 1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-
tetraazacyclododecane; DTPA, diethylenetriaminepentaacetic
acid; EDTA, ethylenediaminetetraacetic acid; Eu, europium;
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo-
[4,5-b]pyridinium 3-oxide hexafluorophosphate; IAM, 2-
hydroxyisophthalamide; IAM(OMe), 2-methoxyisophthala-
mide; ED50, half maximal effective dose; ICP-MS, Inductively
coupled plasma mass spectrometry; Ir, iridium; Ln, lanthanide;
MTT, (3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium
bromide; PBS, phosphate buffered saline; PCC, pyridinium
chlorochromate; Phen, 6-methylphenanthridine; Pt, platinum;
Rh, rhodium; RNA, ribonucleic acid; ROS, reactive oxygen
species; Ru, ruthenium; Tb, terbium
CTb = (Cm/M)/Cp
where Cm is the measured metal concentration (ppb) of the cell
samples, M is the molecular mass of Tb, and Cp is the protein
concentration (mg/mL). Results are expressed as mean SD (n = 3).
Fluorescence Microscopy. L6 myoblasts (passage 9 or 10) were
seeded in a polylysine-coated 8-well glass-bottom plate and allowed to
grow for 24 h to 50% confluence at 37 °C in 5% CO2. Then Tb
complexes [Tb-2]−−[Tb-8] were added in PBS for a final
concentration of 200 μM. Negative control wells containing cells
grown in Tb complex free media (addition of only PBS) were
included. Following a 4 h incubation, the media were removed, and
cells were washed three times with PBS at room temperature by
rocking the culture plate back and forth gently. Cells were fixed with
4% formaldehyde in PBS at room temperature for 15 min.
Additionally, live cell imaging of [Tb-6] was performed in DMEM
phenol red free media. Epifluorescence images of representative cells
were acquired on an Olympus IX81 inverted microscope equipped
with a 60× (N.A. 1.45) oil immersion objective. Excitation was
supplied by an Exfo XCite 120 metal halide lamp source coupled to
the microscope via a liquid light guide, images were collected with a
C9100−01 CCD camera, and HCImage software was utilized to
control the microscope and camera and to process images. An
excitation filter (325−375 nm), dichoric filter (460 nm), and emission
filter (470−750 nm) were employed with exposure times of 0.4 s for
fixed cells and 0.1 s for live cells. Bright field images of cells were also
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