Investigating Intracellular Localisation and Cytotoxicity Trends for Neutral and Cationic Iridium Tetrazolato Complexes in Live Cells

Article abstract of DOI:10.1002/chem.201701352

A family of five neutral cyclometalated iridium(III) tetrazolato complexes and their methylated cationic analogues have been synthesised and characterised. The complexes are distinguished by variations of the substituents or degree of π conjugation on either the phenylpyridine or tetrazolato ligands. The photophysical properties of these species have been evaluated in organic and aqueous media, revealing predominantly a solvatochromic emission originating from mixed metal-to-ligand and ligand-to-ligand charge transfer excited states of triplet multiplicity. These emissions are characterised by typically long excited-state lifetimes (～hundreds of ns), and quantum yields around 5–10 % in aqueous media. Methylation of the complexes caused a systematic red-shift of the emission profiles. The behaviour and the effects of the different complexes were then examined in cells. The neutral species localised mostly in the endoplasmic reticulum and lipid droplets, whereas the majority of the cationic complexes localised in the mitochondria. The amount of complexes found within cells does not depend on lipophilicity, which potentially suggests diverse uptake mechanisms. Methylated analogues were found to be more cytotoxic compared to the neutral species, a behaviour that might to be linked to a combination of uptake and intracellular localisation.

Full text of DOI:10.1002/chem.201701352

DOI: 10.1002/chem.201701352
Full Paper
&
Fluorescent Probes
Investigating Intracellular Localisation and Cytotoxicity Trends for
Neutral and Cationic Iridium Tetrazolato Complexes in Live Cells
[
a]
[b]
[b]
[a]
Chiara Caporale, Christie A. Bader, Alexandra Sorvina, Karen D. M. MaGee,
[
c]
[b]
[a]
[d]
[e]
Brian W. Skelton, Todd A. Gillam, Phillip J. Wright, Paolo Raiteri, Stefano Stagni,
[g]
[b, f]
[b]
[a]
Janna L. Morrison, Sally E. Plush,* Douglas A. Brooks,* and Massimiliano Massi*
Abstract: A family of five neutral cyclometalated iridium(III)
tetrazolato complexes and their methylated cationic ana-
logues have been synthesised and characterised. The com-
plexes are distinguished by variations of the substituents or
degree of p conjugation on either the phenylpyridine or
tetrazolato ligands. The photophysical properties of these
species have been evaluated in organic and aqueous media,
revealing predominantly a solvatochromic emission originat-
ing from mixed metal-to-ligand and ligand-to-ligand charge
transfer excited states of triplet multiplicity. These emissions
are characterised by typically long excited-state lifetimes
aqueous media. Methylation of the complexes caused a sys-
tematic red-shift of the emission profiles. The behaviour and
the effects of the different complexes were then examined
in cells. The neutral species localised mostly in the endoplas-
mic reticulum and lipid droplets, whereas the majority of the
cationic complexes localised in the mitochondria. The
amount of complexes found within cells does not depend
on lipophilicity, which potentially suggests diverse uptake
mechanisms. Methylated analogues were found to be more
cytotoxic compared to the neutral species, a behaviour that
might to be linked to a combination of uptake and intracel-
lular localisation.
(
~hundreds of ns), and quantum yields around 5–10% in
Introduction
II
[
a] C. Caporale, K. D. M. MaGee, Dr. P. J. Wright, Prof. M. Massi
Curtin Institute of Functional Molecules and Interfaces and
Department of Chemistry, Curtin University,
Kent Street, Bentley 6102 WA (Australia)
Luminescent metal complexes of transition [for example, Ru ,
I
III
I
II [1–4]
Re, Ir , Au, and Pt ]
and lanthanoid elements [for example,
III
III
III
Eu and Tb for visible emission; Yb for near-infrared emis-
[5,6]
sion]
have specific photophysical properties that are ideal
E-mail: m.massi@curtin.edu.au
for cellular imaging. These properties have the potential to
solve some of the critical drawbacks associated with more tra-
[
b] Dr. C. A. Bader, Dr. A. Sorvina, T. A. Gillam, Dr. S. E. Plush, Prof. D. A. Brooks
Mechanisms in Cell Biology and Disease Research Group,
School of Pharmacy and Medical Sciences,
Sansom Institute for Health Research,
[7]
ditionally investigated organic fluorophores. Appropriately
designed metal complexes exhibit enhanced photostability,
and therefore do not suffer from rapid photobleaching, allow-
University of South Australia, Adelaide (Australia).
E-mail: sally.plush@unisa.edu.au
[1]
doug.brooks@unisa.edu.au
ing for imaging at longer time scales. Luminescent heavy-
metal complexes are also typically characterised by large
Stokes shifts, which prevents concentration quenching phe-
[
c] Prof. B. W. Skelton
School of Molecular Sciences, University of Western Australia,
3
5 Stirling Highway, Crawley, Perth, WA 6009 (Australia)
[1]
nomena. Furthermore, their relatively longer excited-state
lifetimes make this class of luminophores amenable to time-
gated imaging to improve signal-to-noise ratio by minimising
[
d] Prof. P. Raiteri
Curtin Institute for Computation and Department of Chemistry,
Curtin University, Kent Street, Bentley 6102 WA (Australia)
[8]
[
e] Prof. S. Stagni
interference from fast-decaying autofluorescence.
Department of Industrial Chemistry “Toso Montanari”-
University of Bologna, viale del Risorgimento 4, Bologna 40136 (Italy)
Luminescent cyclometalated iridium(III) complexes are prov-
ing to be exceptional systems for the development of markers
[
f] Dr. S. E. Plush
Future Industries Institute, University of South Australia,
Mawson Lakes, SA 5095 (Australia)
[9,10]
for live cell imaging.
The chemical, photophysical, and bio-
logical targeting properties of these complexes can be readily
varied by altering the coordinating ligands or by chemical cou-
[
g] Prof. J. L. Morrison
[11,12]
Early Origins of Adult Health Research Group,
School of Pharmacy and Medical Sciences,
Sansom Institute for Health Research,
pling.
In addition, their absorption and emission profiles
can be modulated along the spectral range of visible light
from blue to red, and further into the near-infrared region, by
introducing appropriate functional groups or by the variation
University of South Australia, Adelaide, South Australia 5000 (Australia)
Supporting information and the ORCID identification number(s) for the au-
thor(s) of this article can be found under https://doi.org/10.1002/
chem.201701352
[13,14]
of the p conjugation on the aromatic ligands.
Following
similar principles, the lipophilicity of these complexes can be
Chem. Eur. J. 2017, 23, 1 – 15
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adjusted to favour transport across the cellular membrane,
biospecificity to the more popularly investigated cationic rhe-
[1,15]
[54]
while maintaining adequate solubility in aqueous media.
A
nium complexes. For example, we demonstrated unprece-
large number of luminescent iridium complexes have been re-
ported to possess not only the capacity to internalise into
dented staining of polar lipids in live cells using the complex
fac-[Re(CO) (phen)L] (L=5-(4-cyanophenyl)tetrazolate; phen=
3
[
13,16]
[55,56]
cells,
but also the ability to accumulate within specific or-
1,10-phenanthroline).
Furthermore, simple chemical modu-
[
17–22]
[15,23–25]
ganelles such as mitochondria,
lysosomes,
lation of the tetrazolato ancillary ligand has been a viable strat-
egy to impart alternative biological behaviour, with successful
staining of the endoplasmic reticulum and the visualisation of
[
26–30]
[31–34]
nuclei,
and endoplasmic reticulum.
Some iridium
complexes have been conjugated with biologically relevant
[
18,35]
[36–39]
[57]
moieties such as carbohydrates,
biotin/avidin,
amino
membrane events in live cells. We have now focused our at-
[
23]
[40]
acids, and proteins, in an attempt to impart and control
their biospecificity. Furthermore, examples of complexes with
the ability to sense targeted biological species via reversible or
irreversible modulation of their photophysical properties have
tention on the investigation of cyclometalated iridium com-
plexes bound to tetrazolato ligands, which is a family of neu-
tral and analogous cationic iridium species that have not been
previously used as molecular probes for live cell imaging. For
this scope, we prepared an initial library of complexes by
modifying the functional groups on the ligands and by the
extension of the p conjugation (Figure 1). Due to the intrinsic
reactivity of the coordinated tetrazolato ligand towards elec-
[
21,27,34,41–47]
also been reported.
A rationalisation of the structure–activity relationship (SAR)
of luminescent iridium complexes is still difficult to achieve
from the current knowledge base. This is partly due to the fact
that labelling experiments are often carried out with a diverse
range of complexes using different cell lines or model systems.
Moreover, the staining protocols used are often variable when
[11,58,59]
trophiles,
these neutral complexes could also be easily
methylated to form the analogous cationic species. This ap-
proach allowed the preparation of a family of complexes for a
more systematic SAR investigation that can relate how varia-
tions in the chemical structure and charge might influence the
biological behaviour of the iridium markers in relation to cellu-
lar incubation, localisation, and cytotoxicity.
[
48]
comparing data from different research groups. In addition,
a priority has been given to producing cationic iridium com-
plexes, seemingly arising from the fact that they might possess
increased solubility in aqueous media and exhibit more favour-
able membrane transport by exploiting membrane potential
gradients. However, many of these molecular complexes have
exhibited moderate to acute cytotoxicity, a property that can Results and Discussion
be regarded favourably for the joint development of therapeu-
[
22,24,49–51]
Synthesis of the tetrazolato ligands and corresponding
iridium complexes
tics,
but which is not ideal for visualising cellular func-
tions in live cell systems. Notably, only a handful of neutral iri-
dium complexes have been investigated as cellular imaging
The adopted synthetic procedures are shown in Figure 2. The
ligand precursor 2,5-dicyanopyridine was obtained in three
steps starting from commercially available 2,5-pyridinedi-
carboxylic acid. Following a Fisher esterification to form the
[
15,23,25,47,52,53]
agents.
We previously showed that neutral luminescent rhenium(I)
complexes can be used for live cell imaging, offering different
Figure 1. Neutral and cationic iridium tetrazolato complexes synthesised and investigated for live cell imaging.
&
&
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Figure 2. Summary of the synthetic procedures for the preparation of the iridium complexes.
corresponding methyl ester groups, aminolysis with aqueous
ammonia gave the corresponding primary amides. Lastly, dehy-
dration of the amide groups yielded the desired nitrile moiet-
the impure HTzPyCN was used as recovered for complexation
with iridium. The ligands HTzQn and HTziQn were synthesised
following the same methodology.
[
60]
[61]
ies.
Following a methodology developed by Sharpless,
To prepare the family of neutral iridium complexes
[Ir(ppy) (TzPyCN)], [Ir(F ppy) (TzPyCN)], [Ir(ppy) (TzQn)], and
2,5-dicyanopyridine was reacted with sodium azide in the pres-
2
2
2
2
ence of zinc bromide in water at reflux, yielding the HTzPyCN
ligand. Complete separation of HTzPyCN from the ditetrazole
byproduct (present in 5–10% molar amount determined by
NMR analysis) by selective protonation was challenging, and
[Ir(ppy) (TziQn)], the corresponding chloro-bridged iridium
2
dimers (bearing either ppy or F ppy as cyclometalating li-
2
gands) were reacted with a slight excess of the corresponding
[11]
pyridyl or (iso)quinolyl tetrazolato species.
The complex
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[
Ir(ppy) (TzPyPhCN)] was prepared via a different route, involv-
iridium complexes in diverse cellular environments such as cy-
toplasm, endosomes, acidic organelles (e.g. lysosomes), and
lipid-rich compartments like membranes, endoplasmic reticu-
lum, and lipid droplets. The photophysical properties were
measured in air-equilibrated solutions to account for the pres-
ence of oxygen in live cells. However, to confirm triplet multi-
plicity of the excited states, the measurements were also per-
2
ing a Suzuki coupling of the previously reported [Ir(p-
py) (TzPyBr)] with 4-cyanophenylboronic acid. Previous at-
[
11]
2
tempts to react the iridium chloro-bridged dimer with the pre-
formed corresponding tetrazolato ligand were unsuccessful, as
an inseparable mixture of products was obtained. Methylation
of all the neutral complexes was performed by treatment with
methyl trifluoromethanesulphonate, followed by metathesis
[62]
formed in degassed dichloromethane solutions.
[
11]
with ammonium hexafluorophosphate. All the synthesised
complexes were characterised by means of NMR (see the Sup-
The absorption spectra from air-equilibrated dichlorome-
thane solutions are presented in Figure 4 (for the spectral pro-
files in other solvents see the Supporting Information). All the
1
13
porting Information for all the H and C NMR spectra) and IR
spectroscopy as well as elemental analysis.
X-ray structural determination
Single crystals suitable for X-ray diffraction were obtained for
the neutral [Ir(F ppy) (TzPyCN)] and the charged [Ir(p-
2
2
py) (MeTzPyPhCN)][PF ] complexes. These species crystallized
2
6
in the monoclinic I2/a and P2/n space groups, respectively
Figure 3). The coordination arrangement of the ligands is
(
Figure 3. X-ray crystal structures of [Ir(F
2
ppy)
2
(TzPyCN)] (left) and [Ir(p-
+
py)
2
(MeTzPyPhCN)] (right), with displacement ellipsoids drawn at the 50%
ꢀ
6
) are
probability level. Lattice solvent molecules and counter anion (PF
omitted for clarity.
analogous, with the two pyridine rings of the phenylpyridine li-
gands adopting a trans configuration, whereas the two cyclo-
metalated phenyl rings are arranged in cis configuration. The
rest of the coordination sphere is occupied by the two nitro-
gen atoms of the tetrazole and pyridine rings. The [Ir(p-
py) (MeTzPyPhCN)][PF ] complex highlights methylation of the
2
6
tetrazole ring at the N3 position.
Photophysical properties
A summary of the photophysical properties for the synthesised
iridium complexes, measured from dichloromethane and aque-
ꢀ
5
ous (ca. 10 m) solutions are listed in Table 1 (see the Support-
ing Information for photophysical data in PBS, lysosomal pH
fluid solution, and ethyl laurate). The aqueous media contained
about 0.2% DMSO to favour the solubilisation of the iridium
complexes. The various solvents have been chosen to identify
potential modulations of the photophysical properties of the
ꢀ5
Figure 4. Absorption spectra of the iridium complexes from 10 m air-equili-
brated dichloromethane solutions.
&
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ꢀ
5
Table 1. Photophysical data of the iridium complexes from 10 m dichloromethane and aqueous solutions.
[
a]
Complex
Solvent
l
abs [nm]
l
em [nm]
t
t
aer
F
aer
4
-1
-1
(10 e [M cm ])
[
b]
[c]
deaer [ns]
F
deaer
1
5
31
29
0.055
0.397
0.047
CH
2
Cl
2
2
2
2
2
2
2
2
2
2
263 (6.57), 339 (1.59), 382 (1.09), 420 (0.58)
253 (3.52), 377 (0.69)
618
618
542
552
560
580
680
[
[
[
[
[
[
[
[
[
[
2
Ir(ppy) (TzPyCN)]
1
1
2
23 (25), 440 (75)
2
H O
[
d]
[d]
–
1
6
–
40
97
0.042
0.406
0.057
CH
2
Cl
256 (5.20), 364 (0.95)
2 2
Ir(F ppy) (TzPyCN)]
89 (41), 628 (59)
H
2
O
256 (4.83), 302 (3.36), 366 (1.16)
268 (3.95), 290 (3.64), 345 (0.58), 423 (0.27)
286 (1.87), 350 (0.86), 421 (0.34)
266 (8.93), 376 (1.44)
[
d]
[d]
–
–
1
14
80
00 (31), 728 (69)
0.023
0.149
0.055
2
CH Cl
8
Ir(ppy)
Ir(ppy)
2
2
(TzPyPhCN)]
(MeTzPyCN)]
2
H O
[
d]
[d]
–
–
3
3
1 (79), 168 (21)
5 (61), 362 (39)
0.012
0.014
2
CH Cl
+
[
[
e]
e]
[e]
–
–
2
5
–
–
[
e]
H
2
O
260 (4.44), 373 (0.59)
–
[e]
87
51
0.057
0.188
0.005
CH
2
Cl
250 (3.56), 316 (1.28), 356 (0.58)
260 (4.12), 309 (1.55), 353 (0.66)
267 (3.39), 290 (3.14), 378 (0.40)
264 (3.82), 390 (0.35)
600
636
600
605
580
600
575
590
635
630
640
660
+
2 2
Ir(F ppy) (MeTzPyCN)]
1
1 (76), 56 (24)
2
H O
[
d]
[d]
–
1
4
–
84
34
0.057
0.093
0.005
2
CH Cl
+
Ir(ppy)
Ir(ppy)
Ir(ppy)
Ir(ppy)
Ir(ppy)
2
2
2
2
2
(MeTzPyPhCN)]
(TzQn)]
1
7 (22), 320 (88)
2
H O
[
d]
[d]
–
1
8
–
53
31
0.021
0.065
0.039
CH
2
Cl
263 (8.44), 345 (1.97), 428 (0.42)
266 (4.41), 350 (2.35), 425 (0.97)
260 (9.77), 340 (3.13), 424 (0.75)
268 (3.42), 350 (2.00), 435 (0.80)
257 (9.29), 376 (0.95)
3
2
05 (25), 928 (75)
2
H O
[
d]
[d]
–
–
4
97
713
17 (33), 531 (67)
0.036
0.144
0.018
2
CH Cl
6
(TziQn)]
2
H O
[
d]
[d]
–
2
5
–
18
46
0.028
0.043
0.004
2
CH Cl
+
(MeTzQn)]
1
7 (39), 134 (61)
H
2
O
251 (8.30), 307 (2.52), 364 (1.09)
253 (6.96), 303 (2.45), 371 (1.24)
251 (6.64), 291 (2.60), 355 (1.36)
[
d]
[d]
–
2
4
–
08
85
0.049
0.063
0.004
2
CH Cl
+
(MeTziQn)]
9
(53), 93 (47)
2
H O
[
d]
[d]
–
–
[
a] Aqueous solutions contain 0.2% DMSO. [b] For the biexponential excited state lifetime (t), the relative weights of the exponential curves are reported
2
+
in parentheses; tdeaer is reported only for dichloromethane solutions. [c] Measured versus [Ru(bpy)
3
]
2 r
in H O (F =0.028). [d] Measurements in aqueous sol-
utions were only performed in air-equilibrated conditions. [e] Not emissive.
[
12]
iridium complexes show intense absorption bands in the UV
region (250–300 nm), which are associated with ligand-centred
character.
The [Ir(ppy) (TzPyCN)] complex presents a red-
2
shifted maximum at 618 nm, which is ascribed to a stabilisa-
ꢀ
(LC) p–p* transitions, followed by bands of lower intensity tail-
tion of the unoccupied p* orbitals localised on the TzPyCN
ing off into the visible region of the spectra. These lower
energy bands are ascribed to spin allowed (singlet to singlet)
and spin forbidden (singlet to triplet) ligand-to-ligand (LLCT)
ligand caused by the electron-withdrawing nature of the nitrile
[11]
functional group. As expected, the addition of electron-with-
drawing fluoride substituents on the ppy ligands causes a
blue-shift of the emission maximum for [Ir(F ppy) (TzPyCN)]
[11]
and metal-to-ligand charge transfer (MLCT) transitions.
2
2
Excitation of the iridium complexes at 375 nm results in
long-lived orange to red emission in air-equilibrated dichloro-
methane solutions, and the emission spectra measured at
(lem =542 nm), as a consequence of the stabilisation of the oc-
cupied p orbitals of the ligands and iridium 5d orbitals com-
posing the mixed LLCT(F ppy!TzPyCN) and MLCT(Ir!
2
[11]
298 K are shown in Figure 5 (for the emission spectra in other
TzPyCN) transition.
The emission maximum of [Ir(p-
solvents see SI). The neutral complexes [Ir(ppy) (TzPyCN)],
py) (TzPyPhCN)] at 560 nm is again blue-shifted with respect
2
2
[
Ir(F ppy) (TzPyCN)], and [Ir(ppy) (TzPyPhCN)] are character-
to [Ir(ppy) (TzPyCN)], albeit to a lesser extent when compared
2
2
2
2
ised by broad and featureless emission bands, that are typical
for radiative decays from excited states of charge transfer
to [Ir(F ppy) (TzPyCN)]. This trend is ascribed to the fact that
the electron-withdrawing effect of the nitrile group on the
2
2
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emission properties is analogous to that observed for their
neutral counterparts, with the most red-shifted emission origi-
+
nating from [Ir(ppy) (MeTzPyCN)] .
2
The iridium complexes containing quinoline and isoquino-
line-functionalised tetrazolato ligands, [Ir(ppy) (TzQn)] and
2
[
Ir(ppy) (TziQn)], reveal emission bands that are similarly rang-
2
ing between 500 and 750 nm (Figure 5). Compared to the pre-
[11]
ꢀ
viously published complex [Ir(ppy) (TzPy)], in which TzPy is
2
5-(2’-pyridyl)tetrazolate, displaying a structured emission band
with peaks at 481 and 510 nm, the complexes [Ir(ppy) (TzQn)]
2
and [Ir(ppy) (TziQn)] present a red-shifted emission that is ra-
2
tionalised by the increased p conjugation on passing from a
phenyl to a quinolyl or isoquinolyl substituent. The structures
of these two bands appear to be quite different. The emission
profile of [Ir(ppy) (TzQn)] is broad and structureless, whereas
2
that of [Ir(ppy) (TziQn)] appears to be structured with a vi-
2
ꢀ
1
bronic progression spaced around 1300 cm . Furthermore, the
excited-state lifetime of [Ir(ppy) (TziQn)] is significantly longer
2
than that of [Ir(ppy) (TzQn)] (Table 1). These results suggest
2
the presence of a more dominating charge transfer character
[64]
for the emission of [Ir(ppy) (TzQn)]. On the other hand, the
2
excited state of [Ir(ppy) (TziQn)] seems to be strongly influ-
2
[64]
enced by LC character. To confirm this assumption, we per-
formed time-dependent density functional theory (TD-DFT)
analysis on [Ir(ppy) (TzQn)] and [Ir(ppy) (TziQn)] (see the Sup-
2
2
porting Information). The results show that the lower energy
bands are mainly composed of MLCT and LLCT transitions.
However, in the case of [Ir(ppy) (TziQn)], there is also a contri-
2
bution from the HOMOꢀ4!LUMO transition corresponding to
ꢀ
a LC transition localised on the TziQn ligand. A further indica-
tion of the LC character of the excited state of [Ir(ppy) (TziQn)]
2
comes from the significantly elongated excited state decay life-
time in degassed dichloromethane, reaching a value of
[65]
6.713 ms.
As expected, the emission profiles of the methylated [Ir(p-
+
+
py) (MeTzQn)] and [Ir(ppy) (MeTziQn)] complexes appear
2
2
red-shifted, broad, and structureless (Figure 5).
For all the complexes, an elongation of the excited-state life-
time decay (t) and increase of photoluminescence quantum
yield (F) values is observed upon degassing. This behaviour is
indicative of the triplet spin multiplicity of the excited
[12,62]
state.
The analysis of the emission bands in various solvents re-
veals solvatochromic behaviour, with a red-shift of the emis-
sion maxima upon increasing the polarity of the solvent. The
most blue-shifted emission is recorded in ethyl laurate, where-
Figure 5. Emission spectra of the iridium complexes from approximately
ꢀ5
10 m air-equilibrated dichloromethane solutions with excitation at 375 nm.
as the emission red-shifts by ꢁ20–30 nm in dichloromethane,
+
pyridyltetrazole p* system is decreased by the addition
with the exception of [Ir(ppy) (MeTzPyPhCN)] and [Ir(p-
2
+
of a phenyl ring spacer. The emission profiles of the methylat-
py) (MeTziQn)] for which almost no shift is observed. The
2
+
ed
complexes
[Ir(ppy) (MeTzPyCN)]
(lem =680 nm),
and [Ir(ppy)2-
emission bands further shift (ꢁ10–30 nm) towards longer
2
+
[Ir(F ppy) (MeTzPyCN)]
(lem =600 nm),
wavelength in aqueous media. There are no significant differ-
2
2
+
(
MeTzPyPhCN)] (lem =600 nm) are red-shifted compared to
ences in the various aqueous solvents, apart from [Ir(p-
+
their corresponding neutral complexes. Once more, this red-
shift demonstrates that the p* system of the pyridyl tetrazole
ligand acts as an electron acceptor in the charge transfer tran-
py) (MeTzPyPhCN)] that shows a 22 nm red-shift on passing
2
from PBS to a lysosomal pH fluid solution. The methylated
+
complex [Ir(ppy) (MeTzPyCN)] is found to have appreciable
2
[
11,63]
sitions.
When comparing the emission maxima of these
emission only in dichloromethane solution, as the complex is
virtually non-emissive in aqueous solvents. In general, the
three methylated iridium complexes, the trend for the
&
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+
methylated complexes highlight a greater degree of quench-
ing in aqueous solvents with respect to their neutral ana-
logues. Although conclusive explanations cannot be drawn
from these data, potential causes could be ascribed to in-
creased non-radiative decays due to energy gap law and the
higher solubility of the cationic complexes in aqueous solvents.
In fact, we previously observed a similar behavior for neutral
rhenium complexes, in which a decrease of solubility in aque-
ous media showed an enhanced quantum yield and elongated
increased from 0.64 for [Ir(ppy) (MeTzPyCN)] to 1.86 for
2
+
[Ir(F ppy) (MeTzPyCN)] . In contrast, little change was ob-
2
2
served in the logD7.4 value between [Ir(ppy) (TzPyCN)] and
2
[Ir(F ppy) (TzPyCN)], consistently with previously published cy-
clometalated iridium complexes with lipophilicity values
2
2
[24]
around 2.
Cellular uptake and localisation
[
54]
excited state lifetime.
The cellular uptake for the synthesised iridium complexes was
studied by means of ICP-MS measurements, and the results are
reported in Table 2. The H9c2 cells were incubated with com-
plexes at 20 mm in cell culture medium (0.2% DMSO) for 30 mi-
nutes at 378C. The calculated cellular concentration values
were obtained assuming an average volume of a single H9c2
Lipophilicity
The lipophilicity of the iridium complexes was measured by
the shake-flask method using a pH 7.4 buffered PBS solution
[
66]
[72]
and n-octanol.
The calculated logD7.4 values are listed in
cell equal to 2 pL. An analysis of the data reveals that the as-
Table 2. The values obtained are within a range that is similar
sociation between internalisation and lipophilicity does not
follow a simple trend. For example, three of the neutral com-
plexes, [Ir(F ppy) (TzPyCN)], [Ir(ppy) (TzPyPhCN)], and [Ir(p-
2
2
2
Table 2. Distribution coefficient values (logD7.4) for neutral and methylat-
py) (TzQn)] exhibit high iridium intracellular concentrations in-
2
ed iridium complexes.
dicating good cellular internalisation. The remaining two com-
[
b]
plexes, [Ir(ppy) (TzPyCN)] and [Ir(ppy) (TziQn)], exhibit lower
2
2
Complex
logD7.4
Molar amoun- Concentration
[
a]
t
[mm]
intracellular iridium concentrations although their lipophilicity
fell within the range of the aforementioned neutral complexes.
On the other hand, only the cationic iridium complexes bound
to the quinolyl and isoquinolyl tetrazolato ligands exhibit effi-
cient cellular internalisation. The lack of straightforward trends
suggests that the mechanism of internalisation might be differ-
ent for the various complexes.
[fmol]
[
[
[
[
[
[
[
[
[
[
Ir(ppy)
2
(TzPyCN)]
(TzPyCN)]
(TzPyPhCN)]
(MeTzPyCN)]
2.09ꢂ0.06 0.042ꢂ0.004
2.01ꢂ0.05 0.319ꢂ0.049
2.68ꢂ0.08 0.171ꢂ0.042
0.64ꢂ0.03 0.015ꢂ0.002
1.86ꢂ0.02 0.025ꢂ0.007
1.87ꢂ0.08 0.045ꢂ0.005
2.23ꢂ0.04 0.346ꢂ0.070
2.57ꢂ0.05 0.063ꢂ0.011
1.68ꢂ0.05 0.372ꢂ0.038
1.49ꢂ0.06 0.515ꢂ0.085
21.16ꢂ1.85
Ir(F ppy)
2
2
159.71ꢂ46.95
85.46ꢂ20.76
7.35ꢂ0.81
Ir(ppy)
Ir(ppy)
2
+
2
+
Ir(F
2
ppy)
2
(MeTzPyCN)]
12.74ꢂ3.26
22.74ꢂ2.48
173.03ꢂ35.20
31.61ꢂ5.60
186.24ꢂ18.92
257.29ꢂ42.64
+
Ir(ppy)
Ir(ppy)
Ir(ppy)
Ir(ppy)
Ir(ppy)
2
2
2
2
2
(MeTzPyPhCN)]
(TzQn)]
(TziQn)]
(MeTzQn)]
(MeTziQn)]
The cellular localisation of the iridium complexes was inves-
tigated via confocal microscopy. Following the same incuba-
tion protocol described for the ICP-MS measurements, com-
plexes could be detected in cells by single-photon excitation
at 403 nm and were also shown to be compatible with two-
photon imaging modalities, with optimal excitation in the
range of 810–830 nm (see the Supporting Information). All the
complexes could be readily detected within cells in an emis-
sion interval of 525–644 nm, (consistent with emissions record-
+
+
[
a] Average number of moles of iridium complex incubated within a H9c2
cell (see the Experimental Section for incubation conditions). [b] The rela-
tive concentration was calculated assuming an average volume of H9c2
cell equal to 2 pL.
to those reported previously for cyclometalated iridium
ed in the cuvette), allow for co-staining with commercially
[
18,24,26,53]
+
complexes,
and, in the case of the neutral complexes,
available organelle markers. The [Ir(ppy) (MeTzPyCN)] com-
2
fluorophores for the lipid droplets of the BODIPY family (func-
plex was not detected in cells, therefore co-localisation studies
were not performed for this species.
[67]
tionalised 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene cores).
The values for the five neutral complexes are comprised be-
tween 2.01 and 2.68. Methylation of the iridium complexes
lowers the lipophilicity, which is ascribed to the introduction of
the cationic charge, and results in logD7.4 values in the range
All the neutral complexes had similar staining patterns and
evidenced a diffuse network emanating from the nucleus and
extending into the cytoplasm (Figure 6), consistent with the
endoplasmic reticulum (ER). In addition, the methylated cation-
+
1
.49–1.97. The lowest lipophilicity is observed for the complex
ic complex [Ir(ppy) (MeTzQn)] also detected this reticular net-
2
+
[Ir(ppy) (MeTzPyCN)] at 0.64. A comparison of the logD7.4
work in the perinuclear region (Figure 6). Co-staining with ER-
Trackerꢁ revealed relatively high Pearson’s correlation coeffi-
2
values between [Ir(ppy) (TzPyCN)], [Ir(ppy) (TzPyPhCN)], [Ir(p-
2
2
[73]
py) (TzQn)], and [Ir(ppy) (TziQn)], shows that extending the
cients, with values above 0.7, confirming these complexes
localise within the ER (see the Supporting Information).
2
2
conjugation of the tetrazolato ligands increases lipophilicity. A
similar trend also holds true for the methylated analogues,
Although the neutral complexes and the methylated cationic
+
+
+
[
Ir(ppy) (MeTzPyCN)] ,
[Ir(ppy) (MeTzPyPhCN)] ,
[Ir(p-
complex [Ir(ppy) (MeTzQn)] were predominately localised
2
2
2
+
+
py) (MeTzQn)] , and [Ir(ppy) (MeTziQn)] . The introduction of
within the ER, their emission was also detected within com-
partments in the cytoplasm that were not labelled with ER-
Trackerꢁ. The morphology and localisation of these compart-
2
2
fluoride substituents on aromatic moieties is often associated
[
68–71]
with an increase in logD7.4 values.
However, this trend was
ꢂ
only observed in the methylated complexes, in which logD7.4
ments resembled lipid droplets. BODIPY 500/510 C ,C , a fatty
1
12
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Figure 6. Iridium complexes interacting with endoplasmic reticulum (ER) in
Figure 7. Iridium complexes localise with lipid droplets in live H9c2 cells. Mi-
live H9c2 cells. Micrographs of H9c2 cells stained with the iridium complexes
crographs of H9c2 cells stained with the iridium complexes (green) and
(green) and counter-stained with ER Trackerꢁ (red). Yellow/orange colour in
ꢂ
counter-stained with BODIPY 500/510 C
1
,C12 fatty acid analogue (red).
the merge indicates marker co-localisation. The iridium complexes were ex-
cited at 403 nm and the emission was collected in the region 525–644 nm;
ER-Tracker was excited at 561 nm and the emission was collected in the
region 570–620 nm. Scale bars: 10 mm.
Yellow/orange colour in the merge indicates marker co-localisation. The iridi-
um complexes were excited at 403 nm and the emission was collected in
the region 525–644 nm; BODIPY500/510 was excited at 488 nm and the
emission was collected in the region 500–550 nm. Scale bars: 10 mm.
acid analogue that localises with lipid droplets and partially
with the endoplasmic reticulum, produced strong co-localisa-
tion with these complexes (Figure 7). A high Pearson’s correla-
tion coefficients was obtained for these complexes and
ꢂ
BODIPY 500/510 C ,C (ranging between 0.63 and 0.92; see
1
12
[
73]
the Supporting Information), confirming that these compart-
ments were lipid droplets.
+
The methylated complexes [Ir(F ppy) (MeTzPyCN)] and
2
2
+
[
Ir(ppy) (MeTzPyPhCN)] localised to elongated structures
2
throughout the cytoplasm, a staining pattern that is character-
istic of mitochondrial association (Figure 8). Co-staining with
ꢂ
MitoTracker Red CMXRos confirmed this interaction, with
Pearson’s correlation coefficient between 0.65 and 0.71 (see
[
73]
the Supporting Information). It is possible that the positive
charge favours the transport of these complexes across the mi-
tochondrial membrane as a consequence of the intermem-
brane electrical potential gradient. This explanation is consis-
tent with the typical mitochondrial affinity of delocalised lipo-
philic cations such as phosphonium salts, or previously report-
Figure 8. Iridium complexes interacting with mitochodria in live H9c2 cells.
Micrographs of H9c2 cells stained with the iridium complexes (green) and
ꢂ
counter-stained with Mito-Tracker Red CMXRos (red). Yellow/orange colour
in the merge indicates co-localisation between markers. The iridium com-
plexes were excited at 403 nm and the emission was collected in the region
[
74]
ed cationic metal complexes. It is interesting to note that
525–644 nm; MitoTracker was excited at 561 nm and the emission was col-
+
the complex [Ir(F ppy) (MeTzPyCN)]
also stained what
lected in the region 570–620 nm. Scale bars: 10 mm.
2
2
&
&
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appears to be the ER along with the mitochondria, although
co-staining between the complex and ER-Tracker could not be
This suggests that energy dependent pathways are predomi-
nantly involved in the cellular uptake of these complexes, a
result that is consistent with previously studied cyclometalated
performed (see the Supporting Information). However, [Ir(p-
+
[17,26,42,46,76]
py) (MeTziQn)]
also co-located with MitoTrackerꢂ Red
iridium complexes incubated in various cell lines.
2
CMXRos, this charged complex appeared to induce apoptosis,
characterised by the formation of cellular vacuoles and round-
ing of the mitochondria (Figure 8). In this case, ER-Trackerꢁ
was also observed to co-localise with these unusual cellular
Again, the lack of clear trends in Figure 9 suggests variable
mechanism of entrance even for subtle chemical changes. For
example, [Ir(ppy) (TzQn)] is detected well upon incubation at
2
48C, but [Ir(ppy) (TziQn)] is barely visible, even though the
2
structures. An increase of caspase activity in H9c2 cells after in-
two complexes differ only by the orientation of the quinolyl
+
cubation with [Ir(ppy) (MeTziQn)] confirmed the induction of
substituents and have very similar lipophilicity values. Our re-
2
[
75]
[48,77]
apoptosis (see the Supporting Information).
sults reinforce previously reported views
that conclusions
Confocal microscopy was also employed to assess the mech-
anism of cellular internalisation. The intensity of the emission
from the iridium complexes was compared between cells incu-
bated at 37 and 48C (Figure 9), the latter to suppress energy-
mediated mechanisms of entrance. The emission detected
from each complex shows a good correlation with the average
amount of iridium found in cells measured by ICP-MS (Table 2).
Cells incubated with [Ir(F ppy) (TzPyCN)], [Ir(ppy) (TzPyPhCN)],
based exclusively on lipophilicity measurements might not be
adequate, even for families of complexes with very similar
chemical structures.
Cytotoxicity
The cell viability was assessed via MTS assay to highlight cyto-
toxic effects after internalisation of the iridium complexes
(without photoexcitation). The H9c2 cells were incubated for
either 2 or 24 hours with the complexes at 20 mm or 40 mm. An
important trend emerges from the collected results (Figure 10),
highlighting the fact that although the neutral complexes only
exhibit slight cytotoxicity, cell viability is reduced upon incuba-
tion with the corresponding cationic complexes. On comparing
the cytotoxicity and ICP-MS data from incubated cells, it is
clear that cytotoxic effects are not exclusively dependent on
the relative amount of iridium complexes incubated within the
2
2
2
+
+
[
Ir(ppy) (TzQn)], [Ir(ppy) (MeTzQn)] and [Ir(ppy) (MeTziQn)]
2
2
2
show the highest emission intensity, and also record the larg-
est concentration of iridium per cell. The trend in emission in-
tensity also correlates well with the quantum yield values
(
Table 1). For example, the emission detected from [Ir(p-
+
py) (TzPyPhCN)] is comparable with [Ir(ppy) (MeTzQn)] and
[
2
2
+
Ir(ppy) (MeTziQn)] , even though the former was detected at
2
a lower concentration. The emission detected from each of the
cells following incubation at 48C was significantly reduced.
Figure 9. Confocal micrographs of H9c2 cells incubated with iridium complexes at 378C (A) or 48C (C). Scale bars: 50 mm. (B) Histogram showing the emission
intensity of iridium complexes detected from H9c2 cells when incubated at 378C (grey bars) or 48C (black bars) to inhibit energy dependent cell entry.
Chem. Eur. J. 2017, 23, 1 – 15
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Figure 10. Cell viability after 2 and 24 hours incubation with the iridium complexes at 20 and 40 mm. Neutral complexes are represented by blue bars, where-
+
as cationic complexes are represented by green bars. 0) Control (0.2% DMSO in serum-free DMEM media); 1) [Ir(ppy)
2
(TzPyCN)]; 2) [Ir(ppy)
2
(MeTzPyCN)] ;
+
+
+
3
) [Ir(F
2
ppy)
2
(TzPyCN)]; 4) [Ir(F
2
ppy)
2
(MeTzPyCN)] ; 5) [Ir(ppy)
2
(TzPyPhCN)]; 6) [Ir(ppy)
2
(MeTzPyPhCN)] ; 7) [Ir(ppy)
2
(TzQn)]; 8) [Ir(ppy)
2
(MeTzQn)] ; 9) [Ir(p-
+
py) (TziQn)]; and 10) [Ir(ppy)
2
2
(MeTziQn)] .
Photostability
cells. For example, the fluorinated complex [Ir(F ppy) (TzPyCN)]
2
2
does not exhibit significant cytotoxicity compared to its me-
Photobleaching experiments were carried out on H9c2 cells in-
cubated with the iridium complexes at 20 mm for 30 minutes
(see SI). The cells were illuminated continuously at 403 nm for
900 s, and the decrease in emission intensity was compared
with that from cells incubated with ER-Tracker. All the com-
plexes evaluated in cells showed better photostability than ER-
Tracker, with the cationic complexes exhibiting emission inten-
sity above 85% of the initial values after 900 s irradiation. The
neutral iridium complexes proved to be slightly more prone to
photobleaching, but still displayed emission above 70% of the
initial emission intensity after 900 s irradiation. The only excep-
+
thylated analogue [Ir(F ppy) (MeTzPyCN)] , even though the
2
2
concentration of the latter is lower by an order of magnitude.
These results suggest that cytotoxic effect for this family of iri-
dium complexes might be strongly related to their relative cel-
lular localisation.
The only exception to the cytotoxic effect of the cationic
+
complexes seems to be related to [Ir(ppy) (MeTzPyCN)] ,
2
which from the ICP-MS results showed minimal accumulation
within the cells. Unfortunately however, the localisation of this
compound could not be assessed due to its non-emissive
nature in aqueous medium.
tion was the fluorinated [Ir(F ppy)(TzPyCN)], which showed a
2
It is well-known that phosphorescent metal complexes act
photobleaching profile just slightly slower that ER-Tracker. The
data clearly highlight how the cationic complexes exhibit
better photostability than their neutral analogues. The same
general trend was also observed from photobleaching experi-
[
78]
as sensitisers for singlet oxygen. Therefore, during imaging
with confocal microscopy a resting time of at least 30 seconds
is always provided to the cells to recover from a potential in-
crease in reactive oxygen species. This is a protocol we opti-
mised during our previous studies using rhenium complexes
ꢀ
5
ments in cuvettes using 10 m aqueous solutions (0.2%
DMSO), excited using a Pen-Ray Mercury Lamp source at
365 nm (see the Supporting Information for experimental
details).
[
79]
as cellular labels. Using this protocol, and imaging cells incu-
bated for 30 minutes with a 20 mm solution of iridium complex,
blebbing of the cell membrane was never observed. On the
other hand, cells incubated under the same conditions and
continuously illuminated showed signs of blebbing within a
Conclusions
3
0 minute timeframe (see the Supporting Information). These
This work reports the synthesis of iridium tetrazolato com-
plexes and their potential use as probes in live cells. A library
of neutral complexes displaying chemical variations at the cy-
clometalating phenylpyridine and tetrazolato ligands was syn-
thesised. Furthermore, methylation of these species allowed
facile access to the corresponding cationic complexes, so that
results demonstrate that the neutral series of iridium tetrazola-
to complexes possess very promising properties for the devel-
opment of iridium-based markers for imaging of the ER in live
cells. This is in contrast to other cationic iridium complexes
that were previously shown to accumulate in the endoplasmic
reticulum, but which were cytotoxic due to the triggering of
a
SAR for cellular internalisation, localisation, and
2
+
[80]
Ca release into the cytoplasm.
cytotoxicity could be more systematically investigated. The
&
&
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photophysical properties of these species revealed typical
phosphorescent emission from excited states of charge trans-
X-ray crystallography
Crystallographic data for the structure were collected at 100(2) K
on an Oxford Diffraction Gemini diffractometer with Mo Ka or Cu
Ka radiation. Following absorption corrections and solution by
3
3
fer nature, as a mixture of MLCT and LLCT. The emission of
the complexes was found to be in general solvatochromic,
with a red-shift occurring upon increase of medium polarity.
Quantification of cellular incubation via ICP-MS revealed great
variability, with trends that cannot be simply correlated to the
degree of lipophilicity and charge of the complexes. These re-
sults suggest potentially different mechanisms of uptake, and
a comparison of the emission intensity from cells incubated at
2
direct methods, the structures were refined against F with full-
[
83]
matrix least-squares using the program SHELXL-2014. All hydro-
gen atoms were added at calculated positions and refined by use
of riding models with isotropic displacement parameters based on
those of the parent atoms. Anisotropic displacement parameters
were employed throughout for the non-hydrogen atoms. Structure
refinement data are reported in the Supporting Information.
37 and 48C reveals these are predominantly energy mediated.
CCDC 1506221 ([Ir(F ppy) (TzPyCN)])
and
1506222 ([Ir(p-
2
2
The majority of the complexes were internalised into cells and
detection was possible via either one-photon or two-photon
imaging modalities. The neutral species were predominantly
localised within the endoplasmic reticulum and to various ex-
tents within lipid droplets. On the other hand, the cationic spe-
cies exhibited more specificity for mitochondria. A marked dif-
ference between the neutral and cationic probes was found in
terms of cytotoxicity, which was restricted mainly to the
charged complexes during long term experiments. The higher
cytotoxicity of the cationic complexes does not seem to be ex-
clusively linked to the amount of complex internalised within
the cells, and it is potentially highly dependent also on the lo-
calisation. This study has therefore highlighted that neutral iri-
dium tetrazolato complexes are suitable building blocks for
the design of markers for cellular imaging on long timescales,
and more specifically for the imaging of ER and lipid droplets.
However, specific protocols for imaging modalities (e.g., rest-
ing time between consecutive excitation regimes) are essential
to avoid photocytotoxicity. On the other hand, cationic iridium
tetrazolato complexes are good candidates for the design of
mitochondrial markers. However, careful considerations must
be taken for longer time experiments, as these cationic com-
plexes have demonstrated higher cytotoxicity with the respect
to their neutral analogues.
py) (MeTzPyMeCN)][PF ]) contain the supplementary crystallo-
graphic data for this paper. These data are provided free of charge
by The Cambridge Crystallographic Data Centre.
2
6
Photophysical measurements
Absorption spectra were recorded at room temperature using a
Cary 4000 UV/Vis spectrometer. Uncorrected steady state emission
and excitation spectra were recorded on an Edinburgh FLSP980-
S2S2-stm spectrometer equipped with: i) a temperature-monitored
cuvette holder; ii) 450 W Xenon arc lamp; iii) double excitation and
emission monochromators; iv) a Peltier cooled Hamamatsu R928P
photomultiplier tube (spectral range 200–870 nm). Emission and
excitation spectra were corrected for source intensity (lamp and
grating) and emission spectral response (detector and grating) by
a calibration curve supplied with the instrument. According to the
[84]
approach described by Demas and Crosby, luminescence quan-
tum yields (Fem) were measured in optically dilute solutions (O.D.
<
0.1 at excitation wavelength) obtained from absorption spectra
on a wavelength scale [nm] and compared to the reference emitter
by Equation (1):
ꢀ
ꢁꢀ
ꢁꢀ ꢁꢀ
ꢁ
2
A ðl Þ I ðl Þ nx Dx
r
r
r
r
x
F ¼ Fr
2
A ðl Þ I ðl Þ nr
A
r
x
x
x
x
Experimental Section
where A is the absorbance at the excitation wavelength (l), I is the
intensity of the excitation light at the excitation wavelength (l), n
is the refractive index of the solvent, D is the integrated intensity
of the luminescence and F is the quantum yield. The subscripts r
and x refer to the reference and the sample, respectively. The
quantum yield determinations were performed at identical excita-
tion wavelength for the sample and the reference, therefore can-
celling the I(l )/I(l ) term in the equation. The quantum yields of
General Considerations
Unless otherwise stated, all reagents and solvents were purchased
from Sigma Aldrich or Alfa Aesar and used as received without fur-
[81]
[81]
ther purification. The species HTzQn,
HTziQn,
[Ir(ppy) (m-
2
[82]
[82]
2
[11]
Cl)]2,
[Ir(F ppy) (m-Cl)]
2
2
and [Ir(ppy) (TzPyBr)] were prepared
2
according to previously published procedures. Detailed synthetic
procedures for the iridium complexes and corresponding ligands
described in this work are reported in the Supporting Information.
r
x
complexes were measured against an aqueous solution of [Ru(bi-
[85]
py) ]Cl (bipy=2,2’-bipyridine; F =0.028). Emission lifetimes (t)
3
2
r
Nuclear magnetic resonance spectra were recorded using a Bruker
were determined with the time-correlated single-photon counting
technique (TCSPC) with the same Edinburgh FLSP980-S2S2-stm
spectrometer using either a pulsed picosecond LED (EPLED/EPL
377 nm, FHWM <800 ps). The goodness of fit was assessed by
1
Avance 400 spectrometer (400 MHz for H NMR; 100 MHz for
1
3
C NMR) at 300 K. All NMR spectra were calibrated to residual sol-
vent signals. Infrared spectra were recorded using an attenuated
total reflectance PerkinElmer Spectrum 100 FT-IR with a diamond
2
minimising the reduced c function and by visual inspection of the
ꢀ1
stage. IR spectra were recorded from 4000-650 cm . The intensity
of the band is reported as strong (s), medium (m), or weak (w),
with broad (br) bands also specified. Melting points were deter-
mined using a BI Barnsted Electrotermal 9100 apparatus. Elemental
analyses were carried out on bulk samples using a Thermo Finning
EA 1112 Series Flash; the presence of solvents in the bulk samples
weighted residuals. The solvents used for the preparation of the
solutions for the photophysical investigations were of LR grade
and the water was deionised. Degassing of the dichlorometane sol-
utions was performed using the freeze-pump-thaw method. Experi-
mental uncertainties are estimated to be ꢂ8% for lifetime deter-
minations, ꢂ20% for quantum yields, ꢂ2 nm and ꢂ5 nm for ab-
sorption and emission peaks, respectively.
1
was further confirmed by H NMR.
Chem. Eur. J. 2017, 23, 1 – 15
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TD-DFT
ed at 403 nm and the emission was collected in the 525–644 nm
region; ER-Tracker and MitoTracker were excited at 561 nm and
the emission was collected in the region 570–620 nm. BODIPY 500/
Time-dependent density functional theory calculations were per-
formed with GAUSSIAN 09. Prior to these calculations, the struc-
tures were relaxed at the B3LYP level of theory.
were treated with the Stuttgart-Dresden effective core potential,
[86]
5
10 was excited at 488 nm and the emission was collected in the
[
87,88]
The Ir atoms
region 500–550 nm. Image analysis including, co-localisation and
emission intensity was measured using NIS elements V4.50 soft-
ware (Nikon, Japan). The final preparation of the images was con-
ducted with Adobe Photoshop CC (Adobe Systems Inc., USA).
Two-photon microscopy imaging was performed using a Zeiss
LSM710 META NLO inverted microscope supplemented with a two-
photon Mai-Taiꢂ, tunable Ti:Sapphire femtosecond pulse laser
[89]
the Pople 6–311G** basis set was used for all the other atoms, and
the effect of the solvent was mimicked with the PCM solvation
[90]
model, with parameters adequate for dichloromethane. The low-
lying singlet-singlet excitation energies were calculated at the
same level of theory, and the spectra were reproduced as the su-
perposition of Gaussian functions with heights proportional to cal-
culated intensities and a variance of 11 nm.
(710–920 nm, Spectra-Physics). The emission from the iridium com-
plexes was detected using the following settings: two-photon exci-
tation wavelength at 830 nm, beam splitter MBS 690+, emission
interval 520–650 nm. The images were acquired using a Plan-APO-
CHROMAT 63X/ NA1.4 oil immersion objective. The final prepara-
tion of the images was conducted with Adobe Photoshop CC
Cell culture
H9c2 rat cardiomyoblast cells were maintained in high-glucose
ꢀ1
(
4500 mgL ) DMEM medium (Sigma–Aldrich, USA) containing
(Adobe Systems Inc., USA).
1
2
0% fetal bovine serum (FBS; In Vitro Technologies, USA) and
mm l-glutamine (Sigma–Aldrich, USA) at 378C and 5% CO . The
2
2
H9c2 cells were cultured in 75 mm flasks. Cells that had been pas-
saged for no more than 18 times, were used for experiments. For
ICP-MS, H9c2 cells were seeded at 2.7ꢃ10 cellsmL and grown in
T25 flasks for 72 h, when 100% confluence was reached. For live
MTS cell viability assay
4
ꢀ1
The cellular NADPH-dependent redox activity was measured using
ꢂ
CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay
5
(MTS), according to the manufacturer’s instruction (Promega, USA).
The H9c2 cells were stained with the iridium complexes at either
cell imaging and MTS assay, the H9c2 cells were seeded at 1ꢃ10
ꢀ
1
cellsmL and cultured overnight in either ibidi m-slide 8 wells in a
final volume of 250 mL or 96-well microtiter plate in a final volume
of 200 mL.
2
0 mm or 40 mm in DMEM full cell culture medium with 0.2%
DMSO, and held at 378C and 5% CO for 4 or 24 hours. As a con-
2
trol, H9c2 cells were incubated for the same length of time in
DMEM with 0.2% DMSO. Following addition of MTS, the cells incu-
ICP-MS
bation for a further 2 hours at 378C and 5% CO . The absorbance
2
of the formazan dye was measured by EnVision multi-label plate
reader (PerkinElmer, Beaconsfield, UK) at 490 nm. The data are re-
ported as the mean ꢂ SEM of three biological replicates for each
group.
The H9c2 cells were incubated with the iridium complexes at
2
3
0 mm (prepared in DMEM full culture media with 0.2% DMSO) for
0 minutes at 378C and 5% CO2. The cells were washed three
times with phosphate buffered saline (PBS), and then detached
with TripLE Express reagent (Gibco, USA) for 2 minutes. The cells
were then washed from T25 flasks with 3 mL of DMEM full culture
medium, and pelleted by centrifugation at 400 rpm for 10 minutes.
The medium was removed and the cells were resuspended in 2 mL
of PBS for cells counts. The remaining cells were centrifuged at
Supporting Information
1
13
The Supporting Information contains: synthetic details; H and
C
spectra; X-ray refinement data; all the photophysical details in the
various solvent systems including absorption, excitation, and emis-
sion spectra, excited state lifetime decays; TD-DFT calculation for
1
000 rpm for 10 minutes, the medium was then removed leaving
behind the stained cell pellets, which were desiccated for 3.5 h at
58C. The pellets were reconstituted in 36% HCl (400 mL) and left
9
[Ir(ppy) (TzQn)] and [Ir(ppy) (TziQn)], confocal imaging via two-
2 2
at 958C until dry in order to digest organic material. The dry sam-
photon excitation of live H9c2 cells; Pearson’s co-localisation coeffi-
ples were then reconstituted in 2% HCl (2.5 mL) and sonicated for
cient table; caspase assay for cells incubated with[Ir(ppy)(MeT-
ziQn)] ; live cell response to imaging conditions; photostability of
+
1
0 minutes before filtering (pore diameter of 0.2 mm). The total iri-
dium determination was performed with an Agilent 8900x triple
quad ICP-MS (Agilent Technologies, USA), using He gas
the iridium complexes in cells and cuvette.
ꢀ1
(
4 mLmin ).
Acknowledgements
Cell staining
M.M. and P.R. wishes to thank the ARC for funding
H9c2 cells were incubated with the iridium complexes at 20 mm in
DMEM full culture medium with 0.2% DMSO for 30 minutes at
either 378C or 48C. The cells were washed twice with PBS, before
imaging in DMEM. For co-staining experiments, the cells were then
(
FT130100033 and FT130100463). D.A.B. and J.L.M. wishes to
thank the NHMRC for funding (APP1092904 and APP1066916,
respectively). C.C. wishes to thank Curtin University for the
IPRA scholarship. The authors acknowledge the facilities, and
the scientific and technical assistance of the Australian Micros-
copy & Microanalysis Research Facility at the Centre for Micros-
copy, Characterisation & Analysis, at the University of Western
Australia, a facility funded by the University, State and Com-
monwealth Governments. The authors acknowledge Adelaide
Microscopy, University of Adelaide, South Australia. This re-
search was undertaken with the assistance of resources from
ꢁ
ꢂ
incubated with ER-Tracker Red, MitoTracker Red CMXRos, or
ꢂ
BODIPY 500/510 C ,C (Life Technologies Australia Pty Ltd, Austral-
1
12
ia), following manufacturer’s instructions.
Confocal micoscopy
Single-photon live cell imaging was performed using a Nikon A1+
confocal microscope (Nikon, Japan) with a OKOLab Microscope In-
cubator (Okolab USA Inc., USA). The iridium complexes were excit-
&
&
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www.chemeurj.org
12
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Full Paper
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FULL PAPER
A glowing family in cell: Neutral iridi-
um tetrazolato complexes provide ex-
ceptional building block for the design
of molecular probes for the endoplas-
mic reticulum in live cells, while methyl-
ation causes the complexes to shift in-
teraction to the mitochondria and ex-
hibit enhanced cytotoxicity.
&
Fluorescent Probes
C. Caporale, C. A. Bader, A. Sorvina,
K. D. M. MaGee, B. W. Skelton,
T. A. Gillam, P. J. Wright, P. Raiteri,
S. Stagni, J. L. Morrison, S. E. Plush,*
D. A. Brooks,* M. Massi*
&
& – &&
Investigating Intracellular Localisation
and Cytotoxicity Trends for Neutral
and Cationic Iridium Tetrazolato
Complexes in Live Cells
Chem. Eur. J. 2017, 23, 1 – 15
www.chemeurj.org
15
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&
These are not the final page numbers! ÞÞ