Tracking mitochondrial dynamics during apoptosis with phosphorescent fluorinated iridium(iii) complexes

Article abstract of DOI:10.1039/c8dt02918k

Mitochondria are the control centers of apoptosis. To study mitochondrial dynamics during apoptosis, four phosphorescent fluorinated iridium(iii) complexes (Ir1-Ir4) were designed and synthesized. The complexes' emission maxima, phosphorescent quantum yie

Full text of DOI:10.1039/c8dt02918k

View Article Online
View Journal
Dalton
Transactions
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: C. Zhang, K. Qiu,
C. Liu, H. Huang, T. W. Rees, L. Ji, Q. Zhang and H. Chao, Dalton Trans., 2018, DOI:
10.1039/C8DT02918K.
This is an Accepted Manuscript, which has been through the
Volume 45 Number
1 7 January 2016 Pages 1–398
Royal Society of Chemistry peer review process and has been
accepted for publication.
Dalton
Transactions
Accepted Manuscripts are published online shortly after
An international journal of inorganic chemistry
www.rsc.org/dalton
acceptance, before technical editing, formatting and proof reading.
Using this free service, authors can make their results available
to the community, in citable form, before we publish the edited
article. We will replace this Accepted Manuscript with the edited
and formatted Advance Article as soon as it is available.
You can find more information about Accepted Manuscripts in the
author guidelines.
Please note that technical editing may introduce minor changes
to the text and/or graphics, which may alter content. The journal’s
standard Terms & Conditions and the ethical guidelines, outlined
in our author and reviewer resource centre, still apply. In no
event shall the Royal Society of Chemistry be held responsible
for any errors or omissions in this Accepted Manuscript or any
consequences arising from the use of any information it contains.
ISSN 1477-9226
PAPER
Joseph T. Hupp, Omar K. Farha et al.
Efficient extraction of sulfate from water using
framework
a Zr-metal–organic
rsc.li/dalton

Please do not adjust margins
Dalton Transactions
Page 1 of 8
View Article Online
DOI: 10.1039/C8DT02918K
Journal Name
ARTICLE
Tracking mitochondrial dynamics during apoptosis with
phosphorescent fluorinated iridium(III) complexes†
Chen Zhang,‡a Kangqiang Qiu,‡ab Chaofeng Liu,a Huaiyi Huang,a Thomas W. Rees,a Liangnian Ji,a
Qianling Zhang,*b and Hui Chao*ab
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Mitochondria are the control centers of apoptosis. To study mitochondrial dynamics during apoptosis, four
phosphorescent fluorinated iridium(III) complexes (Ir1-Ir4) were designed and synthesized. The complexes emission
maxima phosphorescent quantum yields, and phosphorescent lifetimes are tuned by the degree of fluorination of the
ligand. The complexes exhibit excellent photostability and low (photo)cytotoxicity in HeLa cells. As the complexes are
cationic and lipophilic, they localize in mitochondria and enter cells through an energy-independent pathway. In
comparison with commercially available mitochondrial trackers MTR, Ir1-Ir4 exhibit high specificity to mitochondria even
in fixed cells. Due to these outstanding properties, the complexes were successfully used to track mitochondrial dynamics
during apoptosis.
www.rsc.org/
structural properties.29-31 Among them, phosphorescent Ir(III)
complexes have been shown as promising candidates for bio-
imaging.32-35 Compared to commercial dyes, Ir(III) probes
Introduction
Apoptosis (from the Greek word for leaves falling off trees),
was first used to describe natural cell death in the 19th century
by embryologists and anatomists.1-3 It is prevalent in
eukaryotes,4,5 which could keep the balance between new-
generating cells from mitosis and cell death.6 Apoptosis
defects can cause a variety of malignant diseases, even
cancer.7 The key to triggering or inhibiting apoptosis lies in the
mitochondria.9,10 Mitochondria are the powerhouse of cells11-
possess the advantages of lower cytotoxicities, stronger
phosphorescence intensities, longer phosphorescent lifetimes
and good photostabilities.29-39 Cationic Ir(III) complexes are
particularly of interest as they can specifically target
mitochondria due to the mitochondrial membrane potential of
-180 mV.25,26,31,40 However, most reported mitochondria-
targeting Ir(III) cations have a [Ir(C^N)2(N^N)]+ architecture,
13
mitochondria-targeting terpyridyl Ir(III) complexes with
[Ir(N^N^N)(C^N)Cl]+ structure are rare (Fig. 1).41,42
a
and their protein-related modulation of apoptosis has two
modes, the release or activation of caspase14,15 and the signal
transmission of Bcl-2 family stimuli.16,17 The morphologies of
mitochondria can change during apoptosis and differ from
normal states.18-20 Moreover, the mitochondrial morphologies
are also different in different stages of apoptosis.21-23 To study
the relationship between mitochondrial morphologies and
apoptosis, it is of great importance to track mitochondrial
dynamics during apoptosis.
Fluorocarbons have attracted tremendous attention in
biological applications due to their properties such as
lipophobicity,
hydrophobicity
and
bioinertness.43-45
Interestingly, the good biocompatibility and low toxicity of
fluorinated compounds has been reported in biological
systems.46-48 Herein, a series of terpyridyl Ir(III) complexes (Ir1-
Ir4
) with different fluorine atoms were designed and
synthesized (Fig. 2) for tracking mitochondrial dynamics during
apoptosis. The photophysical properties of the fluorinated
complexes were studied in solution and in cells. With low
cytotoxicities, excellent photostability and the ability to
For biological dynamics tracking, the probes must exhibit
low cytotoxicity and superior photostability.24-26 Organic dyes
have been used as mitochondria imaging agents, even
commercially. But most of them are limited in tracking
because of poor photostability.27,28 Metal-based complexes
could largely overcome photobleaching because of their
a.MOE Key Laboratory of Bioinorganic and Synthetic Chemistry, School of
Chemistry, Sun Yat-Sen University, Guangzhou, 510275, P. R. China.
b.College of Chemistry and Environmental Engineering, Shenzhen University,
Shenzhen, 518055, P. R. China.
Email: ceschh@mail.sysu.edu.cn (H. Chao); zhql@szu.edu.cn (Q. Zhang).
‡ These authors contributed equally to this work.
† Electronic supplementary informaꢀon (ESI) available: additional data. See DOI:
10.1039/x0xx00000x
Fig. 1 Two types of Ir(III) architectures.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 1
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 2 of 8
View Article Online
DOI: 10.1039/C8DT02918K
ARTICLE
Journal Name
reaction. The Ir(III) complexes were purified by alumina
column chromatography with eluents of acetonitrile and
toluene, and characterized with elemental analysis, ES-MS (Fig.
S1-S4), 1H NMR (Fig. S5-S8) and 19F NMR (Fig. S9-S12).
Photophysical properties
The photophysical properties of Ir1-Ir4 at 298 K in phosphate
buffer saline (PBS) were performed (Fig. 3) and the data were
presented in Table S1. A weak absorption was found around
470 nm in the UV-Vis spectra and an intense absorption was
observed at 300-400 nm.49 When excited at 405 nm, Ir1-Ir4
exhibit yellow to orange phosphorescent emissions from 549
to 569 nm. The wavelengths of emission peaks showed a red
Fig. 2 The chemical structures of the iridium complexes Ir1-Ir4
.
specificity target mitochondria, the complexes were developed
as mitochondrial probes for real-time tracking of
mitochondrial dynamics during the early stages of apoptosis.
The uptake mechanisms of the complexes were also
investigated in detail.
shift when the number of
F atoms increased. The
phosphorescent lifetimes of the complexes are 731 ns (Ir1),
655 ns (Ir2), 511 ns (Ir3) and 400 ns (Ir4). Using [Ru(bpy)3]2+ as
a standard, the quantum yields of Ir1-Ir4 in PBS buffer were
measured as 14.3 %, 12.7 %, 9.23 % and 5.56 %, respectively.50
The change in phosphorescent lifetime and quantum yields
could be attributed to variations of dipole moment and
Results and discussion
Design and synthesis
The complexes were synthesized through the synthetic route electron distribution within the complexes, which due to the
(Scheme S1). The fluorinated terpyridyl ligands were obtained electron withdrawing influence of F.51 Furthermore, Ir1-Ir4
firstly. Then Ir1-Ir4 were synthesized by reacting IrCl3•3H2O displayed superior photostablity in buffer solution (Fig. S13)
and the corresponding ligands in ethylene glycol via a two-step during continuous irradiation for 40 minutes, which is a
desirable property for practical applications.
Cellular uptake and intracellular localization
The cellular uptake of the complexes by Human cervix
Fig. 4 Confocal images of HeLa cells co-labeled with the
complexes (10 μM, 1 h) and the commercial mitochondria
imaging agent MTR (50 nM, 0.5 h). The complexes were
excited at 405 nm. MTR was excited at 543 nm. The
phosphorescence/fluorescence was collected at 550 ± 20 nm
and 620 ± 20 nm for the complexes and MTR, respectively. BF:
bright field. The 5th column was the Pearson correlation
Fig. 3 (a) Absorption spectra and (b) emission spectra of Ir1-
Ir4 (10 μM) in PBS.
coefficient. Scale bar: 20 μm.
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 3 of 8
View Article Online
DOI: 10.1039/C8DT02918K
Journal Name
ARTICLE
adenocarcinoma (HeLa) cells was investigated by confocal
microscopy. As shown in Fig. S14, with an increasing
incubation time, the phosphorescent intensities in the images
increased. The intensity of emission of the complexes in cells
was saturated at around 60 minutes and the phosphorescence
distributed in the cell cytoplasm. The distribution of the
complexes was studied via co-localization assay with
commercial organelle-targeting dyes LysoTracker® Red (LTR),
ER-Tracker® Red (ERTR) and MitoTracker® Red (MTR). Low
overlaps were found between the location of the complexes
and LTR (Fig. S15). The Pearson’s correlation coefficients were
approximately 70% for the complexes and ERTR (Fig. S16), but
the coefficients for the complexes and MTR were more than
80%, even more than 90% for Ir1 and Ir3 (Fig. 4). The results
suggested the complexes mainly localize in mitochondria. Their
Fig. 6 Confocal phosphorescence/fluorescence images and
brightfield (BF) images of fixed HeLa cells incubated with 50
nM MTR for 30 min or 10 µM complexes for 1 h at 37 °C. The
complexes were excited at 405 nm. MTR was excited at 543
nm. The phosphorescence/fluorescence was collected at 550 ±
20 nm and 620 ± 20 nm for the complexes and MTR,
respectively. Scale bar: 20 μm.
mitochondrial
localization
was
further
confirmed
quantitatively via inductively coupled plasma mass
spectrometry (ICP-MS) analyses. Mitochondria of the
complexes from treated HeLa cells were extracted and the
iridium contents of the mitochondria were determined to be
74.7% for Ir1, 82.2% for Ir2, 76.5% for Ir3 and 79.4% for Ir4 (Fig.
S17). It has been reported a molecule with a log P value
between 0 and 5 and a change number above zero would
probably target mitochondria.41,52,53 The log P values (Fig. S18)
of the cations Ir1-Ir4 were measured to be 0.322 (Ir1), 0.613
that phosphorescence could still appear clearly in
mitochondria at 4 °C, demonstrating the temperature does not
affect the entry of the iridium complexes. HeLa cells were
pretreated with endocytosis inhibitors (chloroquine and
NH4Cl).58 Subsequent imaging of the cells showed that Ir1-Ir4
could still cross the cell membrane in these conditions, thus
revealing that the cellular uptake process is not endocytosis.
The results infer that all the iridium-based probes in this work
share a similar entry mechanism into cells, which is via an
energy-independent pathway.
(Ir2), 0.862 (Ir3) and 0.557 (Ir4). The combined results pointed
out that the complexes were mainly localized in mitochondria.
Cellular uptake mechanisms
Anti-photobleaching in cells
Usually, small molecules enter cells through energy-dependent
or independent pathways.54,55 In order to discriminate
whether the uptake process utilizes energy, the intracellular
phosphorescence of Ir1-Ir4 at 37 °C and 4 °C were recorded
first (Fig. S19). Then cells were pretreated with metabolic
The cellular microenvironment is very complex and different
from that in buffer solution.59 Besides, in order to compare the
complexes with commercial dyes, their anti-photobleaching
properties in HeLa cells were studied. Ir1-Ir4 and MTR treated
HeLa cells were irradiated under continuous scanning. As
shown in Fig. 5 and S20, the fluorescent intensity of MTR
finally decreased in half after 30 scans, while the
phosphorescence intensities of Ir1-Ir4 were nearly unchanged
inhibitors (2-deoxy-D-glucose and oligomycin) before
incubation with the iridium complexes.56,57 Results showed
Fig. 5 Quantitative photobleaching results of Ir1-Ir4 and MTR
in HeLa cells under irradiation by a light source (405 nm for the
complexes and 543 nm for MTR) of 20.0 mW/cm2.
Fig. 7 Viability of HeLa cells incubated with 10 μM of Ir1-Ir4 for
24 h with or without irradiation (5 J/cm2, 405 nm).
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 4 of 8
View Article Online
DOI: 10.1039/C8DT02918K
ARTICLE
Journal Name
Fig. 8 Real-time imaging of HeLa cells stained with Ir1 (10 μM) for 1 h at 37 °C, followed by treatment with 30 μM CCCP, with
increasing scan time. Phosphorescence images of Ir1 (upper panel), Brightfield images (lower panels). The complex was excited
at 405 nm. The phosphorescence was collected at 550 ± 20 nm. Scale: 20 μm.
compared to initial tests. Dynamic photobleaching changes properties, their application in mitochondrial dynamics
with time could be seen in Video S1. Results above showed tracking during apoptosis was put into practice. Carbonyl
that Ir1-Ir4 possessed good anti-photobleaching capabilities, cyanide m-chlorophenylhydrazone (CCCP) can be used to
and are therefore promising candidates for dynamic tracking in interrupt the proton gradient across the mitochondrial
living system.
membrane, leading to MMP decrease and apoptosis.55 HeLa
cells pretreated with Ir1-Ir4 were exposed to CCCP and the
tracking began. The changes of the mitochondrial morphology
were shown in Fig. 8 (Video S2). The reticulum structure of the
Imaging in fixed cells
During apoptosis, the mitochondrial membrane potential mitochondria was gradually transformed into small and
(MMP) can decrease or even be lost.18-20 Usually, MMP change dispersed fragments, indicating the early stages of apoptosis.21
would affect the specificity of organic commercial dyes like Similar phenomena (Fig. S20-S22, Video S3-S5) were also
MTR.24,27 MMP is totally lost in fixed cells, which can represent found in the experiments with Ir2-Ir4. The results displayed
the physical status of mitochondria after apoptosis. To study the ability of the fluorinated terpyridyl Ir(III) complexes to
the specificity of mitochondria-targeting, the complexes were track mitochondrial dynamics during cell apoptosis.
used to stain mitochondria in fixed cells and the commercial
dye MTR was used as a contrast. As shown in Fig. 6, the whole-
Conclusions
cell staining of fixed cells was observed with MTR. However,
Ir1-Ir4 could stain mitochondria in fixed cells as well as living
In summary, a series of phosphorescent fluorinated Ir(III)
cells, indicating the complexes can target mitochondria even
complexes, Ir1-Ir4, were applied to the study of changes in
the MMP is lost.
mitochondrial morphology during apoptosis. Tuning of the
photophysical properties of the series was demonstrated by
the charge in degree of fluorination. The complexes also
Cytotoxicity measurements
displayed superior photostability in PBS. Analysis by cellular
MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliu
uptake assay demonstrated the iridium-based probes entry
into cells via an energy-independent pathway. Co-localization
and ICP-MS analyses indicated that Ir1-Ir4 locate in the
mitochondria of HeLa cells. The complexes were also shown to
have low cytotoxicities and desirable photobleaching
properties. As a result of these excellent characteristics, the
complexes were successfully used to track mitochondrial
dynamics during apoptosis. We expect our work will facilitate
the introduction of fluorine chemistry and terpyridyl Ir(III)
complexes for the study of organelles during biological events.
bromide) assays were set to study the cytotoxicity of the
complexes with HeLa cells. After incubation for 24 h, the
complexes did not present significant cytotoxicity in HeLa cells.
Photocytotoxicity experiments were made to examine their
potential in cellular real-time tracking. Under irradiation of 5
J/cm2 at 405 nm, HeLa cells subjected to Ir1
, Ir3 and Ir4 still
showed viabilities over 80%, while more than 75% cells
survived in Ir2 assay (Fig. 7). The results indicated that the
complexes will not interrupt normal biological activities in cells
during tracking.
Experimental section
Tracking mitochondrial dynamic during cell apoptosis
General information
Now that Ir1-Ir4 were shown to have good photostablity, low
(photo)cytotoxicity and excellent mitochondrial targeting
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 5 of 8
View Article Online
DOI: 10.1039/C8DT02918K
Journal Name
ARTICLE
The reagents and solvents for chemical synthesis were of (v/v: 1:2) as the eluant. The solvent was removed via rotary
analytical grade, and of biological grade for cell experiments. evaporator, and then the purified orange Ir1 was obtained.
All of them were obtained from commercial sources and used The synthesis methods of Ir2-Ir4 were similar as that of Ir1
as received unless specified. 2-phenylpyridine, 2-acetylpyridine, [IrL1(ppy)Cl](PF6) (Ir1). Yield: 56.1%. Elemental analysis:
4-fluorobenzaldehyde, 3,4-difluorobenzaldehyde, 2,4,6- calcd (%) for C32H22ClF7IrN4P: C, 45.00; H, 2.60; N, 6.56. Found:
.
- +
trifluorobenzaldehyde and 2,3,5,6-tetrafluorobenzaldehyde C, 44.77; H, 2.61; N, 6.53. ES-MS (m/z): 709.1 [M-PF6 ] , found:
- +
were bought from Alfa Aesar (U.S.A.). IrCl3•3H2O was obtained 708.9 [M-PF6 ] . 1H NMR (500 MHz, d6-DMSO) δ 9.88 (d, J = 5.6
from Energy Chemical (China). MTT and CCCP were from Sigma Hz, 1H), 9.26 (s, 2H), 8.95 (d, J = 8.0 Hz, 2H), 8.50 (d, J = 8.2 Hz,
Aldrich. LTR, ERTR and MTR were purchased from Invitrogen 1H), 8.39 (dd, J = 8.8, 5.3 Hz, 2H), 8.30-8.21 (m, 3H), 7.94 (d, J =
(U.S.A.). HeLa cells were received from Experimental Animal 7.9 Hz, 1H), 7.81 (t, J = 7.2 Hz, 1H), 7.69 (d, J = 5.3 Hz, 2H), 7.60
Center of Sun Yat-Sen University (Guangzhou, China). (t, J = 8.8 Hz, 2H), 7.54 (t, J = 7.2 Hz, 2H), 6.92 (t, J = 7.6 Hz, 1H),
Dulbecco’s modified Eagle medium (DMEM) and fetal bovine 6.75 (t, J = 7.9 Hz, 1H), 6.07 (d, J = 7.4 Hz, 1H). 19F NMR (470
serum (FBS) were from Gibco. Ultrapure Milli-Q water was MHz, d6-DMSO) δ -67.40, -70.91, -109.86.
used in all experiments.
[IrL2(ppy)Cl](PF6) (Ir2). Yield: 54.9%. Elemental analysis:
Elemental analysis (C, H, N) was performed via Elementar calcd (%) for C32H21ClF8IrN4P: C, 44.07; H, 2.43; N, 6.42. Found:
- +
Vario EL. Electrospray mass spectra (ES-MS) were acquired C, 43.85; H, 2.44; N, 6.39. ES-MS (m/z): 727.2 [M-PF6 ] , found:
1
- +
from a LCQ system (LCMS-2010A, SHIMADZU, Japan). H NMR 726.9 [M-PF6 ] . 1H NMR (500 MHz, d6-DMSO) δ 9.88 (d, J = 5.1
and 19F NMR spectra were determined on a Bruker AVANCE AV Hz, 1H), 9.28 (s, 2H), 8.94 (d, J = 8.0 Hz, 2H), 8.49 (m, 2H), 8.27
500 NMR spectrometer, using d6-DMSO as solvent at room (m, 4H), 7.94 (d, J = 7.2 Hz, 1H), 7.89-7.79 (m, 2H), 7.70 (d, J =
temperature. UV-Vis spectra were recorded on a Perkin-Elmer 5.0 Hz, 2H), 7.55 (t, J = 7.2 Hz, 2H), 6.92 (t, J = 7.5 Hz, 1H), 6.74
Lambda 850 spectrophotometer, and emission spectra on a (t, J = 6.9 Hz, 1H), 6.06 (d, J = 7.7 Hz, 1H). 19F NMR (470 MHz,
Perkin-Elmer L55 spectrofluorophotometer at room d6-DMSO) δ -69.39, -70.91, -135.06, -135.11, -137.18, -137.23.
temperature, respectively. Phosphorescent lifetime was
[IrL3(ppy)Cl](PF6) (Ir3). Yield: 53.8%. Elemental analysis:
measured via an Edinburgh FLS-920 combined fluorescence- calcd (%) for C32H20ClF9IrN4P: C, 43.18; H, 2.26; N, 6.29. Found:
- +
lifetime and steady-state spectrometer, with excitation source C, 42.96; H, 2.27; N, 6.26. ES-MS (m/z): 745.2 [M-PF6 ] , found:
- +
of hydrogen-filled lamp. Phosphorescence quantum yields 745.0 [M-PF6 ] . 1H NMR (500 MHz, d6-DMSO) δ 9.87 (d, J = 5.2
were performed using [Ru(bpy)3]2+ as reference (0.028 in Hz, 1H), 9.16 (s, 2H), 8.82 (d, J = 8.1 Hz, 2H), 8.51 (d, J = 8.2 Hz,
aerated H2O).50 Cell morphologies and imaging were observed 1H), 8.29 (t, J = 8.6 Hz, 1H), 8.21 (t, J = 8.5 Hz, 2H), 7.96 (d, J =
through Zeiss LSM 710 laser microscopy system. ICP-MS was 7.2 Hz, 1H), 7.82 (t, J = 6.6 Hz, 1H), 7.75-7.63 (m, 4H), 7.56 (t, J
conducted on iCAP-RQ inductively coupled plasma mass = 6.1 Hz, 2H), 6.94 (t, J = 7.9 Hz, 1H), 6.79 (t, J = 7.5 Hz, 1H),
spectrometer (Thermo Fisher).
6.03 (d, J = 7.4 Hz, 1H). 19F NMR (470 MHz, d6-DMSO) δ -69.40,
-70.91, -103.85, -103.87, -103.88, -109.86, -109.87.
[IrL4(ppy)Cl](PF6) (Ir4). Yield: 52.1%. Elemental analysis:
Synthesis of Ir(III) complexes Ir1-Ir4
calcd (%) for C32H19ClF10IrN4P: C, 42.32; H, 2.11; N, 6.17. Found:
- +
The complexes were synthesized by similar methods according C, 42.11; H, 2.12; N, 6.14. ES-MS (m/z): 763.1 [M-PF6 ] , found:
- +
to previous studies.49,60 Firstly, for the synthesis of the ligand, 762.9 [M-PF6 ] . 1H NMR (500 MHz, d6-DMSO) δ 9.87 (d, J = 5.1
4’-(4-fluorophenyl)-2,2’:6’,2”-terpyridyl (L1), a mixture of EtOH Hz, 1H), 9.23 (s, 2H), 8.81 (d, J = 8.0 Hz, 2H), 8.52 (d, J = 8.2 Hz,
(25 mL) , KOH (10 mmol, 0.56 g), 2-acetylpyridine (10 mmol, 1H), 8.32-8.21 (m, 4H), 7.96 (d, J = 7.4 Hz, 1H), 7.82 (t, J = 7.1
1.21 g), 4-fluorobenzaldehyde (5 mmol, 0.620 g) and NH3•H2O Hz, 1H), 7.73 (d, J = 5.5 Hz, 2H), 7.58 (t, J = 7.2 Hz, 2H), 6.94 (t, J
(14.5 mL) were added accordingly into a 100 mL round bottom = 7.5 Hz, 1H), 6.79 (t, J = 7.0 Hz, 1H), 6.04 (d, J = 7.6 Hz, 1H). 19
F
flask and stirred at room temperature overnight. Then the NMR (470 MHz, d6-DMSO) δ -69.40, -70.91, -138.08, -138.11, -
cream white precipitate was filtered and washed with EtOH, 138.13, -138.16, -141.38, -141.41, -141.43, -141.46.
water and diethyl-ether three times. The ligand isolated as a
white solid was dried and saved for subsequent use.
Cell culture
Secondly, a mixture of L1 (0.28 mmol, 91.6 mg) and
IrCl3•3H2O (0.32 mmol, 112 mg) in 10 mL ethylene glycol was HeLa cells were seeded in a medium containing 90% DMEM
heated at 180 °C under argon for 30 minutes. The intermediate and 10% FBS, then cultured at 37 °C in 5 % CO2 incubator.
product was reddish-brown and filtered immediately before
washing with EtOH, water and diethyl ether successively. The
MTT assay
intermediate (after drying) was further reacted with excess 2-
phenylpyridine in ethylene-glycol at 180 °C under argon MTT assay was utilized to determine the cell viability of HeLa
overnight, until the solution became clear. After cooling to cells over time after treatment with the iridium-based probes.
room temperature, the solution was carefully added into HeLa cells were seeded in 96-wells plates for 24 h. Then HeLa
saturated NH4PF6 solution dropwise. An orange precipitate cells were treated with 10 μM of Ir1-Ir4 for 1 h in the dark. For
was formed, isolated by filtration and washed with water and the phototoxicity, an LED system was used to irradiate the cells
ether three times. The crude product was purified by column under 405 nm (5 J/cm2). Then cells in both light and dark
chromatography on neutral alumina, using acetonitrile-toluene assays were incubated in the dark. After 24 h, the cells were
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 5
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 6 of 8
View Article Online
DOI: 10.1039/C8DT02918K
ARTICLE
Journal Name
incubated together with MTT solution (20 μL/well, 5 mg/mL) complexes were excited at 405 nm and the emission signal was
for another 4 hours. Finally, the MTT media was removed and measured at 550 ± 20 nm.
replaced with 200 μL of DMSO in each well. After shaking for 5
minutes, the OD595 values were measured with a Tecan Infinite
Conflicts of interest
M200 monochromator-based multifunction microplate reader.
There are no conflicts to declare.
Colocalization imaging
After incubation with 10 μM of Ir1-Ir4 at 37 °C for 1 h, HeLa
Acknowledgements
cells were stained with 50 nm LTR/MTR or 1 μM ERTR for an
This work was supported by the National Science Foundation
additional 0.5 h. The cells were rinsed with PBS three times
of China (Nos. 21525105, 21471164 and 21778079), the 973
and observed by confocal microscopy. The excitation
Program (No. 2015CB856301), the Fundamental Research
wavelength of Ir1-Ir4 was 405 nm, while the three commercial
Funds for the Central Universities and China Postdoctoral
dyes were excited at 543 nm. The emission signals were
Science Foundation (No. 2018M630988).
collected at 550 ± 20 nm for Ir1-Ir4 and 620 ± 20 nm for
LTR/MTR/ERTR.
Notes and references
1
B. Fadeel and S. Orrenius, J. Intern. Med., 2005, 258, 479-
517.
P. G. Clarke and S. Clarke, Anat. Embryol., 1996, 193, 81-99.
R. A. Lockshin and Z. Zakeri, Nat. Rev. Mol. Cell Biol., 2001, 2,
545-550.
ICP-MS analysis
HeLa cells were seeded in 10 cm culture plates in 6 mL of
DMEM with 10% FBS. After addition and incubation of Ir1-Ir4
(10 μM) for 1 h at 37 °C, the cells were digested with trypsin
(Gibco) and divided into two portions. One sample was
2
3
4
5
6
M. M. Metzstein, G. M. Stanfield and H. R. Horvitz, Trends
Genet., 1998, 14, 410-416.
subjected to
a mitochondrial extraction kit (Beyotime
B. A. Hay, J. R. Huh and M. Guo, Nat. Rev. Genet., 2004,
911-922.
5
,
Biotechnology), and the other was the control group. Then
each portion was digested in 65% HNO3 for one day and
diluted with water to 2% HNO3 solution. The content of iridium
complexes was determined by ICP-MS.
J. F. Kerr, A. H. Wyllie and A. R. Currie, Br. J. Cancer, 1972, 26
239-257.
,
7
8
9
J. C. Reed, Cancer Cell, 2003,
T. G. Cotter, Nat. Rev. Cancer, 2009,
3
, 17-22.
9
Q. L. Deveraux and J. C. Reed, Genes. Dev., 1999, 13, 239-
, 501-507.
252.
Cellular uptake mechanism
10 D. R. Green and J. C. Reed, Science, 1998, 281, 1309-1312.
11 S. Vyas, E. Zaganjor and M. C. Haigis, Cell, 2016, 166, 555-
566.
12 F. Weinberg, R. Hamanaka, W. W. Wheaton, S. Weinberg, J.
Joseph, M. Lopez, B. Kalyanaraman, G. M. Mutlu, G. R.
Budinger and N. S. Chandel, Proc. Natl. Acad. Sci. U. S. A.,
2010, 107, 8788-8793.
13 M. Leist, B. Single, A. F. Castoldi, S. Kühnle and P. Nicotera, J.
Exp. Med., 1997, 185, 1481-1486.
14 N. J. McCarthy, M. K. Whyte, C. S. Gilbert and G. I. Evan, J.
Cell Biol., 1997, 136, 215-227.
15 T. Hirsch, P. Marchetti, S. A. Susin, B. Dallaporta, N. Zamzami,
In temperature tests, the cells were incubated with Ir1-Ir4 (10
μM) for 1 h at 4 °C and 37 °C, respectively. In metabolic
inhibition tests, cells detached from the culture were first
incubated with 2-deoxy-D-glucose (50 mM) and oligomycin (5
μM) in PBS for 1 h at 37 °C. For endocytic inhibition, cells were
pretreated with NH4Cl (50 mM), or chloroquine (50 μM) for 30
min. Then the pretreated samples with each inhibitor were
treated with Ir1-Ir4 (10 μM) in fresh media at 37 °C for 1 h. The
cells were imaged by confocal microscopy after washing with
PBS.
I. Marzo, M. Geuskens and G. Kroemer, Oncogene, 1997, 15
1573-1581.
,
16 G. Lessene, P. E. Czabotar and P. M. Colman, Nat. Rev. Drug
Discov., 2008, , 989-1000.
7
Fixed cells imaging
17 J. Xiang, D. T. Chao and S. J. Korsmeyer, Proc. Natl. Acad. Sci.
U. S. A., 1996, 93, 14559-14563.
18 D. R. Green and G. Kroemer, Science, 2004, 305, 626-629.
For fixed cells, 75% EtOH was used to fix HeLa cells for 0.5 h.
Then fixed cells were incubated with Ir1-Ir4 (10 μM, 1 h) or
MTR (50 nm, 30 min) at 37 °C. After washed by PBS for three
times, fixed cells imaging was performed by confocal
microscopy.
19 K. Okamoto and J. M. Shaw, Annu. Rev. Genet., 2005, 39
503-536.
,
20 D. Arnoult, Trends Cell Biol., 2007, 17, 6-12.
21 M. Karbowski and R. J. Youle, Cell Death Differ., 2003, 10
870-880.
,
22 J. L. Perfettini, T. Roumier and G. Kroemer, Trends Cell Biol.,
2005, 15, 179-183.
Real-time tracking mitochondrial dynamics during cell apoptosis
23 K. Elquss, J. Pakay, M. T. Ryan and C. S. Palmer, Biochim.
Biophys. Acta, 2013, 1833, 150-151.
24 Y. Chen, L. Qiao, B. Yu, G. Li, C. Liu, L. Ji and H. Chao, Chem.
Commun., 2013, 49, 11095-11097.
25 Y. Chen, T. W. Rees, L. Ji and H. Chao, Curr. Opin. Chem. Biol.,
2018, 43, 51-57.
For real-time tracking, HeLa cells were stained with Ir1-Ir4 (10
μM) at 37 °C for 1 h, and then washed by PBS for three times.
Before imaging, CCCP (30 μM) was added into the plates.
Images were taken every 30 s by confocal microscopy. The
6 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Dalton Transactions
Page 7 of 8
View Article Online
DOI: 10.1039/C8DT02918K
Journal Name
ARTICLE
26 K. Qiu, Y. Chen, T. W. Rees, L. Ji and H. Chao, Coord. Chem. 44 Z. Zhang, W. Shen, J. Ling, Y. Yan, J. Hu and Y. Cheng, Nat.
Rev., 2017, DOI: 10.1016/j.ccr.2017.10.022. Commun., 2018, , 1377.
27 Y. Chen, L. Qiao, L. Ji and H. Chao, Biomaterials, 2014, 35, 2- 45 T. Wu, L. Wang, S. Ding and Y. You, Macromol. Biosci., 2017,
9
13.
28 X. Zhou, F. Su and H. Lu, Biomaterials, 2012, 33, 171-180.
17, 1700114.
46 H. J. Lehmler, Expert Opin. Drug Deliv., 2007, 4, 247-262.
29 Q. Zhao, F. Li and C. Huang, Chem. Soc. Rev., 2010, 39, 3007- 47 M. Wang, H. Liu, L. Li and Y. Cheng, Nat. Commun., 2013,
3030. 3053.
30 D. Ma, V. P. Ma, D. S. Chan, K. Leung, H. He and C. Leung, 48 G. Zuo, A. Xie, X. Pan, T. Su, J. Li and W. Dong, ACS Appl.
Coord. Chem. Rev., 2012, 256, 3087-3113. Nano Mater., 2018, , 2376−2385.
31 Y. Chen, R. Guan, C. Zhang, J. Huang, L. Ji and H. Chao, Coord. 49 D. N. Chirdon, W. J. Transue, H. N. Kagalwala, A. Kaur, A. B.
5,
1
Chem. Rev., 2016, 310, 16-40.
32 C. Caporale and M. Massi, Coord. Chem. Rev., 2018, 363, 71-
Maurer, T. Pintauer and S. Bernhard, Inorg. Chem., 2014, 53,
1487-1499.
91.
33 K. K.-W. Lo and K. K.-S. Tso, Inorg. Chem. Front., 2015, 2, 510-
524.
34 Y. You and W. Nam, Chem. Soc. Rev., 2012, 41, 7061-7084.
35 K. K.-W. Lo, Acc. Chem. Res., 2015, 48, 2985-2995.
36 K. Qiu, H. Huang, B. Liu, Y. Liu, P. Zhang, Y. Chen, L. Ji and H.
50 K. E. Erkkila, D. T. Odom and J. K. Barton, Chem. Rev., 1999,
99, 2777-2796.
51 Y. Liu, H. Ma, H. Xu, J. Sun and K. Han, Int. J. Quantum Chem.,
2005, 102, 415-421.
52 R. W. Horobin and F. Rashid-Doubell, Biotech. Histochem.,
2013, 88, 461-476.
Chao, J. Mater. Chem. B, 2015,
37 W. Lv, Z. Zhang, K. Y. Zhang, H. Yang, S. Liu, A. Xu, S. Guo, Q.
3
, 6690-6697.
53 R. W. Horobin, F. Rashid-Doubell, J. D. Pediani and G.
Milligan, Biotech. Histochem., 2013, 88, 440-460.
Zhao and W. Huang, Angew. Chem. Int. Ed., 2016, 55, 9947- 54 C. W. Leung, Y. Hong, S. Chen, E. Zhao, J. W. Lam and B. Z.
9951. Tang, J. Am. Chem. Soc., 2013, 135, 62-65.
38 Z. Chen, K. Y. Zhang, X. Tong, Y. Liu, C. Hu, S. Liu, Q. Yu, Q. 55 T. Nishikawa, D. Edelstein, X. L. Du, S. Yamagishi, T.
Zhao and W. Huang, Adv. Funct. Mater., 2016, 26, 4386-
4396.
Matsumura, Y. Kaneda, M. A. Yorek, D. Beebe, P. J. Oates, H.
P. Hammes, I. Giardino and M. Brownlee, Nature, 2000, 404,
39 T. Huang, Q. Yu, S. Liu, W. Huang and Q. Zhao, Dalton Trans.,
2018, 47, 7628-7633.
40 K. Qiu, Y. Liu, H. Huang, C. Liu, H. Zhu, Y. Chen, L. Ji and H.
Chao, Dalton Trans., 2016, 45, 16144-16147.
41 H. Huang, L. Yang, P. Zhang, K. Qiu, J. Huang, Y. Chen, J. Diao,
787-790.
56 C. A. Puckett and J. K. Barton, Biochemistry, 2008, 47, 11711-
11716.
57 K. Y. Zhang, H. W. Liu, T. H. Fong, X. G. Chen and K. K.-W. Lo,
Inorg. Chem., 2010, 49, 5432-5443.
J. Liu, L. Ji, J. Long and H. Chao, Biomaterials, 2016, 83, 321- 58 J. S. Lau, P. K. Lee, K. H. Tsang, C. H. Ng, Y. W. Lam, S. H.
331. Cheng and K. K.-W. Lo, Inorg. Chem., 2009, 48, 708-718.
42 H. Huang, P. Zhang, K. Qiu, J. Huang, Y. Chen, L. Ji and H. 59 K. Qiu, L. Ke, X. Zhang, Y. Liu, T. W. Rees, L. Ji, J. Diao and H.
Chao, Sci. Rep., 2016, , 20887. Chao, Chem. Commun., 2018, 54, 2421-2424.
6
43 M. C. Z. Kasuya, S. Nakano, R. Katayama and K. Hatanaka, J. 60 J. Wang and G. S. Hanan, Synlett, 2005,
Fluorine Chem., 2011, 132, 202-206
8, 1251-1254.
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 7
Please do not adjust margins

Dalton Transactions
Page 8 of 8
View Article Online
DOI: 10.1039/C8DT02918K
Graphical Abstract
A series of phosphorescent fluorinated Ir(III) complexes, which possess low
cytotoxicity, excellent photostability and the specificity of mitochondria-targeting,
were used for tracking mitochondrial dynamics during apoptosis.