A long-lifetime iridium(iii) complex for lysosome tracking with high specificity and a large Stokes shift

Article abstract of DOI:10.1039/c8tb00666k

Investigating the role of lysosome dysfunction in cancer requires the development of efficient probes for lysosomes. We report herein a cyclometalated iridium(iii) complex (Ir-Ly) as a luminescent probe for visualizing lysosomes in cancer cells. The morpholine and hydroxy moieties within Ir-Ly provide suitable hydrophilicity and responsiveness to pH. Importantly, Ir-Ly exhibits a large Stokes shift, long lifetime and high photostability, which are important advantages for lysosome tracking in living cells.

Full text of DOI:10.1039/c8tb00666k

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A long-lifetime iridium(III) complex for lysosome tracking with
high specificity and a large Stokes shift
Received 00th January 20xx,
Accepted 00th January 20xx
Jin-Biao Liu,a,b,‡ Kasipandi Vellaisamy,b,‡ Guodong Li,c Chao Yang,c Suk-Yu Wong,b and Chung-Hang
Leung,c Shou-Zhi Pu,a,* and Dik-Lung Ma,b,*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract
on fluorescent organic dyes, which suffer from poor
8
Investigating the role of lysosome dysfunction in cancer requires photostability,7,
limiting the time period for observing
the development of efficient probes for lysosomes. We report dynamic changes of lysosome behavior. Furthermore, other
herein
a
cyclometalated iridium(III) complex (Ir-Ly) as
a
long-term fluorescent probes for lysosomes reported in the
luminescent probe for visualizing lysosomes in cancer cells. The literature,
including
dextran-linked
fluorophores,9
morpholine and hydroxy moieties within Ir-Ly provide suitable inorganic/hybrid nanoparticles,10 and organic dyes,11,
hydrophilicity and responsiveness to pH. Importantly, Ir-Ly required excitation with short wavelengths leading to
exhibits large Stokes shift, long lifetime and high photostability, unwanted autofluorescence, photobleaching, photodamage,
which are important advantages for lysosome tracking in living and shallow depth of penetration. Inorganic luminescent
12
cells.
materials have emerged as promising alternatives to
fluorescent dyes for cellular imaging due to their low
phototoxicity, good photostability, deeper penetration, large
Stokes shifts and long luminescent lifetimes.13-15
Introduction
Based on our continuing interest in the development of
luminescent iridium(III) complex probes,16-18 we report in this
work our efforts to develop a iridium(III) complex for lysosome
tracking. We designed and synthesized an iridium(III) complex
Ir-Ly, which incorporates morpholine and hydroxy groups in
the phenanthroline N^N ligand, and also bears two
phenylpyridine C^N co-ligands. The morpholine moiety is
crucial for lysosomal localization,19 as the protonation of Ir-Ly
within the acidic interior of the lysosome converts the complex
into a more hydrophilic species that is retained within the
lysosome. Therefore, the protonated morpholine moiety acts
as a “lock” that allows Ir-Ly to accumulate in lysosomes,
allowing the probe to be used for observing lysosomes in
cancer cells (Fig. 1a). Meanwhile, the hydroxy groups in the
complex can act as a sensing unit in basic solution, and also
confer increased water solubility (Fig. 1b).
Lysosomes are essential organelles that are responsible for
cellular digestion. They break down extracellular materials
taken up by the cell as well as intracellular components under
specific circumstances in order to regulate the cellular dynamic
2
physiological balance.1, There are over 50 hydrolases in
lysosomes which are responsible for carrying out the
degradation activities of lysosomes. As lysosomes and
lysosomal enzymes play important functions in programmed
cell death, considerable attention has been drawn towards the
4
role of lysosomes in cancer.3, These studies demand the
development of new kinds of molecular probes for examining
lysosomal function and dysfunction within the cell.5
Fluorescence microscopy is a widely used technology used for
non-invasive imaging of organelles in living cells. The ability to
track and image lysosomes accurately could be useful for the
monitoring of lysosome behavior in cancer cells.6 However,
most commercially accessible probes for lysosomes are based
a
Jiangxi Key Laboratory of Organic Chemistry, Jiangxi Science and Technology
Normal University, Nanchang , China. E-mail: pushouzhi@tsinghua.org.cn
b Department of Chemistry, Hong Kong Baptist University, Kowloon Tong, Hong
Kong, China. *E-mail: edmondma@hkbu.edu.hk
c State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese
Medical Sciences, University of Macau,
duncanleung@umac.mo
Macao,
China.
*E-mail:
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 20xx
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With the complex in hand, the optical characteristics of Ir-Ly were
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DOI: 10.1039/C8TB00666K
next investigated. Ir-Ly showed excitation and emission maxima of
290 and 590 nm, respectively (Fig. S3†). Its large Stokes shift of 300
nm is larger than the Stokes shift of typical organic dyes, which
reduces the likelihood of self-quenching. Additionally, Ir-Ly’s
luminescence lifetime was relatively long-lived (ca. 4.2 μs) (Table
S1), which is substantially longer than lifetimes of organic dyes
(typically ns). Therefore, the emission of complex Ir-Ly could easily
be distinguished from background autofluorescence through the
use of time-resolved emission spectroscopy (TRES). To verify this
hypothesis, the luminescent properties of Ir-Ly were monitored in
the presence of coumarin-460 (CM460), as a typical fluorescent dye.
In steady-state mode, the luminescence of Ir-Ly was disturbed by
the robust emission of CM460 at about 460 nm, reducing the
precision of determination (Fig. 3a). However, when the delay was
set to 333 ns, which is long after the completion of the fluorescence
decay of CM460 (3.4 ns),20 the emission from CM460 was almost
entirely absent and only the emission of complex Ir-Ly could be
detected (Fig. 3b). These results establish the possible application of
Ir-Ly in naturally fluorescent milieus, via the use of TRES, thanks to
its long-lived luminescence.
Fig. 1 Ir-Ly for lysosome tracking. (a) Ir-Ly enters and is retained in
lysosomes. (b) Schematic of H+ and OH- sensing by Ir-Ly.
Results and discussion
Synthesis of Ir-Ly
Complex Ir-Ly was prepared through four steps as depicted in
Fig. 2. 1,10-Phenanthroline-5,6-dione
methoxybenzaldehyde in simple cyclo-condensation
reaction to produce compound . Then, alkylation of
produced the morpholine derivative followed by
demethylation to reveal the hydroxy groups in the
phenanthroline N^N ligand . Finally, the reaction of N^N
ligand with the organometallated dimer [Ir(ppy)2Cl]2 and
1 was reacted with 4-
2
a
3
3
4
,
5
5
subsequent anion exchange with NH4PF6 generated the
iridium(III) complex Ir-Ly in 65% yield. The intermediates 3-5
Fig. 3. TRES of complex Ir-Ly (20 μM) in the presence of CM460
(2 μM) in PBS buffer (0.1 mM, pH 4.5) under an excitation
wavelength at 355 nm; time gate (a) Delay < 333 ns or (b)
Delay > 333 ns.
1
and complex Ir-Ly were characterized by H NMR, 13C NMR,
and HRMS spectrometry (see SI).
pH effect and specificity study
The luminescent intensity of Ir-Ly was responsiveness to pH.
As pH increased, the emission intensity of Ir-Ly was reduced
(Fig. 4). This is due to the ability of morpholine, as an electron
donor, to quenching the luminescence of the iridium(III) center
through photoinduced electron transfer (PET). However, since
morpholine has a pKa of 5−6,21 it will become protonated in
acidic conditions. This prevents PET, leading to an
enhancement of luminescence emission at low pH.
As specificity is an important characteristic of a probe, we
investigated the selectivity of Ir-Ly by introducing different
concentrations of sodium ions, various cations, anions or
amino acids into a solution of Ir-Ly (Fig. S1). Encouragingly, the
results revealed that Ir-Ly had no obvious response towards
any of the tested interferents. Such emission response of pH
change and specificity are important for indicating the location
and morphology of lysosomes.
Fig. 2. Synthesis of complex Ir-Ly. Reagents and conditions: a)
NH4OAc/AcOH,
reflux,
overnight,
85%;
b)
4-(2-
chloroethyl)morpholine, NaH/DMF, r.t. to reflux, 25 h, 60%; c)
2-aminobenzenethiol, Cs2CO3, NMP, 185 °C, 3 h, 60%; d)
[Ir(ppy)2Cl]2, DCM/MeOH (1:1), r.t., overnight, 65%.
Photophysical properties of Ir-Ly
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Ly could selectively monitor lysosomes in both carcinoma and
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DOI: 10.1039/C8TB00666K
normal living human cell lines.
Fig. 4. Emission intensity of Ir-Ly at 590 nm at different pH
values in PBS buffer.
Visualization of lysosomes in living cells
Lysosomes are more acidic organelles (pH 5.0-5.5) compared
with cytoplasmic or other subcellular compartments (pH ca.
7.4).22 In consideration of responsiveness of Ir-Ly to pH, as well
as the promising luminescent behavior of the complex in
aqueous media, we hypothesized that Ir-Ly could function as
an ideal probe for lysosomes. To study this, the ability of the
probe to track lysosomes in living cells was investigated. First,
the cytotoxicity of complex Ir-Ly towards human cervical
cancer (HeLa) cells was first investigated using an MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
Complex Ir-Ly showed negligible cytotoxicity (IC50 value > 200
μM) after 6 h (Fig. S2), indicating that it could be used for
tracking lysosomes in living cells without adverse effects on
their normal function under the typical time and concentration
parameters employed.
Fig. 5. Luminescence images of HeLa cells incubated with
different concentrations of Ir-Ly (0, 2.5, 5, and 10 μM). After
incubation with Ir-Ly for 1 h at 37 °C, cells were washed three
times with phosphate buffer and stained with Lyso-Tracker
Red (1:15,000) for 30 min and Hoechst 33342 (1:2,000) for 10
min. Luminescence images were captured under an excitation
wavelength of 405 nm.
We next explored the ability of Ir-Ly for lysosome tracking in
living HeLa cells. Cells were stained with the indicated
concentrations of Ir-Ly for 1 h in a CO2 incubator at 37 °C,
followed by a subsequent incubation with the lysosome-
staining Lyso-Tracker dye for 30 min and the nuclear-staining
dye Hoechst 33342 for another 10 min. Cells were washed
three times with phosphate buffer, then luminescence images
were recorded using a confocal laser scanning microscope with
excitation at 405 nm. Ir-Ly exhibited a yellow punctuated
luminescence that was enhanced with increasing complex
concentration (Fig. 5). The co-localization of Ir-Ly with Lyso-
Tracker suggested that Ir-Ly could selectively monitor
lysosomes in living HeLa cells.
The ability of Ir-Ly to monitor lysosomes in different kinds of
living cells was further validated using a human non-small-cell
lung cancer cell line (A549) and a normal human umbilical vein
endothelial cell line (HUVEC) (Fig. 6). Notably, Ir-Ly appeared
to show even higher specificity for lysosomes in both cell lines,
as evidenced by the accumulation of the yellow punctuated
luminescence in the cytoplasm, compared to Lyso-Tracker,
which exhibited slight fluorescence in the nuclear region
where lysosomes are not expected to be found. Taken
together, these results indicate that the luminescent probe Ir-
Fig. 6. Intracellular co-localization of Ir-Ly (10 μM) and Lyso-
Tracker (1:15,000) in cancer (A549) and normal (HUVEC) living
cells. Luminescence images of cells were captured under an
excitation wavelength of 405 nm.
Conclusions
In conclusion, we have designed and synthesized
a
cyclometalated iridium complex Ir-Ly as an effective
luminescence lysosomal tracker. As a consequence to its large
Stokes shift, weak cytotoxicity, and specific localization in
lysosomes, Ir-Ly can be used to track lysosomes in both
carcinoma and normal living cell lines. Further studies for
imaging of other organelles and biomedical applications with
the long-lived luminescent iridium(III) complexes are ongoing
in our laboratories.
Acknowledgement
This journal is © The Royal Society of Chemistry 20xx
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This work is supported by Hong Kong Baptist University 14.
(FRG2/16-17/007, FRG2/17-18/003), the Health and Medical
Research Fund (HMRF/14150561), the Research Grants Council 15.
(HKBU/12301115), the National Natural Science Foundation of
China (21575121, 21775131, 21628502, 21502075 and 16.
21762018), the Postdoctoral Science Foundation of Jiangxi
Province (2017KY22), the Guangdong Province Natural Science
Foundation (2015A030313816), the Hong Kong Baptist 17.
University Century Club Sponsorship Scheme 2017, the
Interdisciplinary Research Matching Scheme (RC-IRMS/15- 18.
16/03 and RC-IRMS/16-17/03), Innovation and Technology
Fund (ITS/260/16FX), Collaborative Research Fund (C5026- 19.
16G), Matching Proof of Concept Fund (MPCF-001-2017/18),
the Science and Technology Development Fund, Macao SAR 20.
(077/2016/A2), the University of Macau (MYRG2016-00151-
ICMS-QRCM and MRG044/LCH/2015/ICMS), and the Natural 21.
Science Foundation of Jiangxi Province (20171BAB213008).
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might include comments relevant to but not central to the
matter under discussion, limited experimental and spectral data,
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