Responsive and mitochondria-specific ruthenium(ii) complex for dual in vitro applications: Two-photon (near-infrared) induced imaging and regioselective cell killing

Article abstract of DOI:10.1039/c0cc01848a

A mitochondria-permeable ruthenium(ii) complex has been designed as a responsive probe which may be used to sensitize the formation of singlet oxygen (Φ_Δ = 0.93) and cause local damage in cellulo when exposed to UV and near-infrared laser excit

Full text of DOI:10.1039/c0cc01848a

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Responsive and mitochondria-specific ruthenium(II) complex for dual
in vitro applications: two-photon (near-infrared) induced imaging and
regioselective cell killingw
Hanzhong Ke,a Hongda Wang,a Wai-Kwok Wong,*be Nai-Ki Mak,c Daniel W. J. Kwong,b
Ka-Leung Wongb and Hoi-Lam Tamde
Received 11th June 2010, Accepted 3rd August 2010
DOI: 10.1039/c0cc01848a
A mitochondria-permeable ruthenium(II) complex has been
designed as a responsive probe which may be used to sensitize
the formation of singlet oxygen (UD = 0.93) and cause local
damage in cellulo when exposed to UV and near-infrared laser
excitation.
two-photon absorption (TPA).7 Lower energy, longer wave-
length photons readily penetrate the surface and if absorbed
simultaneously, their combined energy may be sufficient to
cross the energy gap between the excited-state energy levels
and the ground-state energy levels of the photosensitizer.8
Herein, we describe the synthesis of a new mitochondria-
specific PDT agent, PZn–Ru, an amphiphilic conjugate in
which a porphyrin moiety and a polypyridyl Ru(II) moiety
are covalently linked together via an ethynyl bridge. PZn–Ru
exhibits a strong two-photon absorption cross-section and
Photodynamic therapy (PDT) has received considerable attention
as a promising modality for cancer treatment.1 This therapy
requires a photosensitizer, visible light, and molecular oxygen.
Upon irradiation with a specific wavelength, the photosensitizer
undergoes a transition from its ground state to an excited
singlet state. Subsequently, the excited singlet state may decay
back to its ground state with emission of fluorescence or may
undergo intersystem crossing to its excited triplet state.
The excited triplet state can react with molecular oxygen to
generate reactive oxygen species (ROS), such as singlet oxygen
(1O2) and other radical species, which ultimately cause cellular
and tissue damage. 1O2 is known to be the major cytotoxic
agent responsible for photobiological activity.2
1
is capable of generating O2 through linear and two-photon
excitation. It is also cell permeable and can be excited selec-
tively with high spatial control in cellulo using a laser, giving
rise to high local concentrations of singlet oxygen, which can
cause sufficient damage to trigger apoptosis. The insensitive
PZn-Phen complex was used as negative control for the PDT
therapy of PZn–Ru in vitro.
The precursor complex PZn-Phen can be readily synthesised
via the Sonogashira cross-coupling reaction between 5-ethynyl-
1,10-phenanthroline and 5-bromo-10,15,20-tri(30,40,50-trimethoxy-
phenyl)porphyrinato zinc(II).9 It is then reacted with cis-Ru
(bpy)2Cl2 in THF–ethanol at 80 1C to give the title product
PZn–Ru in 72% yield (see the ESIw for further experimental
details).
Porphyrin-based photosensitizers, such as haematoporphyrin
derivatives (HpD), have been studied extensively3 and used
in clinical work during the past two decades.4 However, their
non-specific localization and toxic UV excitation have still
severely limited their achievable efficiency. In fact, requests
have been made for the development of target specific-vectors
for particular signal pathways and organelles, the mitochondria
in particular.5 By targeting the mitochondria, the power
supply of the cell, the rate of cell death can be increased.
The treatment efficiency of porphyrin-based sensitizers is also
limited by their small extinction coefficient excitation in the
body’s therapeutic window (650–900 nm).6 However, this can be
overcome by a new generation of photosensitizers that absorb
strongly at wavelengths greater than 650 nm through
The linear and two-photon induced photophysical properties
of PZn–Ru in solution were measured at room temperature.
PZn–Ru and showed marked peak-broadening and batho-
chromic shifts of 32 nm in its Soret bands compared to its
intermediate (Fig. S2w). This suggests that there is rather
strong electronic interaction between the porphyrin and the
phenanthroline moieties. In CHCl3, PZn–Ru displays identical
normalized linear and two-photon induced visible emission
spectra with maxima at 646 nm (Fig. 1).
The two-photon absorption cross section s2 value was
measured at 800 nm using the open-aperture Z-scan
method.10 The inset of Fig. 1 shows the Z-scan traces
of PZn–Ru, from which the absolute s2 value can be
determined. PZn–Ru exhibits a large s2 value of 1104 GM
a Faculty of Material Science & Chemistry Engineering,
China University of Geosciences, Wuhan, Hubei 430074
P. R. China
b Department of Chemistry, Hong Kong Baptist University,
Kowloon Tong, Hong Kong, P. R. China.
E-mail: wkwong@hkbu.edu.hk; Fax: 852-3411-5862;
Tel: 852-34117011
(GM
=
10ꢀ50 cm s4 photonꢀ1 moleculeꢀ1), which is
40 times larger than that of tetraphenylporphyrin (H2TPP)
(s2 = 28 GM),11 and also much higher than the recently
reported two-photon bio-available organometallic molecular
probes.12 The large enhancement is mainly due to the expansion
of p-conjugation and the molecular polarization induced by
the asymmetrical (D–p–A) structure between the porphyrin
and Ru(II)-polypyridyl moieties linked with an ethynyl
bridge.
c Department of Biology, Hong Kong Baptist University,
Kowloon Tong, Hong Kong, P. R. China
d Department of Physics, Hong Kong Baptist University,
Kowloon Tong, Hong Kong, P. R. China
e Centre for Advanced Luminescence Materials,
Hong Kong Baptist University, Kowloon Tong, Hong Kong
P. R. China
w Electronic supplementary information (ESI) available: Experimental
details. See DOI: 10.1039/c0cc01848a
c
6678 Chem. Commun., 2010, 46, 6678–6680
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Fig. 3 Flow cytometric analysis of the cellular uptake of PZn–Ru
in the HK-1 cells. The cells were incubated with 1 mM of PZn–Ru for
0 h (control, black line), 6 h (red line) or 24 h (green line) in dark. The
cells were trypsinized and washed twice with PBS. The fluorescence
profiles of the treated cells were analyzed using FACSCalibur (Becton
Dickinson). The 488 nm laser line was used for the excitation
of PZn–Ru and the fluorescence signal was collected using the FL-3
channels equipped with long pass filter (4 650 nm). At least 10 000
events were counted.
Fig. 1 The normalized linear and two-photon induced emission
spectra of PZn–Ru; Inset: Open-aperture Z-scan traces of PZn–Ru
(250 mM DMSO) excited at 800 nm. The average power of the laser
beam used was 0.20 mW, the two-photon absorption cross-section of
PZn–Ru is 1104 GM, lex = 800 nm.
In order to evaluate the photosensitizing efficiency of
PZn–Ru, the 1O2 quantum yields (FD) were determined by
measuring the near-infrared (NIR) phosphorescence intensity
of 1O2 (at 1.27 mm) produced from these compounds upon
irradiation at 424 nm, using H2TPP as a reference (FD = 0.55),
The uptake of a photosensitizer by tumour cells is a critical
determinant of treatment efficacy. The cellular uptake of
PZn–Ru by the human nasopharyngeal carcinoma HK-1 cells
was studied by flow cytometry (Fig. 3). The fluorescence
intensity of the HK-1 cells after incubation with PZn–Ru
for 6 and 24 h was about 7 and 10 times, respectively, higher
than that of the untreated cells. The results clearly indicated
that PZn–Ru was taken up rapidly by the HK-1 cells. This is
due to the amphiphilic structure of PZn–Ru, which has an
appropriate hydrophobic/hydrophilic balance to enhance its
cellular uptake.
1
in CHCl3. The O2 quantum yield of PZn–Ru (FD = 0.93) is
found to be significantly higher than that of H2TPP. The
subcellular localization of PZn–Ru in the HeLa (Fig. 2a and
b), and HK-1 cells (Fig. 2c–f) was studied using both two-
photon and linear fluorescence microscopy. After 40 min
incubation, upon excitation at 850 nm, strongly solid red
emission can be observed apparently in the mitochondria of
the HeLa cells (Fig. 2a and b). To confirm the observed
mitochondria localization, co-localization of PZn–Ru (red
emission) with a commercial mitochondria tracker (green
emission) was investigated and Fig. 2c–f shows the confocal
images of cells co-stained with PZn–Ru and this mitochondria
tracker.
We designed two experiments to evaluate the therapeutic
efficacy of PZn–Ru. Firstly, its photocytotoxicity towards
HK-1 cells was measured by
a MTT-reduction assay
(Fig. 4). It can be seen that PZn–Ru was essentially non-
cytotoxic in the absence of light, but exhibited very high
Fig. 2 (a and b) Two-photon induced in vitro mitochondria staining
microscopy with PZn–Ru for 1 h (HeLa cells, dose conc. = 50 mM,
lex = 850 nm); (c, d, e and f) co-localization experiment with
intracellular fluorescence of HK-1 cells treated with PZn–Ru
(50 mM, 24 h) and Mito tracker (lex = 450 nm).
Fig. 4 Photocytotoxicity of PZn–Ru on HK-1 cells. HK-1 cells were
treated with PZn–Ru (1 mM) for 24 h, then washed with fresh medium,
and irradiated with various doses of light. The MTT-reduction assay
was carried out 24 h after PDT. The results were expressed as the mean
ꢁ S.D. of three separate trials.
c
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Fig. 5a and b). We observed that both PZn–Ru and PZn-Phen
were taken up by the cells and localized in mitochondria and
cytoplasm, respectively. There was no significant cell death
seen in either case. After 5 min flash excitation by 850 nm laser
on the cells loaded with PZn–Ru, 90% of the HeLa cells were
deformed or had lost their integrity. However, the same was
not observed for PZn-Phen. No significant cell death occurred
and there was no observable change in the bright-field images
of the HeLa cells (Fig. 5c and d). The cells dosed with PZn–Ru
and PZn-Phen were further flashed with an NIR laser line for
15 min, after which all the cells incubated with PZn-Phen were
seen to be alive (Fig. 5e).
In summary, a novel ethyne-linked amphiphilic porphyrin–
polypyridyl Ru(II) conjugate with relatively high two-
photon absorption cross-section has been synthesized for
mitochrondra localization. The compound shows a substantial
1O2 quantum yield under visible and NIR excitation. Signifi-
cant cell death occurred upon suitable photoexcitation in the
presence of PZn–Ru, suggesting that PZn–Ru may be a
promising candidate for TPA photodynamic therapy.
The work described in this paper was partially supported
by grants from the Research Grants Council of the Hong
Kong SAR, P. R. China (HKBU 202308 and HKBU 202509)
and
a grant from the Hong Kong Baptist University
(FRG2/08-09/067).
Notes and references
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Fig. 5 Confocal microscopy and bright-field images of HeLa cells
loaded with PZn-Phen (a, c, e) and PZn–Ru (b, d, f). (a and b) Images
were observed with the incubation of PZn–Ru and PZn-Phen for 1 h
before snap flash with a laser at 457 nm; (c and d) confocal microscopy
images observed after flash with a laser at 850 nm for 5 min; (e and f)
after further flash for 15 min with the laser line at 850 nm.
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photocytotoxicity. At the concentration of 1 mM and a light
dose of 3 J cmꢀ2, 80% of HK-1 cells were killed. The light dose
required to kill 50% of HK-1 cells (LC50) was ca. 1.8 J cmꢀ2
.
This remarkably high photocytotoxicity may be attributed to
1
the high cellular uptake, high O2 quantum yield and proper
subcellular localization of PZn–Ru.
Andrasik et al. showed that the quantum yield of two-
photon induced 1O2 generation is around 30–40% even
though the linear process and the mechanism of two-photon
and linear 1O2 generation are the same.13 Thus, to further
show the potential of its therapeutic efficiency, two-photon
induced 1O2 generation was also performed in vitro with
PZn–Ru. 5 mM PZn–Ru or PZn-Phen (negative control,
incapable of generating 1O2 in the NIR region) was incubated
in cervical carcinoma HeLa cells for 1 h and the uptake was
visualized using a confocal microscope equipped with laser
excitation sources at 457 nm (linear) or 850 nm (two-photon
excitation). Cells in the field of view were then excited at
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1
457 nm for a short period of time (to avoid O2 generation,
c
6680 Chem. Commun., 2010, 46, 6678–6680
This journal is The Royal Society of Chemistry 2010