A Mitochondria-Targeted Photosensitizer Showing Improved Photodynamic Therapy Effects Under Hypoxia

Article abstract of DOI:10.1002/anie.201604130

Organelle-targeted photosensitizers have been reported to be effective photodynamic therapy (PDT) agents. In this work, we designed and synthesized two iridium(III) complexes that specifically stain the mitochondria and lysosomes of living cells, respectively. Both complexes exhibited long-lived phosphorescence, which is sensitive to oxygen quenching. The photocytotoxicity of the complexes was evaluated under normoxic and hypoxic conditions. The results showed that HeLa cells treated with the mitochondria-targeted complex maintained a slower respiration rate, leading to a higher intracellular oxygen level under hypoxia. As a result, this complex exhibited an improved PDT effect compared to the lysosome-targeted complex, especially under hypoxia conditions, suggestive of a higher practicable potential of mitochondria-targeted PDT agents in cancer therapy.

Full text of DOI:10.1002/anie.201604130

Angewandte
Communications
Chemie
International Edition: DOI: 10.1002/anie.201604130
German Edition: DOI: 10.1002/ange.201604130
Photodynamic Therapy
A Mitochondria-Targeted Photosensitizer Showing Improved
Photodynamic Therapy Effects Under Hypoxia
Wen Lv, Zhang Zhang, Kenneth Yin Zhang, Huiran Yang, Shujuan Liu, Aqiang Xu, Song Guo,
Qiang Zhao,* and Wei Huang*
Abstract: Organelle-targeted photosensitizers have been
reported to be effective photodynamic therapy (PDT) agents.
In this work, we designed and synthesized two iridium(III)
complexes that specifically stain the mitochondria and lyso-
somes of living cells, respectively. Both complexes exhibited
long-lived phosphorescence, which is sensitive to oxygen
quenching. The photocytotoxicity of the complexes was
evaluated under normoxic and hypoxic conditions. The results
showed that HeLa cells treated with the mitochondria-targeted
complex maintained a slower respiration rate, leading to
a higher intracellular oxygen level under hypoxia. As a result,
this complex exhibited an improved PDT effect compared to
the lysosome-targeted complex, especially under hypoxia
conditions, suggestive of a higher practicable potential of
mitochondria-targeted PDT agents in cancer therapy.
biological functions of the organelles under photoactivation,
[
4,5]
leading to cell death of tumor cells. However, investigation
was performed under normoxia conditions where oxygen
supply was sufficient, which is opposite to the hypoxic solid
tumor cells. To develop highly effective organelle-targeted
PDT drugs, it is urgent and important to evaluate the relation
between organelle-targeting and PDTeffect, especially under
hypoxia conditions.
In this work, two photosensitizers based on iridium(III)
complexes (Ir-P(ph)3 and Ir-alkyl, Scheme 1a,b) were
designed and synthesized, which specifically targeted the
mitochondria and lysosomes (Scheme 1c), respectively. The
complexes exhibited similar photophysical properties and
singlet oxygen quantum yields (F ) owing to the same
D
cyclometallating ligands. As the oxygen concentration could
[8]
greatly influence the PDT effect, imaging experiments of
P
hotodynamic therapy (PDT) is an approved photo-
activated and non-invasive medical technique that has been
widely used for cancer therapy through the generation of
[1]
excessive reactive oxygen species (ROS). Singlet oxygen
1
(
O ), as one of the primary toxic ROS, is generated by energy
2
transfer between the triplet excited state of photosensitizers
[
2]
and the ground state of O2. Organelle-targeted PDT agents
have been developed and their PDT effect has been stud-
[
3–5]
ied.
For example, nucleus-targeted PDT agents caused
great damage to cancer cells, but it is not favorable because of
[3]
the risk of genetic variation. As indispensable organelles,
mitochondria and lysosomes play essential roles in cell
respiration and digestion, respectively. Importantly, both of
[
6,7]
them are closely related to cell apoptosis.
Mitochondria-
Scheme 1. Chemical structures of a) Ir-P(ph) and b) Ir-alkyl, and c) the
process of mitochondria- or lysosome-targeted PDT.
3
and lysosome-targeted PDT agents can rapidly damage the
[*] W. Lv, Z. Zhang, Dr. K. Y. Zhang, H. Yang, Prof. S. Liu, A. Xu, S. Guo,
Prof. Q. Zhao, Prof. W. Huang
HeLa cells treated with the complexes under different oxygen
concentrations were conducted. Interestingly, the cells stained
with Ir-P(ph)3 maintained a relatively high intracellular
Key Laboratory for Organic Electronics and Information Displays
and Institute of Advanced Materials (IAM)
Jiangsu National Synergetic Innovation Center for
Advanced Materials (SICAM)
Nanjing University of Posts and Telecommunications (NUPT)
Nanjing 210023 (P.R. China)
oxygen concentration (icO ) under hypoxic conditions com-
2
pared to the cells loaded with Ir-alkyl, which probably
resulted from respiration inhibition induced by mitochon-
E-mail: iamqzhao@njupt.edu.cn
dria-targeted Ir-P(ph) . The PDT effects of both complexes
3
wei-huang@njtech.edu.cn
were evaluated, and the results indicated that the mitochon-
dria-targeted agent was more practicable in cancer therapy.
The synthetic routes of the complexes are shown in the
Supporting Information (Scheme S1). The complexes exhib-
ited strong absorption bands located at 380–520 nm, which
could be assigned to singlet and triplet metal-to-ligand charge
transfer and ligand-to-ligand charge transfer absorption (Fig-
Prof. W. Huang
Key Laboratory of Flexible Electronics (KLOFE) and
Institute of Advanced Materials (IAM)
Jiangsu National Synergetic Innovation Center for
Advanced Materials (SICAM)
Nanjing Tech University (NanjingTech)
Nanjing 211816 (P.R. China)
ure S1). The emission peaks of Ir-P(ph) and Ir-alkyl were
located at 590 nm and 600 nm, respectively (Figure S2). Upon
Supporting information for this article can be found under:
http://dx.doi.org/10.1002/anie.201604130.
3
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increasing oxygen concentration, their luminescence intensity
decreased and lifetimes shortened (Figure S2, Table S1). The
Stern–Volmer plots revealed a good linear relation (Fig-
[
9]
ure S3). The luminescent quantum efficiencies of Ir-P(ph)3
and Ir-alkyl were 0.13 and 0.15 in water, respectively
(
Table S2). The singlet oxygen quantum yields of Ir-P(ph)3
and Ir-alkyl were calculated to be 0.17 and 0.21 using
[
4a,10]
methylene blue (MB; F = 0.52 in DMF) as a standard.
D
The luminescence spectra (Figure S4) and phosphorescence
lifetimes (Figure S5) of the complexes in phosphate buffered
solution at different pH values remained largely unchanged,
indicating that the complexes are not pH dependent. Both Ir-
P(ph)3 and Ir-alkyl exhibited long emission lifetime
(
Table S1) and excellent O sensitivity. These results indicated
2
their promising applications as oxygen probes and PDT
photosensitizers.
The cell viability was measured through the methyl
thiazolyltetrazolium (MTT) assay. HeLa cells were incubated
with different concentrations of the complexes in the dark for
2
4 h. The cell viability remained high, indicating the low dark
cytotoxicity of Ir-P(ph)3 and Ir-alkyl (Figure S6). Subse-
quently, colocalization experiments involving Mito-Tracker
Green (MTG) and LysoGreen were conducted to confirm the
intracellular distribution of the complexes. The results
showed that Ir-P(ph) specifically localized in the mitochon-
3
dria of the cells, while Ir-alkyl was concentrated in the
lysosomes (Figure S7,S8). The Pearsonꢀs colocalization coef-
ficients of Ir-P(ph) with MTG and Ir-alkyl with LysoGreen
3
were 0.85 and 0.91, respectively. Specific mitochondria and
lysosome staining was further confirmed using other cell lines,
including A549 cells and HepG2 cells (Figure S9–S12).
To evaluate the photostability of the two complexes,
photobleaching experiments in living cells were conducted.
After strong 488 nm light irradiation, the luminescence
Figure 1. PLIM images of a) Ir-P(ph) - and b) Ir-alkyl-loaded fixed and
living HeLa cells. Luminescence lifetime distributions of PLIM imaging
3
of c) Ir-P(ph) - and d) Ir-alkyl-treated fixed cells, and e) the fitted
3
Stern–Volmer plots. The cells were incubated with the complexes
(5 mm) at 378C for 12 h. The images were taken under different oxygen
partial pressures. l =405 nm, f=0.5 MHz. All of the images share
ex
intensity of Ir-P(ph) and Ir-alkyl were slightly decreased
3
the same scale bar of 30 mm. Images were taken at 378C. f) Scheme of
the setup to measure the partial pressure of oxygen in cell culture
medium. g) The partial pressure of oxygen in cell culture medium
under different conditions. The cells were seeded on a 96-well plate at
(
Figure S13a,S14a). In contrast, the luminescence of MTG
and LysoGreen was almost completely quenched (Fig-
ure S13b,S14b), demonstrating the higher photostability of
the two complexes than the commercial dyes. The cellular
378C for 24 h and then treated with the complexes (5 mm) at 378C for
12 h.
uptake mechanism of Ir-P(ph) and Ir-alkyl was investigated
3
by incubating cells with the complexes under different
[
11]
conditions.
Compared with cells incubated at 378C, the
complexes displayed enhanced luminescence with elongated
lifetimes upon decreasing the extracellular oxygen concen-
tration from 21% to 2%, corresponding to a decrease of icO2
from 21% to 2%. The calibration curves were fitted through
the luminescence lifetime distributions of the PLIM imagings
luminescence intensity of cells incubated at 48C or pretreated
with metabolic inhibitors (deoxy-d-glucose and oligomycin)
and endocytosis inhibitors (NH Cl) was much weaker (Fig-
ure S15), indicating that the cellular uptake mechanism of the
complexes was energy-dependent endocytosis.
4
of Ir-P(ph) - and Ir-alkyl-treated fixed cells under different
3
The generation of singlet oxygen depends on the presence
of molecular oxygen, which is often inadequately supplied to
oxygen concentrations (Figure 1c–e). According to the cali-
bration curves, the icO of the living cells treated with the
2
[
12]
solid tumor cells. However, the oxygen concentrations in
various organelles under hypoxia are still unclear. Thus, the
intracellular oxygen concentration of living cells was first
determined using confocal microscopy imaging and phos-
complexes was determined (Table S3). Under ambient con-
ditions, the living cells treated with Ir-P(ph)3 exhibited
a luminescence lifetime of about 562 ns and the mitochondrial
oxygen concentration was about 26%. When the extracellular
oxygen concentration was reduced to 2%, the luminescence
lifetime was elongated to 682 ns and the mitochondrial
oxygen concentration was calculated to be 11%. Similarly,
the lysosomal oxygen concentration determined according to
the microscopy images of living cells treated with Ir-alkyl was
calculated to be 3% when the cells were cultured under 2%
[
13]
phorescence lifetime imaging (PLIM). A calibration curve
for icO2 was constructed using fixed cells because they
provided a similar biological environment to living cells and
the icO in the fixed cells was supposed to be the same as that
2
in the extracellular environment. As shown in Figure S16–S19
and Figure 1a,b, the fixed HeLa cells loaded with the
2
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O extracellular conditions. The higher oxygen concentration
2
in mitochondria than in lysosomes is likely the result of
cellular respiration. The following experiment was performed
to compare the respiration rate of the cells treated with Ir-
P(ph) and Ir-alkyl. As illustrated in Figure 1 f, cells were
3
cultured in a closed microchamber and the oxygen partial
pressure PO2 in the culture medium was measured via XF96
[14]
Analyzer (Seahorse Bioscience). A lower PO2 value indi-
cated more oxygen consumption and thus a faster respiration
rate. Living cells cultured in the microchamber for about
1
5 min resulted in a decrease of oxygen content by 44%
owing to the regulated respiration. When the cells were
pretreated with the complexes, the decrease of oxygen
content in the culture medium became 18% and 29% for
Ir-P(ph) and Ir-alkyl, respectively, indicative of a slowed
3
respiration rate (Figure 1g and Figure S20). Ir-P(ph) exhib-
3
ited a more effective inhibition of mitochondrial respiration
than Ir-alkyl, which led to a higher mitochondrial oxygen
concentration than the extracellular environment and thus
a good PDT performance in hypoxic tumor cells.
Figure 2. Confocal microscopy images and flow cytometry quantifica-
tion of annexin V-FITC- and PI-labeled HeLa cells. The cells were
treated with the complexes (5 mm) at 378C for 12 h. The cells were
then incubated under a,c,d) 21% O
or b,e,f) 5% O and irradiated by
2
2
À2
475 nm light (22 mWcm ) with a xenon lamp for a,b) 30 min or c–
The oxygen concentration inside the solid tumors is
[12]
f) 15 min. Cells were viewed in the green channel for annexin V-FITC
lex =488 nm, lem =500–560 nm) and red channel for PI (l =488 nm,
usually as low as 4%, which seriously limits the PDTeffect.
(
ex
As treatment with Ir-P(ph) led to a relatively higher oxygen
3
lem =600–680 nm), respectively. The images were taken 0, 2, and 4 h
after the irradiation. All the images share the same scale bar of 50 mm.
Images were taken at 258C.
concentrations in the mitochondria, the PDT performance of
the complex was tested under hypoxic conditions. Annexin V-
FITC and PI were used as the indicators for apoptotic and
dead cells, respectively. Cells undergoing early apoptosis
would be stained with green luminescent Annexin V-FITC on
cell membrane, while further staining with red luminescent PI
in the nucleus indicated the apoptosis and cell death. HeLa
À2
irradiation (22 mWcm ) displayed dim green fluorescence as
revealed in the confocal microscopy images (Figure S24).
When the cells were pretreated with the complexes, intense
green fluorescence was observed, indicating the generation of
intracellular ROS (Figure S25,S26). The luminescence inten-
cells treated with the complexes in the dark remained healthy
À2
(
Figure S21). Irradiation at 475 nm (22 mWcm ) under
[15]
normoxia for 30 min led to cell death in 4 h (Figure 2a).
sity of DCF in the Ir-P(ph) -treated cells was stronger than
3
When the cells were cultured under hypoxia conditions (5%
O ), light irradiation of the Ir-P(ph) -treated cells caused cell
that of Ir-alkyl-treated cells, indicative of more ROS produc-
tion. These cells were analyzed by flow cytometry. When Ir-
2
3
death in 4 h, while the PDTeffect of Ir-alkyl was less efficient
Figure 2b), which may be due to the relatively high oxygen
concentration in mitochondria compared to that in lysosomes
and extracellular environments.
The PDTeffect of the complexes was further studied using
flow cytometry. Increasing the dose concentration or irradi-
ation time led to a higher percentage of dead cells (Fig-
ure S22,S23). As expected, irradiation under normoxia
brought about a higher percentage of dead cells compared
to irradiation under hypoxia (Figure 2c–f), owing to the
sufficient O2 supply. Interestingly, 3.0% dead cells were
P(ph) -treated cells were irradiated under normoxia, 80% of
3
(
the cells exhibited intense DCF fluorescence (Figure S27a).
When the irradiation was performed under hypoxia, 63%
cells were green-emissive (Figure S28a). When Ir-alkyl was
used, irradiation under normoxia and hypoxia resulted in
35% and 14% cells, respectively, exhibiting green fluores-
cence (Figure S27b,S28b). Again, the photodynamic ROS
generation was more efficient in the cells treated with the
mitochondria-targeted Ir-P(ph) compared to those loaded
3
with the lysosome-specific Ir-alkyl.
Since early apoptotic cells undergo characteristic depola-
[
16]
detected after treatment of the cells with Ir-P(ph) (5 mm) for
rization of the mitochondria, the mitochondrial membrane
potentials (MMP) were measured using JC-1 as an indicator,
which was converted from aggregation state to monomer
upon decreasing MMP accompanied with a fluorescence
color change from red to green. In a positive control
experiment, carbonyl cyanide m-chlorophenyl hydrazone
(CCCP) was used to induce the decrease of MMP. HeLa
cells treated with JC-1 exhibited red and green fluorescence.
Pretreatment with CCCP caused quenching of the red
fluorescence and enhancement of the green fluorescence
(Figure S29). Similar MMP decreasing has also been observed
3
1
1
2 h followed by irradiation at 475 nm under hypoxia for
5 min and then cultured under ambient conditions for 30 min
(
Figure 2e), while the percentage was reduced to 0.15% when
Ir-alkyl was used (Figure 2 f). This result indicated an
improved PDT effect of the mitochondria-targeted Ir-P(ph)3
compared to the lysosome-specific Ir-alkyl under hypoxia.
To confirm that cell death was induced by the photo-
dynamic generation of singlet oxygen, the production of
intracellular ROS was investigated. 2’,7’-Dichlorofluorescein-
diacetate (DCFH-DA) was used as an indicator, which was
converted to green-fluorescent DCF in the presence of ROS.
HeLa cells loaded with DCFH-DA followed by 475 nm
when the cells were treated with the mitochontria-targeted Ir-
À2
P(ph) followed by 475 nm irradiation (22 mWcm ) under
3
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normoxia and hypoxia (Figure S30a,S31a). In contrast, the Acknowledgements
cells treated with the lysosome-targeted Ir-alkyl did not
exhibit MMP decreasing (Figure S30b,S31b).
The PDT effect of the complexes under normoxia and
hypoxia was evaluated by the MTT assay. The cells incubated
This work was supported by National Natural Science
Foundation of China (51473078, 61136003), National Pro-
gram for Support of Top-Notch Young Professionals, Scien-
tific and Technological Innovation Teams of Colleges and
Universities in Jiangsu Province (TJ215006), Natural Science
Foundation of Jiangsu Province of China (BK20130038,
BM2012010), Synergetic Innovation Center for Organic
Electronics and Information Displays and Priority Academic
Program Development of Jiangsu Higher Education Institu-
tions (YX03001).
with Ir-P(ph) for 12 h maintained 87% cell viability, which
3
dramatically decreased to 7.6% right after light irradiation,
and further to 2.3% in 4 h under normoxia. When the
irradiation was performed under hypoxia, 3.3% cell viability
was obtained in 4 h (Figure 3a), indicating a high PDT
Keywords: cell death · hypoxia · iridium ·
photodynamic therapy · singlet oxygen
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Received: April 28, 2016
Revised: June 8, 2016
Published online: && &&, &&&&
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Communications
III
Photodynamic Therapy
Two Ir complexes, Ir-P(ph) and Ir-alkyl,
3
were designed and synthesized to eval-
uate the mitochondria- and lysosome-
targeted PDT effect under normoxic and
W. Lv, Z. Zhang, K. Y. Zhang, H. Yang,
S. Liu, A. Xu, S. Guo, Q. Zhao,*
W. Huang*
&&&& — &&&&
hypoxic conditions. As Ir-P(ph) -treated
3
cells could maintain a relatively high
mitochondrial oxygen content under
hypoxia, the mitochondria-targeted com-
A Mitochondria-Targeted Photosensitizer
Showing Improved Photodynamic
Therapy Effects Under Hypoxia
plex Ir-P(ph) showed a superior PDT
3
effect than lysosome-targeted complex Ir-
alkyl.
6
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