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Endoplasmic Reticulum-Localized Iridium(III) Complexes as Efficient Photodynamic Therapy Agents via Protein Modifications.
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Article
Endoplasmic Reticulum-Localized Iridium(III) Complexes as
Efficient Photodynamic Therapy Agents via Protein Modifications
Jung Seung Nam, Myeong-Gyun Kang, Juhye Kang, Sun-Young Park, Shin Jung C. Lee, HyunTak Kim, Jeong Kon Seo, Oh-Hoon Kwon, Mi Hee Lim, Hyun-Woo Rhee, and Tae-Hyuk Kwon
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b05302 • Publication Date (Web): 05 Aug 2016
Downloaded from http://pubs.acs.org on August 6, 2016
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Journal of the American Chemical Society
Endoplasmic Reticulum-Localized Iridium(III) Complexes as
Efficient Photodynamic Therapy Agents via Protein Modifications
Jung Seung Nam,†,ǁ Myeong-Gyun Kang,†,ǁ Juhye Kang,†,ǁ Sun-Young Park,‡,ǁ Shin Jung C. Lee,† HyunTak Kim,† Jeong Kon Seo,§ Oh-Hoon Kwon,†,‡ Mi Hee Lim,*,† Hyun-Woo Rhee,*,† and Tae-Hyuk
Kwon*,†
†
Department of Chemistry, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of Korea
Center for Soft and Living Matter, Institute for Basic Science (IBS), Ulsan 44919, Republic of Korea
§
UNIST Central Research Facility, Ulsan National Institute of Science and Technology (UNIST), Ulsan 44919, Republic of
Korea
‡
ABSTRACT: Protein inactivation by reactive oxygen species (ROS) such as singlet oxygen (1O2) and superoxide radical (O2•–) is
considered to trigger cell death pathways associated with protein dysfunction; however, the detailed mechanisms and direct involvement in photodynamic therapy (PDT) have not been revealed. Thereby, we report herein Ir(III) complexes designed for ROS
generation through a rational strategy to investigate protein modifications by ROS. The Ir(III) complexes were effective as PDT
agents at low concentrations with low-energy irradiation (≤ 1 J cm-2) because of the relatively high 1O2 quantum yield (> 0.78),
even with two-photon activation. Furthermore, two types of protein modifications (protein oxidation and photo-crosslinking) involved in PDT were characterized by mass spectrometry. These modifications were generated primarily in the endoplasmic reticulum and mitochondria, producing a powerful effect for cancer cell death. Consequently, we present a plausible PDT modality that
utilizes photo-activation of rationally designed Ir(III) complexes, indicating the feasibility of a better optimized Ir(III) complex for
PDT.
INTRODUCTION
Photodynamic therapy (PDT) has been successfully used to
treat skin cancer by illuminating photosensitizers for over 100
years.1 Because photosensitizers generate toxic reactive oxygen species (ROS) spatiotemporally by light activation in the
treated spaces, PDT has also been applied to many internal
cancers.2 The most commercialized PDT drug is based on a
hematoporphyrin derivative (HPD)3 that generates ROS, leading to tumor cell death. However, limitations of HPD include
inefficient light penetration in tissue, poor ROS generation
under low dioxygen (O2) concentrations in physiological conditions of cancer cells,4 a low molar absorption coefficient
(1170 M−1 cm−1)5 requiring high treatment doses and longer
light exposure time, and side effects including immune response.6 Organometallic complexes based on ruthenium (Ru)
and platinum (Pt) have been developed to cover such limitations. However, Pt(II) complexes utilize labile ligands for cancer therapy, inducing side effects at unintended sites.7 In turn,
the poor cell membrane penetration of Ru(II) complexes results in long cell permeation times and relatively high concentration requirements (ca. 40 µM) for imaging.8 The low quantum yields for phosphorescence (Φp = 0.03–0.1) and singlet
oxygen (1O2) (Φs = 0.1–0.5) in aqueous systems also hinder
image-guided PDT systems.9 Subsequent advances in photosensitizer technology have not adequately addressed issues of
collateral protein damage, side effects, and cellular substrate
reactions effected by ROS.
Herein, we report an effectively developed design strategy
for PDT agents based on Ir(III) complexes by controlling their
energy levels to achieve marked ROS generation.10 Ir(III)
complexes offer multiple advantages, including (i) simple
color tuning, (ii) energy-level control, (iii) long lifetime (µs)
permitting signal discrimination from protein autofluorescence, and (iv) ROS generation under hypoxia conditions via electron (Type I) and/or energy transfer (Type II).11
Furthermore, Ir(III) complexes exert higher anticancer activity
against over 60 cancer cell lines than the U.S. Food and Drug
Administration-approved drugs, oxaliplatin and cisplatin.12
To date, no in-depth research for ROS property enhancement via a molecular design strategy has been performed. Accordingly, we designed four Ir(III) complexes (TIr1–4; Figure
1a) that exhibit different energy levels as well as distinct Φp
and Φs. Our Ir(III) complexes exhibited two-photon absorption, followed by ROS generation, which might represent ideal
conditions for an effective cancer therapy system because of
the potential for deep-tissue imaging.13 Notably, these Ir(III)
complexes were primarily localized in the endoplasmic reticulum (ER). Only TIr3 and TIr4 showed their noticeable PDT
activity for SK-OV-3 ovarian- and MCF-7 breast cancer cells,
likely because they efficiently produce ROS owing to their
well-matched energy levels for 1O2 generation and high Φp.
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Finally, we propose potential mechanisms clarifying prompt
cell death via oxidation of essential proteins (e.g., TRAP1,
PYCR1) in the ER and mitochondria and by arbitrary protein
photo-crosslinking in the vicinity of the Ir(III) complexes
without requiring toxic additives, leading to protein aggregation in living cells (Figure 1b).
RESULTS AND DISCUSSION
Ir(III) complexes as photodynamic therapy (PDT)
agents. Developing effective PDT systems encompasses the
following factors: light activation in the near infrared (IR)
region, high absorption coefficient, rapid and conspicuous
imaging of cancer cells, and ROS generation capability. Considering these criteria, four cationic Ir(III) complexes (TIr1–4;
Figure 1a and Scheme S1) were designed and prepared. We
expected that ROS generation would be dependent on the energy level of the photosensitizers because 1O2 formation occurs by energy transfer. Thus, suitable energy overlaps between photosensitizers (energy donor) and O2 (energy acceptor) in addition to high emission quantum yields would allow
enhanced ROS production through controlling energy levels of
the photosensitizers.14 Accordingly, four different ligands
[difluorophenylpyridine (dfppy), 2-phenylpyridine (ppy), 2phenylquinoline (2pq), and 1-phenylquinoline (1pq)] were
incorporated into the Ir(III) center. Additionally, the bipyridine (bpy) ligand was employed as an ancillary ligand to afford overall cationic Ir(III) complexes that are soluble in
aqueous systems while retaining their high emission quantum
yields.15 To measure the energy bandgaps and extinction coefficients upon metal-to-ligand charge transfer (MLCT) of the
Ir(III) complexes, the UV-Visible (UV-vis) absorption spectra
of TIr1–4 were measured (Figure 2a and Table 1), indicating
MLCT at 350, 375, 448, and 450 nm, respectively, and the
corresponding extinction coefficients (ε) (range 5587–6971
M−1cm−1) that are higher than that of HPD.5 The respective
onset points of TIr1–4 on the UV-vis spectra corresponding to
the energy bandgaps appear at 399, 428, 488, and 494 nm.
Cyclic voltammetry (CV) was conducted to measure the oxidation potentials (Figure 2b), corresponding to the highest
occupied molecular orbital (HOMO) of the Ir(III) complexes.
Based on the onset points of UV-vis and the oxidation potentials measured by CV, the singlet energy levels of the Ir(III)
complexes were as presented in Figure 2c, along with the corresponding energy bandgap order [TIr1 > TIr2 > TIr3 >
TIr4]. The maximum emission wavelengths (λmax) corresponding to the triplet energy levels of Ir(III) complexes were
measured by photoluminescence spectroscopy and, when excitation was performed at the MLCT, they are indicated to be at
531, 590, 562, and 592 nm for TIr1–4, respectively (Figure
2a). The triplet emission from TIr2 was highly dependent on
the solvent polarity; therefore, a large Stokes shift appears in
water compared that in organic solvents (data not shown).
Furthermore, Φp of the Ir(III) complexes were observed to be
highly dependent on their ligands [TIr3 (0.53 ± 0.05) > TIr1
(0.46 ± 0.02) > TIr4 (0.11 ± 0.01) > TIr2 (0.011 ± 0.001)].
Additionally, all Ir(III) complexes present two-photon absorption properties. The two-photon excitation spectra, similar to
the UV-vis absorption spectra, range from 670 to 950 nm,
which is an appropriate near-IR range wherein living cells
would not be damaged by high-energy excitation light. The
emission spectra and non-linear properties for two-photon
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absorption are depicted (Supporting Information Figures S1
and S2).
Images of the HeLa cells upon incubation with each Ir(III)
complex were further obtained by two-photon laser scanning
microscopy. All Ir(III) complexes emit noticeable phosphorescence even at a relatively low concentration (10 µM) upon
incubation for 30 min (Figure 1c). Our imaging investigations
clearly demonstrate the cell-membrane permeability of each
Ir(III) complex and the accompanying critical localization to
the ER vicinity. Furthermore, fluorescence lifetime imaging
microscopy (FLIM) analysis confirmed the extended TIr3
phosphorescence (ca. 500 ns) in the vicinity of ER compared
to the short lifetime (ca. 5 ns) of a nuclear-localized fluorescent protein (H2B-mCherry) (Supporting Information Figure
S3). This long lifetime could be resulted from the relatively
strong spin-orbit coupling by iridium,16 which can boost the
possibility of electron or energy transfer from Ir(III) complexes to O2, resulting in more efficient ROS generation.
Cytotoxicity evaluation of light-activated Ir(III) complexes. The PDT potency for TIr1–4 was monitored in SKOV-3 ovarian-, and MCF-7 breast cancer cells via the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide
(MTT) assay and compared to that of a well-known anticancer
drug, cisplatin17 and a reported photoactivatable reagent,
[Ru(bpy)3]2+ (Figure 3a and Supporting Information Figure
S4).18 Cell viability upon treatment with a relatively low concentration of TIr3 (i.e., 2 µM) where it could be still photoactivated was markedly reduced to 40% and 19%, respectively, with 10 and 60 s irradiation (Figure 3a); TIr4 displayed its
cell viability similar to TIr3 (31% and 18% of cell viability
for the samples treated 10 and 60 s irradiation, respectively).
Without light, cell death was not significantly triggered by
TIr3 (89/84% of cell viability for 0.2/2 µM, respectively) or
TIr4 (91/89% of cell viability for 0.2/2 µM, respectively).
Furthermore, a submicromolar complex concentration (i.e., 0.2
µM) diminished the survival of SK-OV-3 cells to 52% and
41% for TIr3 and TIr4, respectively, after 60 s of light exposure (Figure 3a). This influential PDT effects were observed in
MCF-7 cells as well (Supporting Information Figure S4a). In
addition, IC50 values of TIr3 and TIr4 are summarized in
Figure 3b (SK-OV-3) and Supporting Information Figure S4b
(MCF-7). TIr3 and TIr4 showed 4.01/1.58 and 3.67/0.65 µM
of IC50 values without/with light, respectively, in SK-OV-3
cells (Figure 3b); similarly, in MCF7 cells, 4.89/0.83 and
3.61/0.63 µM of IC50 values are presented for TIr3 and TIr4
in the absence/presence light, respectively (Supporting Information Figure S4b). For cisplatin and [Ru(bpy)3]2+, IC50 values
were not determined up to 50 µM of each complex in both cell
lines. In contrast to TIr3 and TIr4, the PDT activity for TIr1,
TIr2, [Ru(bpy)3]2+, and cisplatin was not noticeable regardless
of light control under our experimental conditions (Figure 3a
and Supporting Information Figure S4). Overall, our studies
indicate the potential of TIr3 and TIr4 to serve as PDT agents
simply using 1 sun light (100 mW cm−2 light; 1 J cm−2) just for
10 s, thus requiring much lower energy than previously reported Ir(III) complexes (12−36 J cm–2).19
The potential use of TIr3 as a two-photon-based PDT agent
was further investigated by visualizing the morphological
changes of SK-OV-3 cells upon co-incubation. Cell shrinkage
was clearly indicated upon TIr3 treatment as a function of
time (5, 30, and 60 min) upon irradiation at 860 nm (Figure
3b). In contrast, no morphological alteration occurred in the
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Journal of the American Chemical Society
absence of TIr3 even with irradiation. Thus, our Ir(III) complexes, particularly TIr3, demonstrate the potential as PDT
agents employing either one- or two-photon irradiation.
Characterization of ROS generation by Ir(III) complexes. The ROS generation ability of Ir(III) complexes according
to their energy levels was identified by three different analyses: triplet state quenching by O2, 9,10-anthracenediylbis(methylene) malonic acid (ABDA) assay for 1O2 generation,20 and dihydrorhodamine (DHR) 123 assay for superoxide
radical (O2•–) generation.21 Triplet state quenching of Ir(III)
complexes by O2 bubbling relates to the amount of formed
ROS, as it strongly depends on 1O2 generation and charge
transfer interactions from Ir(III) complexes to O2, resulting in
oxygen radical formation.22 The quenching rate order is TIr3
> TIr4 > TIr1 > [Ru(bpy)3]2+ > TIr2 (Supporting Information
Figure S5), suggesting that TIr3 is the best photoactivatable
ROS generator for PDT in our system.
Next, the Φs of Ir(III) complexes were obtained using the
absorbance change (∆A) of ABDA according to the irradiation
time from 0 to 5 min with [Ru(bpy)3]2+ (Φs = 0.18 in H2O) as a
reference (Figure 4a and Supporting Information Figure S6).23
TIr3 showed the highest Φs (0.95 ± 0.04) followed by TIr4
(0.78 ± 0.04), TIr2 (0.37 ± 0.01), and TIr1 (0.29 ± 0.04) (Table 1). 1O2 generation relies upon energy transfer; thus, it is
closely related to the difference in the energy level between
the energy donor and acceptor as well as to the emission quantum yield of the energy donor. Previously, it is reported that
one guideline of the oxidation potential of sensitizer for efficient PDT property is 1.10 V vs SCE (= 1.34 V vs NHE),
which corresponds to –5.51 eV of HOMO24 and the deeper
HOMO is preferable.25 To enhance the energy transfer, the
HOMO of energy donor has a lower (down shift) than that of
energy acceptor, and all of our Ir(III) complexes satisfied this
condition under both organic and aqueous media (Table 1).
Additionally, O2 as an energy acceptor has two singlet excited
states with corresponding absorption energies of 762 and 1268
nm.18,24 Matching of these energy levels is critical for efficient
energy transfer; therefore, the minimum triplet energy of
Ir(III) complexes should be higher than 762 nm (i.e., 1.63 eV).
Conversely, excessively high triplet energy [e.g., TIr1; 531
nm (i.e., 2.34 eV)] leads to inefficient energy transfer because
of mismatch with the energy level of O2.24
If the energy level is well-matched, the emission quantum
yield should also be considered.14 For example, both TIr3
(562 nm, 2.21 eV) and TIr4 (592 nm, 2.09 eV) had appropriate energy levels for efficient energy transfer towards groundstate O2. TIr3 exhibits the highest Φs (0.95), followed by TIr4
(Φs = 0.78), because of its higher Φp (0.53 vs. 0.11). Although Φp of TIr1 is high (0.46), its Φs is the lowest (0.29)
because of mismatched energy levels. TIr2 has a reasonable
triplet energy (590 nm, 2.10 eV); however, it has a lower Φs
(0.37) because of its minimal Φp (0.01). To confirm these calculated predictions, we prepared four neutral (Neutral-1–4)
and three anionic (Anionic-1–3) Ir(III) complexes by substituting the ancillary ligand while retaining the main ligand.
These complexes exhibited the same results with the cationic
species (from TIr1 to TIr4). The highest Φp and appropriate
energy level (> 1.63 eV) of the Ir(III) complexes generated 1O2
efficiently (Supporting Information Figure S7).
Based on these findings, we propose that for efficient generation of 1O2, Ir(III) sensitizers should have an energy band gap
between 1.63 and 2.21 eV as major factor a high emission
quantum yield and a reasonable HOMO energy (lower position than −5.51 eV). In the DHR 123 assay, the relative rates
for O2•– production are in order of TIr3 >> TIr4 > TIr1 =
[Ru(bpy)3]2+ > TIr2 (Figure 4b), corresponding to 1O2 generation. Taken together, our studies of ROS generated by Ir(III)
complexes indicate that TIr3 exhibits relatively high oxygen
sensitivity, resulting in its relatively high Φs and generation of
O2 •–, with subsequent photo-controlled cytotoxicity.
Characterization of protein crosslinking by Ir(III) complexes. Following these ROS generation analyses, we evaluated the efficiency of Ir(III) complex for photo-induced protein
crosslinking, which could inactivate protein physiological
functions by aggregation, potentially generating additional
cytotoxicity.26 [Ru(bpy)3]2+ systems require an exogenous
additive such as ammonium persulphate (APS) as an electron
acceptor for initiating crosslinking pathways27 and might have
not been applied in PDT because of the toxicity of APS. Thus,
fast and efficient additive-free protein crosslinking reagents
would be valuable for PDT development.
Generally, photo-activated protein crosslinking by metal
complexes occurs through coupling reactions with tyrosyl
radicals exposed on the protein surfaces.27 We utilized biotinphenol (BP), which has a tyrosine residue, to confirm covalent
dimerization by in vitro crosslinking with photo-activation. BP
crosslinking induced by metal complexes was confirmed by
matrix-assisted laser desorption ionization mass spectrometry
(MALDI-MS). For TIr3, the crosslinked BP (BP-BP + H+,
m/z 725.3) was clearly observed in the presence of O2 with
light exposure (Figure 4c), whereas it was not generated without either O2 or light (Figure 4c and Supporting Information
Figure S8). BP crosslinking using [Ru(bpy)3]2+ also occurred
only in the presence of APS with O2 and light (Supporting
Information Figure S9). These results suggest that TIr3, in
contrast to [Ru(bpy)3]2+, is able to achieve crosslinking in the
presence of O2 and light without requirement of an exogenous
additive.
We also investigated whether our Ir(III) complexes could
induce protein-protein crosslinking in living cells. We transiently expressed the GFP-Sec61B protein, which localizes at
the ER cytosolic membrane, and conducted photo-crosslinking
experiments with TIr1–4 and [Ru(bpy)3]2+. Western blot results with an anti-GFP demonstrated substantial proteinprotein crosslinking (over 180 kDa) by TIr3 and TIr4, which
were more effective upon light activation (Figure 4d, lanes 6
and 7). For TIr1 (lane 4) and TIr2 (lane 5), only mild crosslinking was observed. Crosslinking by the Ir(III) complexes
occurred only with light illumination. Note that Neutral-3 and
Anionic-3 as a representative of neutral and anionic Ir(III)
complexes (Figure S7) did not generate photo-crosslinked
product efficiently, compared with TIr1 and TIr3 (Supporting
Information Figure S10). Notably, no efficient protein crosslinking was indicated with [Ru(bpy)3]2+, even with APS treatment (Figure 4d, lanes 8 and 9), suggesting limited applications in living cells. Overall, our Ir(III) complexes are demonstrated to be better protein photo-crosslinking reagents than
[Ru(bpy)3]2+ both in in vitro and in living cells, and they do
not include toxic additives, implying suitability for use in the
cellular environment. We postulate that the higher performance of Ir(III) complexes might be derived from their oxygen sensitivity and extended excitation lifetime (Table 1),
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implying efficient charge transfer and energy transfer because
of minimized non-radiative decay.
Identification of oxidative modifications of endogenous
proteins by TIr3. In addition to protein–protein crosslinking,
we attempted to identify whether protein oxidation could be
achieved by TIr3. In our analysis of protein oxidation, we
focused on modified proteins that contain oxidized methionine
residues, since mono-oxidized methionine (O-Met, Met + 16
Da) has been characterized as the major oxidative product by
ROS and recent proteomic profiling studies have shown that
over 2000 oxidized methionine sites are globally generated
when cells were exposed to hydrogen peroxide (H2O2).28 By
using TIr3, we expected to observe focused O-Met sites in
proteins proximal to subcellularly localized TIr3 that could be
directly affected by TIr3-generated 1O2 in situ, which has a
lifetime of less than 1 ms.29 This mapping would enable the
detection of specific proteins oxidatively damaged by our PDT
agents, potentially leading to improved understanding of the
underlying biological events.
To identify the additional oxidation by the TIr3, we used
methionine sulfoxide antibody and it shows that TIr3 and
TIr4 generated more oxidized proteins than negative controls
(Supporting Information Figure S11). Surprisingly, western
blot pattern of methionine-oxidized proteins resembled that of
photo-crosslinked products. This result implies that photooxidation and photo-crosslinking reactions by TIr3 and TIr4
may simultaneously occur on the same substrate proteins.
Other Ir(III) complexes showed similar level of oxidized proteins relative to basal level of the negative controls (Supporting Information Figure S11). This result indicates that other
iridium complexes performed negligible photo-oxidation on
the endogenous proteins in a living cell. From this result, we
selected TIr3 sample for profiling of photo-oxidized proteins
by mass spec analysis.
Three samples containing TIr3 with light illumination, as
well as the three other samples as negative controls (control
sample; #1, without light; #2, without TIr3; #3, without both
light and TIr3), were prepared. The cells were lysed in RIPA
buffer and digested with trypsin after loading in SDS-PAGE
gel. The peptides were desalted and analyzed using LTQOrbitrap mass spectrometry. The analyzed peptides were firstly filtered by O-Met (Met + 16 Da) modification, and the modified peptides were secondly filtered by reproducible observation of the same O-Met sites thrice, either in PDT-treated triplicates or in the three negative control samples. Finally, a total
244 O-Met sites were identified in our study; 101 O-Met sites
were exclusively observed in PDT-treated triplicates (Group I)
and 129 O-Met sites were consistently observed both in PDTtreated sample and negative samples (Group II) (Figure 5b).
Thus, Group II sites can be regarded as endogenous oxidized
methionine residues regardless of photo-oxidation and Group I
sites can be considered to be additionally generated O-Met
from the photodynamic reaction by TIr3. We also noted 14 OMet sites that were exclusively observed in the control samples (Group III).
When these O-Met sites were mapped onto the subcellular
compartments, such as mitochondria, ER, and cytoplasm (Figure 5c), significant numbers of Group I sites were mapped
onto mitochondrial proteins (47 out of 101 sites) and a considerable number was mapped onto ER proteins (25 out of 101
sites). This is a rather surprising result when compared to the
subcellular population of Group II sites, in which the majority
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of modified sites were mapped onto cytoplasmic proteins (88
out of 129 sites) composed of cytoskeleton proteins such as
actin (e.g., ACTB, ACTC1), tubulin (e.g., TUBA1C,
TUBB2B, TUBB4B), and heat-shock proteins (e.g.,
HSP90AA1, HSP90AB1, HSP90B1, HSPA1A, HSPA4,
HSPA5, HSPA8, HSPA9) that are known to readily modify OMet by the endogenously generated ROS.30 In contrast, a small
number of modified sites were mapped onto mitochondrial
proteins (18 out of 129 sites) and ER proteins (12 of 129
sites). Similarly, the majority of Group III (12 out of 14) were
mapped onto cytoplasmic proteins, especially actin proteins
(e.g., ACTB, ACTC1, ACTG1), which are known to be readily oxidized by endogenous ROS.30 Groups II and III showed
similar subcellular localized population, and the total spectral
count of Group III sites were significantly lower than that of
Group I or II. Thus, we postulated that Group III sites should
be endogenously oxidized Met residues and that they might be
excluded during the mass analysis of other abundant O-Metmodified peptides in PDT-treated samples. Overall, the spatially resolved protein oxidation by TIr3 with photo-activation
clearly occurred at the mitochondria and ER, indicating TIr3
should be localized at these subcellular compartments.
Primary subcellular localization of TIr3 in living cells.
To determine the subcellular localization of TIr3, we imaged
TIr3 localized region in HeLa cell and HEK293T cell line,
respectively. ER tracker dyes for ER staining31 and immunofluorescence of anti-Tom20 which showed mitochondrial
pattern were employed to confirm the subcellular localization
pattern of TIr3.32 Noticeably, the resultant images clearly
indicated that TIr3 staining region substantively overlapped
with ER-Tacker (Pearson correlation value = 0.96, Figure 5d)
while it showed partially overlapped with immunofluorescence of anti-Tom20 (Pearson correlation value = 0.44, Figure
5d). Additionally, we confirmed TIr3 was mainly localized at
the ER and partially overlapped with mitochondria in
HEK293T cell line (Supporting Information Figure S13).
TIr1, TIr2 and TIr4 also showed well overlapped localized
pattern with ER-Tracker in both HeLa and HEK293T cell
lines (Supporting Information Figures S12 and S13).
These observations might be explained by our results that
the major population of oxidized proteins by TIr3 was found
in mitochondrial proteins (Figure 5c). The ER and mitochondria are interconnected within a few nanometers at the ER–
mitochondria-tethered junction.33 A fraction of 1O2 also reaches the mitochondrial space through the outer mitochondrial
membrane, which allows passage of small molecules of less
than 5 kDa.34 Thus, we postulated that in situ generated 1O2 by
TIr3 at the ER membrane might be diffused into the mitochondrial membrane and oxidize both proximal ER and mitochondrial proteins. We also observed that photo-oxidation
reaction by TIr3 initiated mitochondrial aggregation which is
associated with mitochondrial pathway of apoptosis (Supporting Information Figure S14).35 Consequently, our imaging
studies imply that our Ir(III) complexes, representing predominantly ER-localized PDT agents, could induce protein modifications (i.e., protein–protein crosslinking and protein oxidation) at both the ER and mitochondria upon light activation.
Oxidation of mitochondrial proteins related to mitochondrial physiology by TIr3. Oxidized methionine is
known to be more hydrophilic than methionine, and O-Met
could form hydrogen bonds with other amino acid residues.36
Thus, a significant conformation change could be triggered,
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affecting endogenous protein function and interactions with
their partners.37 Among 41 oxidized mitochondrial proteins in
Group I, TRAP1 was found to be oxidized by TIr3. TRAP1 is
a mitochondrial matrix localized molecular chaperone
protein38 and serves as an oxidative sensor inducing apoptosis
when it is oxidized under oxidative stress.39 Moreover, we also
observed that PYCR1 involved in proline metabolism was
oxidized by TIr3. Mutation of PYCR1 significantly affects
mitochondrial morphology, membrane potential, and increased
apoptosis rate.40 Thus, we postulate that O-Met modification
of PYCR1 might cause a similar effect on the mitochondrial
physiology (Figure 5e). Similarly, in Group I, we found 15
other mitochondrial metabolic proteins that were related to
human disease (e.g., HIBCH, TUFM, ACAT1, AK2,
ADH7A1, CYCS, HADH, HADHA, HSD17B10, LRPPRC,
OAT, PDHA1, SLC25A3) and were severely oxidized by
TIr3, which might have altered their structures and affected
mitochondrial function. Taken together, the photo-oxidation of
proteins near the mitochondrial space by TIr3 can be utilized
to effectively prompt cell death, demonstrating the Ir(III)
complex as a promising PDT agent.
Proposed mode of action of Ir(III) complexes as PDT
agents. Higher-ordered protein-protein crosslinking as triggered by our Ir(III) complexes can further induce protein aggregation. Additionally, excessive oxidation occurs on amino
acid residues such as cysteine and methionine consequent to
noticeable ROS generation, inducing protein dysfunction by
structural change. As depicted in Figure 6, the induction of
these two pathways by our Ir(III) complexes can accelerate
cell death through a synergetic effect, suggesting their high
potential to be utilized for PDT.
CONCLUSIONS
We developed PDT agents composed of Ir(III) complexes
for cancer cells via a molecular design strategy for efficient
ROS generation that accounted for appropriate energy levels
and high emission quantum yields. TIr3 and TIr4 effectively
triggered the death of cancer cells through spatiotemporal cytotoxic activity via superior ROS generation ability (Φs = 0.95
and 0.78, respectively) localized at the ER, even under low
concentration (≤ 2 µM) and weak light energy (≤ 1 J cm-2).
Additionally, TIr3 efficiently induced cancer cell death by
two-photon irradiation. Using MS, we characterized the modes
of action for Ir(III) complexes for both protein crosslinking
and protein oxidation. In living cells, the damage was predominantly found in proteins near the ER and mitochondria with
significant association to cell death pathways. Therefore, these
Ir(III) complexes efficiently functioned as PDT agents in cancer cells. Further optimization of these iridium(III) photosensitizers could lead to rapid cell death following effective protein
disablement. Additionally, practical use of additive-free photocrosslinking through Ir(III) complexes may have applications
in other fields beyond PDT.
ASSOCIATED CONTENT
Supporting Information
Detailed procedures for the synthesis of the four Ir(III) complexes, photochemical studies, cell culturing and transfection, cell
imaging for both one-/two-photon and FLIM, MALDI-TOF-MS
analysis of in vitro BP photo-crosslinking, Western blotting for
photo-crosslinking in living cells and oxidation analysis by LTQ-
Orbitrap MS are explained in the Supporting Information. The
Supporting Information is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
M. H. Lim. E-mail: mhlim@unist.ac.kr
H.-W. Rhee. E-mail: rhee@unist.ac.kr
T.-H. Kwon. E-mail: kwon90@unist.ac.kr
Author Contributions
J.S.N., M.-G.K., J.K., and S.-Y.P. contributed equally to this
work.
Notes
ǁ
The authors declare no competing financial interest.
ACKNOWLEDGMENT
This research was supported by the Ulsan National Institute of
Science and Technology research fund (1.150117.01 to T.-H.K.
and H.-W.R and 1.140101.01 and 1.160001.01 to T.-H.K., H.W.R., and M.H.L.). J.K. acknowledges the support from the
Global Ph.D. fellowship program through the National Research
Foundation of Korea (NRF) funded by the Ministry of Education
(NRF-2015HIA2A1030823). S.-Y.P and O.H.K acknowledge the
Institute for Basic Science (IBS-R020-D1), Korea.
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Figure 1. Protein modification pathways generated by Ir(III) complexes in cellular regions and
optical imaging thereof. (a) Cyclometalated Ir(III) complexes, TIr1, TIr2, TIr3, and TIr4. (b) Schematic representation of protein modifications in the mitochondria (Mito) and endoplasmic reticulum
(ER) by photo-activation of Ir(III) complexes. There are two expected pathways for protein modification, crosslinking and oxidation. (c) Two-photon optical imaging of TIr1, TIr2, TIr3 and TIr4 (from
left to right) using confocal laser scanning microscopy. Scale bar = 10 µm.
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Figure 2. Optical and electrochemical properties of TIr1, TIr2, TIr3, and TIr4 and correlation of
their energy levels with the two singlet oxygen (1O2) excitation state energies. (a) Normalized UVvisible (closed symbol) and photoluminescence (open symbol) spectra upon excitation in the MLCT region of TIr1, TIr2, TIr3, and TIr4. Conditions: [Ir(III) complex] = 20 µM concentration. (b) Cyclic
voltammetry (CV) graph for each Ir(III) complex. Spectra were obtained with a 50 mV/s scan rate in
tetrabutylammonium hexafluorophosphate (TBAPF6-) electrolyte dissolved in acetonitrile. (c) Energy
level of each Ir(III) complex and ground state oxygen (1O2) absorption. The HOMO was measured by
the onset potential of the Ir(III) complex with a ferrocene reference (inset) using the following equation.
HOMO = − (Eonset of Ir(III) vs. Eonset of ferrocene) − 4.8 eV. The energy band gap was measured from the onset
peak of the absorption spectrum and the LUMO calculated as (= HOMO + Energy band gap). Triplet
energy levels corresponded with maximum emission peaks (dashed lines). Two excitation states of singlet oxygen (1Σg for 762 nm and 1∆g for 1268 nm) representing the absorption energy from ground state
oxygen are presented.
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Figure 3. Cell death triggered by Ir(III) complexes and real time tracking of cell morphological
alteration by two-photon activation. (a) Viability of human ovarian cancer SK-OV-3 cells upon
treatment with Ir(III) complexes, cisplatin and [Ru(bpy)3]2+ [IC50 values of TIr3 and TIr4 in the absence and presence of light 1 sun light (100 mW cm-2) for 10 s} (b, top)]. Plots of cell viability as a
function of log(concentrations of complexes) upon light treatment [1 sun light (100 mW cm-2) for 10 s]
(b, bottom). Cytotoxicity was measured by the MTT assay after 24 h incubation of SK-OV-3 cells with
and without light exposure. The cell viability (%) was calculated compared to cells treated with equivalent amounts of dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) only (1%, v/v). Values represent the mean of three independent experiments. (c) Change of cell morphologies upon treatment with
TIr3 with photo-activation. Morphological changes of TIr3-added (top; [TIr3] = 20 µM) and vehicletreated (bottom) SK-OV-3 cells after two-photon photo-irradiation (λ = 860 nm for 30 s). Scale bar = 10
µm.
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Figure 4. Two different ROS assays and photo-crosslinking analysis in vitro (biotin phenol (BP))
and in HEK293T cell culture. (a) Singlet oxygen (1O2) assay using the absorbance attenuation (∆A =
As – Af) of 9, 10-anthracenediyl-bis(methylene)dimalonic acid (ABDA) under light exposure. The slope
corresponds to the absolute amount of 1O2. (b) Superoxide anion radical (O2•–) assay indicating the enhancement in fluorescence by conversion of dihydrorhodamine 123 to rhodamine 123. (c) Dimerization
of biotin-phenol by TIr3, as monitored by MALDI-MS. Conditions: [BP] = 500 µM; [TIr3] = 1 mM;
100 mW cm−2 light; irradiation time = 0, 300, and 600 s. (d) Analysis of photo-crosslinking in living
cells by metal complexes (top) and Ponceau S staining for identifying protein loading quantities (bottom). Conditions: [metal complex] = 2 µM; [APS] = 50 µM; 100 mW cm−2 light; irradiation time = 60
s.
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Figure 5. Identification of the oxidative damage to the proteome induced by TIr3 upon photoactivation through MS/MS detection of methionine oxidation (+ 16 Da) and primary localization
of TIr3 cell organelle labelling. (a) Schematic representation of the experimental process for identification of oxidized proteins by MS. (b) Proteomic profiling of proteins oxidized on methionine residues
(Group I, additionally oxidized species by TIr3; Group II & Group III, oxidized species by endogenously generated ROS). (c) Distribution ratio of oxidized proteins for each group. TIr3 accelerates the oxidation of proteins localized in the mitochondria and ER. (d) Confocal microscopy imaging (λem = 560
nm) of HeLa cells labelled with TIr3 (10 µM, 0.5 h) and its co-localization with ER-Tracker or MitoTom20. 2D histogram graph indicates degree of co-localization, implying Pearson’s coefficient (R).
Note that Mito-Tom20 was obtained in AF647 window, thereby green is false color. Scale bar = 10 µm.
(e) Crystal structure of representative oxidized proteins. Both proteins play a role in mitochondrial function.
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Figure 6. Proposed mode of action of Ir(III) complexes for photodynamic therapy (PDT). (a) Photo-crosslinking pathway through a catalytic cycle initiated by one-electron process from the Ir(III) complex to O2, which can result in cell death via protein aggregation. (b) Protein oxidation pathway from
1
O2 by triplet-triplet energy transfer of the excited state of the Ir(III) complex. Over-expression of oxi-
dized proteins also induces cell death.
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λAbs
a
TIr1
(nm)
354
(nm)
531
TIr2
374
TIr3
Name
a
b
HOMO
(eV)
-5.73
(-5.81)
ET
(eV)
-3.39
LUMO
(eV)
-2.62
590
-5.64
(-5.56)
-3.54
432
562
TIr4
437
592
-5.62
(-5.61)
-5.57
(-5.60)
[Ru(bpy)3]2+
453
611
-
λEmiss
Lifetime
(ns)
374
e
kr
(× 105 s-1)
12.3
knr
(× 105 s-1)
1.44
0.37
± 0.01
72
1.53
13.7
0.53
± 0.05
0.11
± 0.01
0.95
± 0.04
0.78
± 0.04
712
7.44
0.66
702
1.57
1.27
0.063
0.18
398
1.58
2.35
c
Φp
Φs
0.46
± 0.02
0.32
± 0.04
-2.74
0.011
± 0.001
-3.41
-3.08
-3.48
-3.06
-
-
d
e
Table 1. All information of the photophysical and electrochemical properties for each compound
a
λAbs and λEmiss were measured in aqueous media containing 1% DMSO (v/v). bHOMO energy levels were obtained by cyclic voltammetry
under both organic (Acetonitrile, MeCN) and aqueous (DI water containing 1% v/v DMSO) condition. Tetrabutylammonium hexfluorophosphate (TBAPF6-) and potassium chloride (KCl) were utilized as supporting electrolyte for organic and aqueous condition, respectively.
The HOMO values in parentheses indicate HOMO energy level in aqueous solution. cQuantum yield were measured in aqueous media
containing 1% DMSO (v/v) with the reference of FIrpic for TIr1, (ppy)2Irpic for TIr2, and (2pq)2Ir(ppy)+PF6– for both TIr3 and TIr4.
Standard deviation was obtained with three times measurement. Quantum yield of [Ru(bpy)3]2+ is taken from the reference 41.41 dTimecorrelated single photon counting (TCSPC) measurement confirmed excited state lifetime of TIr1, TIr2, TIr3, TIr4 and [Ru(bpy)3]2+.
e
Radiative decay constant (kr) and non-radiative decay constant (knr) was calculated with previously reported equation.42
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Page 14 of 14
Table of Contents
Remarkable photodynamic therapy potency from photo-activation of Ir(III) complexes was clarified by
superior ROS generation, following protein oxidation of crucial protein in physiological condition and
photo-crosslinking that induces protein aggregation.
14 Environment
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