Organoiridium Photosensitizers Induce Specific Oxidative Attack on Proteins within Cancer Cells

Article abstract of DOI:10.1002/anie.201709082

Strongly luminescent iridium(III) complexes, [Ir(C,N)₂(S,S)]⁺ (1) and [Ir(C,N)₂(O,O)] (2), containing C,N (phenylquinoline), O,O (diketonate), or S,S (dithione) chelating ligands, have been characterized by X-ray crystallo

Full text of DOI:10.1002/anie.201709082

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International Edition: DOI: 10.1002/anie.201709082
German Edition: DOI: 10.1002/ange.201709082
Cancer Very Important Paper
Organoiridium Photosensitizers Induce Specific Oxidative Attack on
Proteins within Cancer Cells
Pingyu Zhang+, Cookson K. C. Chiu+, Huaiyi Huang, Yuko P. Y. Lam, Abraha Habtemariam,
Thomas Malcomson, Martin J. Paterson, Guy J. Clarkson, Peter B. OꢀConnor,* Hui Chao,* and
Peter J. Sadler*
Abstract: Strongly luminescent iridium(III) complexes, [Ir-
(C,N)2(S,S)]+ (1) and [Ir(C,N)2(O,O)] (2), containing C,N
(phenylquinoline), O,O (diketonate), or S,S (dithione) chelat-
ing ligands, have been characterized by X-ray crystallography
and DFT calculations. Their long phosphorescence lifetimes in
living cancer cells give rise to high quantum yields for the
generation of 1O2, with large 2-photon absorption cross-
sections. 2 is nontoxic to cells, but potently cytotoxic to
cancer cells upon brief irradiation with low doses of visible
light, and potent at sub-micromolar doses towards 3D multi-
cellular tumor spheroids with 2-photon red light. Photoactiva-
tion causes oxidative damage to specific histidine residues in
the key proteins in aldose reductase and heat-shock protein-70
within living cancer cells. The oxidative stress induced by
iridium photosensitizers during photoactivation can increase
the levels of enzymes involved in the glycolytic pathway.
can also be directed to a range of other tissues using, for
example, fiber optics. Examples of clinical photosensitizers
include hematoporphyrin derivatives (Photofrin)[2] and ami-
nolevulinic acid (ALA, a porphyrin precursor).[3] Two metal
complexes are currently undergoing PDT clinical trials:
TLD1433, an octahedral tris-N,N-chelated RuII complex,[4]
and WST 11, a square-planar PdII bacteriochlorophyll deriv-
ative.[5] Such complexes, together with octahedral IrIII com-
plexes, are often luminescent and might be useful in
theranostic procedures in which optical imaging is used to
monitor the uptake of the sensitizer before subsequent
irradiation then kills the cancer cells.[6]
A key advantage for metal complexes is the heavy-atom
effect, which favors fast singlet-to-triplet intersystem crossing
(ISC), thus giving longer excited-state lifetimes[7] and higher
1
yields of O2 and/or other so-called reactive oxygen species
(ROS). The use of red and near IR light (l = 600–1100 nm)
also enhances the clinical application of PDT because of its
low energy compared to either UV or visible light.[1d] Singlet
oxygen is a dominant mediator of photocytotoxic effects, but
is short-lived (< 200 ns in vitro).[8] Consequently, the 1O2
diffusion distance is short ( ꢀ 1 mm),[9] thus limiting cytotoxic
damage to the immediate subcellular vicinity of the photo-
sensitizer. Proteins are susceptible to oxidation by photo-
S
elective activation of nontoxic photosensitizers in cancer
cells by spatially-directed light is an attractive regimen for
therapy because of the minimal damage to normal cells,
especially if the sensitizer is preferentially taken up by cancer
cells.[1] Indeed, this procedure has been widely used in the last
40 years, largely as photodynamic therapy (PDT) in which
absorption of light by a photosensitizer (e.g., a porphyrin)
promotes formation of a triplet state which can polarize the
1
[10]
chemically derived O2, but there are few previous inves-
3
1
1
spins of the ground-state O2 to generate highly toxic O2.
Surface cancers are particularly accessible to PDT, but light
tigations of protein attack by O2 in living cells.[6d] If photo-
senstiizers can be distinguished by differences in their
oxidative activity towards target sites, then a more rational
basis for their use might emerge.
[*] Dr. P. Zhang,[+] C. K. C. Chiu,[+] Dr. H. Huang, Y. P. Y. Lam,
Dr. A. Habtemariam, Dr. G. J. Clarkson, Prof. P. B. O’Connor,
Prof. P. J. Sadler
Here we compare the photophysical, chemical, and
biological properties of two highly luminescent organoiridium
phenylquinoline (C,N) complexes, [Ir(C,N)2(S,S)]+ (1) and
[Ir(C,N)2(O,O)] (2). Their absorption and emission properties
depend on the chelated dithione (S,S) and diketonate (O,O)
ligands, as well as their effectiveness in killing cancer cells in
both two-dimensional (2D) and three-dimensional (3D) cell
cultures, and the ability to generate 1O2 in cancer cells.
Moreover, the photoactivation in cancer cells leads to specific
oxidative damage in some key cellular proteins.
Department of Chemistry, University of Warwick
Coventry, CV4 7AL (UK)
E-mail: p.oconnor@warwick.ac.uk
p.j.sadler@warwick.ac.uk
Dr. H. Huang, Prof. H. Chao
School of Chemistry, Sun Yat-Sen University
Guangzhou 510275 (P. R. China)
E-mail: ceschh@mail.sysu.edu.cn
Dr. P. Zhang[+]
The synthesis and characterization of the positively
charged IrIII dithione complex 1 and neutral diketonate
complex 2 (Figure 1a) are described in the the Supporting
Information. They were isolated as their thermodynamically
more-stable isomers with carbon atoms from the phenyl-
quinolene trans to the S atoms of the dithione in 1 and trans to
the O atoms of the diketonate in 2. X-ray crystallography
shows that 2 has a distorted octahedral structure (see
Figure S1 and Tables S1 and S2 in the Supporting Informa-
College of Chemistry and Environmental Engineering, Shenzhen
University, Shenzhen 518060 (P. R. China)
T. Malcomson, Prof. M. J. Paterson
Institute of Chemical Sciences, Heriot-Watt University
Edinburgh EH4 4AS (UK)
[+] These authors contributed equally to this work.
Supporting information and the ORCID identification number(s) for
the author(s) of this article can be found under:
https://doi.org/10.1002/anie.201709082.
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the CC isomer. This excitation is to a higher lying singlet state
(S7), which is dark to one-photon absorption (OPA). The two-
photon absorbing state is of mixed character, with both d-d, as
well as significant MLCT features (see Figure S8). The TPA
maximum is shifted to longer wavelengths for the higher
energy isomers.
Phosphorescence quantum efficiencies (Fem 0.069–0.097)
of the complexes in phosphate-buffered saline (PBS) were
higher under N2 than in air (0.032–0.049; see Table S7).
Phosphorescence lifetimes (t) of the excited states were much
shorter in the presence of O2 (241 ns for 1 and 59 ns for 2)
than in the absence of O2 (389 ns for 1, 109 ns for 2) in PBS
(Figure S9). These results suggest that ground-state 3O2
interacts with the triplet excited states of these complexes.
The lifetimes of 1 and 2 in living A549 human lung cancer
cells were determined to be 404 Æ 23 and 1136 Æ 72 ns,
respectively, by using confocal and phosphorescence lifetime
imaging microscopy (PLIM; Figure 1c,d). These lifetimes,
especially that of 2, are about 19 times longer than for
aqueous solutions (in air), thus suggesting that the complexes
reside in a more hydrophobic and hypoxic environment in
living cells, factors known to enhance the phosphorescence
lifetimes of photosensitizers.[12]
Figure 1. a) Structures of the complexes 1 and 2. b) Absorption and
emission spectra of 1 and 2 in PBS solution (with 2% DMSO),
lex =458 nm. c) PLIM images of living A549 lung cancer cells. d) Life-
times of 1 and 2 in living A549 cells (lex =458 nm, f=0.5 MHz).
À
tion) with Ir O bond lengths of 2.13 to 2.15 ꢀ and an O-Ir-O
twist angle of 87.188. Crystals of 1 could not be obtained, but
DFT calculations gave Ir-S bond lengths of 2.58 and 2.56 ꢀ
for the most stable CC isomer (see Table S3). In addition,
they were highly stable in the cell-culture medium (RPMI-
1640) for 48 hours (see Figure S2).
1O2 generation by 1 and 2 under l = 465 nm (blue) light
irradiation was detected by electron paramagnetic resonance
(EPR) spectroscopy using 2,2,6,6-tetramethylpiperidine
(TEMP) as a spin-trap. The characteristic triplet-of-triplets
for the 2,2,6,6-tetramethylpiperidine-1-oxyl radical was
observed under irradiation. An 1O2 signal was neither
observed in the dark nor in control samples under irradiation
(see Figure S10a). The quantum yields for 1O2 generation
[F(1O2)] by 1 and 2 upon irradiation with l = 465 nm light
were determined as 0.73 and 0.81, respectively (see Table S7).
These values are much higher than those for the well-known
[Ru(bpy)3]2+ [F(1O2) = 0.22].[1b]
The complex 2 has an MLCT absorption at longer
wavelength (l = 475 nm) than that of 1 (l = 445 nm), and
the deep-red phosphorescence of 2 (lmax = 620 nm) is shifted
to longer wavelength compared to that of 1 (lmax = 596 nm;
Figure 1b). TD-DFT calculations (Figures S3 and S4 and
Tables S5 and S6) qualitatively reproduce electronic excita-
tions for both complexes although the primary MLCT
absorption band of 2, compared to 1, is around 0.1 eV
higher, while the phosphorescence emission of 2 is around
0.15 eV higher in energy. The MLCT absorption is blue-
shifted on going from the CC to CN to NN isomers of both
1 and 2. For all three isomers, the excitation character for the
first bright MLCT state is from a d-character orbital to a p*
orbital localized on the phenylquinoline ligand (see Fig-
ure S5). All three isomers of 1 support a bound six-coordinate
triplet state (see Table S4). The stability of these triplets
follows that of the lowest singlet states: CC as the most stable,
CN 6.07 kJmolÀ1 higher, and NN 37.92 kJmolÀ1 higher.
The two-photon luminescence properties of 1 and 2 were
investigated by determining the cross-section d (see Fig-
ure S6). The dithione complex 1 exhibited a slightly stronger
To demonstrate that these IrIII complexes can produce
cellular 1O2 after irradiation with l = 465 nm light or l =
750 nm laser, 2D A549 monolayer lung cancer cells and 3D
A549 tumor spheroids were incubated with the IrIII complexes
and the fluorescence probe 2,7-dichlorodihydro-fluorescein
diacetate (DCFH-DA). Cells treated with only the DCFH-
DA control or the iridium complexes in the dark showed no
enhancement of fluorescence. In contrast, a significant
increase in fluorescence from DCFH-DA was observed
following light irradiation of the cells treated with either
1 or 2 (see Figures S10 and S11). These findings suggest that
1
1 and 2 generate O2 efficiently in cancer cells upon light
irradiation.
PDT in cell experiments was first tested by incubating
monolayer cancer cells with the compounds at various
concentrations for 2 hours. There was no loss of cell viability
after irradiation in the absence of Ir complexes (control +
irradiation; see Figure S12). In the dark, 2 was nontoxic to
both A549 lung cancer and MRC-5 normal lung fibroblasts
(IC50 > 100 mm), while 1 showed moderate toxicity to A549
cells (IC50 = 21.2 mm; see Table S8). The potency of both 1 and
2 towards cancer cells increased markedly upon irradiation,
notably sub-micromolar for A549 cells. The phototoxicity
index (PI) of 2 towards to A549 cancer cells is greater than
two-photon absorption (TPA) at l = 750 nm (d750nm
=
115 GM; 1 GM = 1 ꢁ 10À50 cm4 sÀ1 photonÀ1) relative to that
of the diketonate complex 2 (d750nm = 70 GM). The d values
are encouraging for the possible use of 1 and 2 in two-photon
photodynamic therapy, since red light penetrates more deeply
into tissues than light with shorter wavelengths.[11] The two-
photon excitation process was confirmed by its power
dependence (see Figure S7). A theoretical investigation of
TPA by 1 using a quasi-parity conserving, three-state model
indicated a very large TPA cross-section around 740 nm for
2
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333. Under the same experimental conditions, 5-ALA and
met these requirements and were identified. Two-tail t-tests
cisplatin displayed much lower phototoxicity (IC50 > 100 mm).
The 2-photon (2P) photocytotoxicities of 1 and 2 towards
3D multicellular tumor spheroids (MCTSs) were further
investigated. Untreated MCTSs were unaffected by 1P and 2P
irradiation (see Figure S12). The complex 2 showed no
toxicity towards A549 MCTSs in the dark, and also no
toxicity towards MRC-5 MCTSs in either the dark or upon
irradiation. However, the IC50 values for 2 towards A549
spheroids upon 1P and 2P irradiation were very low, 1.0 mm
and 0.23 mm, respectively (see Table S8). Interestingly, 2P
photocytotoxicity is higher than 1P photocytotoxicity.
Although 1 showed good phototoxicity to A549 spheroids, it
was not so selective, thus exhibiting some phototoxicity
towards the MRC-5 spheroids.
The cellular uptake of either 1 or 2 in 3D A549 spheroids
was further characterized by 2-photon confocal laser scanning
microscopy. Strong red luminescence was observed on the
surface of spheroids to a depth of about 130 mm with l =
458 nm excitation and down to about 212 mm with l = 750 nm
excitation. The luminescence images were captured every
16.3 mm along the z-axis. The spheroids exhibited much
stronger luminescence in deeper layers of cells with 2P
excitation, thus indicating deeper penetration of the 2P light
(Figure 2; and Figures S13 and S14).
ensured the consistency of the reference lysozyme peptide
(FESNFNTQATNR, 714.8365 m/z) throughout all samples
(see Table S9). All p values were calculated to be larger than
0.05 (i.e., quantities of the reference lysozyme peptide were
statistically similar, and consistent throughout all data sets).
Hence, the oxidized peptide/lysozyme peptide area ratios
were calculated using t-tests to analyze the significance of
changes in quantities of the oxidized peptides under different
conditions. The levels of five oxidized peptides (detected
three times or more out of the five replicates) were
significantly different (see Table S10). These peptides were
quantified against the standard lysozyme peptide as described
in the Supporting Information. Two unique oxidized peptides
have p values of less than 0.05 (significantly different; see
Table S10 and Figure 3) and were upregulated. The level of
Figure 3. a) Structure of Hsp 70 (PDB:3ATV),[14] with the oxidized
peptide Ala329-Arg342 shown in color (2-oxo-His332, yellow). b) LC
MS/MS (CAD) of the oxidized peptide from HSP70. c) Structure of
aldose reductase (PDB:1US0),[15] with the oxidized peptide Tyr178-
Lys195 shown in color (2-oxo-His188, yellow). d) LC MS/MS (CAD) of
the oxidized peptide from aldose reductase. Fragments with red labels
indicate the presence of oxidation, and an asterisk indicates alkylated
Cys.
Figure 2. Phosphorescence imaging of A549 spheroids. The spheroids
were incubated with 2 (10 mm) for 2 h. a) Comparison of brightfield,
one-photon (lex =458 nm), and two-photon (lex =750 nm) excitation,
lem =620Æ30 nm. 1P (b) and 2P (c) 3D Z-stack images were taken
every 16.3 mm from the top to bottom of the spheroids. Images were
taken under a 10ꢀ objective. Scale bar: 300 mm.
oxidized peptide Ala329-Arg442, AQIHDIVLVGGSTR (m/z
741.4156; Figure 3a; see Table S11), from the 70 kDa heat-
shock protein (Hsp 70), increases by 5.8-fold for drug-treated
cells upon irradiation compared to the drug-treated cells in
the dark. Specific oxidation of His332 to 2-oxo-His332 was
identified by LC-FT-ICR MS/MS. The second oxidized
peptide, Tyr178-Lys195, from aldose reductase, YKPAVN-
QIECHPYLTQEK (m/z 745.3780; with alkylated Cys, Fig-
ure 3c; see Table S12) contained 2-oxo-His188, which
increased by 3.0-fold for drug-treated cells upon irradiation
as compared to that in the dark (Figure 3; see Table S10).
These data appear to be the first report of the formation of 2-
oxo-His[13] after treatment of cancer cells with organometallic
photosensitizers.
A549 cancer cells (ca. 1 ꢁ 109) were treated with 2 (10 mm,
2 h) either in the dark or with l = 465 nm light irradiation.
Proteins from these cells were collected and digested as
described in the Supporting Information. LC-MS/MS data
were searched for oxidative modifications of the side chains of
cysteine, histidine, tyrosine, phenylalanine, methionine, and
tryptophan, as well as asparagine and glutamine deamidation
and serine, threonine, and tyrosine phosphorylation (see the
Supporting Information). With 1% false discovery rate
(FDR) against the database, only proteins which presented
three times or more (in triplicates) under all three conditions
(control, drug-treated with no irradiation, drug-treated with
irradiation) were quantified. 212 proteins and 1500 peptides
Pathway analysis was carried out to investigate the overall
effects induced by 2 on cell metabolism (for methodology,[16]
see the Supporting Information). The most significant result
identified nine unique proteins along the glycolysis pathway
(see Table S13 and Figure S15). The levels of these proteins,
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which are all involved in the conversion of glucose to
pyruvate, increased by factors of about 2.1–5.3-fold on
irradiation of A549 cancer cells treated with 2, with the
highest increase for fructose-bisphosphate aldolase. Cancer
cells have defective mitochondria and increase their rate of
glycolysis as a source ATP and energy to compensate for this
mitochondrial effect. Mitochondria, where oxygen is reduced
to water, are also a major source of ROS in cells.[17] During
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1
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screening against 2D and 3D (spheroid) cancer cell models. In
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Acknowledgements
We thank the EPSRC (grant no. EP/G006792 for P.J.S.,
platform grant EP/P001459/1 for M.J.P.), the NSFC (Nos.
21471164 and 21525105), and the 973 program (No.
2015CB856301) for H.C., the Royal Society Newton Interna-
tional Fellowships for P.Z. and H.H., Dr C. J. Wedge for
assistance with EPR spectrometry, Dr C. A. Wootton with FT-
ICR MS, Mr I. Hands-Portman with confocal microscopy, and
Mr Rod Wesson for constructing the LED arrays.
Conflict of interest
The authors declare no conflict of interest.
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Keywords: bioinorganic · cancer · cells · iridium ·
photosensitizers
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Manuscript received: September 2, 2017
Version of record online: && &&, &&&&
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

Angewandte
Communications
Chemie
Communications
Cancer
In for a shock: A highly luminescent
organoiridium complex generates O2
1
P. Zhang, C. K. C. Chiu, H. Huang,
Y. P. Y. Lam, A. Habtemariam,
T. Malcomson, M. J. Paterson,
G. J. Clarkson, P. B. O’Connor,*
efficiently and oxidizes specific residues
of heat-shock protein-70 and aldose
reductase within cancer cells. The oxida-
tive stress induced by iridium photosen-
sitizers during photoactivation can
increase the levels of the enzymes
involved in the glycolytic pathway.
H. Chao,* P. J. Sadler*
&&&& — &&&&
Organoiridium Photosensitizers Induce
Specific Oxidative Attack on Proteins
within Cancer Cells
6
www.angewandte.org
ꢀ 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2017, 56, 1 – 6
These are not the final page numbers!