A Multi-action and Multi-target RuII–PtIV Conjugate Combining Cancer-Activated Chemotherapy and Photodynamic Therapy to Overcome Drug Resistant Cancers

Article abstract of DOI:10.1002/anie.201916400

Pt^II complexes are commonly used to treat cancer. To reduce their side effects and improve their pharmacological properties, Pt^IV complexes are being developed as prodrug candidates that are activated by reduction in cancer cells. Concomitantly, Ru^II polypyridine complexes have gained much attention as photosensitizers for use in photodynamic therapy due to their attractive characteristics. In this article, a novel Pt^IV–Ru^II conjugate, which combines cancer activated chemotherapy with PDT, is presented. Upon entering the cancer cell, the Pt^IV centre is reduced to Pt^II and the axial ligands including the Ru^II complex and phenylbutyrate are released. As each component has its individual targets, the conjugate exerts a multi-target and multi-action effect with (photo-)cytotoxicity values upon irradiation up to 595 nm in the low nanomolar range in various (drug resistant) 2D monolayer cancer cells and 3D multicellular tumour spheroids.

Full text of DOI:10.1002/anie.201916400

A Journal of the Gesellschaft Deutscher Chemiker
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Accepted Article
Title: A Multiaction and Multitarget Ru(II)-Pt(IV) Conjugate Combining
Cancer Activated Chemotherapy and Photodynamic Therapy to
Overcome Drug Resistant Cancers
Authors: Johannes Karges, Thirumal Yempala, Mickaël Tharaud, Dan
Gibson, and Gilles Gasser
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201916400
Angew. Chem. 10.1002/ange.201916400
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Angewandte Chemie International Edition
10.1002/anie.201916400
RESEARCH ARTICLE
A Multiaction and Multitarget Ru(II)-Pt(IV) Conjugate Combining
Cancer Activated Chemotherapy and Photodynamic Therapy to
Overcome Drug Resistant Cancers
[
a]
[b]
[c]
[b]
[a]
Johannes Karges, Thirumal Yempala, Mickaël Tharaud, Dan Gibson,* and Gilles Gasser*
[
2]
Abstract: Cancer has emerged as one of the deadliest diseases
worldwide. Pt(II) complexes are commonly used to treat this condition.
To reduce their side effects and improve their pharmacological
properties, Pt(IV) complexes are being developed as prodrug
candidates that are activated by reduction in cancer cells.
Concomitantly, photodynamic therapy (PDT) has received increasing
attention over the last years. Among other compounds studied as
photosensitizers (PSs), Ru(II) polypyridine complexes have gained
much attention over the recent years due to their attractive
characteristics. In this article, the first example of a novel Pt(IV)-Ru(II)
conjugate, which combines cancer activated chemotherapy with
photodynamic therapy, is presented. Upon entering the cancer cell,
the Pt(IV) centre is reduced to Pt(II) and the axial ligands including the
Ru(II) complex and phenylbutyrate are released. As each component
has its individual target, the conjugate exerts a multitarget and
multiaction effect with (photo-)cytotoxicity values upon irradiation up
to clinically relevant 595 nm in the low nanomolar range in various
molecules such as histone deacetylase inhibitors (Vorinostat) ,
[
3]
retinoid receptor regulators (vitamin
D
derivatives)
or
microtubule network disturbing compounds (e.g., paclitaxel,
vinblastine)[4] to generate a drug mixture with multiple targets and
actions. Despite improvements in the treatment outcomes, the
application of these drug mixtures is limited as the different
components may reach the target after different circulation times
leading to non-ideal drug doses.[ To overcome these drawbacks,
research efforts are devoted towards the development of novel
Pt(IV) based compounds, which can be activated on site. As the
compound enters the cell, the metal centre can be reduced from
Pt(IV) to Pt(II), releasing its axial ligands. To improve the
efficiency of the treatment, the axial ligands themselves can be
biologically active molecules such as targeting moieties, carbon
nanotubes, protein-binding moieties or receptor-binding
peptides.[6] Recently, the use of phenylbutyrate (PhB), which is a
histone deacetylase inhibitor that de-condenses chromatin to
improve the interaction of cisplatin with DNA, was
5]
(
drug resistant) 2D monolayer cancer cells and 3D multicellular
tumour spheroids.
[
7]
demonstrated. As a consequence, Pt(IV) prodrugs with PhB as
an axial ligand were shown to have a highly improved therapeutic
profile, causing DNA damage and epigenetic effects.[
8]
Introduction
Complementary to chemotherapy, cancer treatment using
photodynamic therapy (PDT) has received increasing attention
over the recent years. In PDT, a photosensitizer (PS) is either
locally or systemically injected. Upon excitation at a specific
wavelength, the PS acts as a photocatalyst to generate reactive
oxygen species (ROS) and ultimately trigger cell death.[9] Worthy
of note, Sadler and co-workers have recently reported the use of
photoactivatable Pt(IV) complexes which can act by non-classical
mechanism.[ Among the investigated classes of compounds,
metal complexes[ and especially Ru(II) polypyridine complexes
have received increasing attention due to their attractive chemical
and photophysical properties including high water solubility, high
ROS production, chemical stability and photostability.[12] Despite
recent research efforts, the majority of studied Ru(II) polypyridine
complexes are excited using blue or UV-A light.[13] Due to light
scattering effects and absorption by biological compartments, the
light penetration depth in the tissue is poor at these wavelengths,
limiting the treatment of deep seated or large tumors.[14] To tackle
this issue, some of us have recently reported the DFT-guided
design of Ru(II) polypyridine complexes with a strong absorption
red shift towards the biological spectral window (600-900 nm). In
To date, the cure and treatment of cancer remains a major
challenge
in
modern
medicine.
Cisplatin
(cis-
diamminedichloridoplatinum(II)) and its platinum based
derivatives oxaliplatin and carboplatin are the clinically most
frequently used anticancer drugs worldwide. Despite their
enormous clinical success, the application of these compounds is
limited due to severe side effects (e.g., kidney damage, nausea,
vomiting and bone marrow suppression), low cancer cell
selectivity and, more worryingly, an increasing number of
platinum resistant tumours.[1] A known concept to improve the
therapeutic outcome of chemotherapy is to administer cisplatin
and its derivatives in combination with other biologically active
10]
11]
[
a]
Dr. J. Karges, Dr. G. Gasser
Chimie ParisTech, PSL University, CNRS, Institute of Chemistry for
Life and Health Sciences, Laboratory for Inorganic Chemical Biology,
75005 Paris, France.
gilles.gasser@chimieparistech.psl.eu; www.gassergroup.com
[
b]
c]
Dr. T. Yempala, Prof. D. Gibson
Institute for Drug Research, School of Pharmacy, The Hebrew
University of Jerusalem, Jerusalem, Israel, 91120,
dang@ekmd.huji.ac.il
this
pursuit,
the
complex
[Ru(4,7-diphenyl-1,10-
2+
phenanthroline)
2
(4,4′-dimethyl-2,2′-bipyridine)] , which showed a
phototoxic effect from 480 nm up to clinically relevant 595 nm,
was unveiled.[
[
M. Tharaud
15]
Université de Paris, Institut de Physique du Globe de Paris, CNRS,
7
5005 Paris, France.
Herein, a novel Ru(II)-Pt(IV) conjugate (Ru-Pt), which combines
cancer activated chemotherapy and PDT by acting on multiple
targets inside the cancer cell with several biological mechanism
to overcome drug resistances, is described. Upon entering cancer
Supporting information for this article is given via a link at the end of
the document.
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Angewandte Chemie International Edition
10.1002/anie.201916400
RESEARCH ARTICLE
2+
cells, the Pt(IV) metal centre is reduced to Pt(II) releasing PhB
and the Ru(II) polypyridine complex. The Ru(II) complex was
found to selectively accumulate in the Golgi apparatus where it is
O
1
N
Ru
N
2
able to generate singlet oxygen ( O ) upon irradiation at various
O
OH
N
N
N
N
Cl
Cl
NH3
NH3
Cl
Cl
NH3
NH3
wavelength ranging from 480 up to clinically relevant 595 nm.
Pt
Pt
O
H
N
O
NH2
O
O
O
Results and Discussion
Ru
Pt-PhB-OAc
Pt-PhB-OH
To ensure that the photophysical and biological properties of each
component are not influenced, the moieties were conjugated with
a long aliphatic linker. The linking unit between the Ru(II) and the
Pt(IV) complex was synthesised starting from 4,4´-dimethyl-2,2´-
bipyridine, which was asymmetrically oxidised to the carboxylic
2
+
N
N
N
N
N
Ru
N
[
16]
acid using SeO
carboxylic acid was treated with SOCl
2
.
The generated 4´-methyl-2,2´-bipyridinyl-4-
to yield the acid chloride
O
H
N
N
H
O
2
NH3
NH3
O
Cl
Pt
O
intermediate, which was used to attach tert-butyl-N-(6-
aminohexyl)carbamate by peptide bond formation.[17] The
generated 2,2´-bipyridine derivative was then coordinated to
dichlorobis(4,7-diphenyl-1,10-phenanthroline)ruthenium(II),
which was previously synthesised from 4,7-diphenyl-1,10-
Cl
O
Ru-Pt
Figure 1. Chemical structures investigated in this work. Ru was isolated as a
-
-
6
PF salt whereas Ru-Pt was isolated as a TFA salt.
3
phenanthroline and RuCl . Finally, the tert-butyloxycarbonyl
nanosecond range (Figure S11-S12) in an air saturated
environment (147-183 ns) and in degassed environment (1003-
protection group was removed using trifluoracetic acid to give the
Ru(II) polypyridine complex Ru. To facilitate the conjugation of Ru
to the Pt-PhB-OH the axial OH group of the Pt(IV) was activated
to form Pt-PhB-MSC (MSC=monosuccinimidylcarbonate), whose
1054 ns). Importantly, the presence of air had a drastic effect on
the lifetime, indicating that the excited state of the complex can
3
interact with molecular oxygen ( O
2
) to generate singlet oxygen
synthesis was prepared in a similar manner to the structurally
related derivatives.[8b] Both carboxylates (link to PhB) and
1
1
( O ). For quantification of the amount of O produced upon light
2
2
IV
[8c]
irradiation, the singlet oxygen quantum yields were determined by
two methods: 1) directly by measuring the phosphorescence
carbamate (link to Ru) detach from the Pt following reduction.
The Ru complex was then coupled with Pt-PhB-MSC in presence
of diisopropylethylamine to yield the final Ru-Pt complex. The
synthesis and characterisation of the complexes are described in
the SI (Scheme S1, Figure S1-S8).
1
1
signal of
2 2
O , 2) indirectly by capturing O with a reporter
molecule and monitoring its change by absorption
spectroscopy.[ The characteristic emission signal at 1270 nm in
19]
the infrared region using the direct method confirmed the
The photophysical properties of Ru were compared with Ru-Pt to
evaluate its potential as a PDT PS and to investigate whether the
conjugation to the Pt(IV) complex influenced them (Table S1). As
expected, the absorption and emission spectra of Ru and Ru-Pt
1
production of O
are able to produce O
2
upon light exposure. Importantly, Ru and Ru-Pt
1
2
upon irradiation at 450 and 540 nm. The
1
measurements revealed high O
2
quantum yields of 45-64 % in
3
CH CN and of 3-6 % in an aqueous solution (Table S2). Overall,
(
Figure S9-S10) showed no significant differences. Importantly,
these studies showed that Ru-Pt has attractive photophysical
properties for applications as a PDT agent and the conjugation to
the Pt(IV) centre did not significantly influence the photophysical
properties of the Ru(II) complex.
the spectra showed an absorption tail towards the biological
spectral window potentially enabling the treatment of deep-seated
and large tumours as the light penetration depth directly
correlates with the used wavelengths. Worthy of note, previous
studies have shown that PSs can show a phototoxic effect
The stability of the conjugate was then investigated as previous
studies have shown that this could be problematic for metal
-
1
-1 [12a,15]
although their extinction coefficients are below 100 M cm .
The conjugate was found to have a large Stokes shift, implying
minimal interference between excitation and emission. The
luminescence quantum yields of Ru an Ru-Pt were found to be
similar (Φem, Ru = 2.0 %, Φem, Ru-Pt = 2.1 %), which is in the same
complexes.[20] The conjugate was incubated in the dark in H
2
O at
room temperature and physiological pH and the absorption
recorded in time intervals up to 48 h (Figure S13). Additionally,
the complex was analysed by HPLC after a 48 h incubation in H O
2
(Figure S14). As no significant differences were observed, the
stability in an aqueous solution is indicated.
[
18]
range as other published Ru(II) polypyridine complexes. Both
compounds were found to have excited state lifetimes in the
After a chemical and photophysical characterisation of the
conjugate, its potential activity as a chemotherapeutic agent and
2
2
Table 1. IC50 values (μM) in the dark and upon irradiation at 480 (10 min, 3.1 J/cm ) and 595 nm (120 min, 22.5 J/cm ) for Pt-PhB-OAc, Ru and Ru-Pt in
comparison to Protoporphyrin IX (PpIX) in cancerous in human cervical carcinoma (HeLa) and non-cancerous retinal pigment epithelium (RPE-1) cells. Average
of three independent measurements.
HeLa
RPE-1
dark
480 nm
-
PI
-
dark (120
min)
595 nm
-
PI
-
dark
480 nm
-
PI
-
dark (120
min)
595 nm
-
PI
-
Pt- PhB-
OAc
18.94 ± 0.83
13.57 ± 0.28
3.26 ± 0.07
>100
-
81.49 ± 6.54
4.22 ± 0.42
4.38 ± 0.57
> 100
-
Ru
1.65 ± 0.11
0.31 ± 0.06
2.50 ± 0.13
8.2
6.13 ± 0.38
1.41 ± 0.13
81.26 ± 8.34
0.58 ± 0.16
0.13 ± 0.07
1.52 ± 0.92
10.9
10.8
53.4
0.82 ± 0.17
0.61 ± 0.29
3.84 ± 0.19
5.1
2.5 ± 0.36
2.47 ± 0.49
83.5 ± 6.71
0.62 ± 0.30
0.45 ± 0.18
1.93 ± 0.36
4.0
5.5
43.3
RuPt
PpIX
10.5
> 40.0
7.2
> 26.3
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
PS for PDT was investigated. The cytotoxicity of cisplatin, Pt-
PhB-OH, Pt-PhB-OAc, Ru and Ru-Pt towards non-cancerous
retinal pigment epithelium (RPE-1) and cancerous human cervical
carcinoma (HeLa) cells in the dark as well as upon irradiation at
various wavelengths between 480 – 595 nm was determined
(
Table 1, Table S3). In both investigated cell lines the conjugate
Ru-Pt (IC50, HeLa = 3.26 ± 0.07 μM, IC50, RPE-1 = 4.38 ± 0.57 μM)
showed a higher cytotoxicity in the dark than Ru (IC50, HeLa = 13.57
±
0.28 μM, IC50, RPE-1 = 4.22 ± 0.42 μM) or the platinum based
complexes cisplatin, Pt-PhB-OH or Pt-PhB OAc (IC50, HeLa = 5.9
18.9 μM, IC50, RPE-1 = 27.7 – 81.4 μM) alone. All Pt(IV) prodrugs
–
and importantly Ru-Pt showed a 1.3 - 4.7 times higher cytotoxicity
in the cancerous cell line in comparison to the non-cancerous cell
line. This effect was previously described in the literature and is
caused by the preferential reduction of the Pt(IV) centre to Pt(II)
in cancer cells, which are hypoxic. Strikingly, upon irradiation at
various wavelengths between 480 – 595 nm, the Ru-Pt conjugate
is able to cause a phototoxic effect. Thanks to the additional PDT
effect, the cytotoxicity improved drastically, with IC50 values in the
nanomolar range (IC50, HeLa = 0.13 – 0.39 μM, IC50, RPE-1 = 0.45 –
0
.61 μM). Overall, these results indicate the beneficial effect of
combing the properties of the Pt(II) complex as
chemotherapeutic agent and the Ru(II) complex as a PS as a
conjugate. Interestingly, Ru-Pt showed slightly higher
a
a
cytotoxicity in cancerous cells in comparison to non-cancerous
cells.
Capitalising on these preliminary results, the potential of Ru-Pt to
overcome resistance was further investigated in depth in human
ovarian carcinoma (A2780) cells and its cisplatin resistant line
Figure 2. a) Cellular uptake mechanism study of Ru-Pt (10 μM) in A2780 cells
in the presence of different inhibitors/conditions by determination of the amount
(
A2780 cis) as well as its doxorubicin resistant line (A2780 ADR).
The uptake mechanism of the Ru-Pt conjugate was studied in
A2780 cells by blocking different cellular pathways and
determining the amount of Ru and Pt inside the cells (Figure 2a)
by inductively coupled plasma mass spectrometry (ICP-MS).[21]
of Ru and Pt inside the cells by ICP-MS. Endocytic inhibition: NH
chloroquine (100 μM), metabolic inhibition: 2-Deoxy-D-glucose (50 mM) and
oligomycin (5 μM), cation transporter inhibition: Et NCl, low temperature:
4
Cl (50 mM) or
4
incubation at 4°C, control: incubation at 37°C. b) Cellular localisation of Ru and
Pt of the conjugate Ru-Pt (10 μM) in A2780 cells inside the major cellular
organelles (endoplasmic reticulum, golgi apparatus, lysosomes, mitochondria,
nucleus, whole cell) after 4 h incubation in the dark, extraction of their cellular
organelles and determination of the amount of Ru and Pt inside each organelle
by ICP-MS.
The incubation with the cationic transporter inhibitor
(tetraethylammonium chloride) did not significantly influence the
cellular uptake, suggesting that this pathway does not majorly
contribute to the internalisation of the compound. The incubation
at lower temperature (4 °C) and with metabolic inhibitors (2-
deoxy-D-glucose and oligomycin) decreased the uptake,
indicating that the internalisation is energy dependent. In contrast,
the incubation with endocytotic inhibitors (ammonium chloride or
chloroquine) drastically decreased the uptake. Overall, these
results indicate that Ru-Pt is internalised primarily by an energy
dependent endocytotic mechanism. Worthy of note, the recently
reported Ru-Pt metallacage was internalised by the same
simultaneously in different cellular organelles. As anticipated, the
vast majority of Pt was found in the nucleus. This finding is in
agreement with the in-depth investigation of the anticancer drug
cisplatin, which is targeting the nucleus and binding to DNA. On
the contrary, the majority of Ru was found in the Golgi apparatus.
Additionally, small amounts of Pt and Ru were also found in the
lysosomes and the endoplasmic reticulum. Taken together, these
results indicate that Ru-Pt is internalised through an engulfing
mechanism, in which the cell membrane draws the complex from
the outside to the inside by formation of intracellular transport
vesicles. These can then interact with the endomembrane system
consisting of the lysosomes, Golgi apparatus and endoplasmic
reticulum, where they release their content. As small amounts of
Ru and Pt were both found in the lysosomes and the endoplasmic
reticulum, it tends to demonstrate that the Ru-Pt conjugate is
majorly released in these organelles. In a further process, the
Pt(IV) centre is reduced and the Ru and Pt moieties localise in
their respective target organelles (Golgi apparatus and nucleus).
[
22]
pathway. Importantly, similar levels of Ru and Pt were detected
inside the cells indicating that the Ru-Pt is stable in the cell culture
medium and was internalised as a single moiety.
Following this, the localisation of the released Ru(II) complex and
Pt(II) complex (cisplatin) were investigated by extraction of the
major cellular organelles (nucleus, mitochondria, lysosomes,
Golgi apparatus, endoplasmic reticulum) and determination of the
amount of Ru and Pt inside of each by ICP-MS (Figure 2b). As
expected, Ru and Pt were found in different cellular compartments,
indicating that the Pt(IV) centre is reduced and the axial ligands
(Ru and PhB) released and that the conjugate is acting
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Angewandte Chemie International Edition
10.1002/anie.201916400
RESEARCH ARTICLE
2
2
Table 2. IC50 values (μM) in the dark and upon irradiation at 480 (10 min, 3.1 J/cm ) and 595 nm (60 min, 11.3 J/cm ) for cisplatin, Pt-PhB-OH, Pt- PhB OAc,
Ru and Ru-Pt in comparison to Protoporphyrin IX (PpIX) in the human ovarian carcinoma (A2780), human cisplatin resistant ovarian carcinoma (A2780 cis) and
human doxorubicin resistant ovarian carcinoma (A2780 ADR) cell lines. Average of three independent measurements.
A2780
A2780 cis
A2780 ADR
dark
480 nm
PI
595 nm
PI
dark
480 nm
PI
595 nm
PI
dark
480 nm
PI
595 nm
PI
cisplatin
4.54 ±
-
-
-
-
-
-
-
-
19.53±
1.11
-
-
-
-
-
-
-
-
8.97 ±
0.71
-
-
-
-
-
-
-
-
0.65
Pt- PhB
OAc
1.39 ±
0.22
6.17 ±
0.36
3.69 ±
0.32
Pt-PhB-
OH
1.07 ±
0.24
-
-
-
-
5.95 ±
0.37
-
-
-
-
2.56 ±
0.34
-
-
-
-
Ru
15.35
2.86 ±
0.52
5.4
4.30 ±
0.66
3.6
17.26 ±
0.90
4.67 ±
0.43
3.7
7.10 ±
0.92
2.4
18.60 ±
0.71
3.41 ±
0.42
5.5
4.47 ±
0.35
4.2
±
0.89
Ru-Pt
PpIX
0.98 ±
.22
0.08 ±
0.04
12.3
22.1
0.16 ±
0.05
6.1
1.38 ±
0.29
0.10 ±
0.03
13.8
14.4
0.19 ±
0.06
7.1
1.13 ±
0.28
0.09 ±
0.03
12.6
14.0
0.17 ±
0.04
6.6
9.6
0
>100
4.53 ±
8.19 ±
1.13
12.2
>100
6.95 ±
1.46
8.94 ±
1.53
11.2
>100
7.12 ±
1.96
10.38 ±
1.62
0.61
To study the efficiency of the conjugate against drug resistant cell
lines, the cytotoxicity of cisplatin, Pt-PhB-OH, Pt- PhB-OAc, Ru
and Ru-Pt in the dark and upon irradiation at 480 (10 min, 3.1
paraptotic inhibitors, indicating a cell death mechanism for the
phototoxic effect involving both pathways. Worthy of note, several
PDT agents were previously reported in the literature to cause cell
2
2
[24]
J/cm ) and 595 nm (60 min, 11.3 J/cm ) against A2780, A2780 cis
and A2780 ADR was investigated. All complexes (Table 2) were
found to have lower cytotoxic values in all ovarian cancer cell lines
in comparison to the HeLa and RPE-1 cell lines (Table 1). These
results show the enormous potential of the conjugate Ru-Pt with
cytotoxicity values in the dark in the low micromolar range (IC50,
dark= 0.98 – 1.38 μM). It is worth mentioning that a PS should be
ideally non-toxic in the dark. The irradiation drastically improved
the cytotoxic profile (IC50, 480nm= 0.08 – 0.10 μM, IC50, 595nm= 0.16
death by apoptosis and paraptosis pathways.
For further examination of the cell death mechanism, its
dependency on caspase 3/7 pathways was investigated using a
Caspase-Glo 3/7 assay. These caspases are known executers of
extrinsic and intrinsic apoptosis mechanism.[ The caspase 3/7
activity was measured in A2780 cells for cisplatin, Ru and Ru-Pt
in the dark and upon irradiation at 480 nm (10 min, 3.1 J/cm2)
using half of their IC50 concentration (Figure 3b). As a positive
control for caspase activity, the kinase inhibitor staurosporin was
used. Cisplatin, Ru and Ru-Pt in the dark did not significantly
increase caspase 3/7 activity, ruling out this pathway for cell death.
The result for cisplatin is in agreement with a recent study in the
25]
–
0.19 μM) with PI values between 6.1 – 13.8. While the
cytotoxicity of cisplatin, Pt-PhB-OH and Pt- PhB-OAc is lower in
the resistant cell lines A2780 cis and A2780 ADR, the Ru-Pt
conjugate overcomes the resistance even in the dark and
strikingly, by irradiation the cytotoxicity in all cell lines is further
improved by a factor of ten. Comparing the cytotoxicity of Ru and
Ru-Pt under irradiation we see that Ru-Pt is significantly more
potent than Ru in all A2780 cell lines. Taken together, these
results confirm the beneficial effect of the conjugation of the Ru(II)
and Pt(IV) complex to the conjugate Ru-Pt.
same cell line that showed that apoptosis is induced by
ERK/p53/PUMA activation.[
26]
In contrast to this, upon light
irradiation and treatment with Ru and Ru-Pt, the caspase 3/7
activity is highly elevated, suggesting that the phototoxic effect is
caused by these pathways. Worthy of note, other Ru(II)
polypyridine complexes were also found to generate a cytotoxic
effect by this pathway.[
27]
Encouraged by these promising results, the mechanism of action
of Ru-Pt was further examined by determining its cell death
pathway. For this study, the cytotoxic profile of cisplatin, Ru in
After evaluation of the (photo-)cytotoxicity on 2D monolayer cells,
the effect of Ru-Pt on 3D multicellular tumour spheroids (MCTS)
was investigated. This is of special interest as many anticancer
agents have failed translation from 2D monolayer cells to in vivo
models due to compromised drug delivery. MCTS can mimic the
pathological conditions found in solid tumors such as proliferation
gradients and its hypoxic centre and can therefore be used for the
assessment of drug delivery.[28] A2780 MCTS with a diameter of
about 400 μM were incubated with increasing concentrations of
Ru-Pt for 12 h. After this time, the cytotoxic effect in the dark and
the dark, Ru-Pt in the dark and upon irradiation at 480 nm (10 min,
2
3
.1 J/cm ) at its IC50 concentration in A2780 cells was measured
in the presence of autophagy (3-methyladenine), apoptosis (Z-
VAD-FMK), paraptosis (cycloheximide) or necrosis (necrostatin-
1) inhibitors (Figure 3a). The incubation with autophagy,
paraptosis and necrosis inhibitors did not significantly influence
the cell viability of cisplatin, Ru and Ru-Pt in the dark, indicating
that these pathways are not primarily accessed for triggering of
their cell death. In contrast, preincubation with an apoptosis
inhibitor increased the survival of the cells treated with cisplatin,
indicating that this mechanism is majorly contributing to the cell
death. This result is in agreement with a study of cell death
2
upon irradiation at 480 nm (10 min, 3.1 J/cm ) and 595 nm (120
2
min, 22.5 J/cm ) was determined upon measurement of the ATP
concentration of living cells inside the MCTS. Ru-Pt showed a
strong cytotoxic effect (IC50, dark= 7.32 ± 0.41 μM), indicating that
the conjugate is able to completely penetrate the MCTS.
Importantly, as the cytotoxicity is shifting to the nanomolar range
(IC50, 480nm= 0.49 ± 0.16 μM, IC50, 595nm= 0.68 ± 0.27 μM) upon
irradiation at 480 nm and 595 nm, the synergetic effect of the
[
23]
pathways of cisplatin in human ovarian carcinoma cells.
In
addition, elevated survival levels for the preincubation with an
apoptotic inhibitor and treatment with Ru in the dark were
detected, suggesting an apoptosis induced cell death mechanism. Pt(IV) complex as a chemotherapeutic agent and the Ru(II)
Therefore, it was not surprising to observe an apoptotic cell death
pathway for Ru-Pt in the dark. Importantly, upon irradiation of Ru-
Pt, the cell survival is highly increased with apoptotic and
complex as a PDT agent is demonstrated.
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Angewandte Chemie International Edition
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RESEARCH ARTICLE
combination of apoptosis and paraptosis pathways,
(photo-)cytotoxicity values in the low nanomolar range were
observed in various 2D monolayer cells as well as 3D MCTS.
Importantly, the conjugate showed a synergetic effect and was
able to overcome drug resistance, which is found in many
clinically treated tumors. We strongly believe that this novel
conjugate and the approach of combing a Pt(IV) complex as a
chemotherapeutic drug and a Ru(II) complex as a photosensitizer
for photodynamic therapy has a great potential for further
development.
Acknowledgements
We thank Dr. Philippe Goldner for access to state-of-the-art laser
apparatus. This work was financially supported by an ERC
Consolidator Grant PhotoMedMet to G.G. (GA 681679), has
received support under the program “Investissements d’ Avenir”
launched by the French Government and implemented by the
ANR with the reference ANR-10-IDEX-0001-02 PSL (G.G.). DG
acknowledges the support of the Israel Science Foundation (grant
1
002/18) and the Alex Grass Center for Drug Design and
synthesis.
Keywords: Bioinorganic Chemistry
• Medicinal Inorganic
Chemistry • Metals in Medicine • Photodynamic Therapy
[1]
a) A.-M. Florea, D. Büsselberg, Cancers 2011, 3, 1351-
371; b) C. Cullinane, G. B. Deacon, P. R. Drago, A. P.
1
Erven, P. C. Junk, J. Luu, G. Meyer, S. Schmitz, I. Ott,
J. Schur, L. K. Webster, A. Klein, Dalton Trans. 2018,
Figure 3. Cell death mechanism study in A2780 cells in the dark or upon
irradiation at 480 nm (10 min, 3.1 J/cm ) a) upon treatment with the IC50 value
2
4
7, 1918-1932; c) Z. Wang, Z. Deng, G. Zhu, Dalton
of the corresponding compound and in the presence of different inhibitors by
determination of the cell viability. Autophagy inhibitor: 3-Methyladenine (100
μM), apoptosis inhibitor: Z-VAD-FMK (20 μM), paraptosis inihibitor:
Cycloheximide (0.1 μM), necrosis inihibitor: Necrostatin-1 (60 μM). b) upon
treatment with a concentration of half of the IC50 value of the corresponding
compound by determination of the caspase 3/7 activity.
Trans. 2019, 48, 2536-2544; d) M. Hanif, C. G.
Hartinger, Future Med. Chem. 2018, 10, 615-617; e) C.
G. Hartinger, in Advances in Bioorganometallic
Chemistry (Eds.: T. Hirao, T. Moriuchi), Elsevier, 2019,
1
57-172; f) D. Cirri, M. G. Fabbrini, A. Pratesi, L. Ciofi,
L. Massai, T. Marzo, L. Messori, BioMetals 2019, 32,
13-817.
8
[
2]
C.-H. Pan, Y.-F. Chang, M.-S. Lee, B. C. Wen, J.-C. Ko,
S.-K. Liang, M.-C. Liang, BMC Cancer 2016, 16, 857.
M. Milczarek, S. ROSIŃSKA, M. Psurski, M.
Maciejewska, A. Kutner, J. Wietrzyk, Anticancer Res.
2013, 33, 433-444.
Conclusion
[3]
In summary, in this article, a novel conjugate (Ru-Pt) combining
a Pt(IV) complex as a chemotherapy drug and a Ru(II) complex
as a photosensitiser for long wavelength photodynamic therapy,
which targets several cellular organelles and acts by various
cytotoxic mechanism, is reported. The conjugate is able to enter
cancerous cells by an energy depended endocytosis mechanism.
While being stable in an aqueous solution, the Pt(IV) centre is
reduced inside cancerous cells to Pt(II) and releases its axial
ligands. Phenylbutyrate acts as a histone deacetylase inhibitor
and can de-condense the chromatin, improving the binding of the
Pt(II) complex (cisplatin) to DNA. The Ru(II) complex was found
to accumulate at the Golgi apparatus. Next to the cytotoxic effect
exerted by the anticancer properties of cisplatin, the Ru(II)
complex can act as a photosensitizer to catalytically generate
singlet oxygen from 480 up to clinically relevant 595 nm. Thanks
to the multitargeting and multiaction mechanisms based on a
[
4]
5]
Y. H. Kim, S. W. Shin, B. S. Kim, J. H. Kim, J. G. Kim,
Y. J. Mok, C. S. Kim, H. S. Rhyu, J. H. Hyun, J. S. Kim,
Cancer 1999, 85, 295-301.
[
a) S. Dasari, P. Bernard Tchounwou, Eur. J. Pharmacol.
2
014, 740, 364-378; b) E. Boros, P. J. Dyson, G.
Gasser, 2020 Chem 6, 41-60; c) A. Sharma, J. F.
Arambula, S. Koo, R. Kumar, H. Singh, J. L. Sessler, J.
S. Kim, Chem. Soc. Rev. 2019, 48, 771-813.
[6]
a) T. C. Johnstone, K. Suntharalingam, S. J. Lippard,
Chem. Rev. 2016, 116, 3436-3486; b) M. D. Hall, T. W.
Hambley, Coord. Chem. Rev. 2002, 232, 49-67; c) W.
H. Ang, I. Khalaila, C. S. Allardyce, L. Juillerat-
Jeanneret, P. J. Dyson, J. Am. Chem. Soc. 2005, 127,
1382-1383; d) K. Suntharalingam, Y. Song, S. J.
Lippard, Chem. Commun. 2014, 50, 2465-2468; e) E.
Petruzzella, R. Sirota, I. Solazzo, V. Gandin, D. Gibson,
Chem. Sci. 2018, 9, 4299-4307; f) R. Ma, Y. Wang, L.
Yan, L. Ma, Z. Wang, H. C. Chan, S.-K. Chiu, X. Chen,
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201916400
RESEARCH ARTICLE
G. Zhu, Chem. Commun. 2015, 51, 7859-7862; g) M. V.
Babak, Y. Zhi, B. Czarny, T. B. Toh, L. Hooi, E. K.-H.
Chow, W. H. Ang, D. Gibson, G. Pastorin, Angew. Chem.
Int. Ed. 2019, 58, 8109-8114; h) J. X. Ong, C. S. Q. Lim,
H. V. Le, W. H. Ang, Angew. Chem. Int. Ed. 2019, 58,
K. Zarschler, B. Goud, H. Stephan, G. Gasser,
ChemBioChem,
2019,
accepted,
DOI:
10.1002/cbic.201900419; i) Y. Ellahioui, M. Patra, C.
Mari, R. Kaabi, J. Karges, G. Gasser, S. Gómez-Ruiz,
Dalton Trans. 2019, 48, 5940-5951; j) B. S. Howerton,
D. K. Heidary, E. C. Glazer, J. Am. Chem. Soc. 2012,
134, 8324-8327; k) J. D. Knoll, C. Turro, Coord. Chem.
Rev. 2015, 282-283, 110-126; l) A. M. Palmer, B. Peña,
R. B. Sears, O. Chen, M. E. Ojaimi, R. P. Thummel, K.
R. Dunbar, C. Turro, Philos. Trans. R. Soc. A 2013, 371,
20120135; m) J. Karges, F. Heinemann, F. Maschietto,
M. Patra, O. Blacque, I. Ciofini, B. Spingler, G. Gasser,
Biorg. Med. Chem. 2019, 27, 2666-2675; n) S. A.
McFarland, A. Mandel, R. Dumoulin-White, G. Gasser,
Curr. Opin. Chem. Biol. 2020, 56, 23-27; o) W. Streciwilk,
A. Terenzi, X. Cheng, L. Hager, Y. Dabiri, P. Prochnow,
J. E. Bandow, S. Wölfl, B. K. Keppler, I. Ott, Eur. J. Med.
Chem. 2018, 156, 148-161.
a) H. Yin, M. Stephenson, J. Gibson, E. Sampson, G.
Shi, T. Sainuddin, S. Monro, S. A. McFarland, Inorg.
Chem. 2014, 53, 4548-4559; b) J. Karges, O. Blacque,
P. Goldner, H. Chao, G. Gasser, Eur. J. Inorg. Chem.,
2019, 3704-3712, DOI: 10.1002/ejic.201900569; c) J.
Karges, O. Blacque, M. Jakubaszek, B. Goud, P.
Goldner, G. Gasser, J. Inorg. Biochem. 2019, 198,
110752.
a) K. Ogawa, Y. Kobuke, Anti-Cancer Agents Me. 2008,
8, 269-279; b) B. C. Wilson, W. P. Jeeves, D. M. Lowe,
Photochem. Photobiol. 1985, 42, 153-162.
J. Karges, F. Heinemann, M. Jakubaszek, F. Maschietto,
C. Subecz, M. Dotou, O. Blacque, M. Tharaud, B. Goud,
E. V. Zahínos, B. Spingler, I. Ciofini, G. Gasser,
ChemRvix, doi: 10.26434/chemrxiv.11336669.
T. Banerjee, S. Rawalekar, A. Das, H. N. Ghosh, Eur. J.
Inorg. Chem. 2011, 2011, 4187-4197.
E. J. McLaurin, A. B. Greytak, M. G. Bawendi, D. G.
Nocera, J. Am. Chem. Soc. 2009, 131, 12994-
13001.[18] a) M. J. Cook, A. P. Lewis, G. S. McAuliffe,
V. Skarda, A. J. Thomson, J. L. Glasper, D. J. Robbins,
J. Chem. Soc. Perkin Trans. 2 1984, 1293-1301; b) A.
Juris, V. Balzani, F. Barigelletti, S. Campagna, P. l.
Belser, A. Von Zelewsky, Coord. Chem. Rev. 1988, 84,
85-277.
164-167; i) J. Z. Zhang, E. Wexselblatt, T. W. Hambley,
D. Gibson, Chem. Commun. 2012, 48, 847-849; j) L.
Cubo, T. W. Hambley, P. J. Sanz Miguel, A. Carnero, C.
Navarro-Ranninger, A. G. Quiroga, Dalton Trans. 2011,
4
0, 344-347; k) L. Ma, N. Wang, R. Ma, C. Li, Z. Xu, M.-
K. Tse, G. Zhu, Angew. Chem. Int. Ed. 2018, 57, 9098-
102; l) G. Thiabaud, L. Harden-Bull, Y.-J. Ghang, S.
9
Sen, X. Chi, J. L. Bachman, V. M. Lynch, Z. H. Siddik,
J. L. Sessler, Inorg. Chem. 2019, 58, 7886-7894; m) N.
Muhammad, N. Sadia, C. Zhu, C. Luo, Z. Guo, X. Wang,
Chem. Commun. 2017, 53, 9971-9974; n) X. Wang, X.
Wang, S. Jin, N. Muhammad, Z. Guo, Chem. Rev. 2019,
1
19, 1138-1192.
a) S. D. Gore, M. A. Carducci, Expert Opin. Inv. Drug.
000, 9, 2923-2934; b) R. R. Rosato, S. Grant, Cancer
[
7]
8]
[13]
2
Biol. Ther. 2003, 2, 31-38.
a) H. Kostrhunova, E. Petruzzella, D. Gibson, J.
Kasparkova, V. Brabec, Chem. Eur. J. 2019, 25, 5235-
[
5245; b) R. Raveendran, J. P. Braude, E. Wexselblatt,
V. Novohradsky, O. Stuchlikova, V. Brabec, V. Gandin,
D. Gibson, Chem. Sci. 2016, 7, 2381-2391; c) J. J.
Wilson, S. J. Lippard, Inorg. Chem. 2011, 50, 3103.
a) D. E. Dolmans, D. Fukumura, R. K. Jain, Nat. Rev.
Cancer 2003, 3, 380-387; b) S. Bonnet, Dalton Trans.
[14]
[15]
[
9]
2
018, 47, 10330-10343; c) F. Heinemann, J. Karges, G.
Gasser, Acc. Chem. Res. 2017, 50, 2727-2736.
a) C. Vallotto, E. Shaili, H. Shi, J. S. Butler, C. J. Wedge,
M. E. Newton, P. J. Sadler, Chem. Commun. 2018, 54,
[10]
13845-13848; b) C. A. Wootton, C. Sanchez-Cano, A. F.
[16]
[17]
Lopez-Clavijo, E. Shaili, M. P. Barrow, P. J. Sadler, P.
B. O'Connor, Chem. Sci. 2018, 9, 2733-2739; c) N. A.
Kratochwil, J. A. Parkinson, P. J. Bednarski, P. J. Sadler,
Angew. Chem. Int. Ed. 1999, 38, 1460-1463.
a) L. K. McKenzie, H. E. Bryant, J. A. Weinstein, Coord.
Chem. Rev. 2019, 379, 2-29; b) L. Zeng, P. Gupta, Y.
Chen, E. Wang, L. Ji, H. Chao, Z.-S. Chen, Chem. Soc.
Rev. 2017, 46, 5771-5804; c) C. Imberti, P. Zhang, H.
Huang, P. J. Sadler, Angew. Chem. Int. Ed., 2020, 59,
[11]
6
1-73; d) V. Novohradsky, A. Rovira, C. Hally, A.
[19]
[20]
a) J. Karges, P. Goldner, G. Gasser, Inorganics 2019, 7,
4; b) J. Karges, G. Gasser, Inorg. Chim. Acta 2019,
119196.
a) U. Basu, J. Karges, F. Chotard, C. Balan, P. Le
Gendre, G. Gasser, E. Bodio, R. Malacea Kabbara,
Polyhedron 2019, 172, 22-27; b) A. K. Renfrew, J.
Karges, R. Scopelliti, F. D. Bobbink, P. Nowak-
Sliwinska, G. Gasser, P. Dyson, ChemBioChem 2019,
20, 2876.
a) J. Karges, U. Basu, O. Blacque, H. Chao, G. Gasser,
Angew. Chem. Int. Ed., 2019, 58, 14334-14340; b) J.
Karges, O. Blacque, H. Chao, G. Gasser, Inorg. Chem.
2019, 58, 12422-12432.
Z. Zhou, J. Liu, T. W. Rees, H. Wang, X. Li, H. Chao, P.
J. Stang, Proc. Natl. Acad. Sci. 2018, 115, 5664-5669.
M. G. Ormerod, C. F. O'Neill, D. Robertson, K. R.
Harrap, Exp. Cell Res. 1994, 211, 231-237.
Galindo, G. Vigueras, A. Gandioso, M. Svitelova, R.
Bresolí-Obach, H. Kostrhunova, L. Markova, J.
Kasparkova, S. Nonell, J. Ruiz, V. Brabec, V. Marchán,
Angew. Chem. Int. Ed. 2019, 58, 6311-6315; e) J.
Pracharova, G. Vigueras, V. Novohradsky, N. Cutillas,
C. Janiak, H. Kostrhunova, J. Kasparkova, J. Ruiz, V.
Brabec, Chem. Eur. J. 2018, 24, 4607-4619; f) A.
Zamora, G. Vigueras, V. Rodríguez, M. D. Santana, J.
Ruiz, Coord. Chem. Rev. 2018, 360, 34-76.
a) S. Monro, K. L. Colón, H. Yin, J. Roque III, P. Konda,
S. Gujar, R. P. Thummel, L. Lilge, C. G. Cameron, S. A.
McFarland, Chem. Rev. 2019, 119, 797-828; b) A. Li, C.
Turro, J. J. Kodanko, Acc. Chem. Res. 2018, 51, 1415-
[21]
[12]
[22]
[23]
[24]
1421; c) J. Liu, C. Zhang, T. W. Rees, L. Ke, L. Ji, H.
Chao, Coord. Chem. Rev. 2018, 363, 17-28; d) F. E.
Poynton, S. A. Bright, S. Blasco, D. C. Williams, J. M.
Kelly, T. Gunnlaugsson, Chem. Soc. Rev. 2017, 46,
a) D. Kessel, Photochem. Photobiol. 2019, 95, 119-125;
b) V. Rapozzi, F. D’Este, L. E. Xodo, J. Porphyr.
Phthalocyanines 2019, 23, 410-418.
J. M. Adams, Genes Dev. 2003, 17, 2481-2495.
H. Song, M. Wei, W. Liu, S. Shen, J. Li, L. Wang, Histol.
Histopathol. 2018, 33, 73-79.
7
706-7756; e) M. Jakubaszek, J. Rossier, J. Karges, J.
Delasoie, B. Goud, G. Gasser, F. Zobi, Helv. Chim. Acta
019, 102, e1900104; f) J. Shum, P. K.-K. Leung, K. K.-
2
[25]
[26]
W. Lo, Inorg. Chem. 2019, 58, 2231-2247; g) K. Qiu, Y.
Chen, T. W. Rees, L. Ji, H. Chao, Coord. Chem. Rev.
2
019, 378, 66-86; h) J. Karges, M. Jakubaszek, C. Mari,
This article is protected by copyright. All rights reserved.

Angewandte Chemie International Edition
10.1002/anie.201916400
RESEARCH ARTICLE
[27]
a) C. Qian, J.-Q. Wang, C.-L. Song, L.-L. Wang, L.-N. Ji,
H. Chao, Metallomics 2013, 5, 844-854; b) H. Huang, P.
Zhang, B. Yu, Y. Chen, J. Wang, L. Ji, H. Chao, J. Med.
Chem. 2014, 57, 8971-8983.
[28]
a) P. A. Netti, D. A. Berk, M. A. Swartz, A. J. Grodzinsky,
R. K. Jain, Cancer Res. 2000, 60, 2497-2503; b) J.
Friedrich, C. Seidel, R. Ebner, L. A. Kunz-Schughart,
Nat. Protoc. 2009, 4, 309.
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Entry for the Table of Contents
Insert graphic for Table of Contents here:
Insert text for Table of Contents here:
The treatment of cancer remains a major challenge in modern medicine, initiating the development of multiple strategies. Herein, a
novel approach combining a Pt(IV) complex as chemotherapeutic drug and a Ru(II) complex as an agent for photodynamic therapy is
presented. The conjugate demonstrated to act on various cellular compartments with different mechanism on 2D monolayer cells as
well as 3D multicellular tumor spheroids.
Institute and/or researcher Twitter usernames:
Johannes Karges: @Johannes_Karges
Gilles Gasser: @GasserGroup
Chimie ParisTech: @ChimieParisTech
Universite PSL: @psl_univ
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