A Dual Killing Strategy: Photocatalytic Generation of Singlet Oxygen with Concomitant PtIV Prodrug Activation

Article abstract of DOI:10.1002/anie.201908511

A ruthenium-based mitochondrial-targeting photosensitiser that undergoes efficient cell uptake, enables the rapid catalytic conversion of Pt^IV prodrugs into their active Pt^II counterparts, and drives the generation of singlet oxygen was designed. This dual mode of action drives two orthogonal cancer-cell killing mechanisms with temporal and spatial control. The designed photosensitiser was shown to elicit cell death of a panel of cancer cell lines including those showing oxaliplatin-resistance.

Full text of DOI:10.1002/anie.201908511

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Accepted Article
Title: A Dual Killing Strategy — Photocatalytic Generation of Singlet
Oxygen with Concomitant Pt(IV) Prodrug Activation
Authors: Daniel Norman, Alessia Gambardella, Andrew Mount, Alan
Murray, and Mark Bradley
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A Dual Killing Strategy — Photocatalytic Generation of Singlet
Oxygen with Concomitant Pt(IV) Prodrug Activation
[a]
[a]
[a]
[b]
[a]
Daniel Norman, Alessia Gambardella , Andrew Mount , Alan Murray and Mark Bradley*
Abstract:
A
Ruthenium-based
mitochondrial-targeting
reducing Pt(IV) prodrugs while simultaneously generating singlet
oxygen (Scheme 1). The Pt(IV) prodrugs were designed to be
“bio-inert” prior to photochemically-induced reduction, while the
photosensitiser (PS-1) was shown to be taken up rapidly by cells
and localized in the mitochondria. Upon irradiation at 470 nm, the
photosensitisers were capable of activating Pt(IV) prodrugs, while
also causing significant oxidative damage to cells, thus affording
a spatially- and temporally-controlled cytotoxic effect. The ability
of this Pt(IV) prodrug activation system to overcome drug
resistance was explored. While the commercial photosensitiser,
photosensitiser that undergoes efficient cell uptake, enables the rapid
catalytic conversion of Pt(IV) prodrugs into their active Pt(II)
counterparts and drives the generation of singlet oxygen was
designed. This duality drives two orthogonal killing mechanisms with
cytotoxicity mediated with temporal and spatial control and was shown
to elicit cell death of a panel of cancer cell lines including those
showing oxaliplatin-resistance.
Photodynamic therapy (PDT) utilizes photosensitisers (PS) in
combination with illumination to generate cytotoxic reactive
oxygen species (ROS); primarily singlet oxygen ( O
only occurs in areas where light is focused, it enables spatially
selective cytotoxic effects. Clinical applications of PDT include
skin tumors[2] and head and neck cancers[3] as these are optically
readily accessible.
Several porphyrin-based photosensitisers have been approved
for clinical use including photofrin , however it has serious side-
3 2
Ru(bpy) Cl , was found to be capable of reducing Pt(IV) species,
it had limited cell uptake. Therefore, a derivative, PS-1, was
synthesized by addition of 1,3,3-trimethyl-2-methyleneindoline to
1
)[1]. As this
2
4
-formylphenyl boronic acid (Scheme 2), followed by Suzuki-
Miyaura coupling with 4-bromo-2,2’-bipyridine to afford the
desired ligand that was treated with Ru(bpy) Cl with replacement
2
2
of the chloro ligands driven by microwave heating.
[
4]
[
5]
effects in that patients may exhibit severe photosensitivity , as
well as non-specific damage to surrounding healthy tissue .
Recently, ruthenium-based photosensitiser, TLD-1433,
[
6]
a
completed Phase Ib clinical studies for bladder cancer treatment.
This trial demonstrated the safety of this PS and also the utility of
PDT at wavelengths outside of the “optimal window” with cancers
[7]
that may not be considered optically accessible .
A related therapeutic concept involves photo-activatable prodrugs,
whereby irradiation generates a cytotoxic drug from an inert
[
8]
prodrug . Of relevance are the Pt-based photo-activatable
prodrugs developed by Sadler et al[9] where irradiation of a
diazido-Pt(IV) complex gives rise to the cytotoxic Pt(II)
counterpart, eliciting a dramatic increase in cytotoxic effect, ideal
for photo-activated chemotherapy applications. Riboflavin has
also been shown to be an effective photocatalyst for the
conversion of Pt(IV) complexes to their cytotoxic Pt(II)
counterparts[10] (for other Pt(IV) approaches see review by
Lippard et al[11]).
Scheme 1. Representation of the photocatalytic conversion of
Pt(IV) prodrugs to their active Pt(II) counterparts by a Ru(II)
1
photocatalyst with simultaneous O
2
generation.
Ruthenium-based
characteristic light absorption profiles
photosensitisers
generally
due to ligand-to-ligand
display
Herein, we report the design, synthesis and evaluation of a
photocatalytic Pt(IV) prodrug activation platform capable of
[12]
(
π-π*) and metal-to-ligand (dπ-π*) charge transfer bands and
these were observed for PS-1 (Figure S1). The singlet oxygen
quantum yield of PS-1 was quantified by the Singlet Oxygen
[
[
a]
b]
Dr. D. J. Norman, Ms. A. Gambardella Prof. A. R. Mount, Prof. M.
Bradley
EaStChem School of Chemistry
University of Edinburgh
David Brewster Road, Edinburgh
E-mail: mark.bradley@ed.ac.uk
Prof. A. F. Murray
[
13]
Sensor Green (SOSG) assay and found to be 0.72 (compared
to 0.88 for Ru(bpy)
Cl [14]) (Figure S2). In the presence of a Pt(IV)
3
2
complex (Pt-c), the quantum yield reduced to 0.37. The photo-
activation of the Pt(IV) species was desired to occur intracellularly
so cellular uptake of PS-1 was determined by ICP-MS. PS-1 was
observed to be taken up well by all three cell lines used, with 62-
School of Engineering
University of Edinburgh
Mayfield Rd, Edinburgh
9
0% of the compound added to the culture media taken up into
the cells after 4 h (see Table 1). The high cellular uptake of PS-1
attributed to the positively charged indoline moiety and the
lipophilic nature of the ligands, facilitating passage across the
Supporting information for this article is given via a link at the end of
the document.
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Scheme 2. Synthesis of the photocatalyst PS-1 and the Pt(IV) prodrugs Pt-a to Pt-g.
negatively charged cell membrane; a feature that can be
accentuated in cancer cells [15]. The stability of PS-1 in complex
media was demonstrated by incubation in 10% FBS in DMEM
for Pt-f and Pt-g (-0.86 V and -0.74 V, respectively), which were
not stable towards biological reductants.
The complex Pt-d exhibited low water solubility and was
discontinued from further studies with Pt-c taken forwards. Pt-c
was also found to be relatively stable in 10% FBS in DMEM, as
measured by HPLC (Figure S7). The difference in cytotoxicity of
Pt-c compared to the clinical drug, oxaliplatin (OxPt), was
analysed in HCT116 and SKOV-3-wt cells, showing that Pt-c
exhibited significantly reduced cytotoxicity compared to the parent
drug with IC50’s of 64 vrs 9 µM for SKOV-3-wt and 97 vrs 6 µM for
HCT116 cells (Figure S8).
The cellular uptake of Pt-c (as quantified by ICP-MS, Table 1)
showed much greater uptake of the Pt(IV) prodrug than of OxPt,
as is commonly observed in comparisons of Pt(II) and Pt(IV)
complex cell uptake, as the Pt(IV) oxidation state affords more
substitution-inert complexes than Pt(II) complexes[19], thereby
enabling passage into cells without degradation or attack by
biomolecules. The increased lipophilicity may also be responsible
for promoting cellular uptake, although trends between cLogP and
cellular accumulation of Pt complexes are often poorly
correlated[20].
(
Figure S3).
The ability of PS-1 to elicit cell death upon illumination of light was
confirmed in SKOV-3-wt cells (Figure S4). In the dark, PS-1 has
negligible effects on cell viability whereas when illuminated it
generates cytotoxic reactive oxygen species (IC50: 17 µM).
To identify a “bio-inert” Pt(IV) prodrug, a series of symmetrical and
non-symmetrical Pt(IV) complexes, Pt-a to Pt-g, were
synthesized using standard conditions and screened against
biological reductants to identify Pt(IV) prodrugs that were resistant
to the biological reductants glutathione (GSH) and ascorbic acid
(
AsA) (Figure S5).
The non-symmetrical Pt(IV) complexes carrying an axial acetate
ligand and either tert-butanoate or benzoate axial ligands (Pt-c
and Pt-d) were stable to reduction by glutathione or ascorbic acid.
Symmetrical complexes with increased steric hindrance, (Pt-f and
Pt-g) were ineffective at preventing reduction. Electron-
withdrawing axial ligands, such as trifluoroacetate (Pt-b and Pt-e)
were unstable towards biological reductants, presumably due to
destabilisation of the Pt(IV) center as has been observed
previously[ . Pt-e has also previously been shown to hydrolyze
in solution to Pt-b expediting further reduction[17]. Prior work
correlating trends of physicochemical properties of Pt(IV), such as
reduction potential or logP have shown possible links to biological
stability or activity[18]. Analyses of the reduction potentials of Pt-a
to Pt-g, showed that Pt-c had the highest reduction potential (-
The ability of PS-1 to photocatalytically reduce the Pt(IV) prodrug
Pt-c into OxPt was confirmed, as shown in Figure 1, with Pt-c
reduced into OxPt by 2 mol% PS-1 by illumination (λ = 470 nm,
16]
-2
0.58 mW.cm ), with 88% conversion observed after 60 min of
illumination (Figures S9-S11) with the conversion of Pt-c to OxPt
also confirmed by NMR (Figure 1b and Figure S12).
To quantify the level of cell death brought on by photoactivation
of Pt-c and the dual oxidative damage inflicted by PS-1 SKOV-3-
wt and HCT116 cell lines were incubated with PS-1 and Pt-c and
irradiated.
0.90 V) which may confer its stability towards biological
reductants (Figure S6 and Table S5). However, this is not the only
determinant factor as similar reduction potentials were observed
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Cells that did not undergo irradiation showed little cell death when
incubated with either PS-1 or PS-1 with Pt-c (Figure 2b).
Whereas when PS-1 was used in conjunction with illumination
there was significantly reduced cell viability, due to the generation
1
of O
2
and other reactive oxygen species. To verify this, the singlet
oxygen generation in cells was measured by co-incubation of
SOSG and the turn-on of fluorescence tracked over time (Figure
S13).
The combination of PS-1 and Pt-c with illumination demonstrated
very high levels of light-mediated cytotoxicity in both SKOV-3-wt
and HCT116 cell lines. Due to the high uptake of PS-1 and the
increased uptake of Pt-c compared to OxPt and the efficacy of
the photocatalysed Pt(IV) prodrug activation, it was hypothesised
that light-mediated photocatalytic activation of Pt-c may be able
to overcome the acquired resistance to OxPt. To explore this,
SKOV-3-wt cells were allowed to accrue resistance to oxaliplatin
by sub-culturing in incremental doses of OxPt over 3 months
(
3
procedure in ESI). The IC50 for OxPt in wild-type SKOV-3 (SKOV-
-wt) cells was 8.8 µM, which increased some 3-fold to 25 µM for
the more resistant cells (SKOV-3-OxR). The Pt(IV) prodrug Pt-c
again exhibited reduced cytotoxicity compared to its Pt(II)
counterpart, OxPt, with IC50 values of 82 vrs 25 µM in the SKOV-
3-OxR cells.
Photocatalytic activation of Pt-c in SKOV-3-OxR cells elicited
substantial cell death, albeit with a slightly diminished cytotoxic
effect than in SKOV-3-wt. Harvesting of the cellular DNA was
performed 24 h post-photocatalytic activation of Pt-c in SKOV-3-
wt and SKOV-3-OxR cells with the Pt content of each analysed
by ICP-MS. There was a marked increase in platinated DNA in
SKOV-3-wt and SKOV-3-OxR cells when PS-1 and Pt-c were
utilised in conjunction with light irradiation compared to controls
Figure 1. Analysis of the reduction of Pt-c by PS-1: (a) HPLC
-2
analysis of the photo-reduction (λ = 470 nm, 0.58 mW.cm ) of Pt-
c (50 µM) by PS-1 (1 µM) in PBS over time. (b) NMR analysis
following the conversion of the Pt(IV) prodrug Pt-c (100 µM) into
2
oxaliplatin (Pt(II)) in D O by monitoring the resonances correlating
to the protons of the diaminocyclohexyl ligands.
(
Figure S14). Interestingly, there was a larger overall platinated
DNA content for SKOV-3-OxR (acquired resistance to oxaliplatin)
than with either OxPt or photocatalysed Pt(IV)-Pt(II) conversion
compared to SKOV-3-wt i.e. naïve cells towards oxaliplatin. As
the photosensitiser was found to primarily localize in the
mitochondria (Figure 2a and S15), cellular fractionation (into
cytosol, mitochondrial and nuclear fractions) followed by ICP-MS
analysis was used to probe the localization of the Pt(IV) prodrug
in SKOV-3-wt cells (Figure S16). Pt-c was found mainly in the
cytosolic fraction but was distributed throughout the cell, with
slightly lower levels in the mitochondria and the nucleus. This ratio
of distribution did not appear to be altered by the co-incubation of
PS-1.
Table 1. Cellular uptake of PS-1 and Pt-c as measured by ICP-
MS
Compound[a]
Cell Line
Cell Uptake (ng/10 cells)[%]
6
[b]
PS-1
SKOV-3-wt
SKOV-3-OxR
HCT116
275 ± 9 (90 ± 3%)
191 ± 3 (63 ± 1%)
190 ± 10 (63 ± 3%)
531 ± 20 (14 ± 0.5%)
384 ± 2 (10 ± 0.04%)
458 ± 12 (12 ± 0.3%)
161 ± 11 (1 ± 0.9%)
150 ± 5 (1 ± 0.4%)
281 ± 10 (2 ± 0.8%)
Finally, to explore the scope of this Pt(IV) prodrug activation
system, the ability of two ruthenium-based photosensitisers to
reduce three different Pt(IV) species was shown (Figure S17).
TLD-1433, a photosensitiser currently under clinical investigation,
was capable of reducing two oxaliplatin Pt(IV) species upon
illumination of light. This was also shown to be possible with other
platinum drugs, such as a Pt(IV) cisplatin derivative (Cpt-Ac).
In conclusion, a photocatalytic platform for the simultaneous
activation of Pt(IV) prodrugs and generation of singlet oxygen has
been developed utilising a ruthenium-based photosensitiser. This
system demonstrated excellent cytotoxic capabilities following
illumination of cancer cell lines. The prodrug activation system
overcame acquired Pt resistance in cells and demonstrated
robustness in each of the cell lines used. The catalytic activation
of Pt(IV) with concomitant singlet oxygen generation thus allows
Pt-c
SKOV-3-wt
SKOV-3-OxR
HCT116
OxPt
SKOV-3-wt
SKOV-3-OxR
HCT116
[
a] Cells were incubated with either PS-1 (1 µM) or Pt-c (20 µM) for 4 h
at 37°C (n = 3)
b] % uptake calculated as proportion of the theoretical maximal
uptake.
[
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Figure 2. (a) Live-cell confocal microscopy showing co-localisation of PS-1 (red) and MitoTracker (green) and nuclei (blue). Yellow
shows overlap of green and red. Cell viability measured by Cell Titre Glo 2.0 for (b) SKOV-3-wt cells, (c) SKOV-3-OxR cells, with
cells either in the dark or with illumination or untreated (control), with incubation with PS-1 (1 µM) or incubation with PS-1 (1 µM)
with Pt-c (20 µM). The data represent the mean ± S.D. *** P<0.001 by one-way ANOVA with Tukey post-test
low dosing of photosensitiser which may help reduce
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absorption wavelength to more therapeutically applicable ranges
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cancer-targeting capabilities of photocatalytic compounds that
can be used for Pt(IV) prodrug activation
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Acknowledgements
This work was supported by funding from University of Edinburgh
and UK Engineering and Physical Sciences Research Council
through the Implantable Microsystems for Personalised Anti-
Cancer Therapy (IMPACT) programme grant (EPK034510/1).
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