Riboflavin as a bioorthogonal photocatalyst for the activation of a PtIV prodrug

Article abstract of DOI:10.1039/c7sc01109a

Encouraging developments demonstrate that few transition metal and organometallic catalysts can operate in a bioorthogonal fashion and promote non-natural chemistry in living systems by minimizing undesired side reactions with cellular components. These catalytic processes have potential for applications in medicinal chemistry and chemical biology. However, the stringent conditions of the cell environment severely limit the number of accessible metal catalysts and exogenous reactions. Herein, we report an unorthodox approach and a new type of bioorthogonal catalytic reaction, in which a metal complex is an unconventional substrate and an exogenous biological molecule acts as a catalyst. In this reaction, riboflavin photocatalytically converts a Pt^IV prodrug into cisplatin within the biological environment. Due to the catalytic activity of riboflavin, cisplatin-like apoptosis is induced in cancer cells under extremely low doses of light, potentially preventing systemic off-target reactions. Photocatalytic and bioorthogonal turnover of Pt^IV into Pt^II species is an attractive strategy to amplify the antineoplastic action of metal-based chemotherapeutics with spatio-temporal control.

Full text of DOI:10.1039/c7sc01109a

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Riboflavin As Bioorthogonal Photocatalyst For The Activation Of A
PtIV Prodrug
Received 00th January 20xx,
Accepted 00th January 20xx
Silvia Alonso-de Castroa, Emmanuel Ruggieroa, Ane Ruiz-de-Anguloa, Elixabete Rezabalb, Juan C.
Mareque-Rivasa,c, Xabier Lopezd, Fernando López-Gallegoa,c, Luca Salassa*a,c,d
DOI: 10.1039/x0xx00000x
Encouraging developments demonstrate that few transition metal and organometallic catalysts can operate in a
bioorthogonal fashion and promote non-natural chemistry in living systems by minimizing undesired side reactions with
cellular components. These catalytic processes have potential for applications in medicinal chemistry and chemical
biology. Nevertheless, the stringent conditions of the cell environment severely limit the number of accessible metal
catalysts and exogenous reactions. Herein, we report an unorthodox approach and a new type of bioorthogonal catalytic
reaction, in which a metal complex is an unconventional substrate and an exogenous biological molecule acts as catalyst.
In such reaction, riboflavin photocatalytically converts a PtIV prodrug into cisplatin within the biological environment. The
catalytic activity of riboflavin induces cisplatin-like apoptosis in cancer cells with extremely low doses of light, potentially
preventing systemic off-target reactions. Photocatalytic and bioorthogonal turnover of PtIV into PtII species is an attractive
strategy to amplify the antineoplastic action of metal-based chemotherapeutics with spatio-temporal control.
www.rsc.org/
biocompatible transformations are highly needed to further
advance this extremely challenging field that is still in its
infancy.
Herein, we describe an original photocatalysis approach to
control the reactivity of transition metal complexes in a
bioorthogonal fashion. In a new type of lightꢀdriven reaction,
the exogenous biological molecule riboflavin (Rf) functions as
Introduction
The combination of catalysis and bioorthogonality1–3 promises
to impact drug discovery and bioimaging by facilitating the
execution of nonꢀnatural chemical reactions in living systems.
Catalytic turnover can boost the efficiency of bioorthogonal
chemical reactions, unveiling new strategies for prodrug
activation and uncaging of molecular probes.4–8
a
bioorthogonal photocatalyst and
a metal complex as
In this context, transition metal and organometallic catalysis
have opened new avenues for the advance of bioorthogonal
catalysis in cells.5,9–16 The laboratories of Meggers,
Mascareñas, UncitiꢀBroceta and Bradley used organoruthenium
and palladium catalysis to deprotect proꢀfluorescent substrates
and activate prodrugs in cancer cells or in their compartments
and surroundings.11–14,16 Rotello devised biomimetic
nanoenzymes for imaging and therapy, by encapsulating
ruthenium and palladium catalysts into waterꢀsoluble gold
nanoparticles and controlling their catalytic activity in HeLa
cells through supramolecular chemistry.15 These pioneering
studies exploited metalꢀbased catalytic uncaging of
allylcarbamateꢀ and propargylꢀprotected amines as viable
unconventional substrate (Scheme 1).
Scheme
1 Transition metal complex acting as substrate and its
bioorthogonal activation by riboflavin which functions as photocatalyst.
strategies for bioorthogonal catalysis, however new This unusual catalyst/substrate pair relies on the photoredox
properties of Rf to enable the selective activation of a PtIV
prodrug of cisplatin with exceptionally low doses of blue light,
and induce apoptotic death in PCꢀ3 human prostate cancer cells.
Metal complexes are typically regarded as catalysts which
convert organic substrates in more valuable compounds,
whereas catalytic transformations of metal complexes are, to
date, practically unknown and represent a paradigm shift in
catalysis.17,18 Their development can expand the scope of
bioorthogonal chemical reactions to inorganic substances and
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metalꢀbased prodrugs, fostering the creation of new inorganic µM
1
in 5 minutes (light dose 0.75 J·cm–2). A Rf concentration
chemistry toolkits for biology and medicine.
as low as 0.13 µM still photocatalyses the transformation of
(120 µM), however more than 2 hours are required to achieve
27% of conversion (Fig. S8†). No reaction between and Rf
occurs in the dark within 1 week (Fig. S9†). Interestingly,
photoconversion of takes place in pure water (pH 5) or in
phosphate buffer (PB, 100 mM, pH 5.5), but never reaches
completion due to poor photostability of Rf in these media
(vide infra). Thus, MES buffer plays a key role in the catalytic
process preventing Rf from undergoing photodecomposition
reactions.
1
1
Results and discussion
As part of our ongoing efforts to design innovative lightꢀ
activation modes for anticancer platinum complexes,19 we
reasoned that Rf and its rich photochemistry would facilitate
1
the
[Pt(NH3)2(Cl)2(O2CCH2CH2CO2)2]2–
of excitation wavelengths appropriate for use in biological
systems (Scheme 2). Complex is a cisplatin prodrug suitable
photoreduction
of
cis,cis,transꢀ
(
1) to cisplatin by means
1
for photochemotherapy because of its high dark stability in
aqueous solutions and its negligible dark cytotoxicity in several
cancer cell lines, e.g. prostate cancer PCꢀ3 cells.20,21
Fig. 1 (a) Absorption spectrum of riboflavin (Rf) and cis,cis,trans-
[Pt(NH3)2(Cl)2(O2CCH2CH2CO2)2]2–
catalysed photoreduction of in MES buffer (18 mM, pH 6) monitored
by 1H NMR. Spectra were recorded for a MES/D2O (9:1) solution of 120
µM and 50 µM Rf upon t = 0 sec, 30 sec, 1 min and 2.5 min of 460-
nm light irradiation (2.5 mW·cm–2). 1H NMR signal labelling:
Pt–
methyl groups of Rf
(1) in aqueous solution. (b) Rf-
Scheme
2
Light-induced
reduction
of
cis,cis,trans-
[Pt(NH3)2(Cl)2(O2CCH2CH2CO2)2]2– (1) promoted by riboflavin (Rf) in MES
1
buffer.
1
Upon UVA light excitation (385 nm)
photochemical activation. However, UVA light is of limited use
in therapy and PtIV complexes such as
rarely display
1
undergoes
●
–
–
OCOCH2CH2CO2
,
●
Pt–OCOCH2CH2CO2 ,
●
–
free –O2CCH2CH2CO2
.
1
isoalloxazine ring,
●
satisfactory absorption features at wavelengths longer than 400
nm (Fig. 1a).
In order to assess the rate law for the Rfꢀcatalysed
photoreduction of
to PtII species, we studied the reaction rate
at different substrate concentrations (120 µM–1.92 mM, i.e.
2.4–38.4 mol equiv of compared to Rf) in 18 mM MES buffer
during 30 sec of irradiation at 298 K (Fig. S10†). The effect of
MES on the reaction rate was evaluated in a separate set of
experiments, in which MES concentration was varied in the 3–
20 mM range (Fig. S11†). Results demonstrate that the rate of
Rf is vitamin B2 and the precursor of biologically important
cofactors such as FMN and FAD, which are essential to
humans and animals due to their redox activity.22 The yellowꢀ
colored Rf absorbs in aqueous media with good extinction
1
1
coefficients (ε446 > 104 M−1 cm−1)23 as far as ca. 500 nm (Fig.
⋅
1a), and can promote a great variety of lightꢀinduced reactions
which depend on its 7,8ꢀdimethylꢀ10ꢀalkylisoalloxazine
fragment.22 Rf has been adopted as photocatalyst in several
organic reactions, including the photooxidation of benzyl
alcohols and alkyl benzenes or the [2+2] cycloaddition of
styrene dienes and bis(arylenones).24–26
the reaction linearly increases with the concentration of
1 and
MES, corresponding to a firstꢀorder reaction for both species.
Importantly, the reaction shows a stronger dependency on the
concentration of
1
than on MES, suggesting that PtIV reduction
is limiting step of the reaction.
Photocatalytic activation of a PtIV prodrug by riboflavin in
solution
Since our experiments employ a large excess of MES and all
reaction steps are irreversible, the rate constant can be
described using the pseudoꢀfirst order model (ESI†). Using 50
µM Rf, we obtained a pseudoꢀfirst order reaction constant (kobs
After confirming blue light has not direct effects on
1 (Fig. S1–
3†), we investigated the capability of Rf to photoactivate the
complex upon 460ꢀnm excitation in MES buffer. Using low
excitation power density (2.5 mW·cm–2), 120 µM solutions of
=
10.0 ± 0.05·10–3 s–1) that increases with the catalyst
1
concentration (Fig. S12† and S13†) and depends on MES
concentration.
A turnover frequency (TOF) value of 0.22 ± 0.06 s–1 was
were photolysed in the presence of Rf at various concentrations
1
(12–120 µM, Fig. S4–7†). The process was monitored via H
NMR by the evolution of diagnostic peaks corresponding to the
Ptꢀbound (triplets) and free (singlet) succinate ligands (Fig. 1b).
Subꢀstoichiometric quantities of Rf are capable to produce full
determined for the conversion of
under light irradiation at 298 K in 18 mM MES buffer. Under
such reaction conditions, the maximum total turnover number
1 (1.92 mM) by Rf (50 µM)
conversion of
1 into its photoproducts under light excitation,
(TTN) value is 38 after 3 minutes of light irradiation, and no
1
demonstrating that Rf does not act as a simple photosensitizer
but is indeed a photocatalyst. The efficiency of this catalytic
process is remarkable since 12 µM Rf converts 100% of 120
decomposition of catalyst is observed by H NMR. The Rf
/
1
catalyst/substrate pair achieves approx. 700–70 times higher
TOF compared to ruthenium(II) organometallic catalysts,
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which catalytically convert NAD+ in NADH or transform Oꢀ Employing ferrioxalate actinometry,30 we determined the
allyl carbamates into their respective amines under biologically photochemical quantum yield for Rf (50 µM / 1.0 mM, 18
relevant conditions and in cells.9,8 High TOF is crucial for mM MES) obtaining a value of 1.4
0.1 (Fig. S15† and S16†).
application in photochemotherapy since it guarantees rapid and Yield values > 1 are rather common in photoredox catalysis
/
1
±
sufficient conversion of
light doses.
1
with short irradiation times and low where radical chain propagation cycles form part of the
catalytic mechanism.31
MES buffer dramatically improves Rf photostability by
preventing that the isoalloxazine unit reacts with the ribityl
Mechanism of photocatalytic activation
Rf is extremely light sensitive and its photochemical reactivity moiety or with molecular oxygen. NMR and UVꢀVis show that
strongly depends on the surrounding environment. Electron MES substantially preserves Rf from decomposition for over
transfer and protonꢀcoupled reactions or singletꢀoxygen 30 min, whereas the catalyst is fully converted to the
generation take place upon light excitation of Rf, depending on photoproduct lumichrome in water within 1 min of light
the availability of electron donors. In addition, light irradiation, and then to 2,3ꢀbutanedione at longer irradiation
decomposes Rf into several fragments through intramolecular times (Fig. S17–19†).32
reactions in which the ribityl chain can be used as the electron The role of the buffer was confirmed using HEPES (18 mM,
source.27
pH 6), an analogue zwitterionic buffering agent (Fig. S20† and
can be ruled out since there S21†), in which Rf and behave similarly to MES in terms of
photocatalytic activity.
The presence of sodium azide (singlet oxygen scavenger) in
Hence, the sensitizing and catalytic capacity of Rf in MES water and PB also improves the efficiency of the photocatalytic
Direct energy transfer from Rf to
1
1
is no overlap between the emission band of the flavin (λem
535 nm) and the absorption spectrum of
=
1
.
buffer reasonably relies on electron transfer processes triggered reaction of Rf with
1 (Fig. S22† and S23†). When added to
by light. In the triplet excited state (3Rf*), Rf is a strong MES buffer, sodium azide does not improve Rf
/1
(Fig. S24†),
oxidant (Eº = 1.77 V)22 capable of efficiently extracting excluding the participation of O2 and other oxygen radicals as
electrons from the abundant MES molecules, and generate the major actors in the catalytic mechanism. On the other hand, O2
twoꢀelectron reduced RfH2/RfH– species (pKa ~ 6) together partially deactivates Rf, since under inert Ar atmosphere the
1
with morpholino radicals28 which eventually evolve to the photoconversion of
oxidized Nꢀoxide form of MES (Fig. S14†).29
1 is faster (Fig. S25†).
Fig. 2 Proposed mechanism for the photocatalytic activation of 1 by Rf. (a) Computed structure and frontier molecular orbitals (DFT:PBE0/def2-SVP) of a
selected 1-RfH2 adduct. Intermolecular H-bonds in 1-RfH2 are highlighted with magenta lines (top). Isodensity surfaces are plotted with the isovalue of 0.02
e−·bohr−3. Atoms color code: Pt grey, Cl green, O red, N blue, C pale brown (1) or yellow (Rf), H white. (b) Rf absorbs 460-nm photons to generate the triplet
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excited state (3Rf*) which oxidizes two MES molecules to give the reduced species RfH2/RfH–. Next, complex 1 forms stable adducts with either RfH2 (shown
in Fig. 2a) or RfH– and undergoes photoreduction and elimination reactions upon absorption of more photons, liberating cytotoxic PtII species and
regenerating the Rf catalyst.
Importantly, complex
1
(1.8 mM) does not affect the 1H NMR data showed that 3 min of blue light irradiation (light
to PtII species in
fluorescence lifetime of Rf in MES (Fig. S26† and S27†), dose 1.08 J·cm–2) can fully convert 1.92 mM
1
indicating that the active catalyst is not likely to be a Rf the presence of 50 µM Rf and 3 mM MES, without any
excitedꢀstate species. Therefore, photooxidation of MES significant side reaction affecting either medium components or
ultimately leads to the formation of reduced (groundꢀstate) the catalyst (Fig. S38†). Under such conditions the TOF and
RfH2/RfH–, whose low redox potential (ca. –0.2 V)22 cannot TTN for the Rf catalyst are as good as in pure MES buffer
directly promote reduction of
1
(–0.9 V).33
solutions, indicating that the catalytic process is bioorthogonal
Yet, as suggested by density functional theory (DFT) modelling in cell culture medium.
(PBE0/def2ꢀSVP) and consistently with the results obtained
The antiproliferative activity of Rf/1 against PCꢀ3 cancer cells
under Ar atmoshpere,
1 is capable to form adducts with either
was investigated in the dark and under 460ꢀnm light irradiation
by coꢀadministering the catalyst/substrate pair at a molar ratio
of 1:4 and using three different concentrations of complex (40,
RfH2 (Fig. 2a) or RfH– (Fig. S28–30†) by means of Hꢀbonding
interactions between its succinate and amino ligands, and the
isoalloxazine and ribityl groups of Rf. FMN and FAD also
80, 120 ꢁM). In our cell experiments, Rf prevalently activates
1
photocatalyze the PtIV conversion of
1, displaying an efficiency
in the extracellular space since we performed light irradiation
after a short preꢀincubation period and replaced culture medium
after 6 h. Once photocatalytically generated, PtII species can be
internalized by cells and exert their antiproliferative action.
MES is well tolerated by cells (Fig. S39†) and was hence
employed during cell viability assays as an electron donor.
Under these conditions, a short light irradiation period (1 min)
and an extremely low light dose (0.36 J·cm–2) are sufficient for
comparable to Rf (Fig. S31–34†). FAD, however, is somewhat
less active, possibly due to steric constrains introduced by its
adenine moiety which would disfavour Hꢀbonding between the
complex and the flavin.
Computed
on the Rf isoalloxazine rings, while LUMO and LUMO+1 are
ꢀantibonding orbitals of . Absorption of a second photon and
1ꢀRfH2 and 1ꢀ
RfH– adducts have HOMO localized
σ
1
subsequent lightꢀinduced population of the dissociative LUMO
orbitals can trigger photoreduction and ligand elimination
reactions,34 ultimately promoting the formation of cisplatin and
other PtII species. Nevertheless, we cannot exclude at this stage
that these strong and specific interactions could significantly
the full photoconversion of
the absence of MES the photoactivation of
1
by Rf (Fig. S40† and S41†). In
still takes place,
1
although less efficiently (Fig. S42†), likely because other
biological components of the medium act as electron donors.
Against PCꢀ3 cells, the Rf
doseꢀ dependent lightꢀinduced toxicity comparable to cisplatin
in the dark. Remarkably, 120 ꢁM and 30 ꢁM Rf induce a 55 ±
/1 catalyst/substrate pair displays
lower the redox potential of
prodrug once the Rfꢀadducts are formed.35,36 Calculated binding
energy for RfH2 and
RfH– adducts are in range 52–69
kcal·mol–1, indicating that these transient species are strongly
1 and cause direct reduction of the
1
1ꢀ
1ꢀ
5% reduction in cell biomass under light irradiation, against a
65 ± 5% caused by cisplatin at the same concentration. Control
experiments indicate that Rf/1 does not reduce viability of PCꢀ3
cells when kept in the dark, neither does any of the components
when irradiated individually (Fig. 3a).
stabilized and may bestow unique selectivity to the Rf/1
catalyst/substrate pair (vide infra).
A pHꢀdependency profile for the photoreaction at fixed lightꢀ
irradiation time (2.5 min) shows that complete photoconversion
of
1 occurs above pH 6 in MES, whereas at lower pHs the The antiproliferative action of Rfꢀactivated 1 is associated with
photocatalysis is less efficient (Fig S35†). The finding is in the formation of cisplatin as one of the major cytotoxic
agreement with the prevalence of the RfH2/RfH– forms of photoproducts. Initial evidence was gathered from binding
reduced Rf at pHs higher than 6.22
experiments with the RNA and DNA base model 5′ꢀguanosin
On the basis of the described evidence, we have schematized a monophosphate (GMP). 1H NMR shows that irradiated Rf/1
tentative photocatalytic mechanism for the Rf solutions incubated with GMP (0–24 h) present the diagnostic
/1
catalyst/substrate pair in Fig. 2b, although further investigations peak, corresponding to the cisplatin mono GMPꢀPtII adduct
will be needed for its complete elucidation.
(Fig. S43† and S44†).37 When incubated with the pET28b as
model of double stranded circular DNA (24 h, MES 1.5 mM,
Photocatalysis in the biological environment
pH 6), lightꢀactivated Rf/1 (2.5:10 µM) inhibits the polymerase
chain reactions (PCR). Thirty sec of light irradiation are
sufficient to stop DNA amplification and reach PCR inhibition
level comparable with cisplatin (10 µM), hence confirming the
capacity of this biorthogonal system to produce DNAꢀtargeting
species (Fig. S45†).38,39
Fluorescence microscopy of PCꢀ3 cells treated with either
irradiated Rf/1 (30:120 µM) or cisplatin (120 µM) is in
agreement with this scenario (Fig. 3b and Fig. S46†). In both
cases, images obtained after 48 h of incubation show increased
To test the potential of Rf photocatalysis as a bioorthogonal
tool for photochemotherapy, we studied next the activation of
by Rf in cell culture medium and its effects in PCꢀ3 cancer cells
(in which has no dark toxicity). At first, photocatalysis
1
1
experiments and controls (Fig. S36† and S37†) were performed
in Ham's Fꢀ12K medium supplemented with fetal bovine
serum, in which biological components such as growth factors,
antibodies, aminoacids, vitamins, and inorganic salts are
present at concentrations ranging from µM to mM.
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percentage of apoptotic versus viable cells, together with Double staining with Pacific Blue™ Annexin V / SYTOX®
changes in cell morphology that are characteristic of apoptosis, allowed differentiating between earlyꢀstage and lateꢀstage
i.e. cell shrinkage and rounding. Nonꢀtreated cells and cells apoptosis. Upon treatment with irradiated Rf
treated with Rf in the dark included as controls do not induce of early apoptotic cells is 16.7 ± 2.8 % against 19.9 ± 1.3 %
appreciable cell death. obtained for cisplatin. Rf and Rf in the dark exhibit no
Consistently, flow cytometry results confirm that PCꢀ3 cells significant population of cells in either stage of apoptosis after
exposed to the Rf mixture and light (30:120 µM) die through 48 h of incubation (Fig. 3c).
/1, the percentage
/
1
,
1
/1
/1
apoptosis 48 hours after irradiation. Cisplatin induces
comparable levels of apoptosis under the same conditions.
Fig. 3 Antiproliferative activity in human prostate cancer PC-3 cells. (a) Percentage cell viability of PC-3 cells following treatment with Rf, 1, Rf/1 and cisplatin
with and/or without light activation (460 nm, 0.36 J·cm–2). (b) Fluorescence microscopy images showing the effects of Rf/1 on PC-3 cells upon light
irradiation. (A, B) untreated PC-3 cells, (C, D) Rf/1 (30:120 µM) in the dark, (E, F) Rf/1 (30:120 µM) activated by 460-nm light and (G, H) cisplatin (120 µM) in
the dark. Top row: cell nuclei (green); bottom row: apoptotic cells (red). (c) Quantification of early apoptotic PC-3 cells (Annexin V+ / SYTOX–) treated by Rf
(30 µM), Rf/1 (30:120 µM) and cisplatin (120 µM) with and/or without light activation. Cell viability and flow cytometry data are presented as mean ± SEM of
at least three independent measurements. ***P < 0.001, **P < 0.01, ns = non-significant by two-way ANOVA followed by Bonferroni’s test (a) or by one-way
ANOVA followed by Tukey’s test (c).
In summary, we have described the first photocatalytic
Conclusions
activation of a metalꢀbased prodrug in solution and in a
biological environment. Coꢀadministration of the Rf
catalyst/substrate couple enables photoconversion of
/
1
1
as
into
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biologically active species by light irradiation at 460 nm, a parallel experiments were performed with Rf
wavelength that is ineffective when directly applied to the kept in the dark. A homeꢀmade LED plate was employed to
prodrug. In addition, the Rf prodrug activation strategy irradiate cells in 96ꢀwell plates (Fig. S47†).
induces an anticancer activity comparable to cisplatin with light PCꢀ3 cells exposed to
doses as low as 0.36 J·cm–2, that is ca. 15–35 times lower than 30:120 µM Rf
were viewed using a Zeiss Axio Observer
what is typically used for UVA and blue light activation of wide field fluorescence microscope (Carl Zeiss). Cells were
analogue platinum complexes.40,41
plated in an ibidi µꢀSlide 0.4 (13500 PCꢀ3 cells/channel) and
The photocatalytic turnover of PtIV into PtII species is an allowed to adhere overnight before they were treated with Rf
attractive prospect to amplify the antineoplastic action of metalꢀ as described for cell viability experiments (2 mM MES, 6 h of
based prodrugs in a locoregional manner. This is particularly treatment plus 42 incubation). Analysis of cellular
/1 and cisplatin
/
1
F
LUORESCENCE MICROSCOPY ASSAYS.
/
1
/
1
h
relevant for platinum anticancer agents, which have some of the morphological alteration was performed using a cellꢀpermeable
poorest absorption properties amongst photoactivatable metal green fluorescent dye from the “Live and Dead Cell Staining
complexes, but are widely tested in preclinical work and nearly Kit” (Abcam) for cell nuclei (green channel) and SYTOX®
indispensable in clinical practice.
AADvanced™ (Invitrogen™) for dead cells (red channel).
In principle, photocatalysis can help expanding the therapeutic Cells were stained following commercial protocols and using
potential of platinum prodrugs. Efficient light activation of PtIV binding or 10 mM PBS buffer at the end of the incubation
complexes through catalysis may help localize the cytotoxic period (48 h). Images were acquired with a Plan Apochromat
effects of Pt drugs, increase their dosing at the tumour target 20× objective and a multiꢀband pass Colibri filter to collect
and reduce their systemic toxicity. Rf (vitamin B2) is a highly fluorescence emission signals. Control experiments in the dark
biocompatible molecule and its capacity to function in a (no light irradiation) were performed on untreated cells and on
bioorthogonal fashion may serve to enhance the selectivity of cells exposed to 30:120 µM Rf
/
1
or 120 µM cisplatin.
PCꢀ3 cells were seeded in 96ꢀ
well plates (2000 cells per well) and treated with Rf (30:120
metalꢀbased drugs by minimizing side reactions.
FLOW CYTOMETRY ANALYSIS.
/
1
µM) and MES (2 mM) as described above. After 48 hours of
incubation, 7 wells for each sample were pooled into cytometer
tubes, cells were washed with 10 mM PBS and stained using
100 ꢁL per tube of the Pacific Blue™ Annexin V/SYTOX®
AADvanced™ flow cytometry kit (Invitrogen™). Early and
late apoptosis were measured using a FACS Canto II (BD
Biosciences) and results analysed using the FlowJo, LCC
software. The PCꢀ3 population was electronically gated based
on the forward and side scatter parameters and the nonꢀsingle
events leaved out based on forward area and height scatter
parameters. Inside this final population, live cells were gated as
negative for both dyes; early apoptotic cells were defined as
Pacific BlueꢀAnnexin V positive cells and SYTOX® negative;
and late apoptotic cells were gated as double positive for both
dyes. Nonꢀlabelled and singly labelled samples were included
as control and as compensation samples, respectively.
Experiments were repeated three times. Besides untreated cells,
Experimental section
M
ETHODS
.
NMR, UVꢀVis and fluorescence characterization of
lightꢀirradiated Rf and controls, PCR and microscopy data,
/
1
photochemical quantum yield by actinometry, setup for cell
work under light irradiation, computational methodology and
instrumentation details are described in the ESI†.
L
IGHT
-
IRRADIATION
EXPERIMENTS
IN
SOLUTION.
Photocatalysis studies on Rf/1 were performed under different
solution conditions (buffers, cell culture medium and in
presence of coꢀreactants). All reactions were carried out in air at
298 K and pH 6, employing an LED light source (λexc = 460
nm, 2.5 mW·cm–2). Reaction kinetics, law rates of the reaction,
turnover frequency (TOF) and total turnover number (TTN)
were calculated by varying both reactants and catalyst
concentration and quantifying via 1H NMR the amount of
photoconverted 1.
CELL VIABILITY STUDIES. The antiproliferative activity of Rf/1
control experiments included cells treated with 30:120 µM Rf
and 120 µM cisplatin in the dark (no light irradiation), and
lightꢀirradiated Rf and alone.
/1
was determined in human prostate cancer cells PCꢀ3 (ATCC) in
the dark and under light irradiation by coꢀadministering Rf and
1
1
at a fixed molar ratio (1:4), and using three different
concentrations (Rf : 10/40 µM, 20/80 µM, 30/120 µM). MES
/
1
was employed in all cell experiments at a final concentration of
2 mM. PCꢀ3 cells were seeded 24 h before the experiment in
96ꢀwell plates with a density of 4000 cells per well and grown
under standard conditions (Ham's Fꢀ12K medium supplemented
with 10% fetal bovine serum and 1% penicillin/streptomycin at
Acknowledgements
This work was supported by the Spanish Ministry of Economy
and Competitiveness (grant CTQ2016ꢀ80844ꢀR and BESꢀ2013ꢀ
065642), the Department of Industry of the Basque Country
(grant ETORTEK). L.S. thanks the MICINN of Spain for the
Ramón y Cajal Fellowship RYCꢀ2011ꢀ07787 and the MC CIG
fellowship UCnanomat4iPACT (grant no. 321791). L.S., F.L.G.
and J.M.R. thank Ikerbasque for funding. We are also grateful
to the members of the European COST Actions CM1105,
CM103 and CM1403 for stimulating discussions.
37˚C, 5% CO2 and 90% humidity). Stock solutions of Rf/1
were added and incubated with cells for 1 h, light irradiated for
1 min at 460 nm (light dose 0.36 J·cm–2), and then incubated for
other 6 h. Finally, cells were washed in fresh medium and
grown for other 42 h. The Sulforhodamine
colorimetric assay was used for cell density determination. As
controls, Rf and were tested under identical conditions, while
B (SRB)
1
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