Mitochondrial targeted osmium polypyridyl probe shows concentration dependent uptake, localisation and mechanism of cell death

Article abstract of DOI:10.1039/c9dt02967b

A symmetric osmium(ii) [bis-(4′-(4-carboxyphenyl)-2,2′:6′,2′′-terpyridine)] was prepared and conjugated to two mitochondrial-targeting peptide sequences; FrFKFrFK (r = d-arginine). The parent and conjugate complexes showed strong near infra-red emission c

Full text of DOI:10.1039/c9dt02967b

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DOI: 10.1039/C9DT02967B
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PAPER
Mitochondrial Targeted Osmium Polypyridyl Probe Shows Concentration
dependent Uptake, Localisation and Mechanism of Cell Death.
Karmel Sofia Gkika, Aisling Byrne and Tia E. Keyes*
Received 00th January
20xx,
A symmetric Osmium(II) [bis- (4’-(4-carboxyphenyl)-2,2’ :6’, 2”- terpyridine)] was prepared and
conjugated to two mitochondrial-targeting peptide sequences; FrFKFrFK (r= D-arginine). The parent
and conjugate complexes showed strong near infra-red emission centred at λmax 745nm that was
modestly oxygen dependent in the case of the parent and oxygen independent in the case of the
conjugate, attributed in the latter case, surprisingly, to a shorter emission lifetime of the conjugate
compared to the parent. Confocal fluorescence imaging of sub-live HeLa and MCF 7 cells showed
the parent complex was cell impermeable whereas the conjugate was rapidly internalised into the
cell and distributed in a concentration dependent manner. At concentrations below approximately
30 µmol, the conjugate localised to the mitochondria of both cell types where it was observed to
trigger apoptosis induced by the collapse of the mitochondrial membrane potential (MPP). At
concentrations exceeding 30 µmol the conjugate was similarly internalised rapidly but distributed
throughout the cell, including to the nucleus and nucleolus. At these concentrations, it was
observed to precipitate a caspase- dependent apoptotic pathway. The combination of
concentration dependent organelle targeting, NIR emission coincident with the biological window,
and distribution dependent cytotoxicity offers an interesting approach to theranostics with the
possibility of eliciting site dependent therapeutic effect whilst monitoring the therapeutic effect
with luminescence imaging.
Accepted 00th January
20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
species that are upregulated in cancer cell mitochondria to
achieve preferential targeting of cancer cells or accumulate
within the organelle matrix environment depending on
Introduction
Mitochondria are the centres of cellular metabolism and like
the nucleus are repositories for DNA in eukaryotic cells.
Mitochondrial dysfunction, which often arises from mutation
to mitochondrial DNA, is associated with numerous disease
states including inflammatory diseases and cancer.1,2
Consequently, molecular targeting to the mitochondrial has
attracted significant attention, both in the fields of
imaging/sensing and in the field of medicinal
chemistry/therapeutics.3,4 Cancer cells differ from normal
cells in terms of their energy metabolism, ATP production,
reactive oxygen species levels and present many dysfunctions.
Therefore, targeted agents may be driven to interact with
compound design and overall charge. A key role of
mitochondria is ATP production driven by a membrane-based
proton pump which generates an electrochemical gradient.
This transmembrane electrical potential gradient, known as
ΔΨm, is between -80 and -180 mV. Depolarization of the inner
membrane of the mitochondrion typically results in loss of
ΔΨm, due to release of proteins triggering apoptosis. Small
molecules such as silver (I) complexes, pancratistatin alkaloid
or rhodamine 123 are known to penetrate the mitochondria
where they induce apoptosis via depolarization of the
mitochondrial membrane potential (MMP).5 Metallo-
anticancer therapeutic agents have attracted interest since
the breakthrough with cisplatin and DNA interaction in
1978.6,7 Although cisplatin is used in cancer treatment, it is
inevitably limited by resistance phenomena and several side
effects such as nephron- and neuro-toxicity owing to the
mode of action of the drug.8 Ruthenium (III) compounds of
a. School of Chemical Sciences, National Centre for Sensor Research, Dublin City
University, Glasnevin, Dublin 9, Ireland. E-mail: tia.keyes@dcu.ie
†Electronic Supplementary Information (ESI) available: Spectra of parent
complexes, lifetime decays, oxygen studies, 1H NMR analysis, HPLC analysis
See DOI: 10.1039/x0xx00000x
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similar structure to cisplatin have been investigated for their ligand field strength of tpy, that facilitates populVaietwioAnrticolefO3MnlinCe
DOI: 10.1039/C9DT02967B
anticancer activity including some that have been clinically states from the MLCT states at room temperature. This leads
developed: KP10199, NKP1339 (IT- 139)10,11 and NAMI-A12,13
.
to rapid nonradiative decay and relatively poor photostability,
Although more inert toward ligand substitution, osmium- which in the context of the cellular environment, will be
based compounds have been shown to induce cell death via exacerbated at temperatures typically used for cell culture.
several pathways including inhibitory activity against kinases Hanan, Campagna and co-workers, have each reported clever
14,15, endoplasmic reticulum stress and DNA damage16–18 and strategies for improving the photophysical properties of Ru(II)
redox dependent activation in targeted mitochondria of terpy complexes through extension and rigidification of the
ovarian cancer cells.19 Work by Sadler, Dyson and Keppler and terpy ligand.39–41 Nonetheless, in contrast to biscoordinated
their respective groups have revealed the potent ligand systems, osmium terpyridine complexes offer
antiproliferative activity of organometallic osmium significant advantages over their ruthenium analogues in
complexes.7,19,20 More recently,21 the capability of osmium terms of quantum yield, photostability and emission
polypyridyl complexes in imaging and in phototherapy has wavelength.42 We previously reported that in Ruthenium and
started to be explored.22–26 These studies emphasize that Os(II) tris-ligand systems coordinated to polyarginines, Os(II)
along with Ru- complexes, osmium structures can also offer showed superior uptake/permeability, and notably lower
additional coordination chemistry and rich redox and cytotoxicity than their ruthenium analogues. We were
photochemistry leading to alternative pathways for inducing interested to explore the performance of Os(II)terpy
antiproliferative effects.27,28 We have focused on the complexes in this regard to understand if they can be driven
development of peptide conjugated metal complexes capable to organelles using peptides exploited in ruthenium
of organelle selective targeting.29–31 The importance of complexes and also to exploit the symmetric nature of the
efficient localization for multimodal use of transition-metal terpy-like ligands to conjugate two peptides to a single
complexes has been highlighted by MD Ward in a recent complex.
publication
concerning
mitochondria-targeted
Ir(III)
Herein, we report on the preparation of a symmetric Osmium
(II)- terpyridine conjugated to two mitochondrial targeting
peptides [Os(tpybenz-Ahx-MPP)2]8+. We examined its
photophysical behavior, uptake and toxicity in live cells and
compared behavior to its parent. The conjugate shows
precision targeting to the mitochondria was relatively
cytotoxic. The effect of the OsII MPP probe on the
mitochondrial membrane potential was studied in HeLa and
MCF 7 cells and was found to be cytotoxic. Interestingly,
however, the mechanism of cytotoxicity changed with
concentration as distribution of the probe changed. This to
our knowledge is the first metal complex baring two
mitochondrial penetrating peptides (MPP) to be studied in
cells and first MPP-driven Os (II) complex.
complexes.32 Luminescent mitochondrial or nuclear- targeting
metal complexes can act as real-time imaging/sensing probes
and as therapeutic/theranostic agents.33–35 Although emission
quantum yields are frequently lower, osmium polypyridyl
complexes share many photophysical advantages with their
ruthenium (II) analogues with the additional benefits of NIR
emission maxima in spectral region coincident with the
biological optical window and they exhibit outstanding
photostability. Consequently they may be good alternatives
to their ruthenium analogues as imaging probes because of
their photostability.36 To date there have been no examples
of terpyridine-based ruthenium or osmium complexes applied
in the context of cell imaging or therapy although such
complexes have structural advantages compared to
tris(bidentate) ligand bound complexes as they do not form
stereoisomers. While stereoisomerism tends not to affect
photophysical properties they can, in the biological context,
affect recognition, which in turn can affect cytotoxicity as
exemplified recently by Keene et al.37 Furthermore, terpy
ligands offer stepwise coordination permitting spatial
separation of functionality at the 4 positions. In contrast to
other polypyridyl ligands, terpyridine-based osmium
complexes tend to be more luminescent than their ruthenium
analogues. For example, whereas prototype [Ru(terpy)2]2+
barely emits at room temperature [Os(terpy)2]2+ emits
relatively strong.38 This has been attributed to relatively weak
Materials and Methods
Materials
All chemicals and reagents, cell culture media and
corresponding components were purchased from Sigma
Aldrich (Ireland) and were used as received. The MPP peptide
sequence was purchased from Celtek Peptides, Franklin, USA.
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Co-localising dyes were purchased from Life Sciences and solution was transferred to a large beaker and acidified to pH
View Article Online
Resazurin agent from PromoKine.
2 by addition of 1M hydrochloric acidDwOIh: i1l0e.10s3ti9r/rCin9DgTf0o2r96170B
minutes. The resulting white precipitate was collected by
vacuum filtration, rinsed with ice water and allowed to dry
overnight yielding pure white solids of tpybenzCOOH (297mg,
73%)
Instrumentation
1H and 13C NMR spectra were recorded on a 400MHz Bruker
Spectrophotometer and processed and calibrated against
solvent peaks using Bruker Topspin (v2.1) software. High
Resolution LCMS with ESI was performed at the Mass
Spectrometry Facility, NUI Maynooth. Thin layer
chromatography (TLC) was performed on glass silica gel
(Merck, 250μm thickness) or C18 plates (Sorbent
1H NMR (400MHz, TFA-d): δ (ppm) 9.20 (dd, 2H, J= 5.2Hz), 8.98
(d, 2H, J= 0.8Hz), 8.90- 8.85 (s, dd, 4H, J*), 8.42 (dd, 2H, J=
1.2Hz, 9.2Hz), 8.28 (dd, 2H, J = 6.4Hz), 8.04 (dd, 2H, J = 1.2,
9.6Hz). 13C NMR (600MHz, TFA-d): 154.44, 148.33, 147.49,
146.65, 142.49, 140.64, 131.46, 130.63, 128.21, 127.36.
Technologies, 250μm thickness).
Analytical HPLC was
Preparation of [Os(4'-(4-Carboxyphenyl)-2,2' :6', 2''-
terpyridine))2][PF6]2 , [Os(tpybenzCOOH)2][PF6]2
performed on a Varian 940-LC with a Photometric Diode Array
(PDA) detector for peak detection monitoring 280 nm and 490
nm channels. Gradient elution was applied, using 0.1% v/v
TFA in MeCN and deionised water using HICHROM C18
column (4.6 x 250mm). The mobile phase was of HPLC grade
quality, filtered and purged with nitrogen prior to use. Prior
to sample injection, samples were filtered (0.8μm pore size).
Typical chromatographic run-times were 15-20 minutes at a
flowrate of 1ml/min.
To a suspension of tpybenzCOOH (217mg, 0.615mmol) in
deaerated hot ethylene glycol (20ml) was added OsCl3.3H2O
(91.2mg, 0.308mmol), and the reaction was allowed to reflux
for 4d under N2. Reaction progress was monitored using TLC
(MeCN /H2O/20% KNO3; 80:20:1). Following cooling at room
temperature, a saturated aqueous solution of NH4PF6 was
added. The dark precipitate was filtered and washed with
water and dried with diethyl ether. Column chromatography
on silica using the aforementioned mobile phase gave a dark
maroon solid (100mg, 27.3%)
Electronic absorption spectra were acquired on a Jasco V670
UV/vis NIR spectrophotometer using a quartz cuvette with a
pathlength of 1 cm. Fluorescence spectra were collected on a
Varian Cary Eclipse Fluorescence Spectrofluorometer with
background correction. All photophysical measurements
were performed at room temperature (293 K). Luminescent
lifetime data were acquired up to 10,000 counts using a Time
Correlated Single Photon Counting (TCSPC) system by
1H NMR (400MHz, DMSO-d6): δ (ppm) 9.55 (4H, s), 9.10 (4H,
d, J=7.2Hz), 8.60 (4H, J=6.8Hz), 8.31-8.36 (m, 4H), 7.96 (q, 4H,
7.6Hz), 7.46 (4H, d, J= 5.6Hz), 7.23 (q, 4H, 6Hz). HR-MS(ESI-
TOF) m/z: calculated for C44H30N6O4Os [M2+]: 449.0966; found:
449.0529.
PicoQuant with laser excitation source
a 450 nm.
Measurements were performed in triplicate and PicoQuant
NanoHarp software was used for data analysis and fitting.
Preparation of Os(II) bis- peptide conjugates:
[Os(tpybenz-Ahx-MPP)2].2(Cl-), OsII MPP
Preparation of 4’- (4-Carboxyphenyl)-2,2’ :6’, 2’’- terpyridine
, (tpybenzCOOH)
[Os(tpybenzCOOH)2][PF6]2 (4.46mg, 1equiv) was weighed out
directly into a glass vial followed by the addition of PyBOP
(15.6mg, 8equiv), DIPEA(40equiv) and qualitative transfer of
MPP peptide (20mg, 4equiv) with DMF (700μl). The mixture
was allowed to stir at room temperature in dark overnight.
4-formylbenzoic acid (179 mg, 1.2 mmol) was dissolved in
stirring CH3OH (8ml) followed by the addition of 2-
Acetylpyridine (289 mg, 2.4 mmol). The mixture was allowed
to stir for 5 minutes followed by the slow addition of 15%
potassium hydroxide (7.2ml) and conc. ammonium hydroxide The reaction mixture was added dropwise to stirring
(0.8ml) resulting in a bright green solution which turned saturated NH4PF6 (aq) to obtain dark brown solids that were
yellow over time. The mixture was allowed to stand at room collected via vacuum filtration, washed with water and dried
temperature for 3 days. The yellow/white emulsion that with diethyl ether. The solids were dissolved in minimal
formed was filtered off and washed with cold chloroform acetone and added to
a
tetrabutylammonium
(2x2ml) and cold methanol/water (1:1) to yield a white chloride/acetone solution in order to obtain the chloride salt
product which was suspended in methanol/water (80:20) and of the conjugate. The solids were washed with plentiful
sonicated at 35 °C until a yellow solution was obtained. The acetone and dried with diethyl ether yielding OsII MPP. Purity
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of the conjugate relative to the parent complex was confirmed added in triplicate at the following final concentVriaewtiAornticsle: O1n5li0ne,
DOI: 10.1039/C9DT02967B
by analytical Reverse Phase-HPLC and High-Resolution LCMS. 100, 50, 25, 10, 1 and 0.1 μΜ. Control samples were prepared
with 1% and 0.5% DMSO. Following 24 h incubation of the
1H NMR (600MHz, MeOH-d4): δ (ppm) 9.35 (s, 4H), 8.89 (d,
complex, 10 μl of Resazurin reagent was added to each well
4H), 8.40 (s, 8H), 8.21-7.88 (m, 7H), 7.46 (m, 4H), 7.22 (m,
and incubated for 7h in the dark at 37°C. The Alamar Blue
40H), 4.58-3.31 (m, 31H), 3.23-2.81 (m, 20H), 2.34-2.12 (s,
47H), 2.08-1.06 (m, 64H). HR-MS (+)-MALDI m/z: calculated
for C180H230N42O22Os indicative of [M-MPP5++Cl-]: 739.7249,
assay was used to estimate viable cells based on the
absorbance measured at 570nm with a background measured
at 600nm using a Tecan 96-well plate reader. The cytotoxicity
study was carried out in triplicate.
found: 739.4184.
Photophysical Methods
Confocal luminescent Imaging
Stock solutions (1mM) of the Os (II) parent and conjugate
HeLa cells were seeded at 1.5 x 105 cells in 35mm glass-
compound were prepared in MeCN or DMSO respectively and
bottom culture dishes (Ibidi, Germany) of 2mL total volume.
diluted in PBS for the preparation of working solutions. For
Cells were allowed to grow for 48h at 37°C at 5% CO2. The
oxygen measurements, the working solutions were purged
growth medium was removed, and specific concentration of
with Nitrogen for 15minutes for de-aerated conditions and
the complex was added and allowed to incubate for 4h at 37°C
at 5% CO2 in the dark. The dye/media solution was removed,
and cells were washed with supplemented PBS (1.1mM MgCl2
were allowed to re-aerate over time. The [O2]/ μmol L-1 was
measured using a PreSense Oxygen probe. The emission
spectra and lifetimes were recorded at O2 saturated and de-
and 0.9mM CaCl2). For live cell imaging, cells were directly
aerated conditions. Photostability studies were performed
imaged using a Leica TSP DMi8 confocal microscope (100X oil
using a fan-cooled 150 W Orwell Xenon-Arc lamp on solutions
immersion objective lens) with a heated stage at 37°C. OsII
of Os-complex in PBS pH 7.4. All photophysical studies were
MPP was excited using a 490 nm white light laser and the
emission range was set to 650 and 800 nm. DRAQ7, a nuclear
carried out at room temperature.
staining dye was added (3μM) to distinguish intact live cells
from permeabilized/ dead cells. The 633 nm laser was used
Cyclic Voltammetry
to excite DRAQ 7 and emission was collected between 635-
800nm. MitoTracker Deep Red, a cell permeable probe used
to selectively stain mitochondria, was excited at 644nm and
emission was collected between 655-720 nm.
A CH 660 electrochemical analyzer was used to record cyclic
voltammetry (CV). Stock solution of the complex was
prepared as 1mM/ 0.1 M TBAPF6 (supporting electrolyte) in
anhydrous acetonitrile. The electrochemical cell used a
Ag/AgCl reference electrode, Pt- wire counter and glassy
Mitochondrial Depolarization Assay
carbon working electrode.
CV measurements were
performed in triplicate using positive scan polarity and 5mV
s-1 scan rate.
The MitoPT TMRE Assays (ImmunoChemistry Technologies)
were carried out on HeLa and MCF 7 cells. Cell populations
were cultivated at 5 x 105 cells/ mL and MitoPT spiked
suspensions of >3 x 106 cells/mL in 100μl/ well aliquots were
prepared. The following control populations were prepared
for each cell line: (Α) negative control 1 (non-treated) (B)
negative control 2 DMSO (100μM/ 1h), (C) positive control
CCCP (20μM/ 1 h). Experimental populations were exposed to
OsII MPP at concentrations and incubation periods indicated
by confocal imaging. Samples were subsequently incubated
with MitoPT TMRE dye for 30 minutes at 37°C and washed.
Aliquots were analyzed in triplicate (3 x 100 μl) in a black
bottomed 96-well plate using Tecan Plate fluorescence plate
reader set at 540nm excitation and 574 nm emission. The
amount of orange fluorescence from TMRE was measured as
an indication of metabolically stressed cells and mitochondrial
depolarization. Data were quantified and expressed as the
Cell culture
Minimum Essential Medium Eagle (MEME); supplemented
with 10% foetal bovine serum and 1% penicillin/ streptomycin
and L-Glutamine (2 mM) was used as HeLa cell culture media.
Dulbecco’s Modified Eagle Medium (DMEM); supplemented
with 10% foetal bovine serum, 1% penicillin/ streptomycin
and L-Glutamine (2mM) was used to culture the MCF 7 cell
line. Cells were grown at 37°C with 5% CO2 and harvested or
split at 90% confluency (using 1X Trypsin for 5 min at 37°C).
Cytotoxicity
HeLa cells were seeded in a 96- well plate in 100μl media at
104 cells/ well for 24h at 37°C under 5% CO2. OsII MPP was
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percentage cell viability decline based on loss of
mitochondrial membrane potential relative to the control cell
populations.
View Article Online
DOI: 10.1039/C9DT02967B
Results and discussion
Caspase Activity
Polycaspase
FAM-FLICA
assays
(ImmunoChemistry
Synthesis
Technologies) were carried out on HeLa and MCF 7 cells. Cells
were cultivated at 3 x 105 cells/well. The following control
populations were prepared for each cell line: negative control
1 (non-treated) (B) negative control 2 DMSO (100μM/ 1 h) (C)
positive control (staurosporine 1μΜ/ 3 h). Experimental
populations were exposed to OsII MPP at specific
concentration and incubation times based on confocal
imaging findings. Samples were subsequently spiked with 30X
FAM-FLICA for 45 minutes at 37°C. The loss of non-adherent
cells during washing was accounted for by spinning down the
overlay media and recombining the washed cell pellets with
the overlay buffer prior to analysis. Samples were analyzed in
triplicate (3 x 100 μl) in a black bottomed 96-well plate using
Tecan Plate fluorescence plate reader set at 488 nm excitation
We exploited a tridentate, terpyridine-based ligand to
facilitate the synthesis of an achiral Os (II) parent complex
which, with carboxyl termini, allow for the bis-conjugation of
the complex without the formation of multiple isomers. The
synthetic route, and the molecular structures of the ligands
and complex are summarized in Scheme 1. TpybenzCOOH
was prepared according a modification of a previously
reported syntheses.43,44 And, [Os(tpybenzCOOH)2]2+ was
synthesized by modifying a procedure reported for the
synthesis of another tridentate Os(II)-complex.45 Peptide
conjugation to [Os(tpybenzCOOH)2]2+ was accomplished by
amide coupling aided by PyBOP/DIPEA to yield bisconjugated
[Os(tpybenz-Ahx-MPP)2]8+.35,46 The MPP sequence used is a 8-
N
N
O
N
H
N
Os2+
N
N
O
OH
HO
O
OsCl3.3H2O
N
N
N
(i) tpybenzCOOH (2.2 equiv);
Ethylene Glycol; N2
HN
8+
O
H2N
+H2N
NH
NH
O
O
(ii) MPP(4equiv), PyBOP (8eq),
DIPEA (40eq); DMF, stir RT,
24 h in dark
H2N
HN
+H3N
HN
O
O
O
O
N
H
NH
O
NH
HN
O
MPP
NH
HN
N
N
NH2
+H2N
HN
NH
O
Os2+
N
N
O
O
N
N
Scheme 1: Synthetic scheme for the preparation of OsII MPP conjugate complex
and 520 nm emission. Poly caspase activity was monitored by amino acid mitochondrial localization sequence FrFKFrFK (r=
the increase of relative fluorescence units (RFU) of the green D-arginine) originally reported by Kelley et al47,48 which we
fluorescent signal in the apoptotic cell populations. Data were have found to be highly effective in driving metal complexes
quantified and expressed as the percentage of apoptotic cells to the mammalian mitochondria.49 The lysine (K) and arginine
based on the positive control cell populations.
residues provide positive charge and phenylalanine (F)
imparts lipophilicity.
1H NMR spectroscopy, Mass
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recorded at 25μM (PBS Buffer pH 7.4) under aerated and deaerated
spectrometry and HPLC analysis confirmed the structure and
purity of the parent complex and peptide conjugate. RP-HPLC
in absence of gradient confirmed formation of the complex
and conjugate with well resolved peaks with retention time of
2.33 min for the parent complex and 3.13 min for the
conjugate with no evidence for free peptide or unreacted
parent in the latter case (ESI). With gradient elution, using
0.1% v/v TFA in MeCN and deionised water (Table 1) the
retention time of the peptide conjugate was extended to 8.30
min but showed band broadening with peak splitting
tentatively attributed to ion-pairing but. The corresponding
chromatograms and UV/vis spectra, collected using diode
array detection, confirm the band only contains metal
complex, with no evidence for free peptide or unreacted
starting complex (shown in the ESI).
View Article Online
conditions with excitation and emission slit widths of 10nm and emission
DOI: 10.1039/C9DT02967B
excitation wavelength of 490 nm.
exponential emission decay but, surprisingly, is somewhat
shorter-lived than the parent complex with a lifetime of 94.1
± 0.4 ns in air saturated PBS that increases only to 110 ± 0.5
ns upon deaeration and a quantum yield of 0.0084 + 0.0005.
The emission spectra were also compared under air saturated
and upon deaerated conditions under nitrogen. As shown,
corresponding to the lifetime data, the emission intensity of
the conjugate does not respond significantly to change in [O2].
Table 2 summarizes the photophysical properties of the
parent and conjugate complex. This behaviour contrasts with
our previous reports on ruthenium polypyridyl peptide
conjugates, including complexes bound to the same MPP
sequence.49,35 There, the lifetime of emission invariably
increased in the peptide conjugate compared to the parent.
The origin of the decrease with the osmium complex is
unclear, as the thermodynamics of electron transfer seem to
preclude quenching so the effect may be due to steric strain
exerted on the terpy ligand in the conjugate, that increases
non-radiative decay. Nonetheless, the effect is relatively
weak, and the osmium peptide conjugate remains sufficiently
luminescent for imaging, without the complication of being a
significant generator of singlet oxygen. As photostability is an
important issue in bioimaging, this was evaluated for
[Os(tpybenzCOOH)2]2+ in PBS buffer by monitoring its
absorbance spectrum during visible irradiation. Following 3
hours continuous irradiation by a 150 mW arc lamp (using a
>400 nm cut off filter) less than 5% photodegradation had
occurred (ESI†). This renders the 3MC state thermally
inaccessible for Os(II) complexes in contrast to their Ru(II)
analogues, particularly those of terpyridine complexes, that,
as described tend to exhibit poor photophysical properties
and photoinstability.
Photophysics and Electrochemistry
The absorbance and emission spectra of the peptide
conjugate, OsIIMPP, is shown in Fig. 1, analogous
optical/photophysical behaviour was observed in the parent
complex [Os(tpybenzCOOH)2]2+ (ESI†). The Os(dπ)-
tpybenzCOOH (π*) metal to ligand charge transfer transitions
are observed at λmax 490 nm (1MLCT) and 663 nm (3MLCT).
Excitation at 490 nm into the singlet transition of the parent
complex results in a relatively intense emission centered at
750 nm, and in aerated acetonitrile the quantum yield was
determined as 0.0115 ± 0.0012. Under aerated conditions,
the parent complex exhibits a lifetime of 129.9 ± 0.2 ns in
aqueous PBS (pH 7.4) which is increased to 183.3 ± 0.3 ns on
deaeration at room temperature. The photophysical data for
the parent are consistent with those reported for a related
phenyl derivative [Os(phtpy)2]2+ albeit with red-shifted
emission maxima and modification to lifetime attributed to
the carboxylate substitution of the phenylterpyridine ligands
here.45 The peptide conjugate, OsII MPP exhibits mono-
Figure 1: Absorbance and emission spectra of OsIIMPP. Spectra were
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View Article Online
DOI: 10.1039/C9DT02967B
Table 1 Summary of photophysical properties of Os(II)- complexes in MeCN and PBS under aerated and deaerated conditions.
τ /ns ± SD
λabs (ε)/
Compound
Solvent
CH3CN
λem /nm
nm (x 104 M-1 cm-1)
Aerated
120 ± 0.2
Deaerated
194.6.1 ± 0.7
[Os(tpybenzCOOH)2]2+
404(0.83), 490 (2.54),
642(0.51), 670 (0.61)
750
418 (1.90), 505(2.67),
648(1.32), 679(1.54)
745
746
129.9 ± 0.2
94.1 ± 0.4
183.3 ± 0.3
PBS
PBS
[Os(tpybenz-Ahx-MPP)2]8+
418(0.26), 505(0.47),
648(0.14), 679(0.15)
110 ± 0.5
incubation, highly localized emission was observed from small
structures thought to be the mitochondria. The distribution of
the dye remained unchanged over the next 1 to 2 hours but
as shown in Fig. 3, by 4 hours, morphological changes to the
cells were evident including plasma membrane blebbing and
retraction of pseudopods, indicating cell death by apoptosis.
During this process, the probe started to leach from the
mitochondria and at cell death (confirmed by DRAQ7 assay),
the probe was evident in the cytoplasm attributed to loss of
integrity of the mitochondrial membrane. At higher
concentrations (Fig. 4, 50μM) the uptake dynamics and
distribution were significantly different. Uptake was far more
rapid and complete in under 1 hour. Confocal Z- scan shows
(Fig. 4A) that at higher concentrations OsII MPP was no longer
confined to the mitochondria but present throughout the
cytoplasm where it had penetrated the nuclear envelope and
localised in the nucleoli. DRAQ 7 was applied to cells pre-
incubated with the osmium conjugate to assess cell viability.
DRAQ 7 is a far-red fluorescent probe which only stains the
nuclei of damaged or dead cells. As shown in Fig. 4B, nuclei
staining in blue shows that DRAQ 7 has entered the nuclei of
essentially all cells indicating extensive cell death. Uptake of
the dye and cell blebbing was observed when cells were
incubated with complex above 40μM. This was surprising
given the stability of the probe and lack of oxygen dependent
emission. As they may offer therapeutic prospects we carried
out more detailed investigation into the origin of the
cytotoxicity.
Electrochemistry of the parent complex is shown in Fig. 2. The
complex shows well-behaved electrochemistry with
a
reversible anodic process attributed to the OsII/OsIII oxidation
at E1/2 656 mV versus Ag/AgCl in CH3CN/ nBu4NPF6. The
observed photostability, large stokes shift and modest O2
quenching, marked these compounds as potentially attractive
for cellular imaging applications. In particular, since in
comparison to another reported Os(II) mono- conjugate,
applied to cell imaging, the quantum yield of this conjugate is
significantly higher (φ = 0.0025 ± 0.0008).46
Figure 2: Cyclic Voltammogram (n=3) of 1mM [Os(tpybenzCOOH)2]2+. Sample
in deaerated CH3CN containing 0.1M nBu4NPF6. Scan rate 50mV s-1.
Cell Uptake studies
The uptake of [Os(tpybenzCOOH)2]2+ and OsII MPP were
evaluated in two cell lines: HeLa and MCF, as a function of
conjugate concentration between the range 5μM and 50μM.
While the parent complex does not permeate the cell
membrane across this concentration range (see ESI†), the
peptide conjugate is taken up by the cells at 30μM within 1 to
2 hours incubation in the absence of light (Fig. 3). Within 2h
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OsIIMPP 30μM
t120 min
View Article Online
DOI: 10.1039/C9DT02967B
Control
t60 min
t 4 h
Figure 3: Confocal luminescence images of OsΙΙ ΜPP in live HeLa cells where the conjugate and overlay channel are shown. Cells were incubated in the
absence of light with 30μM for: 60 min, 120 min and 4 hr. The bis-MPP complex was excited using 490 nm white light laser and the emission was collected
between 590 and 800nm.
or thermally- activated process. In contrast, we observed
strong confinement of the dye below 30μM to the
mitochondria and distribution does not change under
irradiation. Distribution is found to change over extended
intervals where coincident with DRAQ 7 permeation, the
probe is found to leach into the cytoplasm and around the
Figure 4: Confocal imaging: (A) OsIIMPP at increased concentration (50μM) in
HeLa cells following 1hr incubation in the absence of light; z-scan reveals
nucleoli staining (b) co- staining with DRAQ 7 and (c) overlay image
OsII MPP : Co- localization with MitoTracker Deep Red in live
HeLa cells
Co-localization with MitoTracker Deep Red confirmed the OsII
MPP probe localized at the mitochondria at concentrations
below 30μM/ 2 h. Fig. 5 shows separate and super-imposed
images of OsII MPP and MitoTracker Deep Red and the
corresponding line profile scanning in the x-y direction (B).
Absence of nuclear staining by DRAQ 7 (ESI†) confirms viability
of the cells. The line profile across the cell and the
corresponding fluorescence intensity profile confirms co-
localisation of the osmium conjugate and MitoTracker Deep
Red in HeLa cells (Fig. 5B). In a previously reported
[Os(bpy)2(pic-arg8)]10+ conjugate, partial localization in both
mitochondria and lysosomes in CHO cells was observed.46 In
addition, it was observed that the conjugate penetrated the
nuclear envelope of SP2 myeloma and CHO cells via a photo
Figure 5: Co- localization of OsII MPP with MitoTracker Deep Red where: (A)
Confocal imaging of OsII MPP (red), MitoTracker (purple) and merged image
(B) Fluorescence intensity profiles obtained from the line profile across the
cell (Image J). The cells were also co-incubated with DRAQ7 and absence of
8 | J. Name., 2012, 00, 1-3
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nuclear staining confirms cells are live.
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nuclear envelope, potentially the endoplasmic reticulum (ER). death was investigated using the Alamar Blue viVaiebwilAitrtyiclaesOsnalinye.
Whereas this is the first , mitochondrial directed osmium Reduction of resazurin dye to resorufiDnOiIs: 10u.s1e03d9/aCs9DaT0d2i9r6e7cBt
luminophore to be reported, Ruthenium-MPP conjugates indicator of metabolically active cells. Both HeLa and MCF 7
previously reported have shown mitochondrial confinement cell lines presented poor tolerance to the OsII MPP at
following 2 h incubation.35,49. The concentration dependent concentrations above 40μM (see ESI†). This coincides with
uptake observed here was not seen in comparable ruthenium the confocal imaging studies where 40 - 100μM of dye
complexes which exhibited only moderate toxicity in the dark induced cell death within 60 min incubation. HeLa cells were
following 24 h exposure over a range of concentrations. For shown to be more tolerant to OsII MPP at lower
example, HeLa cells were tolerant to the MPP-bridged concentrations (5-30μM), with an IC50 value of 30.61μM, in
dinuclear Ru (II) at 75μM over periods of 4-6 h of imaging and comparison to MCF 7 cells where viability of 35% was
in the case of the Ru-dppz, cell death was induced only under observed upon incubation with the probe. These findings
intense photoirradiation.
confirm that cell death is triggered by OsII MPP in a dose
dependent manner that seems to follow the trend observed
OsII MPP cytotoxic effects: photo-induced or intracellular in the concentration dependent uptake. We evaluated the
triggered process?
mitocondrial potential and caspase activity to understand if
the cytotoxicity observed originates at the mitochondria and
if this switches at higher concentration.
OsII MPP : Photo-toxicity studies in live HeLa cells
Given the phototoxicity of MPP-Ru complexes, phototoxicity
was assessed in order to understand if the morphological
changes observed during confocal imaging originated from
Mitochondrial depolarization assay (Mito PT TMRE) and
Caspase Activity
photo-induced effects. In cells pre-incubated with 30μM OsII In healthy cells, an electrochemical potential is generated
MPP, single cells were selected and irradiated over time with across the mitochondrial membrane due to the redox activity
increasing laser power (from 0.2μW to 0.9μW). As shown in of the mitochondrial electron transport chain.50 During
Fig. 6, no DRAQ 7 was found to enter the nuclei of individual apoptosis, the loss of mitochondrial membrane potential
cells following continuous irradiation for 20 minutes at 490 (MMP) coincides with the opening of the mitochondrial
nm. This confirmed that the is cytotoxicity is not light induced. permeability transition pores, leading to the release of
This is not surprising given the remarkable photostability of cytochrome c into the cytosol, which in turn triggers other
the complex and weak oxygen dependence.
downstream events in the apoptotic cascade. The
mitochondrial membrane potential of cells can be assessed
using the MitoPT TMRE assay. This assay uses a lipophilic and
highly soluble rhodamine-based dye, TMRE, that penetrates
live cells and accumulates within healthy mitochondria
exhibiting fluorescence upon excitation. With loss of
mitochondrial ΔΨm, TMRE is released into the cytosol
resulting in a reduced fluorescence signal. Therefore, TMRE
dye is used to detect apoptotic cell populations and to assess
the mitochondrial membrane potential under varying
conditions.51,52 We performed MitoPT TMRE assays on HeLa
and MCF 7 cells. Cell populations were prepared and then
exposed to the assay conditions (ESI†). Two negative controls
were prepared: a non-exposed cell population and a cell
population exposed to DMSO. A positive control was also
prepared by exposing the cells to CCCP, an agent known to
cause mitochondrial depolarization. Experimental cell
populations were exposed to OsII MPP at a low concentration,
30μM/ 2 h at 37°C, where confocal imaging confirmed
mitochondrial targeting and at at a higher concentration,
100μM/ 1 h at 37°C, where imaging and cytotoxicity studies
Figure 6: Phototoxicity of OsII MPP (30μM) in HeLa cells using excitation with
490nm and scanning using increased laser power. (A, D) Normal imaging
conditions (10% laser power) before scanning, (B, E) after 10 minutes scanning
at 30% laser power and (C, F) additional 20 minutes scanning at 50% laser
power; Observed in blue is the emission of MitoTracker Deep Red due to co-
excitation of MitoTracker Deep Red at 633 nm.
Cytotoxicity
The viability of mammalian cells treated with OsII MPP was
assessed by incubating HeLa and separately, MCF 7 cells, with
the conjugate in the absence of light overnight at 37°C. Cell
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showed wide probe distribution and poor cell viability.
Following exposure to the experimental conditions, cell
populations were spiked with MitoPT dye and incubated for
30 minutes at 37°C. Fig. 7 summarizes the results of the assay.
As expected, the percentage of viable cells decreased
significantly upon exposure to the CCCP agent, validating the
assay. Decreased cell viability was also observed for the
population of cells exposed to OsII MPP 30μM/ 2 h indicating
that cells were undergoing metabolic or apoptotic stress that
can be attributed to collapse of the mitochondrial potential.
Confocal imaging under these conditions had shown
mitochondrial targeting and initiation of blebbing but that
cells were still viable according to DRAQ 7 co-staining. Over
extended exposure collapse of the mitochondrial membrane
potential gradually leads to irreversible mitochondrial
damage triggering cell death and thus leakage of the OsII MPP
probe from the cells. Interestingly, consistent with
concentration dependent localization observed from confocal
imaging, although some mitochondrial depolarization was
also evident for cells exposed to OsII MPP at 100μM/ 1 h, the
number of apoptotic cells due to collapsed ΔΨm, was
significantly lower for OsII MPP 100μM/ 1 h than for 30μM/ 2
h. Therefore, we conclude that the altered localization of the
probe at increased concentrations elicits the cytotoxic effect
from a mechanism different to that observed at lower
concentrations.
View Article Online
DOI: 10.1039/C9DT02967B
Figure 7: Relative Fluorescence Units obtained by MitoPT Assay (n=3)
expressed as a percentage of the negative control. HeLa and MCF 7 cells were
exposed to DMSO (100μM/ 1 h) (negative control 2) and CCCP depolarizing
agent (positive control). Experimental populations were exposed to OsII MPP
under the conditions previously described. All samples were incubated with
MitoPT TMRE for 30 minutes at 37°C and washed. Aliquots were added in a
black 96-well plate in triplicate and analysed by Tecan fluorescence plate
reader set at 540 nm excitation and 574 nm emission. Healthy cells exhibited
a high level of orange fluorescence whereas metabolically stressed cells
(positive control and OsII MPP treated) exhibited a reduced fluorescence
signal indicating MMP depolarization. Illustrated is the percentage cell
viability decline based on loss of mitochondrial membrane potential relative
to the non-exposed (negative control) cell populations.
Caspase Activation assay
In the caspase assay applied here, caspase activity is
monitored via the green fluorescence signal from the FLICA
probe, a carboxyfluoroscein fluorophore with a fluoromethyl
ketone unit that bonds covalently to an active caspase
enzyme. Unbound FLICA diffuses out of the cell and is
removed during the wash steps prior to analysis. Experimental
populations were prepared where staurosporine, known for
caspase-3 activation,53 was used to induce caspase activity
(positive control) in both cell lines. Identical conditions of
OsIIMPP concentration and incubation times were used here
as for the MitoPT Assay. As shown in Fig. 8, OsII MPP at 30uM/
2 h stimulated caspase activity in the MCF 7 cell line which
corresponds with the confocal imaging which showed that
imaging of mitochondrial targeting was possible for HeLa cells
without cell damage whereas MCF 7 cells showed higher
sensitivity to the OsII MPP complex. In addition, a higher
apoptotic percentage was observed for the 100μM/ 1 h probe
incubation. Therefore, according to the MitoPT and FLICA
assays findings, at OsII MPP concentrations exceeding 40 µMl
is a moderate release of apoptotic factors owing to collapse
of ΔΨm but localization of the probe in other regions of the
cell triggers activation of caspase enzymes leading to
Mitochondrial
apoptotic
effectors
released
upon
mitochondrial depolarization can be triggered outside the
mitochondria by initiation of a cascade of caspase activation
leading to apoptosis.5 Given that the cytotoxicity is greater at
higher OsII MPP concentrations but that mitochondrial
depolarization, coincident with localization of OsII MPP to the
mitochondria is more prevalent at lower concentrations we
examined the caspase activity induction ability of OsII MPP in
cells using the FLICA assay to evaluate caspase activity as a
function of probe concentration.
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apoptosis. At lower concentrations (30μM/ 2 h), when the A novel achiral Os (II) complex [Os(tpybenzCOOH)2]2+ was
View Article Online
complex localizes at the mitochondria, this stimulates the synthesized and characterised. BiDscOoI:n1j0u.1g0a3t9i/oCn9DT02967B
a
of
release of mitochondrial apoptotic effectors, owing to mitochondrial penetrating peptide (MPP) to both
depolarization of MMP, and caspase activity is observed to a conjugatable sites of the parent complex was achieved by
lesser extent.
amide coupling reaction. Both parent and conjugate
exhibited relatively intense NIR-emission that coincides well
with the biological window. Excellent photostability and good
quantum yield rendered the probe attractive for cellular
imaging application. However, rather unusually, the conjugate
showed modest reduction of its emission lifetime and
quantum yield compared to the parent.
Cell uptake studies were explored in live HeLa and MCF 7 cells.
While the parent complex was cell impermeable, confocal
imaging and co-localisation studies with MitoTracker Deep
Red showed that the bis-MPP Os (II) system is membrane
permeable and targets the mitochondria at concentrations
below 50μM. With increased concentration, OsII MPP showed
rapid uptake and wider distribution including penetration of
the nuclear envelope and localisation in the nucleoli
structures. Extensive cell death was confirmed with DRAQ 7
staining. Photocytotoxicity studies did not show any evidence
for photo-induced cytotoxic effects. Loss in TMRE signal
indicating mitochondrial depolarization was observed at OsII
MPP 30μM/ 2h where the probe localises solely at the
mitochondria, caspase activity at these conditions was
moderate with increased activity observed in MCF 7 over HeLa
cells. At higher concentrations (100μM/ 1 h), the MitoPT and
FLICA assays showed increased caspase activity and lower
degree of mitochondrial depolarization indicating a switch in
cell death mechanism with delocalisation of the probe.
Figure 8: The percentage of cells showing caspase activity indicative of
apoptosis obtained by FLICA Assay (n=3). Both cell lines were exposed to
DMSO as a second negative control (S.I) and Staurosporine as the positive
control. Experimental populations were exposed to OsII MPP at 30μM/ 2 h and
100μM/ 1 h. All samples were incubated with 30X FLICA reagent for 45
minutes at 37°C followed by two washing steps and re-suspension of spun
down pellets. Aliquots were analyzed in a black 96-well plate by Tecan
fluorescence plate reader set at 488 nm excitation and 520 nm emission.
Healthy cells exhibited minimal green fluorescence whereas apoptotic cells
due to caspase activity exhibited a green fluorescence signal.
It is plausible that this causes initiation of the mitochondrial
(intrinsic) apoptotic pathway leading to cell death but allows
for confocal imaging of the mitochondrial structures between
2
to
3 h incubation period. In contrast, increased
Conflicts of interest
concentrations of the conjugate lead to rapid uptake and wide
distribution of the OsII MPP probe, in both HeLa and MCF 7 There are no conflicts to declare.
cells, which leads to mitonuclear communication and in turn,
release of mitochondrial apoptotic effectors and caspase
Abbreviations
AIF
cascade activation.54,55 Related RuII polypyridyl complexes
have been rerouted from nuclear DNA to mitochondrial
targeting by controlling the delivery and uptake mechanism of
the probes54 thus highlighting the potential of these
complexes in achieving change in activity based on cellular
delivery and localisation.
Apoptosis-inducing factor
Carbonyl cyanide m-chlorophenyl
hydrazone
Dimethysulfoxide
Deoxyribonucleic Acid
Phenylalanine
CCCP
DMSO
DNA
F
IRF
K
Instrument Response Function
Lysine
Conclusions
MLCT
MMP
Metal-ligand charge transfer
Mitochondrial membrane potential
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Sigel, H. Sigel, E. Freisinger and R. K. O. SigVeile,wDAreticle Online
Gruyter, Berlin, Boston, 2018, pp. 141–170.
MPP
PBS
PyBOP
Mitochondria targeting peptide FrFKFrFK
Phosphate buffer saline
benzotriazol-1-yl-
oxytripyrrolidinophosphonium
hexafluorophosphate
DOI: 10.1039/C9DT02967B
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Arginine
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terpyridine
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KSG and TEK gratefully acknowledge the Irish Research Council
Scholarship: IRC Government of Ireland Postgraduate
Scholarship GOIPG/2016/702. This material is based upon work
supported by Science Foundation Ireland under Grant No.
[14/IA/2488] and [18/TIDA/5862]. The Programme for Research
in Third Level Institutions (PRTLI) Cycle 4 and 5 is acknowledged
for instrumentation under the NBIPI and NRF at Dublin City
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Regional Development Fund (ERDF), part of the European Union
Structural Funds Programme 2007–2015. Professor Richard
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‡ Footnotes relating to the main text should appear here. These
might include comments relevant to but not central to the matter
under discussion, limited experimental and spectral data, and
crystallographic data.
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