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Arene ruthenium dithiolatoecarborane complexes for boron neutron
capture therapy (BNCT)
Isolda Romero-Canel
on
a
, Ben Phoenix
b
, Anaïs Pitto-Barry
a
, Johanna Tran
a
,
Joan J. Soldevila-Barreda
a
, Nigel Kirby
c
, Stuart Green
b
,
*
, Peter J. Sadler
a
,
*
,
Nicolas P.E. Barry
a
,
*
a
Department of Chemistry, University of Warwick, Gibbet Hill Road, Coventry CV4 7AL, UK
b
School of Physics and Astronomy, University of Birmingham, Birmingham B15 2TT, UK
c
Australian Synchrotron, 800 Blackburn Road, Clayton, Victoria 3168, Australia
article info
Article history:
Received 6 March 2015
Received in revised form
7 May 2015
Accepted 8 May 2015
Available online xxx
Dedicated to Professor Georg Süss-Fink on
the occasion of his 65th birthday
Keywords:
Arene ruthenium
Carborane
Pluronic
Micelles
Boron neutron capture therapy
abstract
We report the effect of low-energy thermal neutron irradiation on the antiproliferative activities of a
highly hydrophobic organometallic arene ruthenium dithiolatoecarborane complex [Ru(p-cymene) (1,2-
dicarba-closo-dodecarborane-1,2-dithiolato)] (1), and of its formulation in Pluronic
®
triblock copolymer
P123 coreeshell micelles (RuMs). Complex 1was highly active, with and without neutron irradiation,
towards human ovarian cancer cells (A2780; IC
50
0.14
m
M and 0.17
m
M, respectively) and cisplatin-
resistant human ovarian cancer cells (A2780cisR; IC
50
0.05 and 0.13
m
M, respectively). Complex 1was
particularly sensitive to neutron irradiation in A2780cisR cells (2.6 more potent after irradiation
compared to non-irradiation). Although less potent, the encapsulated complex 1as RuMs nanoparticles
resulted in higher cellular accumulation (2.5), and was sensitive to neutron irradiation in A2780 cells
(1.4more potent upon irradiation compared to non-irradiation).
©2015 Elsevier B.V. All rights reserved.
Introduction
Boron neutron capture therapy (BNCT) has raised considerable
interest for the treatment of high-grade gliomas and either cuta-
neous primaries or cerebral metastases of melanoma [1]. This bi-
nary method consists of the nuclear reaction of nontoxic and
nonradioactive
10
B atoms and low-energy thermal neutrons that
produces high-energy
4
He
2þ
a
-particles and
7
Li
3þ
ions. The dissi-
pation of the high kinetic energy of these particles is achieved in a
small distance (less than one cell diameter), which allows accurate
destruction of the targeted cells [2].
Dicarba-closo-dodecarboranes are a class of boron-rich com-
pounds with globular structure and diameter of ca. 1 nm (diameter
of a rotating phenyl) that possess unusual properties, including
high symmetry and remarkable stability [3]. These clusters contain
ten boron atoms; they possess a rather low cytotoxicity and are
extremely stable in biological media. They are well suited to boron
neutron capture therapy [4,5], but also have potential in other fields
of drug discovery, molecular imaging, and targeted radionuclide
therapy [6]. However, effective delivery of boron agents is still a
critical issue which impairs their further clinical development [7].
We have recently discussed how the combination of arene ruth-
enium(II) complexes and carboranes has unexplored potential in
medicine [8]. Such complexes also exhibit unusual chemistry: co-
ordination of the bulky, electron-deficient carborane ligand 1,2-
dicarba-closo-dodecarborane-1,2-dithiolato to an arene-Ru metal
center leads to the isolation of a stable 16-electron complex [Ru(p-
cymene) (1,2-dicarba-closo-dodecarborane-1,2-dithiolato)] (1)[9].
However, since this complex is highly hydrophobic, exploration of
its biological applications is hampered by the lack of solubility in
water [10]. To exploit the chemistry of carborane-containing arene
ruthenium complexes in aqueous solution, and to take advantage of
their unique properties, we have encapsulated the 16-electron
complex 1in Pluronic
®
triblock copolymer P123 micelles (Fig. 1).
We have recently shown that although entrapment of the 16-
*Corresponding authors.
E-mail addresses: Stuart.Green@uhb.nhs.uk (S. Green), P.J.Sadler@warwick.ac.uk
(P.J. Sadler), N.Barry@warwick.ac.uk (N.P.E. Barry).
Contents lists available at ScienceDirect
Journal of Organometallic Chemistry
journal homepage: www.elsevier.com/locate/jorganchem
http://dx.doi.org/10.1016/j.jorganchem.2015.05.011
0022-328X/©2015 Elsevier B.V. All rights reserved.
Journal of Organometallic Chemistry xxx (2015) 1e9
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electron complex 1in Pluronic
®
micelles (RuMs) leads to a reduc-
tion in its anticancer potency towards ovarian cancer cells A2780,
the micelles exhibit enhanced selectivity towards cancer cells
compared to normal cells (up to a factor 8) [11]. This formulation
was fully characterised by using a combination of analytical tech-
niques, including synchrotron small-angle X-ray scattering, high-
resolution transmission electron microscopy, and light scattering
methods [11]. Polymer encapsulation of metal carborane com-
plexes provides the potential for delivering high amounts of boron
to cells which is of interest for BNCT [12]. We report here the effect
of low-energy thermal neutron irradiation on the antiproliferative
activity of both complex 1and RuMs particles in the A2780 ovarian
cancer cell line, and in A2780cisR cisplatin-resistant cancer cell line.
Results
Synthesis and characterisation
The organometallic half-sandwich Ru
II
arene complex [Ru(p-
cymene) (1,2-dicarba-closo-dodecarborane-1,2-dithiolate)] (1)was
synthesised as reported previously [13]. This complex has a pseudo-
octahedral structure, with a
p
-bonded arene occupying 3 coordina-
tion sites, a S-bound chelated dithiolato dicarba-closo-dodecarbor-
ane ligand, and a vacant 6th site (Fig. 1). It is a 16-electron complex
and therefore electron-deficient at the metal [14]. Complex 1is
highly hydrophobic and insoluble inwater [15]. Toachieve dispersion
in water [16], we encapsulated complex 1in the water-soluble
amphiphilic triblock copolymer P123 (poly(ethylene glycol)-block-
poly(propylene glycol)-block-poly(ethylene glycol)) (PEO-b-PPO-b-
PEO), according to a previously reported procedure (Fig.1)[11].
To gain further insight into the structure of RuMs in RPMI cell
culture medium, and to compare the sizes of the assembly in RPMI
versus water at ambient temperature and at 35
C, solutions of
RuMs were analysed by synchrotron small-angle X-ray scattering
(SAXS; Fig. 1). The experimental profiles were fitted using IgorPro
software [17] to a coreeshell spherical micelle model Poly-
CoreShellRatio [18] (PCR) according to a previous procedure for
similar micelles [19]. Some aggregation was observed for all the
samples (high turn at low q values), however the PCR model fitted
excellently for all micellar solutions from 0.2 Å
1
with very low
dispersity parameters (between 0.13 and 0.16, 0 being an ideal
mono-disperse system; Table 1).
Cell testing
We studied the time-dependence of the antiproliferative activ-
ity of complex 1and micelles RuMs and P123Ms (micelles made of
Pluronic
®
copolymers without complex 1) in A2780 human ovarian
cancer cells (Table 2). Cells were exposed for variable times (1, 4, 16,
24, 48 and 72 h) to complex 1(dissolved in 5% dimethyl sulfoxide
(dmso)/95% saline:RPMI and further diluted in cell culture medium
until working concentrations were achieved) or to RuMs micelles
(dissolved in 100% saline:RPMI, further diluted with cell culture
medium to working solutions). After this, drugs were removed and
cells were washed and placed in fresh growth medium for a further
72 h as a recovery period. Cell viability was then assessed using the
sulforhodamine B (SRB) colorimetric assay. Complex 1was found to
be highly potent towards A2780 cells (Table 2), particularly after
24 h of drug exposure (IC
50
170 nM), and it is also 39 more potent
than RuMs micelles, which still exhibit good (micromolar) activity
towards cancer cells.
Since the optimum time for drug exposure was 24 h, we
determined the IC
50
values of complex 1and micelles RuMs in
A2780cisR cells after 24 h of drug exposure. Complex 1was found
Fig. 1. (a) Self-assembly formation of RuMs (purple dots in 1are BeH vertices). (b) and (c) Small-angle X-ray scattering (SAXS) experimental profiles and fitting with spherical
coreeshell micelle model of micelles RuMs at 25 C and 35 C in water and at 25 C and 35 C in RPMI, respectively; 5 mg/mL aqueous solutions. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
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to be more active towards the cisplatin resistant cancer cell line
(A2780cisR), as noted in Table 3, whilst micelles RuMs exhibit
similar cytotoxicity in both cell lines (resistance factor 1.1).
We then investigated the cellular accumulation of ruthenium
from A2780 cells exposed to complex 1or to the RuMs using
equimolar Ru concentrations of 0.5
m
M. For this experiment, cells
were exposed for 24 h and no recovery time was allowed. The
metal content was determined by inductively coupled plasma
mass spectrometry (ICP-MS) after cells had been digested over-
night in concentrated nitric acid. Intracellular ruthenium in sam-
ples with RuMs was 2.5 higher than that from complex 1
(15.6 ±0.3 ng of Ru 10
6
cells for RuMs versus 6.2 ±0.4 ng Ru for
complex 1).
Finally, we investigated whether complex 1and RuMs micelles
induced apoptosis in A2780 cells. For this experiment, we used 24 h
of drug exposure time and no-recovery time. Fig. 2 shows there is
no significant apoptosis after the first 24 h, highlighting the
importance of the recovery time in the mechanism of action of the
ruthenium compounds.
Boron neutron capture experiments
We then studied the antiproliferative activity of complex 1and
RuMs micelles in A2780 and cisplatin-resistant A2780cisR human
ovarian cancer cells, after thermal neutron irradiation. The mea-
surements were performed in the thermal neutron field available at
the Dynamitron accelerator in Birmingham, United Kingdom. This
field has a large epithermal neutron fluence and a low gamma-dose
rate contamination, approximately 1 Gy/h at an accelerator current
of 1 mA. The plates containing the cancer cells incubated with
complex 1and RuMs micelles were sealed in a plastic bag con-
taining culture medium, and the bag was clipped on a holder
submerged in a tank of water (Fig. 3). This water tank provides
further neutron moderation, which results in a peak in the thermal
neutron flux at several centimetres depth. On-line monitoring of
the neutron beam was provided by a pair of fission chambers
(Centronic FC05A/500/U235) embedded in the beam shaping as-
sembly near to the beam port. The relationship between the count
rate in these chambers and the thermal neutron flux at depth in the
water phantom has previously been well characterised via foil
activation measurements [20]. At a proton beam current of 1 mA,
the thermal neutron flux at 20 mm depth in water is
3.60 10
8
cm
2
s
1
.
The water tank was positioned in front of the beam, and the cells
were irradiated for ca. 90 min at a nominal beam current of 600
m
A.
Exact beam-on time in the cell irradiations was controlled based on
the chamber counts to ensure that each irradiation provided an
equal cumulative thermal neutron flux. The integrated thermal
neutron flux was 1.38 10
12
cm
2
at the position of the cells. We
also incubated cells with boric acid (1 mM concentration) as a
positive control.
After 90 min of neutron irradiation, drugs were removed and
cells were washed without recovery or placed in fresh growth
medium for a further 72 h as a recovery period. Cell viability was
then assessed using the SRB colorimetric assay. Fig. 4 shows the
concentration dependence of A2780 and A2780cisR cell-survival
upon incubation of complex 1and RuMs micelles with and
without neutron irradiation, and with and without recovery
(Table 4). Since 72 h recovery time offered the best conditions for
A2780 cells, we investigated the antiproliferative activity of com-
plex 1and RuMs micelles in cisplatin-resistant A2780cisR cells only
after 72 h recovery.
Discussion
Design and stability of complex 1and of the ruthenium micellar
system
Inorganic compounds offer different mechanisms of drug action
depending on the metal used, their structures and redox properties
[21e61]. Thus, they can be utilised for the design of novel drugs in
the treatment of a broad range of diseases [62]. Ruthenium com-
plexes have been recognised as particularly promising drug can-
didates for the treatment of cancer since the beginning of the 1990s
[63]. In 1992 [64], Tocher and co-workers observed an increase of
the hypoxic cell cytotoxicity of metronidazole [1-
b
-(hydroxyethyl)-
2-methyl-5-nitro-imidazole] after coordination to a benzene
ruthenium dichlorido fragment [65]. Since then several groups
have explored the anticancer activity of air-stable and water-
soluble arene ruthenium(II) complexes [66,67]. The combination
of the remarkable properties of half-sandwich complexes with the
unique features of dicarba-closo-dodecarborane clusters results in
Table 1
Physical characteristics of RuMs determined by SAXS at 5 mg/mL in water and RPMI, at 25
C and 35
C.
Parameter Water (25
C) Water (35
C) RPMI (25
C) RPMI (37
C)
Radius core (nm) 7.53 ±0.02 7.32 ±0.02 7.93 ±0.20 7.55 ±0.06
Thickness shell (nm) 0.80 ±0.04 0.60 ±0.03 0.90 ±0.39 1.81 ±0.10
Total radius (nm) 8.33 ±0.06 7.92 ±0.05 8.83 ±0.59 9.36 ±0.16
Dispersity 0.14 ±0.01 0.13 ±0.01 0.13 ±0.02 0.16 ±0.02
Table 2
Time-dependence of IC
50
values for complex 1and RuMS in A2780 ovarian cancer
cells. All experiments included 48 h of pre-incubation time, 72 h of recovery time in
drug-free medium and variable drug exposure times.
Treatment IC
50
(
m
M)
Exposure time 1 h 4 h 16 h 24 h 48 h 72 h
Complex 1>50 >50 20.4 ±0.8 0.17 ±0.02 0.16 ±0.08 0.16 ±0.05
RuMs >50 >50 18 ±2 6.7 ±0.3 5.4 ±0.3 5.2 ±0.5
Table 3
IC
50
values (
m
M), resistance factors ((IC
50
(A2780cisR)/IC
50
(A2780)), and cellular accumulation of complex 1and micelles P123Ms and RuMs for A2780 human ovarian cancer
cells, and A2780cisR cisplatin-resistant human ovarian cancer cells after 24 h of drug exposure.
Compound IC
50
(
m
M) Cellular accumulation (ng of Ru 10
6
cells)
A2780 A2780cisR Resistance factor A2780
P123Ms >100 >100 ee
10.17 ±0.02 0.130 ±0.008 0.76 6.2 ±0.4
RuMs 6.7 ±0.3 7.93 ±0.04 1.1 15.6 ±0.3
cisplatin 1.2 ±0.1 12.4 ±0.3 10.3 e
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interesting new molecules [8]. Applications of these in organo-
metallic synthesis, catalysis, or bioinorganic chemistry, for
example, can be envisaged, although their high hydrophobicity
impairs their further development as anticancer drug candidates.
We have recently shown that the combination of nanotech-
nology tools with medicinal inorganic chemistry has the potential
to offer several advantages for drug formulation and delivery [68].
Control of drug solubility by increasing the aqueous solubility of
highly lipophilic complexes or decreasing the solubility of com-
plexes which might otherwise be rapidly excreted provides a ‘slow-
release’strategy that may engender less toxicity and improve the
therapeutic response than a burst release. Modulation of drug
distribution may also be achieved. The uptake of drugs encapsu-
lated in nanoparticles is likely to depend on the shape, size and
surface recognition of the nanoparticles by cells rather than on the
characteristics of the drug. The nanoparticle might be designed so
that it has vectors on its surface which can target specific cell
receptors as well as having the capacity to encapsulate the drug, so
reducing side effects and limiting attack to target cells or organelles
only. Also nanomedicines may provide multidrug delivery and
theranostic compounds since more than one drug can be encap-
sulated for combination therapy and reporter groups can be con-
jugated onto particles [16].
Synthetic polymer therapeutics are of particular interest in
medicine, due to their synthetic versatility, as well as their tunable
properties [69]. A number of biologically-active polymeredrug
conjugates and polymeric formulations, such as micelles, hydrogels
and polymer-coated nanoparticles, are currently in clinical devel-
opment [70]. Among the most commonly used polymers for ap-
plications in medicine, the ABA Pluronic
®
triblock block copolymers
are particularly suitable for the design of bio-inspired, bio-
engineered and biomimetic polymer nanoparticles [10]. The uti-
lisation of Pluronic
®
block copolymers as drug delivery systems
[71e76], biological response modifiers [77e81], pharmaceutical
Fig. 2. Flow cytometry analysis of A2780 cells exposed to complex 1or RuMs micelles compared to a negative control. FL1 reads Annexin fluorescence and FL2 reads propidium
iodide fluorescence.
Fig. 3. Experimental set-up used for the neutron beam irradiation of complex 1and RuMs micelles and P123Ms in cells. Right: Irradiation chamber; Left: Cell plate in the water tank
positioned in front of the neutron beam.
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Fig. 4. Concentration dependence of A2780 cell survival upon incubation of complex 1(a) without and (c) with 72 h recovery, and of RuMs micelles (b) without and (d) with 72 h
recovery. Concentration dependence of A2780cisR cell survival upon incubation (e) with complex 1and (f) of RuMs micelles, with 72 h recovery.
Table 4
IC
50
values (
m
M), resistance factors ((IC
50
(A2780)/IC
50
(A2780cisR)) of complex 1and micelles P123Ms and RuMs towards A2780 human ovarian cancer cells, and A2780cisR
cisplatin-resistant human ovarian cancer cells after neutron irradiation. Ratio of IC
50
values (non-irradiated versus irradiated) for 1and RuMs in both cell lines.
Recovery time (h) Compound IC
50
(
m
M) IC
50
ratio (/þIrrad)
A2780 A2780cisR A2780 A2780cisR
Non-irradiated Irradiated Non-irradiated Irradiated
010.60 ±0.08 0.55 ±0.04 ee1.1 e
RuMs 8.12 ±0.05 9.70 ±0.03 ee0.8 e
72 10.177 ±0.002 0.140 ±0.003 0.130 ±0.008 0.050 ±0.004 1.3 2.6
RuMs 6.7 ±0.3 4.8 ±0.2 7.93 ±0.04 9.33 ±0.09 1.4 0.8
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ingredients [72,82,83], and steric stabilisers to lyotropic liquid
crystalline particles [84e86], has led to recent advances in
biochemistry [10].
The SAXS analyses demonstrated that RuMs self-assembly is the
same in water and in RPMI media and leads to core-shell micelles
with a core radius of around 7.7 nm, and a shell thickness of around
1.1 nm. Although the thickness of the shell is very thin, the same
model used with no shell did not provide an acceptable fit, which
thus confirms the presence of a shell. It is also anticipated that the
core seen by SAXS is not only composed of the entire core of the
micelle, but also of the part of the PEO corona which is poorly hy-
drated. The shell seen by SAXS is more likely to reflect the part of
the corona which is fully hydrated. Interestingly, the diameters of
RuMs micelles in RPMI and water are similar at ambient temper-
ature and at 35
C.
Antiproliferative activity of the ruthenium systems without neutron
irradiation
We have recently shown that not only does the entrapment of
the 16-electron complex 1in Pluronic
®
micelles lead to a retention
of the anticancer activity of 1, but also that a certain selectivity
between cancer and healthy cells is achieved by the utilization of a
nanocarrier (selectivity factor of 8 for the micelles, compared to 2
for the complex alone) [11]. This might be due to a passive targeting
of cancer cells via the “enhanced permeation and retention”(EPR)
effect [87]. This effect is widely used in oncology since the dis-
covery made by Maeda et al., who in the 1980's demonstrated the
principle of passive targeting of colloidal particles to tumours
[88e90]. EPR is most effective for colloidal material of molecular
weight above 40 kDa and can occur even in the absence of targeting
ligands on nanoparticles [91].RuMs are made of 66 ±4P123
monomers of individual average weight 5800 g/mol, and 59 ±14
complexes 1of molecular weight 441 g/mol [11], so the molecular
weight of RuMs is about tenfold greater than this threshold.
Nonetheless, the size of the micelles is relatively small (ca. 19 nm in
diameter), which may impair the passive targeting of the micelles
via the EPR effect. However, the class of Pluronic
®
copolymers offers
a pool of more than 50 materials with various molar mass ratio
between the PEO and PPO blocks, and there is a wide scope for
adapting this combination of organometallic complexes and Plur-
onic
®
copolymers for designing bigger particles and for increasing
the selectivity factor.
Importantly, the IC
50
value of complex 1remains unchanged
after 24 h of drug exposure (Table 2), while the value for the RuMs
micelles further improves at 72 h. We hypothesise that this could
be related to the release of the complex from the micelles and into
the cancer cells. Furthermore, according to the cellular uptake
studies, the ruthenium accumulation is more efficient with RuMs,
since at the same administered Ru concentration there is a two-fold
greater accumulation of Ru for the micelles compared to the
complex alone (15.6 ±0.3 ng of ruthenium 10
6
cells for RuMs
versus 6.2 ±0.4 ng for complex 1). Finally, formulating complex 1in
polymer micelles also allows their dispersion in water in a manner
suitable for administration to cancer cells (without the need to add
dmso).
Here, we also showed that complex 1is highly potent towards
A2780 cells (Table 2), but is more active towards cisplatin-resistant
A2780cisR cells than towards the parent A2780 cells (0.13 ±0.02
versus 0.17 ±0.01
m
M; resistance factor 0.76), whilst RuMs micelles
exhibit similar cytotoxicity towards both cell lines (6.7 ±0.3 versus
7.93 ±0.04; resistance factor 1.1). These results suggest that both
complex 1and RuMs micelles have a different mode of action from
that of cisplatin, a tendency observed previously for arene ruthe-
nium metal-based drugs [92,93].
Antiproliferative activity of the ruthenium systems after neutron
irradiation
Boron neutron capture therapy is the traditional area for
application of dicarba-closo-dodecarborane molecules in medicine.
This binary method consists of the nuclear reaction of nontoxic and
nonradioactive
10
B atoms and low-energy thermal neutrons that
produces high-energy
4
He
2þ
a
-particles and
7
Li
3þ
ions. The dissi-
pation of the high kinetic energy of these particles is achieved in a
small distance (less than one cell diameter), which allows accurate
destruction of the targeted cells. Therefore, the efficiency of this
therapy depends on the number of boron atoms delivered to cancer
cells, while the selectivity strongly depends on the preferential
accumulation of boron in tumour tissues rather than in normal
tissues [94]. Dicarba-closo-dodecarboranes contain ten boron
atoms; they possess a rather low cytotoxicity and these clusters are
extremely stable in biological media. These characteristics explain
why dicarba-closo-dodecarborane clusters have the potential to be
efficient BNCT agents. However, dicarba-closo-dodecarborane
clusters on their own do not possess the ability to target cancer cells
selectively.
To increase the selectivity of dicarba-closo-dodecarboranes
towards cancer cells and therefore to increase the clinical feasi-
bility of boron neutron capture therapy, various approaches have
been developed. A first strategy is to attach borane clusters to
cellular building blocks. Indeed, most solid tumours are known to
possess a hypervasculature, a defective vascular architecture, and
an impaired lymphatic drainage [95]. Thus, while the normal
endothelial layer surrounding the blood vessels feeding healthy
cells restricts the amount of constituents (amino acids and
nucleic acid precursors for example) necessary for cell replica-
tion, the endothelial layer of blood vessels in diseased tissues
allows an elevated quantity of such nutrients to enter the cells
[6]. Another strategy is to attach the borane cluster to tumour
antibodies that can target specificcelltypes[96].Athird
approach is to use nano-containers such as lipoproteins and li-
posomes [97]. Encapsulation of hydrophilic borane compounds in
aqueous cores of liposomes, or incorporation of boron-containing
lipids in liposome bilayers can lead to a selective delivery of BNCT
therapeutics to tumours.
Here, we have studied the polymer encapsulation of a metal
carborane complex, its antiproliferative activity alone and in poly-
mer micelles, with and without activation by neutrons to assess its
potential for BNCT. Complex 1is highly potent towards cisplatin-
resistant A2780cisR cancer cells, with IC
50
values in the nano-
molar range. When irradiated for 60 min with low-energy thermal
neutrons, the antiproliferative effect of complex 1was 2.6 higher.
This dramatic enhancement of the cytotoxicity of 1after neutron
irradiation unambiguously demonstrates the potential of this
organometallic compound for BNCT, in particular for treating tu-
mours having developed resistance mechanisms toward cisplatin,
one of the most-used drugs in cancer chemotherapy. It is also
apparent from Fig. 4 that the optimum conditions for achieving
enhanced effects from neutron capture activation of the ruthenium
compound is to allow the cells to recover for a period of 72 h after
irradiation. It is known that the antiproliferative effects of neutron
irradiation are not immediate [98], and our results seem to confirm
this delayed effect. Although the IC
50
values with and without
neutron irradiation of RuMs micelles are in a similar range, in all
cases the effect of the neutron irradiation on the antiproliferative
activity of RuMs micelles is the strongest at 1
m
M concentrations of
ruthenium. A dramatic effect of the neutron irradiation on the
potency of the RuMs micelles is observed for the two cell lines (up
to 32% difference in cell survival). Interestingly, this effect is less
important in the antiproliferative activity of complex 1, which
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might be related to the more efficient accumulation of Ru observed
with RuMs compared with complex 1.
Conclusions
BNCT has been investigated as an alternative anticancer therapy
in clinical trials in Japan, Europe, and the United States using
especially sodium borocaptate (Na
2
B
12
H
11
SH). However, selective
and effective delivery of boron agents is still a critical issue. For this
reason it is of central importance to explore new concepts able to
take advantage of these unique pharmacophores. We showed that
the combination of dicarba-closo-dodecarboranes with half-
sandwich complexes of ruthenium formulated in micelles made
of polymers can provide new agents which are potentially useful in
boron neutron capture therapy. Our results demonstrate that the
formulation of complex 1in micelles leads to a two-fold increase in
accumulation of metal complex in cells, and that the effect of
neutron irradiation on the antiproliferative activity of the micelles
is dramatic at a micromolar concentration in ruthenium. Our
strategy is highly versatile, with choice of the metal complex and
polymer used for the self-assembly of the micelles. The natural
abundance of the activate isotope
10
B is only 20% and it will be
interesting in future work to investigate the neutron capture ability
of
10
B-enriched carborane-containing complexes in micelles.
Materials and methods
Materials
The preparation of the complexes [Ru(p-cym)(1,2-dicarba-closo-
dodecaborane-1,2-dithiolato)] (1) was based on a published pro-
cedure [13]. The purity of complex 1was assessed by
1
H NMR
spectroscopy in CDCl
3
and was in accordance with previous reports
[9,11,14]. The preparation of the RuMs micelles was carried out as
previously described [11].
Instrumentation
Inductively coupled plasma-mass spectrometry: Ruthenium con-
tent was determined using an ICP-MS Agilent technologies 7500
series instrument. Calibration curves were prepared using Ru
standard solutions in double-deionised water (ddw) with 3% nitric
acid, ranging between 50 and 0.5 ppb (9 points). Samples were
freshly prepared in ddw with 3% nitric acid. Readings were made in
no-gas mode with a detection limit of 1 ppt.
Small-angle X-ray scattering (SAXS): Measurements were carried
out on the SAXS/WAXS beamline at the Australian Synchrotron
facility at a photon energy of 11 keV. The samples in solution were
in 1.5 mm diameter quartz capillaries. The data were collected at a
sample-to-detector distance of 3.252 m to give a qrange of
0.004e0.2 Å
1
, where qis the scattering vector and is related to the
scattering angle (
q
) and the photon wavelength (
l
) by the following
Equation (1):
q¼4psinðqÞ
l(1)
The scattering from a blank solution (H
2
O or RPMI) was measured
in the same location as sample collection and was subtracted for each
measurement. Data were normalised for total transmitted flux using
a quantitative beamstop detector and absolute-scaled using water as
an absolute intensity standard. The two-dimensional SAXS images
were converted in one-dimensional SAXS profiles (I(q)versus q)by
circular averaging, where I(q) is the scattering intensity. Functions
were used from the NCNR package. Scattering length densities were
calculated using the “Scattering Length Density Calculator”provided
by NIST Center for Neutron Research.
Cell culture
A2780 human ovarian carcinoma cells and its cisplatin-resistant
derived cell line A2780cisR were obtained from the European
Collection of Cell Cultures (ECACC). Both cell lines were grown in
Roswell Park Memorial Institute medium (RPMI-1640) supple-
mented with 10% of fetal calf serum,1% of 2 mM glutamine and 1%
penicillin/streptomycin. All cells were grown as adherent mono-
layers at 310 K in a 5% CO
2
humidified atmosphere and passaged at
ca.70e80% confluency.
In vitro growth inhibition assays.
A) Assays including 72 h recovery time. The antiproliferative
activities of complex 1and RuMs were determined for A2780
and A2780cisR human ovarian cancer cells. Briefly, 96-well
plates were used to seed 5000 cells per well. The plates
were left to pre-incubate with drug-free medium at 310 K for
48 h before adding different concentrations of the com-
pounds to be tested. A drug exposure period of 24 h was
allowed. After this, supernatants were removed by suction
and each well was washed with PBS. A further 72 h was
allowed for the cells to recover in drug-free medium at 310 K.
The SRB assay was used to determine cell viability. IC
50
values, as the concentration which causes 50% cell death,
were determined as duplicates of triplicates in two inde-
pendent sets of experiments and their standard deviations
were calculated.
B) Assays with no recovery time. Experiments were carried out
as described above, with the following modification. After
the 24 h exposure time, drugs were removed by suction, each
of the wells was washed with PBS and the SRB assay was run
immediately. In both cases (assays with and without recov-
ery time) stock solutions of complex 1were prepared by
dissolving the solid in a mixture of dmso (5% v/v) and 1:1
saline:RPMI-1640 (95% v/v), working solutions were ach-
ieved by dilution of the stock with cell culture medium. Stock
solutions of RuMs were prepared similarly but without
dmso. Exact metal concentrations for all stock solutions were
determined using ICP-MS.
BNCT experiments
The in vitro growth inhibition assays were carried out as
described above using A2780 and A2780cisR ovarian cancer cells
with the following experimental modifications. After drug exposure
the 96-well plate was sealed in a plastic bag containing media, and
the bag was clipped on a holder submerged in a tank ofwater which
acts as a neutron moderator (Fig. 2). The water tank was positioned
in front of the beam, and the cells were irradiated for 60 min.
Ruthenium accumulation in cancer cells
Briefly, 1.5 10
6
cells/well were seeded on a 6-well plate. After
24 h of pre-incubation, complex 1and separately RuMs were added
to give final concentrations equal to 0.5
m
M Ru and a further 24 h of
drug exposure was allowed. After this time, cells were washed,
treated with trypsin-EDTA, counted, and cell pellets were collected.
Each pellet was digested overnight in concentrated nitric acid (73%)
at 353 K; the resulting solutions were diluted using double-distilled
water to a final concentration of 5% HNO
3
and the amount of Ru
I. Romero-Canel
on et al. / Journal of Organometallic Chemistry xxx (2015) 1e97
Please cite this article in press as: I. Romero-Canel
on, et al., Journal of Organometallic Chemistry (2015), http://dx.doi.org/10.1016/
j.jorganchem.2015.05.011
taken up by the cells was determined by ICP-MS. These experi-
ments did not include any cell recovery time in drug-free media;
they were all carried out as duplicates of triplicates and the stan-
dard deviations were calculated.
Inductively coupled plasma-mass spectrometry (ICP-MS)
Cellular ruthenium content was determined using an ICP-MS
Agilent technologies 7500 series instrument. Calibration curves
were prepared using Ru standard solutions in double deionised
water (ddw) with 3% nitric acid. Samples were freshly prepared
after nitric acid digestion in ddw to 3% nitric acid dilution.
Induction of apoptosis
Flow cytometry analysis of apoptosis in A2780 cells caused by
exposure to complex 1and RuMs, was carried out using the
Annexin V-FITC Apoptosis Detection Kit (Sigma Aldrich) according
to the manufacturer's instructions. Briefly, A2780 cells were seeded
in 6-well plates (1.0 10
6
cells per well), pre-incubated for 24 h in
drug-free medium at 310 K, after which they were exposed to
either complex 1or RuMs (concentration equal to IC
50
). Cells were
harvested using trypsin and stained using PI/Annexin V-FITC. After
staining, cell pellets were analysed in a Becton Dickinson FACScan
Flow Cytometer. For positive-apoptosis controls, A2780 cells were
exposed for 2 h to staurosporine (1
m
g/mL). Cells for apoptosis
studies were used with no previous fixing procedure as to avoid
non-specific binding of the annexin V-FITC conjugate.
Acknowledgments
We thank the Leverhulme Trust (Early Career Fellowship No.
ECF-2013-414 to NPEB), the University of Warwick (Grant No.
RD14102 to NPEB), the University of Birmingham/EPSRC Follow-on-
Fund (Grant No UOBFOF026 to BP), the ERC (Grant No. 247450 to
PJS), EPSRC (EP/F034210/1 to PJS). We also thank COST Action
CM1105 for stimulating discussions, Dr Magdalena Moss for tech-
nical assistance with the cell culture, the Australian Synchrotron
and the University of Monash for allocation of beamtime on the
SAXS/WAXS beamline and funding.
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