Electrodrugs: An electrochemical prodrug activation strategy

Article abstract of DOI:10.1039/c8cc04151b

The term electroceutical has been used to describe implanted devices that deliver electrical stimuli to modify biological function. Herein, we describe a new concept in electroceuticals, demonstrating for the first time the electrochemical activation of metal-based prodrugs. This is illustrated by the controlled activation of Pt(iv) prodrugs into their active Pt(ii) forms within a cellular context allowing selectivity and control of where, when and how much active drug is generated.

Full text of DOI:10.1039/c8cc04151b

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Electrodrugs: an electrochemical prodrug activation strategy
Daniel J. Norman[a], Eva González-Fernández[a], Jessica Clavadetscher[a], Lulu Tucker[a], Matteo
Staderini[a], Andrew R. Mount[a], Alan F. Murray[b] and Mark Bradley*[a]
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The term electroceutical has been used to describe implanted handles for functionalising the metal centre that can be used
devices that deliver electrical stimuli to modify biological function. to increase lipophilicity or introduce adjuvant bio-active
Herein, we describe
a new concept in electroceuticals, ligands. Activation of these Pt(IV) prodrugs is generally
demonstrating for the first time the electrochemical activation of accepted to be via reduction by biological reductants such as
metal-based prodrugs. This is illustrated by the controlled glutathione, ascorbic acid or cytochrome c, generating the
activation of Pt(IV) prodrugs into their active Pt(II) forms within a active Pt(II) form9. However, the widespread presence of such
cellular context allowing selectivity and control of where, when biological reductants in vivo means selectivity towards
and how much active drug is generated.
cancerous tissue will always be fraught with limited local
control of the Pt(IV)-Pt(II) conversion.
The emerging field of bio-electronic medicine or
electroceuticals – a term coined by GSK in 20131
For our proof-of-concept studies we used a prodrug of
oxaliplatin, a drug currently approved for use in advanced
colorectal cancer and in clinical trials for an array of other
–
encompasses the potential for diseases to be treated through
the use of implantable devices capable of modulating
biological functions using electrical signals2. To date, the focus
of electroceuticals has been the mimicking of natural neural
signals present in living systems. Devices such as pacemakers3
and cochlear implants4 are commonplace but recent
applications of electroceuticals are more ambitious and
include so-called “closed-loop” systems — devices that can
cancers10, 11
.
receive and interpret electrical nerve signals from the body
2
and elicit
a
modulatory effect in response1,
. This
electroceutical approach directs biological function via
electrical stimulation. Here, we present a new type of
electroceutical and report for the first time an electrochemical
prodrug activation strategy, leading to the site-selective
generation of the anticancer agent, oxaliplatin.
Scheme 1 Prodrug
1 electrochemically activated to the active
anti-cancer drug oxaliplatin,
transfer by [Ru(NH3)6]2+.
2 mediated through electron
Certain ligand types attached to Pt(IV), such as
carboxylates, can be effective in preventing inner-sphere
reduction by biological reductants in comparison to halide and
pseudo-halide axial ligands12, 13. To improve selectivity,
targeting moieties have been incorporated as axial ligands,
such as oestrogen, to exploit active uptake into cancer cells
that overexpress the oestrogen receptor14. More exotic drug
delivery systems have been designed for similar purposes –
this includes Pt(IV) complexes tethered to carbon nanotubes
that enter cells via endocytosis, with reduction of the Pt(IV)15,
up-converting nanoparticles with DNA-bound Pt(IV) complexes
shown to release the Pt(IV) complex upon illumination in the
presence of ascorbic acid16 while Pt(IV)-NaYF4 nanoparticles
Platinum-based cancer therapies have been studied
extensively with the aim of improving their toxicity profiles
while increasing selectivity5, 6. Pt(IV) compounds show reduced
cytotoxicity compared to the “parent” Pt(II) complexes, where
the axial ligands can function as protecting groups until they
are released upon reduction of the Pt(IV) compound7, 8. In
addition, the two additional axial ligands provide convenient
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decorated with folic acid have been used to enable folate
receptor mediated targeting17.
Despite the elegancy of recent developments, they are all
reliant on biological processes that are innately highly variant
between tissue types. Pt(IV) carboxylate complexes typically
exhibit irreversible reduction potentials in the range of −0.3 to
−1.0 V (vs. Ag | AgCl | Cl-)18, which presents a unique
opportunity to develop a prodrug activation system that is
reliant, not upon a biological stimuli, but upon an externally
controlled electrochemical stimulus.
Herein, we report the first electrochemically-activated
prodrug system, where an electrode was used to
electrochemically activate a Pt(IV) prodrug allowing the
generation of an active anticancer Pt(II) complex with spatial
and temporal control. This was enabled by the use of a
ruthenium-based redox mediator (identified through empirical
screening of redox mediators with suitably catalytic redox
potentials) coated onto an electrode surface that acts as an
electron shuttle between the electrode surface and the Pt(IV)
prodrug. This enabled its reduction to the active Pt(II) drug (at
much lower applied potentials when compared to the
potential required for its direct reduction at the electrode
surface), leading to electrochemical-mediated controlled cell
Fig. 1. (a) Overlay of the cyclic voltammograms of [Ru(NH3)6]Cl3
(100 µM) in PBS (dashed line) alone, and following the addition
of prodrug
1 (2 mM). A glassy carbon electrode was used as
death. A Pt(IV) derivative
1 that contained two succinate axial
working electrode. Potentials are relative to the Ag/AgCl
reference electrode employed. Processes (Ia) and (Ic)
correspond to the reversible oxidation and reduction of the
mediator, respectively, and (II) corresponds to the irreversible
ligands was synthesised which, when subjected to an
electrochemical activation/reduction process utilising a redox
mediator, efficiently generated oxaliplatin
2 (Scheme 1). The
anionic nature of the succinate ligands at physiological pH is
likely to contribute to the low cytotoxicity of the prodrug in
relation to the Pt(II) species, by preventing passage through
the cell membrane. This also afforded a “bio-inert” prodrug, as
reduction of prodrug
electro-catalytic mechanism for the reduction of the Pt(IV)
prodrug , where the ruthenium complex shuttles electrons
1 (solid line); (b) Scheme showing the
1
from the electrode surface to the prodrug. The reduced
ruthenium complex ([Ru(NH3)6]2+) is electrochemically
regenerated through the continuous application of the
reduction potential characteristic for the redox couple
[Ru(NH3)6]3+/[Ru(NH3)6]2+; (c) The immobilisation of
[Ru(NH3)6]3+ onto the carbon ink screen-printed electrodes
(SPCE) was carried out by coating the working electrode with
the sulfonated fluoropolymer Nafion®, followed by cation
exchange entrapment of [Ru(NH3)6]3+. The successful
entrapment of the redox mediator was confirmed by CV
analysis in PBS with the immobilised [Ru(NH3)6]3+ showing the
expected reversible redox behaviour (Fig. S4, ESI†); (d) and (e)
1
was not efficiently reduced by biological reductants19. In
addition, prodrug 1 was kinetically inert towards biological
reductants, in comparison to other Pt(IV) compounds with
higher reduction potentials (Fig. S1, ESI†).
Cyclic voltammetry (CV) of the Pt(IV) derivative
1 (2 mM in
PBS) showed an irreversible reduction with a peak potential of
−0.65 V (vs. Ag | AgCl | KCl (3 M), Fig. S2, ESI†), corresponding
to the reduction of Pt(IV) to Pt(II) with the loss of both axial
ligands. This potential indicated that direct electrochemical
reduction of prodrug
1 would require a relatively high
reduction potential that in the context of a biological
environment would be inefficient due to the interference of
redox-active biomolecules, hindering specificity of the system
towards the prodrug. However, CV analysis of a solution
containing the redox mediator, hexaammineruthenium
chloride [Ru(NH3)6]Cl3 (100 µM) in PBS showed reversible
Electroactivation of prodrug
1 by the application of -0.4 V with
modified electrode (MED) or un-modified electrode (SPCE) in
PBS with HPLC monitoring of: (d) %Pt(IV) and (e) the Pt(II)-
GMP bisadduct (n = 3). The data represent the mean ± S.D. ***
P<0.001 by one-way ANOVA with Tukey post-test.
redox behaviour. With the addition of
1 (2 mM) to the same
solution, the current associated with the reduction process of
the mediator (IC) increases, as would be expected of an
electro-catalytic system indicating generation of more oxidised
We thus devised a redox-mediated system to allow
electrochemical activation of prodrug
1 at a lower reduction
potential via [Ru(NH3)6]Cl3 electrostatically bound within a
sulfonated fluoropolymer Nafion® film that was able to shuttle
electrons from the electrode surface via electron hopping to
reduce the Pt(IV) species in solution (Fig. 1b and 1c). Successful
entrapment of the redox mediator was confirmed by CV
mediator via reduction of
application of a reductive potential (-0.4 V) afforded good
conversion of (Fig. S3, ESI†).
1 (Fig. 1a and 1b). Moreover,
1
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analysis in PBS with the immobilised [Ru(NH3)6]3+ showing the
expected reversible redox behaviour (Fig. S4, ESI†).
The oxidised form of the redox mediator, [Ru(NH3)6]3+, was
reduced at the electrode surface by applying a potential of -0.4
V to afford [Ru(NH3)6]2+, which was able to reduce the Pt(IV)
species, becoming re-oxidised in the process and closing the
electro-catalytic cycle.
consisting of
and [Ru(NH3)6]3+ (subjected to a constant
potential of -0.4 V) was analysed by HPLC and showed good
conversion of to the active drug in PBS (Fig. S3, ESI†).
A
bulk electrolysis experiment
1
1
2
The ability of the modified electrode to function in
biological media was assessed in 10% foetal bovine serum
(FBS) and in colorectal carcinoma HCT 116 cell lysate. CV
analysis over 24 h showed that the modified electrode
retained the reversible redox behaviour characteristic of the
Ru(III)/Ru(II) couple (Fig. S5, ESI†), with only incubation in cell
lysate showing a change in redox processes, attributed to
protein binding affecting the redox mediator and its
interactions within the Nafion® layer. Despite this, the
electroactivation of
affected. In addition, prodrug
stable in 10% FBS at 37 °C (16% degradation after 24 h, n = 3,
1
in cell lysate was not significantly
1
was found to be relatively
Fig. S6, ESI†) and towards reduction by glutathione and the reduction of a Pt(IV) prodrug to its active Pt(II) form at a
ascorbic acid with <10% decrease in Pt(IV) levels after 24 h at lower reduction potential
37 °C (Fig. S1, ESI†). Leaching of the mediator from the
electrode into solution was minimal in both solution and in Fig. 2 (a) Set-up used for the electroactivation of
cellular environments as shown by ICP-OES (Fig. S7 and Table culture, showing the modified electrode immersed in cell
S1, ESI†). The total content of the ruthenium complex culture and connected to a 4-channel portable potentiostat;
1 in cell
electrostatically bound in the Nafion® layer was quantified by (b) Cell viability of HCT 116 cells treated with
1 (50 µM), with a
ICP-OES and the proportion that was redox-active was reduction potential of -0.4 V applied for 1 h, and subsequently
determined by chronoamperometry (experimental details in incubated for 3 days (MTT assay, n = 3). *** P<0.001 by one-
supporting information, Fig. S8 and S9, ESI†). The total content way ANOVA with Dunnett post-test, compared to the
of mediator per electrode was 1.08 ± 0.02 µg, of which 0.84 µg untreated control group; red line shows the quantification of
or around 78% of the total content was found to be redox- Pt in the HCT 116 cells after treatment with
active.
V for 1 h, and incubation for 4 h, after which the cells were
The conversion of Pt(IV) to Pt(II) was investigated using the harvested and the Pt content in the DNA isolated (Qiagen
modified electrode for the electroactivation of by applying a DNeasy blood and tissue kit) determined by ICP-MS; (c) Cell
reduction potential of -0.4 V for 24 h at 37 °C in PBS. viability of HCT 116 spheroids treated with
(50 µM) and -0.4
1 (50 µM) and -0.4
1
1
Guanosine monophosphate (GMP) was included in the V applied for 1 h, and incubated for 3 days (CellTiter Glo 3D
solutions to aid analysis as the generated active species assay, n = 3). The data represent the mean ± S.D. *** P<0.001
oxaliplatin, 2, following aquation of the oxalate ligand is highly by one-way ANOVA with Dunnett post-test, compared with the
thermodynamically-driven to complex the N7 position of the untreated control group; (d) Cells were incubated as above
guanosine monophosphate, allowing both Pt(IV) and Pt(II) and stained with the green-fluorescent calcein-AM to indicate
species to be readily visualised by HPLC (Fig. S10, ESI†).
Electroactivation of in PBS with the modified electrodes homodimer-1 to show loss of plasma membrane integrity
resulted in a clear increase of Pt(II) in solution, with 49% (LIVE/DEAD® assay) and imaged by fluorescence microscopy:
conversion of prodrug after 24 h (Fig. 1d and 1e). The (i) live cells (green), (ii) dead cells (red) and (iii) merged image.
modified electrodes were also capable of electroactivating in
10% FBS and HTC 116 cell lysate (Fig. S11, ESI†). The disparity compared to the non-mediated direct reduction. As the direct
between the conversion of Pt(IV) and the resultant yield of reduction of prodrug
has been established to have a pH-
intracellular esterase activity and red-fluorescent ethidium
1
1
1
1
Pt(II) with the non-modified electrodes is attributed to dependent reducꢀon potenꢀal (Fig. S12, ESI†), we confirmed
undesired reactions occurring at the electrode surface. With that no significant decrease in pH occurred during the
the modified electrodes, where there is an electron shuttle electroacꢀvaꢀon process (Table S2, ESI†).
between the electrode surface and
consistently into oxaliplatin . These results illustrate the electrode were assessed (see set-up in Fig. 2a) by immersing
ability of the proposed system to electrochemically mediate the modified electrode into a culture of HCT 116 cells, and
1, the conversion occurs
Cell compatibility of the components of the modified
2
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applying a reduction potential of -0.4 V (Fig. 2b). This resulted
in no cytotoxicity.
Notes and references
Importantly,
thus displaying a good toxicity selectivity ratio between the
Pt(IV) and Pt(II) states (the IC50 of with HCT 116 cells was 0.19
1 exhibited limited cytotoxicity up to 50 µM,
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1
1
5.
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1
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8.
9.
In order to mimic the 3D tumour environment multi
cellular tumour spheroids were subjected to the
electroactivation process. Spheroids were generated from HCT
20, 21
116 cells by the hanging drop method
and treated with 1
followed by electroactivation at -0.4 V for 1 h on day 5. Cell
viability was measured after days which showed, as
expected, no decrease in cell viability in spheroids treated only
with ; however, a 50% decrease in viability and increased
number of apoptotic cells was observed when was activated
electrochemically (Fig. 2c and Fig. S16, ESI†). To further
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on 3D tumour spheroids, live and dead cells were stained after
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In summary, an electrochemical prodrug activation system,
that is stable in biological complex media, for platinum-based
prodrugs has been devised and its efficacy in generating
cytotoxic Pt(II) species examined in cell culture. The system
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16.
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electrochemical devices within a tumour, as part of the
Implantable Microsystems for Personalised Anti-Cancer
Therapy (IMPACT) project, could provide unequivocal control
over prodrug activation in a site-selective, time-dependent and
localised mediated manner. Further investigation will be
focused on improving rate of reduction of Pt(IV) species by the
nano-structuring of the electrode surface to increase active
surface area. Expansion of the scope of this electrochemical
prodrug activation technology is also being explored.
20.
21.
This work was supported by funding from University of
Edinburgh, UK Engineering and Physical Sciences Research
Council through the Implantable Microsystems for
Personalised Anti-Cancer Therapy (IMPACT) programme grant
(EP K034510/1) and the Rosetrees Trust (A865).
Conflicts of interest
There are no conflicts to declare.
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