Inorganica Chimica Acta 374 (2011) 5968

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Inorganica Chimica Acta

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Review

Electrochemical analysis of the interactions and reactivity of ferrocene-based

drugs with a lipid environment: A qualitative overview
O. Mertins a, P. Messina a, E. Labb a, V. Vivier b, S. Arbault a,1, F. Lematre a, O. Buriez a,, C. Amatore a,
a
UMR CNRS 8640 PASTEUR and LIA CNRS XiamENS NanoBioChem, Ecole Normale Suprieure, Dpartement de Chimie, Universit Pierre et Marie Curie-Paris 6,
24 rue Lhomond, 75231 Paris Cedex 05, France
b
LISE, CNRS UPR 15, Universit Pierre et Marie Curie, CP 133, 4 Place Jussieu, 75252 Paris Cedex 05, France

a r t i c l e i n f o a b s t r a c t

Article history: Transport, reactivity and interactions of anti-tumor ferrocene-based compounds within lipid environ-
Available online 24 April 2011 ments have been investigated electrochemically at a glassy carbon electrode (GCE) modied with a lipid
bilayer lm. Cyclic voltammetry performed in the presence of an adsorbed bilipid lm in a H2O/EtOH (8/
Dedicated to Professor W. Kaim
2) mixture, clearly showed that the afnity of the starting neutral ferrocifen derivatives towards the lipi-
dic bilayer depends on both their size and their polarity. Likewise, the electrogenerated ferrocenium
Keywords:
derivatives were expulsed reversibly from the bilayer in agreement with their positive charge. The reac-
Supported lipid membrane
Ferrocene
tivity of ferrocifen compounds was also investigated under the same conditions and in the presence of a
Drug base in order to trigger an intramolecular ferrocene-mediated (2e 2H+) process within the lipidic lm.
Membrane transfer Under these conditions, two distinct sequential electrochemical behaviors were observed depending on
Ferrocifen the base availability in the lipid layer, the magnitude of the corresponding two electrons wave being lim-
ited by the base concentration in the lm. A full two-electron oxidation process, was observed as long as
the amount of base present into the lipid layer was high enough to titrate the two protons released in the
two-electron process. When this was sub-stoichiometric a second wave was observed at higher potential
values representing the one electron oxidation of the ferrocifen excess.
2011 Elsevier B.V. All rights reserved.

Omar Mertins is graduated in Chemistry by the Chemistry Institute of Federal University of Rio Grande do Sul (Brazil) and received his PhD in
Physical Chemistry by the same Institute with one year exchange period at the Charles Sadron Institut (France). He did a rst one year postdoc in
Electrochemistry at the Ecole Normale Superieure de Chimie (Paris) and a second one year postdoc in Physical Chemistry of nano and micro
colloids at the Max Planck Institute of Colloids and Interfaces (Germany). He is currently working on biophysics of phospholipidic membranes at
the Physics Institute of University of Sao Paulo (Brazil).

Corresponding authors.
E-mail addresses: olivier.buriez@ens.fr (O. Buriez), christian.amatore@ens.fr
(C. Amatore).
1
Present address: Institut des Sciences Molculaires, UMR 5255 CNRS, Groupe
NanoSystmes Analytiques, Universit Bordeaux 1, ENSCPB, France.

0020-1693/$ - see front matter 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.ica.2011.04.030
60 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968

Pierluca Messina was born in Desio (Italie) in 1986. He obtained his M. Sc. Degree from the Universit degli Studi di Milano in 2009. He worked
on Innovative catalytic system without Pt for PEM fuel cells under the supervision of Prof. Leonardo Formaro. In 2010, he joined the group of Dr
Christian Amatore at the Ecole Normale Suprieure of Paris as a Ph.D student where he is currently working on the electrochemical investigation of
drug vectorisation through biological membranes.

Eric Labb was born in Brittany, France, in 1965. After a PhD in 1992 supervised by J. Devynck, he obtained a Matre de Conferences position in the
Laboratory headed by Pr J. Perichon and carried out his research activity in the electrochemical determination of nickel- and cobalt catalysed C-C
bond formation mechanisms. In 2006, he joined Christian Amatores group at Ecole Normale Superieure and focuses on topics devoted to the
control of the reactivity of transient intermediates in supramolecular assemblies.

Vincent Vivier was born in Saint-Maur (France) in 1971. He received his PhD in 2000 under the supervision of Dr. L.T. Yu. In 2002, he joined the
Laboratoire Interfaces et Systmes lectrochimiques (CNRS - UPR 15). His current research eld concerns the characterization of heterogeneous
interface reactivity by means of local electrochemical techniques and impedance spectroscopy.

Stphane Arbault is a principal research scientist at CNRS since 1998. After studying Photography and History of Arts, he chose to study Biology
and Physical Chemistry (University Paris 7). During his Ph.D. in the laboratory of Dr. C. Amatore (ENS-Paris), he developed an electrochemical
method to study oxidative stress on single human cells and defended his thesis in 1996. He made a post-doc in the framework of a European project
and has worked on the Chemistry and Biology of carcinogenesis. Stphane received several french and international young investigator Prizes from
the Societies of Electrochemistry or Nitric Oxide.
 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968 61

Frdric Lematre, 33, was born in Saint-Rmy, Burgundy, France. He did a joint thesis between the Universit de Bourgogne (France) and the
Universit de Sherbrooke (Canada). He received a double Ph. D. degree in 2003. His thesis works dealt with electrochemistry of palladium clusters.
After doing postdoctoral research in the Dr. Amatores group at the Ecole Normale Suprieure de Paris (palladium catalysis), he has been obtained
an Assistant Professor position (Matre de Confrences) at the Universit Pierre et Marie Curie in September 2004. His research works are still
performed in the Dr. Amatores group at ENS and concern the detection of secretion processes at the single cell level.

Olivier Buriez was born in Livin (France) in 1968. He received his PhD in 1996, in Molecular Electrochemistry, under the supervision of Drs C.
Amatore and J.N. Verpeaux at the Ecole Normale Suprieure (ENS-Paris). After a postdoctoral research in the group of Dr J.B. Kerr at the University
of California at Berkeley (USA), he obtained a CNRS permanent position of researcher in Pr. J. Prichons group in 1999 where he investigated cobalt
catalyzed C-C bond formation mechanisms by electrochemistry. Since 2006, his research activity is performed in the Dr. Amatores group at ENS-
Paris and concerns the reactivity of molecules possessing biological properties.

Christian Amatore, 58, is a Full Member of the French Acadmie Des Sciences and of the High Council of Science and Technology, advising the
French President on scientic matters. His ultramicroelectrodes have allowed electrochemical investigations of organic, inorganic, and organo-
metallic chemistries or living cells. These works correspond to over 400 publications with over 13500 citations (H = 60). He has received many
renowned international prizes and distinctions and is Doctor Honoris Causa of many universities in Europe and Asia.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
2. Experimental . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.1. Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
2.2. Formation of the supported lipid film at the glassy carbon electrode surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.1. Formation and characterization of the DMPC lipid films at a GCE surface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63
3.2. Electroactivity and distribution of ferrocene/ferricinium species at the lipid layer/solution interface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.3. Voltametric behavior of complex (4) at a DMPC-modified GCE in the absence and the presence of imidazole as a base . . . . . . . . . . . . . . 67
4. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
62 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968
1. Introduction
Grafting a ferrocenyl unit onto the tamoxifen skeleton offers an
entry for the preparation of a new and promising class of
anti-breast cancer drug candidates [14]. Compared to tamoxifen,
well known for its anti-estrogenic properties, these new complexes
(ferrocifens) possess not only endocrine modulating properties, but
also interesting cytotoxic effects against hormone-independent
cancer cell lines [14].
Structureactivity investigations have demonstrated the impor-
tance of not only the ferrocene moiety as an intra-molecular
electronhole reservoir but also of the conjugated p system as an
electron-conjugating module. It was also pointed out that the
presence of an ethylallyl phenol moiety is required as the ulti-
mate electron donor since giving rise ultimately to a quinone met-
hide, the biologically active species that may damage targeted cells
by adduct formation with DNA, GSH, or proteins [510]. Previous
electrochemical experiments have established that the initial elec-
tron uptake occurs from the ferrocene unit, upon which further
reaction in the presence of a base leads to a two-electron oxidation
via the formation of a pseudo phenoxy radical whose oxidation af-
fords ultimately the sought quinone methide (Scheme 1). This spe-
cies is produced along a ferrocene-mediated proton coupled
electron transfer triggered by bases having pKa values close to 7
that roughly correspond to the pKa of peptides or DNA nitrogen-
bases present in the cellular medium [11]. Conversely, in absence
Scheme 1. Ferrocene-mediated proton coupled electron transfer leading to a quinone methide (shown for species (4), see Scheme 2).
OH
Fe Fe
OH
(1) (2)
Scheme 2. Chemical structures of the four ferrocenyl complexes investigated in this study.
of base the ferrocifen voltammetry is reversible and concerns only
the ferrocene moiety.
Yet, one of the main problems to solve for the medical use of
such drugs is related to their poor solubility in aqueous media,
and therefore their availability in biological uids. Though their
hydrophobic moieties are expected to allow a facile penetration
through lipidic cellular membranes, they present formulation
_challenges for intravenous injection. Previous works have demon-
strated that cyclodextrins (CDs) can be used to solubilize and vec-
torize ferrocifen derivatives that suffer such reduced solubility in
water thanks to the inclusion of the ferrocene moiety in the non-
polar cyclodextrin cavity [12]. These works also conrmed that,
under these conditions, the electron transfer activation pathway
(Scheme 1) as well as the biological activity remained unchanged,
evidencing that cyclodextrin complexes are sufciently labile to
act as carriers of the neutral ferrocifen derivatives only [12]. We re-
cently investigated the interactions between ferrocifen complexes
and non-polar molecular architectures such as cyclodextrin cavi-
ties and/or unsubstituted n-alkyl chains, with respect to the hydro-
phobic architectures/barriers which are common to drug vectors
(CDs) as well as cell membranes. This previous strategy consisted
in immobilizing either the organometallic complex or the non-po-
lar architectures at the electrode surface [13].
This work reports about the second stage of such vectorization,
viz., concerns the interactions of ferrocifens with cellular lipid
membranes. In order to get further insight on interactions, trans-
OH
Fe Fe
(3) (4)
 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968
A B
Scheme 3. (A) Chemical structure of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). (B) Schematic representation of the glassy carbon electrode (GCE) modied with
DMPC lm used in the investigation of the interactions between ferrocene (1) and the ferrocenyl-tamoxifen adducts ((2), (3), (4)) with a model membrane. See Scheme 2 for
the chemical structures of the ferrocenyl complexes.
port, as well as reactivity of these potent species, we thus charac-
terized the electrochemical behavior of various ferrocifenyl deriv-
atives (Scheme 2) at glassy carbon electrodes modied with a
bilayer of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC)
often used as model of cell membranes (Scheme 3).
The rst part of the following contribution deals with
electrochemical impedance spectroscopy and cyclic voltammetry
characterizations of the DMPC lipid membrane, in water and in a
H2O/EtOH (8/2) mixture, at a glassy carbon electrode (GCE) using
Fe(CN)64/3 as the probe compounds. The use of a charged com-
plex was particularly interesting to evidence the possible presence
of pinholes in the lipid membrane. Indeed, multi-charged species
are assumed to be repelled from the lipid layer but may be
detected by their passage through the bilayer pinholes, if any,
through which the electrode is directly exposed to the solution.
This section was also used to verify that the presence of alcohol,
which was required to solubilize the ferrocene derivatives, did
not alter signicantly the lipid lm structure or stability. The re-
sults of this section obtained in water/ethanol solvent validated
the present strategy for investigating the electrochemical behavior
of the ferrocifen complexes within the lipidic lm in the absence or
in the presence of imidazole as a base. Hence, this experimental
work offers a qualitative framework delineating phenomena asso-
ciated to the vectorization of drugs of a representative ferrocifen
family (Scheme 2).
2. Experimental
2.1. Materials
Cyclic voltammetry and electrochemical impedance spectros-
copy (EIS) experiments were performed at room temperature un-
der an argon atmosphere in a three-electrode cell using an
Autolab potentiostat (Type III) and KCl (0.1 M Acros) as the sup-
porting electrolyte. The reference electrode was a saturated calo-
mel electrode (SCE KCl saturated, Tacussel), which was
separated from the solution by a bridge compartment lled with
the same solvent/supporting electrolyte solution as used in the
cell. The counter electrode was either a 1 cm length gold wire or
a platinum grid (Goodfellow). The glassy carbon working electrode
was purchased from Radiometer Analytical (3 mm diameter). The
preparation of the gold and glassy carbon electrodes is described
in the next section. A solution of 2.5 mg/ml of 1,2-dimyristoyl-
sn-glycero-3-phosphocholine (DMPC) (99%, Sigma) in chloroform
was previously prepared in a Schlenk ask and constantly kept un-
der argon and stored in a freezer. The ferrocenyl compounds (2),
(3), and (4) were provided to us by Prof. G. Jaouen (UMR CNRS
63
Electrolyte + (1 - 4)
pinhole
Glassy Carbon Electrode
7223) and have been prepared as described elsewhere [3,11,
14,15]. Solutions of ferrocene (98%, Fluka) and of the ferrocenyl
compounds were prepared, under vigorous stirring, in ethanol at
a concentration of 0.1 mM each. Imidazole (99%, Aldrich) was
added in the cell when required. All solvents and reagents were
of analytical grade. Water was highly puried (resistivity = 18
MX cm; Mill-Q system; Millpore, Billerica, MA, USA).
2.2. Formation of the supported lipid lm at the glassy carbon
electrode surface
The modication of the GCE with the lipid was achieved follow-
ing the procedure reported by Wang and coworkers [16] who
characterized their lms as structurally dened bilipidic mem-
branes (BLMs). Briey, the GCE was rst polished (Mecaprex
2400 and 4000) then sonicated for 1 min in water and acetone suc-
cessively to avoid any inorganic and organic contaminations. The
electrode was then immersed in a deoxygenated 0.1 M KCl aque-
ous solution and biased at 1.5 V/SCE during 3 min. After polariza-
tion, the GCE was dried under a puried argon ux.
Subsequently, a 5 ll aliquot of the lipid solution (DMPC in CHCl3)
was dropped onto the surface of the electrode by means of a micro-
syringe. Immediately after chloroform evaporation, the electrode
was transferred back into the electrochemical cell containing the
KCl solution allowing the formation of the supported lipid mem-
brane. Due to the hydrophilic polarized surface of the electrode
and the amphiphilic character of phospholipids, the molecules
are reported to spontaneously self assemble into a lipid lm struc-
tured as a bilayer covering the electrode surface [16].
3. Results and discussion
3.1. Formation and characterization of the DMPC lipid lms at a GCE
surface
Glassy carbon electrodes (GCEs) coated with bilayer lipid mem-
branes were prepared according to the procedure previously de-
scribed by Wang and coworkers [16] (see Section 2). Following
these authors, the molecules then spontaneously self-assembled
and formed a lipid lm over the electrode. The lipid lm was rst
qualitatively characterized by comparing the voltammetry of
Fe(CN)63 in water/KCl (0.1 M) at a bare glassy carbon electrode
or at modied electrode by a DMPC lm (Fig. 1).
In agreement with similar studies, the voltammetric current
intensity of the electrochemical process decreased in the presence
of phospholipids [17]. This behavior revealed a near-complete
blocking of the GCE evidencing that the electrode surface was
64 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968

than in Fig. 1A and the characteristic sigmoidal shape was less

8 A H2O
apparent evidencing that the pinhole density had increased suf-
ciently to allow a partial overlap of their diffusion layers [18].
OFe Importantly, either in water or in water/ethanol mixture the
4
voltametric response remained unchanged after 4 h showing the
(a)
stability of the lipid membrane under either condition.
I*

0 (b) Electrochemical impedance spectroscopy was also used to

quantitatively characterize the lipid lm covering the electrode
GCE / DMPC (Fig. 2).
-4
Fig. 2A and B show the Nyquist representation and the Bode
plot of the impedance response of the electrode at the open circuit
RFe E / SCE potential in the presence of equimolar 1 mM Fe(CN)64/3 in 0.1 M
-8
-0.2 0 0.2 0.4 0.6 KCl solution. The shape of the diagram was a attened semi-circle,
which corresponds to two time constants. Considering the pres-
3 OFe ence of pinholes, the description of the interface using electrical
B H2O / EtOH (8/2)
equivalent circuit that accounts for such a behavior is presented
(a)
in Fig. 2C. Under these conditions, a direct electrochemical reaction
1
(faradaic impedance Z1) is supposed to occur at pinholes due to the
(b) high charge of the reactant. This impedance is in parallel with the
double layer capacitance, which is represented by a constant phase
I*

-1
element (CPE) [Qd, ad] that accounts for the frequency dispersion of
GCE / DMPC
the system [19]. However, most of the surface is covered by the li-
-3 pid layer, characterized by a lm capacitance (also represented by
RFe a CPE, [Qf and af]) in parallel with the lm resistance Rf. This lm
E / SCE impedance is in parallel with the faradaic impedance.
-5 A non-linear regression method (simplex algorithm) was used
-0.4 -0.2 0 0.2 0.4 0.6
to determine the value of the parameters of the circuit presented
Fig. 1. Cyclic voltammograms of K3Fe(CN)6 (5 mM) in H2O (A) and in H2O/EtOH in Fig. 2C, and the results are plotted in Fig. 2A and B (solid lines).
(8/2) (B) + KCl (0.1 M) at a bare glassy carbon electrode (GCE) (3 mm diameter) or at The high frequency limit yielded the electrolyte resistance
a GCE modied with a DMPC lipid lm. v = 50 mV s1. I: normalized current, I = I Re = 171 X, which is in agreement with the working electrode
(lA)/Cbulk (mM).
dimension and the electrolyte conductivity. The ad and Qd param-
eters were found equal to 0.91 and 4  106 F s0.09, respectively.
essentially covered by the lipid lm. Though, the observation of a
The equivalent capacitance value (Cd = 27.5 lF cm2) could be thus
characteristic near-sigmoidal small voltammogram for Fe(CN)63
calculated from the Brug formula (Eq. (1)) [20].
in the presence of the lipid structure indicated the presence of pin-
holes [18]. The stability of this DMPC-modied electrode was then
C Q 1=a R1
e
a=a
1
tested in a H2O/EtOH (8/2) mixture since these conditions were re-
quired to investigate the electrochemical behavior of the ferrocene The same analysis was done for af = 0.91 and Qf = 8.8 
derivatives ((2)(4)). As shown in Fig. 1B, a similar behavior to that 108 F s0.09 leading to a lm capacitance of 4.15  107 F cm2.
obtained in water was observed in the presence of 20% ethanol. From this capacitance value, the thickness d of the lipid membrane
The peak current intensities due to pinholes were slightly higher was nally estimated according to Eq. (2)

Fig. 2. (A) Nyquist and (B) Bode plots of supported DMPC-modied glassy carbon electrode (GCE 3 mm diameter) in 1 mM FeCN4=3 6 + 0.1 M KCl solution at the open
circuit potential with a 10 mV sine wave perturbation; (C) electrical equivalent circuit used for the description of the interface.
 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968
e  e0
C 2
d across the lipidic lm so as to be reduced into Fc mostly dis-
where e0 is the vacuum permittivity (e0 = 8.85  1014 F cm1) and
e is the dielectric constant of the membrane (e = 2.05 [21]). A value
of about 4.35 0.4 nm was obtained indicating that the electrode
surface was mostly covered by one lipid bilayer. The ratio of the
charge transfer resistance for a bare electrode (data not shown)
and the same resistance measured for a DMPC modied electrode
indicates that pinholes represent about 2% of the total surface of
the electrode, a result which is compatible with the behavior
observed in cyclic voltammetry (Fig. 1) [18].
In conclusion, the quality of the bilayer formed and used in this
work was found consistent with those reported before [16]. How-
ever, voltammetry of Fe(CN)63 at these modied electrodes evi-
denced the presence of pinholes, though these contributed to
much smaller currents than those observed at the bare electrodes.
3.2. Electroactivity and distribution of ferrocene/ferricinium species at
the lipid layer/solution interface
Let us rst focus on the voltammetric behavior of ferrocene
(compound (1)) and its ferricinium cation ((1+), introduced in
its hexauorophosphate salt form) at the DMPC-coated electrode
since they represent the redox couple common to all systems
envisioned here in its most simple version. Fig. 3A and B display
the CV of (1) and (1+), respectively, at the modied GCE. Note
that the observed oxidation peaks were considerably higher than
those for Fe(CN)63 reduction/oxidation (Fig. 1A and B). This evi-
denced that the pinholes played a negligible role in these vol-
tammetries. The probable reason for this change will be
discussed below.
On both voltammograms, the oxidation of (1) proceeded
through an intense adsorption peak, while the backward reduction
of (1+) featured a plateau shape of low intensity. This did not arise
because of a would-be unstability of (1+) under these conditions,
since the reductive voltammetry of an authentic sample of (1+)
led to the same phenomenon (Fig. 3B), leading on the backward
scan to an oxidation plateau for (1) whose current intensity is
coherent with the amount of (1+) reduced (see below and
Fig. 3B). These unsymmetrical redox processes need then to be as-
cribed to a considerably different partition of (1) and (1+) between
the lipid lm and the solution. Ferrocene is lipophilic and expected
to dissolve in the lipid layer; conversely, the charged ferricinium
cation should be expelled from it. This observation is in agreement
with the electrochemical behavior of ferrocene-substituted mole-
cules at electrodes either modied with SAMs or derivatized with
the covalent grafting of amine or diazonium aromatic molecules
[13,22].
16
A O(1)
12
8
I*
4
0
-4
R(1)
E / SCE
-8
-0.2 0 0.2 0.4
Fig. 3. (A) Cyclic voltammograms of (1) (0.2 mM) in H2O/EtOH (8/2) + KCl (0.1 M) at a GCE (3 mm diameter) modied with a DMPC lipid layer. v = 50 mV s1. (B) Cyclic
voltammograms of FcPF6 (1+) (3 mM) in H2O/EtOH (8/2) + KCl (0.1 M) at a GCE (3 mm diameter) modied with a DMPC lipid layer. v = 50 mV s1. I: normalized current, I = I
(lA)/Cbulk (mM).
65
Yet, Fig. 3B proves that Fc+ may reach the electrode surface
solved into the bilayer. This may proceed through a CE-type se-
quence where the antecedent C step corresponds to the up-hill
crossing through the bilipid layer. Alternatively, since Fig. 1A
and B showed that the bilayer was not perfect, this may proceed
through pinholes when any are present. However, the normal-
ized current plateaus in Fig. 3A and B appears to be signicantly
larger than that of Fe(CN)63 reduction. Since both species have
similar diffusion coefcients, a pinhole-mediated process should
lead to similar currents in both cases [18]. We are thus inclined
to consider that the reduction of Fc+ in Fig. 3A and B proceed
through a CE-type sequence involving a cross-over of the bilayer
lm by the ferrocenium cations. This difference with Fe(CN)63 is
certainly related to the large charge difference of the two ions
and also the lesser lipophobicity of the Fc+ cation but also to a
change of the bilayer structure following its loading by Fc.
The behaviors of the ferrocifen species (2), (3) and (4) were inves-
tigated under the same conditions and compared to the one ob-
served with ferrocene (1). As shown in Fig. 4, the voltammetric
signature of the oxidation of the ferrocene center remained well de-
ned for the three complexes and the characteristic shape of each
oxidation peak was again consistent with the oxidation of immobi-
lized species at the electrode surface.
As for 1/1+, the reduction peak observed on the backward scan
resulted always smaller than the oxidation one. For (2) the overall
shape and peak intensity of the cyclic voltammogram (Fig. 4A)
were very similar to that of (1) (Fig. 3A). This is interesting to note
since for an adsorptive voltammetry there is no dependence with
the diffusion coefcient, hence with the size. Thus, this evidenced
that the loading of the bilayer membrane by (2) or by (1) was sim-
ilar. The same was true for (3) (Fig. 4B) even when the loading time
was made much larger (4 h versus 5 min. for (1)). Conversely, for
(4) whose size is the largest among the three ferrocifens investi-
gated here, the oxidation peak current (Fig. 4C) was close to that
observed for (1). This series of experiments showed qualitatively,
that the partition between the lipid layer and the bulk solution
(H2O/EtOH, 8/2) decreased in the order (1)(2) > (4)  (3). This is
interesting because it shows that the molecular size is not the main
factor controlling the loading. A possible interpretation is that for
ferrocifens the presence of two hydroxyl groups certainly prone
to interact with the dipolar head group (as in (2)) compensates
somewhat the steric constraints due to the increased molecular
size. In agreement with this rationale, the presence of only one hy-
droxyl group surrounded by two apolar methyl substituents as in
(4) led to a less efcient loading. Complex (3) displays the lowest
loading evidencing that two phenyl groups cannot be accommo-
dated within the bilayer except when at least one is equipped with
6
B
O(1+)
4
2
I*
0
-2
R(1+)
E / SCE
-4
0.6 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7
66 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968

16 A 3.0 A O(4)
O(2)
12
2.0
time
8
1.0
I*

I*
4
0.0
0
-1.0
-4
R (2) R(4) E / SCE
E / SCE -2.0
-8 -0.2 0.0 0.2 0.4 0.6 0.8
-0.2 0 0.2 0.4 0.6 0.8

3.5
B
6 B O(3)
3

(x 10-10 mol.cm-2)
2.5

2 Compound (4)
2
I*

Compound (3)
0 1.5

1
-2
R (3)
E / SCE 0.5
-4
-0.2 0 0.2 0.4 0.6 0.8 Time (min)
0
0 50 100 150 200 250 300
O(4)
15 C Fig. 5. (A) Cyclic voltammograms of (4) (0.2 mM) recorded at a glassy carbon
electrode (3 mm diameter) modied with a DMPC lm as a function of the
10 equilibration time (0, 45, 90, 150, and 210 min) in H2O + EtOH (20%) + KCl (0.1 M)
and at v = 100 mV s1. (B) Evolution of the apparent coverage of the DMPC modied
GCE electrode for (3) and (4) (0.2 mM each in EtOH (8/2) + KCl (0.1 M)) as a function
5 of the time of incubation in the corresponding ferrocifen solution. I: normalized
I*

current, I = I (lA)/C (mM).

0
-5
R(4)
E / SCE
-10
-0.2 0 0.2 0.4 0.6
Fig. 4. Cyclic voltammograms of (2), (3), and (4) (0.2 mM each) in H2O/EtOH (8/2) +
KCl (0.1 M) at a GCE (3 mm diameter) modied with a DMPC lipid layer.
v = 50 mV s1. Cyclic voltammograms recorded after exposition of the modied
electrode to (2), (3), and (4) for 5, 240, and 150 min, respectively. I: normalized
current, I = I (lA)/C (mM).
a polar substituent prone to allow H-bonding with the bilayer head
groups. Though qualitative, the series (14) thus stigmatize that
the loading of the bilayer is modulated by several constraints
which may play antagonistely (e.g., (4) versus (3)).
This was further investigated by monitoring the oxidation peak
growth as a function of time for compounds (3) and (4), (Fig. 5A),
which represents somehow the apparent coverage of the electrode,
U, evolution with time (Fig. 5B).
Note that here and above we implicitly considered that within
the slow scan rate range used in this work, a species loaded into
the nanometric-thick bilayer structure is prone to be oxidized
sufciently rapidly so that the system is equivalent to a classical
electrode coverage. However, strictly speaking, we consider that
all the species are loaded in the bilayer volume so that the system
is not two-dimensional but rather three-dimensional where the
third dimension, perpendicular to the electrode, is nanometric.
As observed in Fig. 5B, compounds (3) and (4) exhibited extre-
mely distinct kinetic features towards their incorporation within
the lipid lm. Possible favorable interactions between the phenolic
hydroxyl group in compound (4) and the polar extremities of the
lipid molecules may explain the faster incorporation of (4) with
respect to (3). Considering the 5  1010 mol cm2 coverage of a
0.8 surface as a reference for a close-packed layer of comparable ferro-
cene molecules in SAMs [23], the saturation observed at ca. 3 
1010 mol cm2 for compound (4) shows that the loading of (4)
in the bilipid lm is certainly large enough to drastically
disorganize the original bilayer structure. The presence of small
molecules such as anesthetics (chloroform for instance) is well
known to induce a strong re-organization, viz. typically swelling
of membranes [24]. This swelling is in fact at the origin of the anes-
thetic effect through the increased pressure created onto ion-chan-
nels crossing the nerves outer membrane. This explains why it was
not relevant for this study to perform a full characterization of the
pinhole sizes and distributions [18] in the bilayer membrane in the
absence of (14). Indeed, whatever these results they are necessar-
ily drastically altered after their loading by ferrocene or the three
ferrocifens tested here. Presumably, the swelling induces a lateral
pressure into the membrane as for chloroform in nerve membranes
[24] so that the initial pinholes are closed or much reduced in size
and concentration. This is perfectly coherent with the disappear-
ance of the characteristic voltammetric features related to the
presence of pinholes in Figs. 3, 4 and 5A, compared to Fig. 1A
and B. In the presence of (1), (2), (3) or (4) the bilayer is then cer-
tainly drastically reorganized, most likely lipids and ferrocene-cen-
tered species interacting with each other to organize in a 2D-stable
packed assembly.
Interestingly, for (4), though the nal loading approaches that
of ferrocene, the loading kinetics is comparatively much slower.
 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968 67

This evidences that even if one phenolic hydroxyl leads to a

A O*4 O4
rather stable structure for the loaded 2D-system (compare to 4.0

(2) or (3), see above) the reorganization is certainly more drastic (e)
3.0 (d)
than for (1).
(c)
The peak current intensities featuring the reduction of the fer- 2.0
ricinium derivatives generated upon oxidation of compounds

I / A
(b)
((2)(4)) were much weaker than their anodic counterparts. As al- 1.0
(a)
ready commented for compound (1), the complex shape of waves
0.0
R((2)(4)) could arise either from a CE mechanism, the chemical step
being the up-hill crossing of the lm by the cation. Yet it is clear -1.0
that the shapes of waves R(3) and R(4) differs from the sigmoidal R4 E / SCE
-2.0
ones for R(1) and R(2) which indicates a change of kinetics [25]. -0.2 0.0 0.2 0.4 0.6 0.8
These voltammetric investigations of the interactions between
ferrocene/ferrocifen complexes with GCE-adsorbed lipid lms have 6.0 O3
evidenced distinct behaviors, related to the polarity, geometry and B
time
size of the molecules. These results appear of importance with re- 4.0
spect to the establishment of a reference behavior to assess the
transport of ferrocifen drugs across articial membranes. Interest- 2.0

I / A
ingly, they establish that within the limit that a DMPC is a suf-
ciently correct model of a cell membrane, large uxes of 0.0
ferrocifens are expected to disrupt cell membranes. This may be
an important factor to consider in view of side-toxicity of these -2.0
species at sufciently high concentration. In other words, if their R3 E / SCE
delivery to cell membranes is highly desirable it should not be -4.0
-0.2 0.0 0.2 0.4 0.6 0.8
excessive unless the delivery is properly targeted to tumor cells.

3.3. Voltametric behavior of complex (4) at a DMPC-modied GCE in C

the absence and the presence of imidazole as a base 3.0 O4
(c)
2.0 O*4
As already established, the cytotoxic effect of ferrocifen com-
(a)
pounds does not arise only from the presence of a ferrocenyl group, 1.0 (b)
I / A

but also depends on the position and structural molecular

backbone in which it is attached. The [Fc][conjugated spacer]
[p-phenol] structure has proven to be crucial for giving rise to
strong cytotoxic effect on various cancer cells [11,2629]. Provided
the structure allows a correct p-bonding electronic communication
between the ferrocene moiety and the phenolic oxygen, the one-
electron oxidation of the ferrocene center may initiate a sequence
(Scheme 1) yielding ultimately a quinone methide that may dam-
age cells by adduct formation with DNA, GSH, proteins, etc. Yet, we
also showed previously that if an extended electronic coupling is
necessary it is not sufcient per se since the presence of an ade-
quate base is required to allow for the formation of the phenoxy
radical. Though, it is not yet known whether the base acts follow-
ing a classical ECE sequence or in a concerted fashion though pre-
interaction with the phenolic proton so as to lower the oxidation
potential of the phenol and simultaneously deprotonating its cat-
ion radical. Since the above study has revealed the occurrence of
interactions between the bilipid lm and ferrocifens, it was of
interest to check whether such interactions favored or altered
the mechanism in Scheme 1.
For this purpose, the electrochemical behavior of ferrocifen (4)
was investigated in the presence of imidazole as a base at DMPC-
coated GCE. In the absence of a base, the cyclic voltammogram of
(4) obtained at a GCE modied with DMPC was depicted in
Figs. 4C and 5A.
Imidazole (20 mM) was added in a H2O + KCl (0.1 M) solution
and displayed no electroactivity at the DMPC modied GCE
(Fig. 6A(b)) even when cyclic voltametry experiments were per-
formed 30 min after the addition of base in order to allow for
any slow equilibration with the phospho-lipidic layer. Complex
(4) was then added in the same solution at a concentration of
0.2 mM. As clearly shown in Fig. 6A, the O4/R4 system was still ob-
served but was preceded by a new irreversible oxidation wave (O4 )
which developed at the expense of O4. Importantly, no equivalent
to wave O4 was observed when the same experiment was per-
0.0
-1.0
R4 E / SCE
-2.0
-0.2 0.0 0.2 0.4 0.6 0.8
Fig. 6. Cyclic voltammograms of (4) (A and C) and (3) (B) (0.2 mM each), recorded
at a glassy carbon electrode (3 mm diameter) modied with a DMPC lm in
H2O + EtOH (20%) + KCl (0.1 M)  v = 100 mV s1. (A) In the absence of (4) before (a)
and 30 min after (b) the addition of imidazole; complex (4) was added after (b) and
cyclic voltammograms were then recorded as a function of time (60(c), 120 (d), 180
(e) minutes). (B) Variations as a function of time (60, 120, 180, 240, 300, 360, and
400 min) of the voltammetry of (3) added after exposition of the DMPC lm to
imidazole (20 mM) during 30 min. Compare to Fig. 4B for (3) alone. (C) Voltam-
metry in the presence of (4) and imidazole (20 mM) alone (a) and immediately (b)
after HCl has been added to the solution (pHbulk = 5) or after 60 min (c).
formed with (3) instead of (4) (Fig. 6B). In this case, one observes
only a progressive accumulation of complex (3) within the lipid
lm (compare Fig. 5B) and essentially the same redox behavior
as in the absence of base (Fig. 4B). For (4), the oxidation wave O4
totally disappeared upon addition of aqueous HCl to the solution
(used to protonate imidazole) whereas the second electrochemical
system was not affected (Fig. 6C(b)). These results clearly indicated
that O4 was related to the presence of both the phenolic moiety
and the imidazole base, revealing a ferrocene-mediated proton
and electron transfer of the type sketched in Scheme 1, but taking
place inside the electrode-coated lm. Importantly, when longer
equilibration times were allowed the intensity of O4 grew to reach
a maximum current value. Beyond that point, the peak current ra-
tio IO4 /IO4 started to increase with time. These results showed that
the base-triggered two-electron pathway observed at O4 was lim-
ited by the amount of base available within the lm. When this
amount was sub-stoichiometric (i.e., [base] < 2 [ferrocifen],
compare Scheme 1) the base was titrated so the excess ferrocifen
was oxidized at O4 as in the absence of the base. Since the lm
68 O. Mertins et al. / Inorganica Chimica Acta 374 (2011) 5968
replenishment in base was too slow for changing during one vol-
tammetric scan (see above), the current intensity of wave O4 was
limited as the lm progressively lled up with ferrocifen (compare
to Fig. 5A). Hence, the excess of ferrocifen was oxidized at a more
positive potential value through the same 1-electron reversible
process described above in absence of base (wave O4, compare
Fig. 4C). At longer times, (4) continued to ll up within the lm
(compare Fig. 5B) while presumably the base had already reached
its maximum loading. From this point wave O4 was limited by the
constant amount of base present in the lm, while the additional
(4) could be oxidized at O4 resulting in its progressive growth.
4. Conclusion
The electrochemical behavior of ferrocifen derivatives at a
glassy carbon electrode modied with a bilipidic layer of DMPC
has been investigated to provide further insight on the interac-
tions, transport, as well as the redox behavior of these potent spe-
cies when incorporated in articial membranes. The results clearly
showed that the interaction between these compounds and the
bilipid lm depended on their size, polarity, and their geometry
which conditioned the possibility of H-bonding. The rather dense
accumulation of ferrocifens in the DMPC-modied GCE strongly
suggested that this involved a drastic structural re-organization
of the initial bilipid layer. Expectedly, the afnities of the starting
neutral complexes for the lm were stronger than those of the cat-
ionic electrogenerated ferrocenium species which were rapidly ex-
pelled from the lm. Yet, this latter process was reversible and the
lm could be replenished with ferrocifen upon reduction of its cat-
ion, presumably through a CE process limited by the unfavorable
partition of the cation in the lm.
Moreover, the reactivity of these complexes in the presence of
an imidazole base was shown to depend on the limited amount
of the base which could enter in the lipid layer, and therefore on
its afnity towards the DMPC lm. Accordingly, only a fraction of
the phenolic ferrocifens present in the lipid lm could undergo a
full two-electron oxidation sequence, the excess giving rise to
the same redox behavior as in the absence of base.
Acknowledgments
This work has been supported in part by the CNRS (UMR 8640),
the Ecole Normale Superieure, UPMC, the French Ministry of
Research, and the Agence Nationale de la Recherche (No. ANR-
06-BLAN-0384-01, FerVect). The authors also thank Prof. G. Jao-
uen (UMR CNRS 7223, Paris) and his group for supplying the fer-
rocifen complexes synthesized in their laboratory.
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