Supramolecular effects on the antifungal activity of cyclodextrin/di-n- decyldimethylammonium chloride mixtures

Article abstract of DOI:10.1016/j.ejps.2012.02.017

Candidiasis infections are growing problem worldwide especially for the immunocompromised individuals. Di-n-decyldimethylammonium chloride is one of the most common antifungal agents used to clean medical devices. The current study examines the antifungal mechanism of di-n-decyldimethylammonium cation and its cyclodextrin inclusion complexes. Depending on the type of cyclodextrin (α-, β- or γ-CD), inclusion complexes can be as active as ammonium alone in terms of microorganism death (fungicidal activity). Moreover, with β-CD inclusion complexes, synergism is observed against fungus growth (fungistatic activity). Based on molecular dynamics, we propose a mechanism supported by cell number, selective electrode and ζ-potential measurements as a function of time. The mechanism involves four steps: (i) the positively-charged complex diffuses through the solution, (ii) it adsorbs onto the fungus membrane surface by electrostatic interaction, (iii) then it dissociates and the ammonium inserts in the microorganism membrane, and (iv) the change of the cell surface charge induces cell lysis.

Full text of DOI:10.1016/j.ejps.2012.02.017

European Journal of Pharmaceutical Sciences 46 (2012) 336–345
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences
journal homepage: www.elsevier.com/locate/ejps
Supramolecular effects on the antifungal activity
of cyclodextrin/di-n-decyldimethylammonium chloride mixtures
Loïc Leclercq a, Quentin Lubart a, Anny Dewilde b, Jean-Marie Aubry a, Véronique Nardello-Rataj a,
⇑
a Université Lille 1, EA Chimie Moléculaire et Formulation 4478, Equipe ‘‘Oxydation et Physico-chimie de la Formulation’’, Bât. C6, F-59655 Villeneuve d’Ascq Cedex, France
b Institut de Microbiologie, Université Lille 2, Faculté de Médecine, CHRU Lille, Bd du Professeur Leclercq, F-59037 Lille Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 January 2012
Received in revised form 20 February 2012
Accepted 21 February 2012
Available online 1 March 2012
Candidiasis infections are growing problem worldwide especially for the immunocompromised individ-
uals. Di-n-decyldimethylammonium chloride is one of the most common antifungal agents used to clean
medical devices. The current study examines the antifungal mechanism of di-n-decyldimethylammoni-
um cation and its cyclodextrin inclusion complexes. Depending on the type of cyclodextrin (
a-, b- or
c
-CD), inclusion complexes can be as active as ammonium alone in terms of microorganism death (fun-
gicidal activity). Moreover, with b-CD inclusion complexes, synergism is observed against fungus growth
(fungistatic activity). Based on molecular dynamics, we propose a mechanism supported by cell number,
selective electrode and f-potential measurements as a function of time. The mechanism involves four
steps: (i) the positively-charged complex diffuses through the solution, (ii) it adsorbs onto the fungus
membrane surface by electrostatic interaction, (iii) then it dissociates and the ammonium inserts in
the microorganism membrane, and (iv) the change of the cell surface charge induces cell lysis.
Ó 2012 Elsevier B.V. All rights reserved.
Keywords:
Cyclodextrins
Dialkyldimethylammonium cation
Antifungal surfactant
Supramolecular chemistry
Host–guest chemistry
1. Introduction
Indeed, the formulation involves the use of other surfactants
(e.g. polyethoxylated alcohols). In binary and multicomponent
Candida albicans is a commensal fungus which lives in the hu-
man mouth and gastrointestinal tract. Under normal circum-
stances, C. albicans lives in 80% of the human population with
no harmful effects. Sometimes, C. albicans overgrowth results in
oral and genital candidiasis (Ryan and Ray, 2010; d’Enfert and
Hube, 2007). Candidiasis, also known as ‘‘thrush’’, is a common
condition, usually easily cured. However, the treatment is much
more difficult in immunocompromised individuals (AIDS, chemo-
therapy, organ transplantation, etc.) and patients in intensive
care units. In some case, Candidiasis results in septicemia. There-
fore, C. albicans, which form biofilms on the surface of implant-
able medical devices, emerged as important causes of mortality
in these patients. To avoid nosocomial infections, hospitals have
sanitation protocols (e.g. equipment sterilization, etc.). One of
the most widely used biocidal agents to clean chirurgical instru-
ments is a double-tailed cationic surfactant: the didecyldimethy-
surfactant mixtures, there is a spontaneous self-aggregation
which inhibits the biocidal activity due to the reduction of free
[DiC10] cations. To prevent this inhibition, cyclodextrins (CDs)
can be used to form highly water-soluble complexes (Leclercq
et al., 2010a,b, 2009, 2007, 2005; Leclercq and Schmitzer,
2008; Uekama et al., 1998; Funasaki et al., 2008; Jeulin et al.,
´
2008; Kopecky et al., 2004, 2002, 2001; Lehner et al., 1993). In-
deed, naturally CDs are six, seven or eight-membered-1,4-linked
cyclic oligomers of
D-glucopyranose (a-, b- and c-CD) and are
described as shallow truncated cones with an hydrophobic cavity
(Uekama et al., 1998). Moreover, CDs are biocompatible and
have already been used in a wide range of applications including
food and pharmaceutical industries (Funasaki et al., 2008; Jeulin
´
et al., 2008; Kopecky et al., 2004, 2002, 2001; Lehner et al.,
1993). However, depending on the complex structure, the bio-
cidal activity can be switched: encapsulated biocides can be sig-
nificantly more or less active than the biocides alone (Leclercq
et al., 2010a,b; Schmidt et al., 1996; Simpson, 1992). Despite a
large number of publications in this field, the delivery mecha-
nism of encapsulated [DiC10] cations remains unexplored. In this
article, we report on the antifungal mechanism of various CD/
[DiC10] aqueous mixtures based on in silico (molecular properties
calculation and molecular dynamics) and in vitro analyses (anti-
fungal activity against C. albicans, potentiometry and zetametry).
´
lammonium chloride, [DiC10][Cl] (Šuljagic, 2008; Russell et al.,
1999), which exhibits an optimal activity in comparison with
other dialkyldimethylammonium salts (Cybulski et al., 2008; Per-
nak et al., 2006; Pernak and Feder-Kubis, 2005; Pernak and
Chwała, 2003). However, the [DiC10] cation is not used alone.
⇑
Corresponding author. Tel.: +33 032 033 6369.
E-mail address: veronique.rataj@univ-lille1.fr (V. Nardello-Rataj).
0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.ejps.2012.02.017

L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
337
2.5. Antifungal activity
2. Experimental section
All biocidal activity tests were performed using C. albicans ATCC
10231. The minimum biocidal concentrations (MBC) were deter-
mined by the European standard NF EN 1275 (2006): Chemical dis-
infectants and antiseptics – Quantitative suspension test for the
evaluation of fungicidal activity or yeasticidal basic chemical disinfec-
tants and antiseptics – Test method and requirements. A sample of
the product was diluted with water and a test suspension of yeast
cells was added. The number of yeast cells in the suspension was
adjusted between 1.5 and 5.0 ꢀ 106 cfu/mL (6.17 6 log N0 6 6.70).
The mixture was maintained at 20 1 °C for 15 min 10 s. At the
end of this contact time, an aliquot was taken; the biocidal activity
in this portion was immediately neutralized (phosphatidylcholine
2.1. Materials and general information
All the reagents were purchased from Sigma–Aldrich chemicals
and used without further purification. b-CD was a generous gift
from Roquette Frères (Lestrem, France). NMR spectra were re-
corded in CDCl3 (Euriso-top). Chemical shifts are given in ppm
(d) and measured relative to CDCl3. The following abbreviations
are used to explain the multiplicities: s = singlet, d = doublet,
t = triplet, q = quartet, p = pentet, m = multiplet. All products were
dried in a Freeze Dryer (Alpha 1-2 LD plus). In all experiments,
the water used was Millipore (Simplicity 185;
m and
j
= 5.5 ꢀ 10ꢁ5 mS/
r
= 72.0 mN mꢁ1 at 20.0 °C). All measurements were taken
3 g, Tween 80 30 mL, sodium thiosulfate, 5H2O 5 g, L-histidine
at 20 0.1 °C and repeated at least three times. Electromotive
forces (emf) were measured with a Meterlab PHM250 Ion Analyzer
(Radiometer Analytical) using a sensitive electrode prepared from
commercial pH-glass electrode as previously reported (Collinet-
Fressancourt et al., 2011; Leclercq et al., 2010a,b). For all measure-
ments, the sensitive electrode was used in potentiometric cells in
conjunction with a reference electrode of KCl-saturated calomel
protected from amphiphile diffusion by a saline agar-agar gel made
from 2 mol Lꢁ1 KCl solution contained in a Teflon capillary tube. f-
Potentials were measured using a Zetasizer Nanosizer (Malvern).
Conductance measurements were taken with a CDM210 conduc-
tivity meter (Radiometer). The optical density was measured using
a UV–visible Cary 50 Probe (Varian).
chlorhydrate 1 g, saponin 30 g, Tryptone salt 9.5 g, water qsp.
1 L). The number of surviving yeasts in each sample was deter-
mined. Samples were serially diluted from 10ꢁ1 to 10ꢁ6 and each
dilution was plated in duplicate on Agar Sabouraud Dextrose. After
48 h incubation of the plates at 37 °C, the colony-forming units par
millimeter (cfu/mL) were counted. The reduction was defined as
the ratio between the number of cfu/mL in the test suspension at
the beginning of the contact time and the number of survivors
per mL. The lowest concentration of ammonium giving a reduction
of viable cells of four in a logarithm scale compared to the control
was defined as the minimum biocidal concentration (MBC). The
MBCs were estimated for several diluted aqueous solutions (0.1,
0.5, 1, 2.5, 5, 10, 20 and 40 v/v%) prepared from a stock solution
of [DiC10][Cl] and cyclodextrin. It is noteworthy that the [DiC10][Cl]
concentration was fixed at 1 mM (CA) for each test but the cyclo-
2.2. Synthesis
dextrin concentration was adjusted to 2.5 ꢀ CA for
a-CD or
The synthesis methodology have been previously described (see
Leclercq et al., 2010a). 1H NMR (300 MHz, CDCl3, 25 °C, TMS): d
(ppm) = 0.85 (t, 6H; CH3, 3J = 6.9 Hz), 1.23–1.32 (m, 28H; CH2),
1.66 (m, 4H; N–CH2–CH2), 3.38 (s, 6H; NCH3), 3.45 (m, 4H;
NCH2). 13C NMR (75 MHz, CDCl3, 25 °C): d (ppm) = 14.1, 22.6,
22.7, 26.2, 29.2, 29.4, 31.8, 51.3, 63.5. Anal. Calc. for (C22H48N;
Cl); 9/7(H2O): C, 68.59%; H, 13.23%; N, 3.64%; O, 5.34%; Cl, 9.20%.
Found: C, 68.74%; H, 13.10%; N, 3.64%; O, 5.37%; Cl, 9.23%.
m.p. = 88 °C. (Yield: 95%).
1.2 ꢀ CA for b- and -CDs. The minimum inhibitory concentrations
c
(MIC) were measured using UV–visible spectroscopy. Various
aqueous solutions of free or complexed ammonium were prepared.
C. albicans was added to each solution at a concentration such that
an optical density reading of 0.1–0.2 at 620 nm. The optical density
readings of microorganism solutions were measured as a function
of time. The MIC corresponds to the concentration at which no
growth is observed. Broth containing cells alone was used as a con-
trol. The tests were repeated at least three times.
2.6. Molecular Dynamic Study
2.3. CD/[DiC10] interaction
The initial configurations of CDs and [DiC10][Cl] have been ob-
tained from PM6-DH+ semi-empirical calculations. The molecular
dynamic (MD) simulations were carried out in a cubic simulation
box with periodic boundary conditions. In the cubic box, a
phospholipid bilayer membrane model was formed with dipalmi-
toylphosphatidylcholine molecules. This bilayer was located be-
In order to assess the energy content of various molecules,
semi-empirical quantum calculations were undertaken using the
PM6-DH+ method with COSMO (conductor-like screening model)
water solvation parameters as implemented in MOPAC2009TM
(ÓStewart Computational Chemistry). The geometries are fully
optimized with the PM6-DH+ semi-empirical SCF-MO method
without restriction to know dispersion and H-bond energy contri-
bution. For water simulation, a relative permittivity of 78.4 was
employed with up to 92 surface segments per atom for the COSMO
model being used to construct a solvent accessible surface area
based on van der Waals radii. All structures were optimized to a
gradient inferior to 0.1 using the eigenvector following method.
tween two aqueous phases. Then, a free or complexed [DiC10
]
cation was placed in different initial relative orientations in the
upper phase. An initial MD run was performed, for a period of
100 ps at 300 K with HyperChem 8.0. This solvent equilibration
phase should be sufficiently extensive to enable the solvent to
readjust completely to the potential field of the solute. The cut-
off for non-bonded interactions was taken to be 12 Å throughout
all the simulations. At the beginning, we carried out high temper-
ature annealed MD simulations starting at 1000 K (2 ps) annealing
to 0 K (100 ps). The temperature of 1000 K is necessary to enable
the molecule to overcome energy barriers between different con-
formations and to prevent the system from getting stuck in a par-
ticular region of the conformational space. Simulations at lower
temperatures yielded in very similar conformations. The simula-
tions in aqueous solution were relaxed using the steepest descent
method until a gradient different of 0.01 kcal/mol was reached.
After energy minimization of the system at 0 K, the MD simulation
2.4. Molecular properties of free and complexed [DiC10] cation
Vega ZZ 2.4.0 (Ó Alessandro Pedretti and Giulio Vistoli) was
used to visualize and analyze the physicochemical properties (sur-
face area, polar surface area, logP, lipole and virtual logP) of the
single or complexed [DiC10] cation (Pedretti et al., 2004). The lipo-
philic surface of single or complexed cation was calculated using
the MLP surface (molecular lipophilicity potential) with the default
values. MOPAC2009TM was used for all others properties.

338
L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
to the binding of some of the counterions to the aggregate, the slope
in the pre-aggregation region is higher than that in the post-aggre-
gation one. The degree of binding, b, defined as the amount of chlo-
ride counterion per micelles can be calculated according to Eq. (2)
(Rosen, 2004).
was initialized using a time step of 1 fs for a time period of 1000 ps.
The temperature was kept constant at 300 K yielding a canonical
ensemble (NVT).
3. Results and discussion
S>CMC
S<CMC
b ¼ 1 ꢁ
ð2Þ
3.1. Physicochemical properties of [DiC10] alone
[DiC10][Cl] was obtained by a SN2 reaction involving dim-
ethyldecylamine and 1-decylbromide, followed by ion exchange
with a hydroxide counterion and finally the aqueous solution of
[DiC10][OH] was neutralized with an aqueous hydrochloride solu-
tion (Leclercq et al., 2010a). The product was characterized by 1H
and 13C NMR, and elemental analysis confirming that the salt is
very pure (>99.9%) but highly hygroscopic. Thus, the salt was kept
in a glove box under argon atmosphere. Before considering the
antifungal properties, we have fully characterized the surfactant
properties of [DiC10][Cl] salt. To determine the CMC, [DiC10]-selec-
tive electrode have been used (Fig. 1a).
where S>CMC and S<CMC are the slopes above and below the CMC in
the vs. [DiC10][Cl] concentration (CA), respectively. From the
j
experimental data, 40% of chlorides are bound to the cationic mi-
celles. This value is close to the published values (Hiramatsu
et al., 2003).
3.2. In vitro antifungal properties of free or complexed [DiC10] cations
The minimal fungicidal concentrations (MBCs) and the minimal
inhibitory concentrations (MICs) against C. albicans of aqueous
solutions of [DiC10] cations with or without CD (
have been determined. No activity was observed for native CDs
alone in a concentration range from 5 to 1880 M. In contrast, free
[DiC10] cation clearly shows a biocidal activity above 79 M thus
leading to a minimum bactericidal concentration (MBC) of 79
a-, b- or c-CD)
The electrode displays a pseudo-Nernstian behavior (Eq. (1)) in
the concentration range from 10ꢁ2 and 1.7 mM, i.e. the emf linearly
increases with the logarithm of the [DiC10][Cl] concentration due
l
l
to the increase of free [DiC10] concentration ([DiC10 free). The slope
]
l
M
(55.1 mV) is close to the value obtained for similar surfactants such
as [DiC10][Br] (58.3 mV) and suggest a pseudo-Nernstian behavior
(Funasaki and Neya, 2000).
(Fig. 2). This value is consistent with the literature value (Hough-
Troutman et al., 2009).
As reported in the literature, the free [DiC10] cation activity is
not limited to a specific class of bacteria or fungi, i.e. its spectrum
biocidal activity is broad (Ferraz et al., 2011; Hough-Troutman
et al., 2009; Cybulski et al., 2008; Hough et al., 2007; Pernak
emf ¼ 224:6 þ 55:1 ꢀ log½DiC10ꢂfree
ð1Þ
Above 1.7 mM, the emf decrease is due to the micellization (for
more details see Leclercq et al. (2010a)). To confirm the CMC value,
complementary experiments have been performed by f-potential
measurements. As depicted in Fig. 1b, there is a significant increase
of the f-potential just below the CMC reported above. A maximum
is reached around 2.0 mM, from which, the f-potential decreases. It
is noteworthy that the maximum (75 mV) is similar to the value re-
ported by del Burgo et al. (2007) (73 mV). Moreover, the same pro-
file has already been reported in the literature for
dodecyltrimethylammonium bromide [C12][Br] (Sabaté et al.,
2000). Authors concluded that the maximum of the f-potential cor-
responds to the CMC and the increase below the CMC suggests the
formation of pre-aggregates. We note that the CMC determined by
the f-potential measurement is very close to the emf value (2.0 vs.
1.7 mM). The reduction of the f-potential above the CMC is ex-
plained by an increase in the number of the counterions adsorbed
on the aggregate (Sabaté et al., 2000; Rodenas et al., 1994; Cocera
et al., 1999). To confirm the adsorption of some counterions on
the cationic micelles, the conductivity as a function of concentration
et al., 2006; Pernak and Feder-Kubis, 2005). However, the [DiC10
cation has a better effectiveness against P. aeruginosa than against
C. albicans (55 vs. 79 M) (Leclercq et al., 2010a). The same obser-
vations have been reported in the literature and can be explained
by the nature of cell walls and cytoplasmic membranes (Russell
et al., 1999). We focus here on the antimicrobial activity of CD/
[DiC10] mixtures. The MBCs have been estimated for several di-
luted aqueous solutions prepared from a stock solution of ammo-
]
l
nium (1 mM) and CD (2.5 eq. for
a-CD or 1.2 eq. for b- or c-CD
due to their association constants, see below). A significant reduc-
tion of the antimicrobial activity (i.e. MBC increase) is obtained for
the three CD/[DiC10] mixtures compared to free [DiC10]. However,
the MBC increase by a factor two from
c-CD to b-CD to a-CD (85 to
176 to 345 M) due to the decrease of the cavity size. The same
l
behavior has been previously reported for P. aeruginosa (Leclercq
et al., 2010a). Surprisingly, for the MIC, a minimum is observed
for the b-CD/[DiC10] mixture whereas MIC values for
a- and c-
CD/[DiC10] mixtures are similar to that of free [DiC10] cation. There
is a synergy for the b-CD/[DiC10] mixture with respect to the
growth of C. albicans. This behavior can be due to interaction of
CDs and the phospholipids (Debouzy et al., 1998; Fauvelle et al.,
has also been reported (Fig. 1c). We clearly observed that
j presents
a break point at 1.3 0.1 mM. This value is close to that obtained in
the literature (1.3 vs. 1.33 mM) (del Burgo et al., 2007). Indeed, due
80
60
40
20
0
250
200
150
100
0.16
(b)
(a)
(c)
0.12
0.08
0.04
0
0.1
1
10
0.01
0.1
CA (mM)
1
10
0
0.5
1
1.5
CA (mM)
CA (mM)
Fig. 1. Electromotive force (emf), zeta potential (f) and conductivity (j) plotted against total [DiC10][Cl] concentration (CA) at 20 0.1 °C.

L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
339
Fig. 2. Minimal biocidal concentrations (MBC) and minimal inhibitory concentrations (MIC) for various CD/[DiC10] mixtures in comparison to free [DiC10] cation (2.5 for
a-CD
and 1.2 eq. for b- and c-CD and). The maximum error limit of MBC and MIC measurements was 13%.
1997). However, the control experiments performed with CDs
alone do not show any sign of membrane damage (i.e. no cell lysis)
and any reduction of the MIC in the 1–400 lM concentration
range. The plausible mechanism is described in section 3.4 (see
favorable interactions with all CDs (Eint P ꢁ8 kcal molꢁ1). Despite
2:1 complexes are also stabilized by intermolecular H-bonds be-
tween the two CDs, dispersion energies are the complexation driv-
ing force. It is noteworthy that these theoretical results are
supported by the experimental binding constants (Leclercq et al.,
2010a).
below).
To get a better understanding of these docked complexes, vari-
3.3. Physicochemical properties of CDꢃ[DiC10] complexes
ous physicochemical properties of the free or complexed [DiC10
]
cation have been calculated from molecular modeling. As the
lipophilicity can be correlated to the membrane permeation when
H-bonding and electrostatic effects are not rate-determining, the
octanol–water partition coefficients (logP) can be easily calculated
from the Broto–Moreau lipophilicity atomic constants (Zloh and
Gibbons, 2007; Rahman et al., 2011). The logP of free [DiC10] cation
is 4.3, which suggests that it is a hydrophobic biocide which has a
higher affinity for an organic phase (i.e. a biomembrane). It is
apparent that upon complexation, the logP sharply decreases and
the phenomenon is strongly pronounced for the 2:1 stoichiome-
tries. As depicted in Table 1, the logP values are in the order:
As described in our previous work, [DiC10] cation easily forms
inclusion complexes with various CDs in aqueous solution (Lecl-
ercq et al., 2010a). As the interaction energy and complex geome-
tries are important criteria for their biocidal application, molecular
modeling has been performed with the PM6-DH+ semi-empirical
(Korth, 2010) method coupled with COSMO water solvation
parameters as implemented in MOPAC2009TM (Klamt and Schü-
mann, 1993). The PM6-DH+ method was parameterized to allow
the description of non-covalent interactions, specifically the Lon-
don dispersion energy and H-bonding while the COSMO method
(conductor-like screening model) is useful for determining the sta-
bility of various species in an aqueous environment. The results are
presented in Table 1 as well as the stoichiometries and the associ-
ation constants determined experimentally in our previous study
(Leclercq et al., 2010a). It was found that [DiC10] cation exhibits
a-CD2ꢃ[DiC10] < b-CD2ꢃ[DiC10] < b-CDꢃ[DiC10] <
c
-CDꢃ[DiC10] ꢄ -CDꢃ
a
[DiC10] <<< [DiC10]. However, the logP values are totally indepen-
dent of the molecular geometries: the calculation is based on group
contribution. Therefore, the virtual logP has been developed in the
Table 1
Interaction parameters and molecular properties of various CDꢃ[DiC10] complexes in comparison to free [DiC10] cation.a
No CD
CDꢃ[DiC10
]
a-CD
b-CD
c-CD
Interaction parameters
Molecular properties
Stoichiometryb
Geometries
–
–
1:1
2:1
1:1
2:1
1:1
b
K (Mꢁ1
)
–
–
–
–
4.3
5.3
5.4
ꢁ49
0.0
517
23.8
26000
ꢁ8
7500
ꢁ51
ꢁ31
ꢁ7
9700
ꢁ20
ꢁ30
+2
ꢁ21.0
ꢁ9.9
1.2
ꢁ89
51.6
1701
20.1
2900
ꢁ17
ꢁ32
ꢁ8
7600
ꢁ50
ꢁ22
+2
ꢁ17.5
ꢁ8.2
1.3
ꢁ101
38.8
1880
22.6
Eint (kcal/mol)c
Edisp (kcal/mol)c
EH-bond (kcal/mol)c
LogPd
ꢁ16
+3
ꢁ16.5
ꢁ3.0
1.8
ꢁ79
37.1
1543
10.7
ꢁ40.5
ꢁ9.8
0.6
ꢁ33.4
ꢁ6.9
0.8
Virtual LogPd
Lipoled
D
Hhydr (kcal/mol)c
ꢁ100
43.6
2512
5.1
ꢁ110
40.8
2876
3.9
%PSAd
Vm (Å3)c
Dipole (D)c
a
The following abbreviation are used: K is the association constant, Eint, Edisp, and EH-bond, are the interaction, the dispersion and the H-bond energies, respectively, LogP is
the octanol–water partition coefficient, lipole and dipole are the lipophilic and the dipole moment, respectively,
DHhydr is the hydration enthalpies, %PSA is the polar surface
area coverage and Vm is the molecular volume.
b
Taken from Leclercq et al. (2010a) from [DiC10]-selective electrode at 25 °C.
Based on PM6-DH+/COSMO calculation (MOPAC2009™).
Calculated by the method implemented in Vega ZZ.
c
d

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L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
a cubic equation (Eq. (8)) that can be solved to obtain the free CD
concentration ([CD]) using Maple 12.0 (Ó Maplesoft).
last decade to overcome this defect. Although the same behavior is
reported for the logP and the virtual logP, the decrease of this last
is more moderate. The result is more realistic because the values
are calculated by the projection of the Broto–Moreau lipophilicity
atomic constants onto the accessible surface of complexes
(Gaillard et al., 1994). The virtual logP values are in the order: b-
3
2
K1K2½CDꢂ þ K1ð1 þ K2ð2CA ꢁ CCDÞÞ½CDꢂ þ ð1 þ K1ðCA ꢁ CCDÞÞ½CDꢂ ꢁ CCD ¼ 0
ð8Þ
The real solution of Eq. (8) can be injected in Eq. (7) to obtain
the free ammonium concentration ([U]; Eq. (9)).
CDꢃ[DiC10] ꢄ
a
-CD2ꢃ[DiC10] <
c
-CDꢃ[DiC10] < b-CD2ꢃ[DiC10
]
<<< a-
CDꢃ[DiC10] <<< [DiC10]. We have then calculated the lipophilicity
CA
distribution (Lipole) defined as the sum of local values of logP
½Uꢂ ¼
ð9Þ
2
(Eq. (3)).
1 þ K1½CDꢂ þ K1K2½CDꢂ
P
Lipole ¼
ð3Þ
Finally, [CD1ꢃU] and [CD2ꢃU] concentrations can be calculated
from the two previous solutions using Eqs. (4) and (5), respectively.
As the CCD/CA ratio is fixed for each biocidal assay, the variation of
[DiC10] species in the CD/[DiC10] mixtures can be calculated as a
function of total ammonium concentration, CA (Fig. 3).
As depicted in Fig. 3, for a given CCD/CA ratio, depending on the
binding constants, the prevalent [DiC10] species, i.e. free or 1:1 or
2:1 complexed [DiC10] is clearly different. As the biocidal activity
is obtained for concentrations greater than the MBC, it is possible
to know the prevalent species in the active range concentration
riꢀli
where ri and li are the distance between atom i and the origin of mol-
ecule and the atomic value of the lipophilicity of atom i, respectively.
The lipole is less influenced by the complexation. However, the 2:1
complexes exhibit a weak lipophilic moment compared to the 1:1
complexes:
a
a
-CD2ꢃ[DiC10] < b-CD2ꢃ[DiC10] < b-CDꢃ[DiC10] ꢄ
-CDꢃ[DiC10] <<< [DiC10]. The lipophilicity decrease
Hhydr
c-
CDꢃ[DiC10] <
uponcomplexationisconfirmed bythehydrationenthalpies(
D
)
and the percentage of polar surface area coverage (%PSA): it is clear
that the complexed [DiC10] cation presents a better affinity for water
due to an increase of PSA. Moreover, the molecular volume (Vm) in-
creases after complexation when compared to the free [DiC10] cation.
Finally, dipole moments are affected by the complexation: e.g. for all
2:1 complexes, a clear decrease is observed, while for the 1:1
complexes, the dipole moment remains almost constant, except for
for each mixtures: (i) for
complex is prevalent, (ii) for b-CD/[DiC10
CD1ꢃ[DiC10] complex is in equilibrium with the b-CD2ꢃ[DiC10] one,
and (iii) for -CD/[DiC10] mixtures, the
-CDꢃ[DiC10] species is in
equilibrium with free [DiC10]. Despite that complexes are probably
a
-CD/[DiC10] mixtures, the
a
-CD2ꢃ[DiC10
]
]
mixtures, the b-
c
c
unable to penetrate the membrane, b-CD1ꢃ[DiC10] and
c
-CDꢃ[DiC10
]
complexes have similar dipole moments as the free [DiC10] cation
(see above, Table 1). Therefore, these two complexes are able to fix
onto the fungal membrane after which the complex can be disso-
ciated to allow the insertion of the free [DiC10] cation: the complex
complexes act as a reservoir of [DiC10] molecules which are readily
a
-CD due to the binding of one of the two alkyl tails. As depicted in
Table 1, the dipole values are in the order: b-CD2ꢃ[DiC10] < -CD2ꢃ[Di-
C10] < -CDꢃ[DiC10] ꢄ [DiC10].
-CDꢃ[DiC10] < b-CDꢃ[DiC10] <
a
a
c
As reported in the literature, there is a relationship between the
molecular lipophilicity and the permeability through the mem-
brane (Mälkiä et al., 2004). So, we supposed that the complexed
[DiC10] are unable to pass through a biomembrane with ease (Arun
et al., 2008; Loftsson et al., 2003; Stella et al., 1999; Stella and
Rajewski, 1997). Indeed, lypophilicity (logP, virtual logP, lipole)
available for adsorption onto the cell. For
a
-CD2ꢃ[DiC10] and b-
CD2ꢃ[DiC10] complexes, the antifungal activity is probably only
due to the free [DiC10] cations. In the presence of a biological tar-
get, the free [DiC10] concentration, [DiC10 free
with [DiC10] adsorbed onto the cell membrane, [DiC10 cell
[DiC10 complexed, [DiC10 complex Therefore, the decrease of
[DiC10 free, due to the insertion in the cell membrane, altering the
[DiC10 free/[DiC10 comp equilibrium and the complexed [DiC10] is
progressively released in solution:
]
, is in equilibrium
]
, and with
and hydration enthalpy (DHhydr) decrease while the polar surface
]
]
.
area coverage (%PSA) increases. Moreover, the complexes are too
large to intercalate in a biomembrane (see Vm in Table 1). However,
as complexes are in equilibrium with free ammonium and as they
have a potential dipole moment, we can suppose that these com-
plexes are able to recognize the biological target (i.e. the mem-
brane) without permeation.
]
]
]
½DiC10 complex¡½DiC10 free¡½DiC10ꢂcell ! cell lysis
ꢂ
ꢂ
To get a better insight in the mechanism involved in the anti-
fungal properties of free or complexed [DiC10] cations, molecular
modeling has been performed. The molecular dynamic (MD) simu-
lations were carried out in a cubic simulation box with periodic
boundary conditions in all directions. In the cubic box, phospho-
lipid bilayer membrane model is formed with dipalmitoylphospha-
tidylcholine molecules. This bilayer is located between two
aqueous phases. By convention, the upper phase represents the
external environment while the lower represents the internal
medium (Fig. 4).
Several 1000 ps runs were performed with different initial rela-
tive orientations of free or complexed [DiC10] cations in the exter-
nal phase (Fig. 4). As depicted in Fig. 5, the free [DiC10] cations
enter the phospholipid bilayer within the first 200 ps of the simu-
lation run and remain in a stable position. The biocidal mechanism
of free [DiC10] cation is formed by three-steps: (i) diffusion in the
external solution, (ii) adsorption onto the cell membrane surface
by electrostatic interaction, and (iii) insertion of [DiC10] cation
which causes the cell lysis. This mechanism is in good agreement
with the published mechanism (Denyer, 1995; Walsh et al.,
2003; Obłak et al., 2001; Merianos, 2001; Balgavy and Devinsky,
3.4. In silico antifungal mechanism
For total surfactant concentrations below the CMC, the com-
plexation takes place via a bimolecular stepwise mechanism:
Kn0
½CD1 ꢃ Uꢂ
U þ CD ! CD ꢃ U K1
¼
ð4Þ
½Uꢂ½CDꢂ
Kn0
½CD2 ꢃ Uꢂ
CD ꢃ U þ CD ! CD2 ꢃ U K2 ¼
ð5Þ
½CD ꢃ Uꢂ½CDꢂ
where Kn is the binding constant of the ith equilibrium, [U] and [CD]
are the free ammonium (unimer) and cyclodextrin concentrations
respectively, and [CDnꢃU] is the complex concentration of the nth
equilibrium. The mass balance equation for each reagent, expressed
as a function of binding constants, K1 and K2, provides two equation
systems (Eqs. (6) and (7)):
2
CCD ¼ ½CDꢂ þ K1½Uꢂ½CDꢂ þ 2K1K2½Uꢂ½CDꢂ
ð6Þ
2
CA ¼ ½Uꢂ þ K1½Uꢂ½CDꢂ þ K1K2½Uꢂ½CDꢂ
ð7Þ
where CA and CCD are the total ammonium and cyclodextrin con-
centrations respectively. Combination of Eqs. (6) and (7) provides
1996; Ashman et al., 1986). For b-CD1ꢃ[DiC10] and
c
-CDꢃ[DiC10
]
complexes, the same mechanism can be proposed. However, a

L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
341
Fig. 3. Prevalent [DiC10] species as a function of total ammonium concentration (CA) in various CD/[DiC10] aqueous mixtures for CCD/CA ratio fixed in antimicrobial assay (I
-CD, (b) b-CD, and (c) -CD. The dotted line is the MBC of the CD/[DiC10] aqueous mixture. The green double arrow
= free [DiC10], II = CD1ꢃ[DiC10] and III = CD2ꢃ[DiC10]): (a)
a
c
represents the active concentration range active against C. albicans. (For interpretation of the references to color in this figure legend, the reader is referred to the web version
of this article.)
Fig. 4. Schematic representation of membrane model in a periodic box. The inset represents the membrane bilayer colored by lypophilicity (MLP surface as implemented in
VEGA ZZ).
Fig. 5. Snapshots of the MD simulation as function of time for: (a, b, c and d) free [DiC10] cation; (a0, b0, c0 and d0) b-CD1ꢃ[DiC10] inclusion complex in water. Water molecules
and the inner phospholipids layer have been removed for clarity.

342
L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
new step is observed: the complex must be dissociated before the
insertion of [DiC10] cation.
suspension, there is a clear relationship between the decrease of
cells number and the decrease of the [DiC10] concentration which
confirms the antifungal activity of free [DiC10] cations for concen-
trations above MBC. As the C. albicans membrane is negatively
charged (see above), the free [DiC10] cation decreasing is due to
the insertion in the cell membrane. This insertion initiates the cell
lysis. Moreover, the free [DiC10] concentration decreases during the
first minutes before to reach a plateau. The concentration differ-
ence between the initial concentration and the value obtained at
The other complexes remain in the aqueous external layer with-
out adsorption onto the membrane model (not presented here). In-
deed, the 2:1 complex remains in the external aqueous layer due to
the absence of dipole moment (see Table 1). In these cases, the ob-
served biocidal activity is only due to the various equilibria be-
tween free and complexed [DiC10] cations. Therefore, the value of
binding constants (i.e. the affinity of the [DiC10] cation for the
CD), the complex stoichiometry and geometry, the molecular prop-
erties (i.e. dipole moment) are key parameters to obtain a biocidal
activity. Finally, one difference can be highlighted: for b-
CD1ꢃ[DiC10] inclusion complex, when the [DiC10] cation is inserted
in the membrane model, the b-CD remains bound to the phospho-
lipid layer (Debouzy et al., 1998; Fauvelle et al., 1997). This obser-
vation is probably at the origin of the MIC decrease observed for b-
the plateau is about 100
[DiC10] cations obtained from the biocidal assays (79 10
More surprisingly, Fig. 6 shows a sharp decrease of the f-potential
over time at 200 M. The reduction of the f-potential is explained
lM. This value confirms the MBC of free
l
M).
l
by an increase in the number of the chloride anions adsorbed on
the cells due to the insertion of [DiC10] cations in the membrane.
Indeed, when [DiC10] cations are intercalated between the phos-
pholipids, the surface potential becomes more positive and some
chlorides are adsorbed on the cell surface which decreases the f-
potential (see above). Therefore, the loss of cell viability is due to
the insertion of some [DiC10] cations in the cell membrane which
disrupts the surface cell potential that leads to a deterioration of
CD/[DiC10] mixtures (see above, Fig. 2). Indeed, when the [DiC10
]
cation is inserted into the cell membrane, the b-CD1ꢃ[DiC10] inclu-
sion complex is only partially disrupted because the presence of
the ammonium leads to a local modification of the cell membrane
(see above: the f-potential). The effect is not sufficient to induce
the cell lysis but allows maintaining the CD near the membrane,
thus influencing the cell growth. This assumption is supported by
the inefficiency of the CD alone because the membrane does not in-
sert [DiC10] cations (see above).
the membrane. For a [DiC10] concentration of 50
[DiC10] concentration above the MIC and below the MBC), they is
a slow decrease of the cell number compare to 200 M. On the
other part, there is a decrease of free [DiC10] concentration (around
30 M in one hour) and a sharp decrease of the f-potential. There-
lM (i.e. a
l
l
fore, the microorganism death kinetic rate is clearly dependant of
the free [DiC10] concentration. It is noteworthy that for concentra-
3.5. In vitro antifungal mechanism
tion below the MIC (i.e. [DiC10] <5 lM in Fig. 6), no significant
To confirm experimentally the mechanism of the biocidal activ-
ity of the free [DiC10] cation, we have recorded the mean colony
count data (log10 of the number of cfu/mL), the emf and the f-po-
tential over time of aqueous suspensions of C. albicans with or
without [DiC10][Cl] (Figs. 6–8). It is noteworthy that the initial
[DiC10][Cl] concentration (CA) is fixed at 5 (CA < MIC), 50 (MIC < -
change is observed for all quantities measured. All the results ob-
served for C. albicans suspension with [DiC10] cations are highly
compatible with the proposed mechanism (see above).
Now, we can consider the antifungal mechanism of CD/[DiC10
]
mixtures. The results are presented in Figs. 7 and 8 for MIC
< CA < MBC and MBC < CA, respectively.
It is clear that for [DiC10][Cl] total concentrations comprised be-
tween the MIC and the MBC, there is no or very weak cell lyses be-
cause all initial free [DiC10] concentrations are lower than the MBC
CA < MBC) and 250 lM (MBC < CA) (see Fig. 6). These concentra-
tions are below the CMC (i.e. only unimers are present).
Therefore, the emf can be easily converted in the free [DiC10] con-
centration ([DiC10 free) from Eq. (1). Moreover, the f-potential evo-
]
of free [DiC10] cation (79
observed for the f-potential behavior over time. Indeed, a clear de-
crease is observed for -CD/[DiC10] mixture that can be related to
lM). The most interestingly results are
lution over time can be related to the cellular surface charge.
Without [DiC10] cation, the C. albicans cell number remains con-
stant (i.e. there is no cellular death or growth during the experi-
ence time). Moreover, the f-potential is not time dependent and
its average mean is slightly negative (ꢁ2.4 0.2 mV). Therefore,
as described in the literature, the C. albicans membrane is globally
negatively charged (Henriques et al., 2004). As depicted in Fig. 6,
c
the loss of free [DiC10] cation due to the insertion of [DiC10] cations
in the cell membrane which disturbs the membrane potential and
leads to microorganism growth inhibition. In contrast, for b-CD/
[DiC10] mixture, the f-potential increases due to the presence of
CDs, in the vicinity of cells, which shielded the chloride negative
when 200 lM of [DiC10] are added to the aqueous C. albicans
Fig. 6. Cell viability, free [DiC10] concentration and f-potential as a function of time for C. albicans suspension at 20 0.1 °C with various [DiC10][Cl] total concentrations. The
standard deviations are 16%, 8% and 3% for cell viability, f-potential and free [DiC10] concentrations, respectively.

L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
343
0
62.5
6.0
4.0
2.0
0.0
-2
50
34 µM
-4
-6
37.5
25
-8
12.5
-10
0
0
20
40
60
0
20
40
60
0
20
40
60
Time (min)
Time (min)
Time (min)
Control
α-CD /[DiC10](625/250 µM)
γ-CD /[DiC10](60/50 µM)
β-CD /[DiC10](150/125 µM)
Fig. 7. Cell viability, free [DiC10] concentration and f-potential as a function of time for C. albicans suspension at 20 0.1 °C with various CD and [DiC10][Cl] total
concentrations. The [DiC10][Cl] total concentration is comprised between the MIC and the MBC of the considered mixture. The standard deviations are 16%, 8% and 3% for cell
viability, f-potential and free [DiC10] concentration, respectively.
150
100
50
0
-2
6.0
4.0
-4
72µM
-6
2.0
0.0
-8
0
10
0
20
40
60
0
20
40
60
0
20
40
60
Time (min)
Time (min)
Time (min)
Control
β -CD /[DiC10] (300/250µM)
α -CD /[DiC10] (1250/500 µM)
γ-CD /[DiC10] (300/250µM)
Fig. 8. Mean values for log10 of the numbers of cfu/mL, free [DiC10] concentration and f-potential vs. time for C. albicans suspension at 20 0.1 °C with various CD and
[DiC10][Cl] total concentrations. The [DiC10][Cl] total concentration is above the MBC of the considered mixture. The standard deviations are 16%, 8% and 3% for cell viability,
f-potential and free [DiC10] concentration, respectively.
[DiC10
]
cell wall
cytoplasmic
membrane
a
c
b
a
CandidaAlbicans
Fig. 9. Biocidal activity on C. albicans of various aggregates formed in CD/[DiC10] mixtures: (a) electrostatic interaction of free or complexed [DiC10] with the cell membrane,
(b) insertion of [DiC10] in the cell wall or cytoplasmic membrane, (c) cell wall and cytoplasmic membrane lysis. The green and red circles are active and inactive species in the
mixture.

344
L. Leclercq et al. / European Journal of Pharmaceutical Sciences 46 (2012) 336–345
charges. For
a
-CD/[DiC10] mixture, the f-potential remains con-
leading to a loss of efficiency. The developed approach can be gen-
eralized to other types of amphiphilic or organic biocides. More-
over, due to the current interest in the development of new
biocidal formulations, work is currently underway in our labora-
tory to obtain more information on the self-assembly mechanism
of [DiC10] with CDs in the presence of surfactant mixtures such
as polyethoxylated alcohols.
stant due to the weak amount of free [DiC10] cations inserted in
the cell membrane.
As depicted in Fig. 8, when the [DiC10][Cl] total concentrations
are higher than the MBC of the considered mixtures, cell lyses oc-
cur. For b- and
centrations (76 and 120
MBC of free [DiC10] cation (79
organism death) is due to a sufficient amount of free [DiC10] cat-
ions. However, the initial free [DiC10] concentration of -CD/
[DiC10] mixture is lower than the MBC of free [DiC10] cation (13
vs. 79 M). The observed antifungal activity is due to a progressive
release of [DiC10] cations by the complexes. This assumption is cor-
roborated by the loss of free ammonium cations: for -CD/[DiC10
mixture only 1 M is lost. Therefore, it is clear that the complexed
c
-CD/[DiC10] mixtures, the initial free [DiC10] con-
M, respectively) are higher than the
M): the biocidal activity (i.e. micro-
l
l
Acknowledgments
a
We are grateful to Université Lille 1 and to Fonds Européen de
Développement Régional (FEDER) for financial support. We are
grateful to Chrystèle Pluchart (Laboratoires Anios, Sainghin,
France) for antifungal experiments. b-CD was a generous gift from
Roquette Frères (Lestrem, France).
l
a
]
l
cations are released and directly incorporated in the cell mem-
brane. The same behavior can be invoked for b-CD/[DiC10] mixture:
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