RESEARCH ARTICLE
Elaboration of antibiofilm surfaces functionalized with
antifungal-cyclodextrin inclusion complexes
Aı̈cha Gharbi1, Vincent Humblot2, Frédéric Turpin3, Claire-Marie Pradier2, Christine Imbert1 &
Jean-Marc Berjeaud1
Laboratoire de Chimie et Microbiologie de l’Eau – UMR 6008 CNRS, UFR Sciences fondamentales et Appliquées, IBMIG, Université de Poitiers,
Poitiers Cedex, France; 2Laboratoire de Réactivité de Surface CNRS, UMR CNRS 7197, Université Pierre et Marie Curie – UPMC Paris VI, Paris
Cedex, France; and 3Biocydex SAS, IBMIG – UFR Sciences fondamentales et Appliquées, Poitiers Cedex, France
IMMUNOLOGY & MEDICAL MICROBIOLOGY
1
Correspondence: Jean-Marc Berjeaud,
Laboratoire de Chimie et Microbiologie
de l’Eau – UMR 6008 CNRS, UFR Sciences
fondamentales et Appliquées, Université
de Poitiers, IBMIG, 1 rue Georges Bonnet,
86022 Poitiers Cedex, France. Tel.:
+33 549 454 006; fax: +33 549 453 503;
e-mail: [email protected]
Received 3 October 2011; revised 4 January
2012; accepted 19 January 2012.
Final version published online 9 May 2012.
DOI: 10.1111/j.1574-695X.2012.00932.x
Editor: Gianfranco Donelli
Keywords
antibiofilm material; Candida albicans;
cyclodextrins; anidulafungin; thymol.
Abstract
To tackle the loss of activity of surfaces functionalized by coating and covalently bound molecules to materials, an intermediate system implying the noncovalent immobilization of active molecules in the inner cavity of grafted
cyclodextrins (CDs) was investigated. The antifungal and antibiofilm activities
of the most stable complexes of Anidulafungin (ANF; echinocandin) and thymol (THY; terpen) in various CDs were demonstrated to be almost the same
as the free molecules. The selected CD was covalently bond to self-assembled
monolayers on gold surfaces. The immobilized antifungal agents reduced the
number of culturable Candida albicans ATCC 3153 attached to the surface by
64 ± 8% for ANF and 75 ± 15% for THY. The inhibitory activity was persistent for THY-loaded samples, whereas it was completely lost for ANF-loaded
surfaces after one use. However, reloading of the echinocandin restored the
activity. Using fluorescent dying and confocal microscopy, it was proposed that
the ANF-loaded surfaces inhibited the adherence of the yeasts, whereas the
activity of immobilized THY was found fungicidal. This kind of tailored
approach for functionalizing surfaces that could allow a progressive release of
ANF or THY gave promising results but still needs to be improved to display a
full activity.
Introduction
Over the past 30 years, fungi have emerged as significant
causes of many human diseases, with attendant morbidity
and mortality (Edmond et al., 1999; Enoch et al., 2006).
Candida spp. are one of the most common causes of
hospital-acquired infections. Candidaemia are frequently
associated with intravascular indwelling medical devicesrelated infections, which are usually correlated with a biofilm formation (Raad et al., 2007). These infections are
caused by microorganisms present on the medical device
after sterilization and/or from contact with the skin or
mucosa of the patient at the moment of insertion
(Dwyer, 2008). The surface of most polymers can be colonized by bacteria and fungi, ultimately leading to biofilm formation. The microorganisms adhere to the
surface, proliferate, and produce an extracellular matrix,
resulting in the formation of a structured community.
FEMS Immunol Med Microbiol 65 (2012) 257–269
Microbial cells residing in a biofilm (sessile cells) show
marked genotypic and phenotypic differences when compared with their planktonic counterparts, including
increased antimicrobial resistance (Mah & O’Toole, 2001;
Schierholz & Beuth, 2001). To reduce the number and/or
impact of device-related infections, prophylactic strategies
leading to reduced colonization and proliferation are
required (Schierholz & Beuth, 2001; Raad & Hanna,
2002; von Eiff et al., 2005).
To prevent biofilm formation on medical devices,
such as catheters, various technologies have been or are
being developed, each strategy having its own particular
constellation of potential inconveniences and advantages.
These approaches can be broadly divided into two
groups: (1) prevention of biofilm formation by treatment with an active solution instilled into the lumen of
catheters, known as the lock-therapy strategy, and (2)
coating catheters, luminal and external surfaces, with
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258
antibiofilm agents that inhibit the microbial attachment
process.
In the lock-therapy strategy, the lumen of the catheter
is washed with an antimicrobial solution, often mixed
with an anticoagulant, which is removed after a few hours
(Raad et al., 2007). A few studies have independently
shown a significant reduction in vascular catheter-related
bloodstream infections with the use of taurolidine, EDTA,
or various antibiotics used as lock solutions: linezolide,
eperezolide, and vancomycine (Shah et al., 2002; Curtin
et al., 2003; Kite et al., 2004; Percival et al., 2005). Considering particularly catheter-related infections due to
Candida albicans biofilms, it was shown that the used antimicrobials have to be active against sessile yeasts to be
efficient (Schinabeck et al., 2004). Indeed, fluconazole,
which is poorly active against sessile yeasts, was unable to
sterilize the intraluminal surface of catheters (Schinabeck
et al., 2004). Unfortunately, this strategy minimizes the
risk of luminal colonization but not that of the external
surface of the catheter.
In the case of the coating approach, the catheter itself
is impregnated with a broad-spectrum antimicrobial
agent that elutes from the device and impairs bacterial
growth through traditional bactericidal or bacteriostatic
mechanisms. Here, the antimicrobial agents are used prophylactically, preventing the biofilm formation by eradicating even the first microbial pathogens to contaminate
the device. This general approach is also the one that has
progressed furthest in clinical development, with some
antimicrobial-impregnated devices currently used in clinical settings (Danese, 2002). Antiseptic, antibiotic-coated,
and silver-impregnated catheters have been approved in
the USA for use in patients (Raad et al., 2007). Various
agents were used for the coating of catheters and some of
them are currently found on commercial catheters such
as the antiseptics, chlorhexidine and sulfadiazine, the
antibiotics, rifampicin and minocycline, as well as silver
associated with platinum and carbone or silver sulfadiazine (Raad et al., 2007).
The coating approach, however, suffers from recurrent
problems. The release of the active compound is temporary, thus a toxic substance leaches into the environment,
and the gradually decreasing level of the released compound provides perfect conditions for resistance development. Some authors proposed to create a permanently
sterile, nonleaching material by covalently functionalizing
its surface with an antimicrobial compound. Various
strategies of grafting by covalent binding and different
types of antimicrobials were investigated, more or less
successfully, for preventing microbial attachment on substrates (Tiller et al., 2001; Lewis & Klibanov, 2005; Donelli
et al., 2007). Recently, we have tested the antimicrobial
activity of gold surfaces functionalized by grafting the
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
A. Gharbi et al.
antimicrobial peptides Magainin (Humblot et al., 2009)
and Gramicidin (Yala et al., 2011). The grafted samples
displayed an antibacterial activity but only 50–80% of the
attachment of bacteria was inhibited. It was postulated
that the activity of the grafted peptides was bacteriostatic
but not bactericidal because it was not able to aggregate
and form pores across the bacterial membrane, as they
usually do when not immobilized. The mode of action of
the bound peptides is probably different once covalently
immobilized (Yala et al., 2011).
Thus, most of the antimicrobial molecules need to be
released from the surface and interact with the microbial
cell to be effective. The aim of the present study was to
elaborate and then to explore the antibiofilm properties of
a functionalized gold surface which results from the chemical grafting of cyclodextrins (CDs), and the inclusion of
an antimicrobial compound [anidulafungin (ANF) or
thymol (THY)] into the CD hydrophobic cavities.
CDs are cyclic (a-1,4)-linked oligosaccharides of a-Dglucopyranose containing a relatively hydrophobic central
cavity and hydrophilic outer surface. The most common
CDs are a-CD, b-CD, and c-CD, which consist of six,
seven, and eight glucopyranose units, respectively. The
central cavity of the CD molecule is lined with skeletal
carbons and ethereal oxygens of the glucose residues. It is
therefore lipophilic. It provides a lipophilic microenvironment into which suitably sized drug molecules may enter
and be included. No covalent bonds are formed or broken during drug–CD complex formation, and in aqueous
solutions, the complexes are readily dissociated. Free drug
molecules are in equilibrium with the molecules bound
within the CD cavity. The parent CDs, in particular
b-CD, have limited aqueous solubility. Substitution of
any of the hydrogen bond forming hydroxyl groups, even
by hydrophobic moieties such as methoxy functions,
may result in a dramatic increase in water solubility
(Frömming & Szejtli, 1994). For example, the aqueous
solubility of b-CD is only 1.85% (w/v) at room temperature but increases with increasing degree of methylation.
The highest solubility (500 g L1) is obtained when twothirds of the hydroxyl groups (Pitha et al., 1987; Fujiwara
et al., 1990; Hashimoto, 1991; Miyazawa et al., 1995) are
methylated, but then falls upon more complete alkylation.
Other common CD derivatives are formed by other types
of alkylation or hydroxyalkylation of the hydroxyl groups
(Hashimoto, 1991; Frömming & Szejtli, 1994). For example, (2-hydroxypropyl)-b-CD is obtained by treating a
base-solubilized solution of b-CD with propylene oxide,
resulting in an isomeric system that has an aqueous solubility well in excess of 60% (w/v; Pitha et al., 1987).
To be active, the drug included in the CD must be
released, and this step can be advantageously delayed
because the antimicrobials, instead of being simply coated
FEMS Immunol Med Microbiol 65 (2012) 257–269
259
Antibiofilm surfaces grafted with cyclodextrins
on the material, will be included in the hydrophobic
cavity of CDs. A similar strategy was proposed previously
by loading miconazole in CDs grafted on polyethylene
and polypropylene (Nava-Ortiz et al., 2010). These
biomaterials were shown to reduce significantly the formation of biofilm by C. albicans. However, miconazole, as
fluconazole, poorly inhibited sessile yeasts (Schinabeck
et al., 2004) and the antifungal mode of action of the
functionalized biomaterial was not studied. More recently
(Blanchemain et al., 2011), a textile polyester vascular
graft modified with methyl-b-CD was studied for the
releasing of an antibiotic agent from prosthesis. It was
shown that the ciprofloxacin release was significantly
delayed when this molecule was encaged in the immobilized CDs. However, the antimicrobial activity was indirectly studied through agar diffusion test, and no evidence
of an antibiofilm activity of such material was assayed.
ANF is a recent cyclic lipopeptide antifungal agent of
the echinocandin class. Echinocandins inhibit the synthesis of 1,3 b-D-glucan polymers in fungal cell walls (Chiou
et al., 2000). The echinocandins are known to have
potent and lasting antifungal and antibiofilm activity
(Katragkou et al., 2008) which has already been demonstrated in vitro and in vivo (Kuhn et al., 2002; Seidler
et al., 2006; Cateau et al., 2011). The in vitro efficacy of
echinocandin lock solutions with respect to C. albicans
biofilm growth was investigated in recent studies (Cateau
et al., 2008, 2011). It was concluded that, used in combination with systemic medication, echinocandin lock
therapy may contribute to controlling candidiasis in catheterized patients (Cateau et al., 2011).
THY, which is a terpen, is a broad-spectrum antimicrobial agent with well-known antifungal as well as antibacterial activities which were demonstrated against planktonic
cells (Cowan, 1999; Dorman & Deans, 2000; Burt, 2004;
Lopez et al., 2007). In a previous study (Dalleau et al.,
2008), it was demonstrated that THY displayed an antifungal activity against planktonic cells as well as biofilms.
Moreover, THY was shown to be efficient in the prevention
of microbial colonization of impregnated urinary catheters
(Mansouri & Darouiche, 2008).
Immobilizing ANF or THY that are poorly water soluble into CDs through the formation of inclusion complexes could be an innovative solution to avoid the rapid
release of molecules associated with the ‘coating’ strategy
that leads to a quick loss of activity of the surface or to
prevent the undesired loss of activity of molecules that
are covalently bound to the sample. The aim of the present assays is to design an intermediate system that could
lead to a progressive and controlled release of active
agents that could be lethal on approaching pathogens. To
reach this goal, two postulates were initially made. First,
the affinity of the molecule for the CD should be stronger
FEMS Immunol Med Microbiol 65 (2012) 257–269
than the affinity for the aqueous environment surrounding the surface, to delay the release of the molecule.
Secondly, the affinity of the antifungal agent for its targets
on the microbial membrane should be stronger than the
affinity for the CD itself so that it could be released only
when the pathogen cells approach close to the surface, to
exert its lethal activity.
In the present work, we investigated the inclusion of
ANF and THY in various CDs through solubilization tests
and checked the antimicrobial activity of the resulting
complexes. The selected CD was then grafted on gold surfaces, and after loading with antifungals, the antibiofilm
activity of these samples was studied by CFU counting as
well as microscopic observations to understand the mode
of action of such functionalized surfaces.
Materials and methods
All the replicates, mentioned below, were made by using
different Candida cultures as well as CD/antifungal agent
complex preparations.
Organisms and growth conditions
The yeast strain used in this study was C. albicans (ATCC
3153). This strain was first grown for 48 h at 37 °C on
Sabouraud Glucose agar with Chloramphenicol (Fluka,
Saint-Quentin Fallavier, France) to obtain a culture of
synchronous stationary-phase yeast. A loopful of this culture was transferred to 25 mL of Yeast Nitrogen Medium
(YNB; BioChemika Sigma Aldrich, Saint-Quentin Fallavier,
France) supplemented with 30 mM of glucose (Sigma
Aldrich) (YNB-Glc 30 mM) and incubated for 16 h at
37 °C without shaking. Blastospores were then harvested
and washed twice in phosphate-buffered saline (PBS; pH
7.2) and adjusted to the desired concentration.
Chemicals
Standard antifungal powder of ANF (purity 82.4%) was
kindly provided by the manufacturer (Pfizer Inc, Groton,
CT). ANF was prepared as a stock solution at 0.9 mM in
dimethylsulfoxide (DMSO), aliquoted (50 lL), and stored
at 80 °C. THY was purchased from Sigma Aldrich and
kept at 4 °C. THY working-solutions in methanol were
freshly prepared before use. Alpha-CD (Acros organics;
Fisher Scientific, Illkirch, France), b-CD (Roquette,
Lestrem, France), c-CD (Acros organics, Fisher Scientific),
and three CD derivatives namely the 2-Hydroxypropylb-CD (HP-b-CD; Roquette), the Randomly Methylatedb-CDs (RAMEB; Wacker Chemie, Munich, Germany),
and a low methylated and partially crystallized CDe, the
CRYSMEB (Roquette) were used. 11-mercaptoundecanoı̈c
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260
A. Gharbi et al.
acid (MUA) and 1-(3-dimethylaminopropyl)-N0 -ethylcarbodiimide hydrochloride (EDC) were purchased from
Sigma Aldrich. All solvents were reagent grade. Reagents
were used without any further purification. Experiments
were carried out at room temperature if not specified
otherwise.
product (3) (5.8 g, 4.3 mmol, 50%) thus obtained was
vacuum-dried in a dessicator.
A solution of (3) (3 g, 2.3 mmol) in 30 mL of DMF
was treated with triphenylphosphine (1.5 g, 5.7 mmol).
The mixture was stirred for 2 h at room temperature.
Concentrated ammonium hydroxide (9 mL) was added
and stirring was continued for 18 h at room temperature.
The solvent was evaporated and water (10 mL) was
added. After elimination of the precipitates of triphenylphosphine and of the corresponding oxide by filtration,
the aqueous solution was concentrated under reduced
pressure. The resulting solid was purified by liquid chromatography (silica gel, CHCl3 : CH3OH, 20 : 1) to obtain
6-monoamino-RAMEB (4) (2.1 g, 1.6 mmol, 70%) as a
white powder.
The final compound (4), named RAMEB-NH2, was
further characterized by NMR analysis (1H and 13C) performed in CDCl3 and by electrospray ionization mass
spectrometry (ESI-MS, low and high resolution; data not
shown).
Modified CD preparation
The different steps of the modified CD preparation are
presented in Fig. 1. The starting material mono-6-deoxy6-(p-tolysulfonyl)-b-CD (1) was synthesized as reported
previously (Zhong et al., 1998). It was converted to
mono-6-azido-6-deoxy-b-CD (2) by SN2 reaction of (1)
with excess sodium azide in DMF followed by purification by complex formation (Melton & Slessor, 1971).
Thereafter, a mixture of (2) (10 g, 8.6 mmol) and
sodium hydroxide (65 g, 1.6 mol, 180 M proportions
based on CD) was dissolved in 150 mL of water. Dimethylsulfate was slowly added to this solution (147 mL,
1.6 mol) and the mixture was stirred at room temperature for 17 h. At the end of the reaction, unreacted
dimethylsulfate was decomposed by the addition of
200 mL of concentrated ammonium hydroxide followed
by mixing at room temperature for 6 h. Methylated CD
(3) was extracted with chloroform, and the organic layer
was washed with water, until the washings were neutral
pH, and dried over anhydrous sodium sulfate. After distilling off the solvent from the dehydrated solution, the
residue was treated with ethanol and then with water. The
mixture was concentrated to dryness, and the white solid
Cyclodextrin-antifungal complexes
characterization
HPLC quantification of ANF and THY
ANF and THY were quantified by reverse-phase HPLC
(Series 200 Ic Pump, 785A UV/VIS Detector; Perkin
Elmer) using a C18 column (Symmetry, 4.6 9 150 mm;
5 lm; Waters SAS, Guyencourt, France). The mobile
phase was composed of 50% water and 50% acetonitrile
H 3C
HO
OTs
HO
6
N3
6
9 5%
OH
5 0%
OH
OH
7
1
N3
(CH3)2SO2
NaOH, H 2O
NaN 3
DMF
OH
O x
OH
O
7
CH 3 y
2
3
PPh 3
DMF
NH 4OH
70%
H 3C
O x
NH2
OH
O
CH 3 y
4
Fig. 1. Reaction scheme of the RAMEB-NH2 synthesis.
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Published by Blackwell Publishing Ltd. All rights reserved
FEMS Immunol Med Microbiol 65 (2012) 257–269
261
Antibiofilm surfaces grafted with cyclodextrins
[HPLC grade, Acros Organics; + 0.01% of trifluoroacetic
acid (TFA), Acros Organics]. The flow rate was
1 mL min1. The wavelength of detection was 300 nm
for ANF and 215 nm for THY. Calibration curves were
generated to relate the measured peak area to the concentration (data not shown).
Solubilization assays and CD selection
A 5 mM methanolic solution of ANF was prepared and
divided into 500 lL aliquots that were freeze-dried after
the evaporation of methanol. ANF was then resuspended
in 500 lL of a freshly prepared 10 mM CD solution.
THY was directly weighed in the microtubes so as to
reach a working concentration of 10 mM and the concentration of the added CD solution was adjusted to
20 mM. The mixture was shaken in the thermomixer®
apparatus (Eppendorf, Le Peck, France) for 24 h. The
sample was centrifuged 1 min at 13 000 g, the supernatant was filtered (20 lm filter; Millipore, Molsheim,
France), and solubilized ANF and THY were quantified
by HPLC. Solubility factors in the presence of CD were
then calculated related to the solubility of the antifungal
without CD: 0.001 mg mL1 for ANF and 0.94 mg mL1
for THY (data not shown).
Phase solubility diagrams
Five solutions of CDs were prepared at concentrations 20,
10, 7, 4, and 1 mM. The concentrations of ANF solutions
depended on the CD. The molar ratio of the complex
HP-b-CD/ANF was fixed at 5/1 and 1/1 for the complex
RAMEB/ANF. As described above, appropriate quantities
of ANF were dried in microtubes before being resuspended with CD solutions. In all cases, working concentration of THY was fixed to 33 mM for each the
following CD concentrations: 3.2, 5.6, 8, 12 and 16 mM.
The quantities of solubilized ANF and THY were determined using HPLC.
Minimum inhibitory concentrations (MICs)
The tested concentrations for complexed ANF with CDs
ranged between 2.6 9 109 and 9.4 9 107 M and
between 2 9 105 and 5.3 9 103 M for THY. Solutions
of ANF and THY without CD were also prepared in
YNB-Glc 30 mM and tested at the same concentrations.
Candida albicans (ATCC 3153) inoculum was prepared to
a final concentration of 5 9 103 CFU mL1 in YNB-Glc
30 mM. The MICs of ANF and THY alone or complexed
to CDs were determined according to the broth microdilution method after being incubated 48 h at 37 °C without shaking (Imbert et al., 2002). The MIC was defined
as the lowest drug concentration at which there was no
visible fungal growth (Imbert et al., 2002).
Antibiofilm activity
The tested concentrations for complexed ANF ranged
between 1.3 9 108 and 2.2 9 107 M and between
2 9 105 and 5.3 9 103 M for THY. Solutions of ANF
and THY without CD were also prepared in YNB-Glc
30 mM and tested at the same concentrations. 96-well
polystyrene culture plates (Corning, NY) were impregnated with fetal bovine serum (FBS; Sigma Aldrich), 1 h
at 37 °C, then filled with 300 lL of Candida suspension
(5 9 106 yeasts mL1) and incubated 1 h at 37 °C to
allow cells to adhere (Dalleau et al., 2008). Wells were
then washed twice with PBS to remove nonadherent cells
and were incubated for 24 h at 37 °C with 250 lL of
YNB-Glc 30 mM and 50 lL of solution of ANF or THY
complexed or not to CD. Control wells contained 300 lL
of YNB-Glc 30 mM. The metabolic activity was assessed
using Tetrazolium (XTT) assay as previously described
(Cocuaud et al., 2005). Inhibition percentages of biofilm
growth were calculated as following: Inhibition percentage
(%) = (1 (Mean A450 nm in wells with antifungal)/
(Mean A450 nm in control wells without molecule)) 9 100.
Functionalization of gold samples
Antifungal and antibiofilm activities
Grafting steps
Preparation of the inclusion complex solutions
Solutions of ANF and THY complexed to RAMEB or
HP-b-CD were prepared as described previously for the
solubilization assays. The complexation was conducted
with a concentration of 1 mM for ANF and 15 mM for
RAMEB or 40 mM for HP-b-CD. The complexation concentrations for THY, as well as the CDs, were all fixed at
33 mM. The filtered solutions of complexed molecules
were serially diluted in YNB-Glc 30 mM.
FEMS Immunol Med Microbiol 65 (2012) 257–269
The used surfaces were purchased from Arrandee (Werther, Germany). They were constituted of glass substrates
(11 9 11 9 1 mm) and coated successively with a 50 Å
thick layer of chromium and a 200 nm thick layer of
gold. The gold-coated substrates were annealed in a
butane flame to ensure a good crystallinity of the topmost
layers and rinsed first in a bath of absolute ethanol for
5 min and then twice in water for 5 min with shaking.
The substrates were immersed in 10 mL of a 0.01 M
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262
A. Gharbi et al.
solution of MUA in absolute ethanol for 3 h, to ensure
an optimal homogeneity of the adlayer (Briand et al.,
2006a, b), and thoroughly rinsed in ethanol and water,
and dried under a flow of dry nitrogen. Activation of acid
groups into ester groups (EDC at 30 mM in water) and
immobilization of RAMEB-NH2 (1.04 9 102 mM in
water) on gold surfaces was carried out in one step by
depositing a 150 lL drop of RAMEB-NH2/EDC solution
on the MUA-Au-modified substrates at room temperature
for 24 h. After the immobilization step, the surfaces were
vigorously rinsed in water under shaking for 5 min and
finally dried under a flow of dry nitrogen (Fig. 2). These
surfaces were immersed in 2 mL of an aqueous solution
of ANF at 0.02 mM or an aqueous solution of THY at
0.03 mM in a 12-well polystyrene microplate (Nunc®;
Thermo Fisher Scientific, Illkirsh, France) and were shaken at 80 r.p.m. for 24 h. During these 24 h incubation,
the surfaces were sonicated, four times, for 15 min at
60 W. Finally, the functionalized surfaces were quickly
dipped and rinsed in a water bath, then dried under a
flow of dry nitrogen.
two-channel electronic device that generates the sum and
difference interferograms. Those are processed and
undergo Fourier transformation to produce the ‘Polarization Modulation – Reflection Absorption Infrared Spectroscopy’ (PM-RAIRS) signal (DR/R0) = (Rp Rs)/(Rp + Rs).
Using a modulation of polarization enabled us to perform
rapid analyses of the sample after treatment in various
solutions without purging the atmosphere or requiring a
reference spectrum.
Adhesion of yeasts on gold samples
Adhesion phase of yeast cells
A Petri dish was partially filled (around 7 mL) with a
kappa carrageenan gel (15 g L1; Sigma Aldrich) and
cooled to room temperature. One sample was then carefully deposited per dish, the functionalized gold face
upwards. A 100 lL drop containing 105 yeast cells was
deposited on the gold surface. After 3 h at 37 °C, each
sample was washed three times in physiological sterile
solution (0.9% NaCl).
PM-RAIRS measurements
Enumeration of adherent yeast cells
The gold samples were placed in the external beam of
FT-IR instrument (Nicolet Nexus 5700 FT-IR spectrometer), and the reflected light was focused on a nitrogencooled Mercury-Cadmium-Telluride wide band detector.
The infrared spectra were recorded at 8 cm1 resolution,
with co-addition of 128 scans. A ZnSe grid polarizer and
a ZnSe photoelastic modulator to modulate the incident
beam between p and s polarizations (HINDS Instruments,
PEM90, modulation frequency = 36 kHz) are placed
prior to the sample. The detector output was sent to a
H 3C
O
O O
HO
CH 3
O
H3C
O HO
O
CH3
O
After the 3 h adhesion phase and the washing step, the
gold surfaces were transferred into a sterile tube containing 2 mL of physiological sterile solution (0.9% NaCl),
then sonicated 3 min at 60 W. After the removing of the
gold sample, the yeast cells were pelleted by centrifugation
at 13 000 g for 5 min. Then, 1.8 mL of supernatant was
removed cautiously, and the cells were resuspended by
vortexing. The suspension was then diluted 50 and 100
times; 50 lL of each dilution was plated on Sabouraud
O
O
O
H 3C O
HO
NH2
O
H3 C O
O
H 3C
O
OH
OH
O
O CH 3
O
O
OH CH 3
O O
H 3C
OH O
O
CH 3
O
O
O
C
OH O
S
C
OH O
S
OH
C
CH3
O
O CH 3
O
RAMEB , 0.0104 mM
S
Au
EDC , 30 mM
24 H, RT
C
OH O
S
C
OH O
S
NH
C
S
Au
Fig. 2. Reaction scheme of the two consecutive steps leading to the immobilization of RAMEB on Au surface, via esterification of the acidic
functions of MUA by EDC.
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FEMS Immunol Med Microbiol 65 (2012) 257–269
263
agar plates, in duplicate for each dilution, using a spiral
plater WASP (AES, France). The plates were incubated at
37 °C for 24 h before counting the colonies. Samples were
washed in pure water and kept for 1 month, at 4 °C
before assaying adhesion again on already used modified
surfaces using the protocol described above, on the same
set of two samples (control Au-MUA and with RAMEB/
antifungal complexes immobilized).
Microscopic analysis
Viability of adherent yeasts to the surfaces was evaluated
using LIVE/DEAD® Bacterial Viability Kit (BacLight®).
The two BacLight stains, Syto9 (at 2.34 mM) and propidium iodide (at 20 mM) were diluted 10 times, in physiological sterile solution (0.09% NaCl), as stock solutions
and were kept at 20 °C, in the dark. BacLight mixture
used for the microscopic observation was freshly prepared
before microscopic observations by mixing 1.5 lL of both
stain stock solutions with 97 lL of distilled water. After
the 3 h adhesion phase and the washing step described
above, a 10 lL drop of this BacLight mixture was deposited on the surface of the sample, a slide was put on the
drop and the whole set was incubated for 15 min in the
dark, at room temperature. Samples were then examined
with a confocal FV-1000 station installed on an inverted
microscope IX-81 (Olympus, Tokyo, Japan). Images were
acquired with an Olympus UplanSapo 9 60 water,
1.2 NA, objective lens (800 9 800 pixels images with
0.13 mm per pixel corresponding to Nyquist criteria for
optimal sampling). Multiple fluorescence signals were
acquired sequentially to avoid cross talk between image
channels. Fluorophores were excited with the 488 nm line
of an argon laser (for Syto9) and the 543 nm line of an
HeNe laser (for propidium iodide). The emitted fluorescences were detected through spectral detection channels
between 500–530 and 555–655 nm, for green and red fluorescence, respectively.
Several photographs (from 5 to 10), of different areas
with same surfaces, were taken on the same sample. Average numbers of adherent yeast cells and adherent permeabilized (red) cells were calculated.
Results and discussion
Inclusion of antifungals by modified CDs
Solubilization of antifungals by various CDs
According to its hydrophobic nature, ANF is poorly soluble in water (about 8.8 9 107 M) and is consequently a
good candidate for interacting with CDs. Solubilization of
ANF was measured in the presence of different natural
FEMS Immunol Med Microbiol 65 (2012) 257–269
Multiplication factor of anidulafungin solubility
Antibiofilm surfaces grafted with cyclodextrins
4000
3459
3000
2000
1221
1176
1000
0
616
8
1
α CD
β CD
γ CD
HPβCD
RAMEB CRYSMEB
Fig. 3. Improvement of ANF solubility in water with different CDs.
Intrinsic solubility = 0.001 mg mL1 (8.7 9 104 mM; n = 1).
(a, b and c-CD) and chemically modified b-CD (Fig. 3).
The solubility of the echinocandin was not significantly
modified by natural a and c-CDs, while it was enhanced
in the presence of b-CD (multiplication factor = 616),
indicating that the size of the hydrophobic cavity of the
CD is important for the inclusion of ANF. The tested
chemically modified CDs all derived from b-CD and in
all cases improved the solubilization of ANF. The randomly methylated b-CD (RAMEB) showed the highest
affinity for ANF because the multiplication factor of solubilization was equal to 3459 in the presence of this CD
(Fig. 3).
The interaction between THY and b-CD or hydroxypropyl (HP) b-CD was already demonstrated (Mulinacci
et al., 1996; Demian, 2000; Ponce-Cevallos et al., 2010)
but not with RAMEB, which best interacted with ANF.
Thus, we decided to compare solubilization in aqueous
solution of THY using those three CDs (Fig. 3). Surprisingly, the solubility of THY was not enhanced by inclusion in b-CD and was only doubled with both HP b-CD
and RAMEB. However, this terpen was naturally a thousand times more water soluble (6.3 9 103 M; Beer
et al., 2007) than ANF (0.001 mg mL1).
The phase solubility diagrams for the two antifungals
showed that the solubility of both molecules in aqueous
medium increased as a function of the RAMEB concentration (data not shown). In both cases, solubility curves
corresponded to 1 : 1 inclusion complexes.
Finally, RAMEB was selected to form inclusion complexes with both antifungals to test the biological activity
of such complexes. However, it was necessary to verify
that the complexes kept the antifungal activity required
for the elaboration of antibiofilm surfaces.
Antifungal activities of inclusion complexes
The MIC of ANF against Candida spp. has been reported
to be variable between Candida species and even between
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
264
A. Gharbi et al.
strains from the same species (Pfaller et al., 2010). The
MIC of ANF against C. albicans ATCC 3153 was measured (Table 1) using the microdilution method developed by the Clinical and Laboratory Standards Institute
(Messer et al., 2009) and found to be 1.1 9 107 M,
which corresponds to the range of values of the literature
(Pfaller et al., 2010). The MIC of THY was found to be
2.7 9 103 M. Same measurements were then realized
with solutions containing inclusion complexes antifungalRAMEB (Table 1). The MICs were found to be identical
for complexed or free ANF (Table 1) against C. albicans
ATCC 3153. However, the value for THY-RAMEB complex (1.33 9 103 M) was lower than for free THY
(2.7 9 103 M, Table 1). The difference between MIC of
complexed and free THY was not significant, corresponding to only one dilution. Nevertheless, the difference
could be related to the molecular size of both antifungals.
Indeed, THY is a small molecule (MW = 150.2 Da)
which is completely included in the CDs, whereas ANF
(MW = 1140.2 Da) is more than seven times bigger than
THY and cannot be totally included in the cavity of the
CD. Thus, we could hypothesize that RAMEB complexed
THY was more available to interact with cells and subsequently inhibited the growth of C. albicans ATCC 3153.
Free RAMEB was tested in the same conditions and
showed no antifungal activity (data not shown).
In parallel, the inclusion complexes were tested for the
inhibition of the biofilm formation of C. albicans ATCC
3153. Results presented in Table 1 are expressed as the lowest antifungal concentration leading to a 90% inhibition of
biofilm growth. Both antifungals displayed an antibiofilm
activity (4.4 9 107 M L1 for ANF and 5.3 9 103 M
for THY). Interestingly, similar antibiofilm activities were
found when inclusion complexes were used for both antifungals (Table 1). As observed for the MIC of THY, the
antibiofilm activity of complexed ANF (2.2 9 107 M)
was found to be lower than for the free molecule
(4.4 9 107 M). Once again, the difference corresponds to
only one dilution and is probably not significant.
Taken together, these results indicated that ANF and
THY can interact with RAMEB without losing their antifungal and antibiofilm activities. Thus, we could hypothesize that surfaces functionalized with immobilized
RAMEB loaded with ANF or THY would present, as
expected, an anti-Candida activity.
Cyclodextrins grafting and surface
characterization
To immobilize the chosen CD on gold surfaces, it was necessary to synthesize a chemically modified RAMEB containing a reactive function dedicated to the covalent
binding with the thiolated Self-Assembled Monolayers
(SAMs) grafted on the gold surface. Thus, it was decided to
add an amine function on the RAMEB. The synthesis steps
are presented in Fig. 1. The starting material mono-6deoxy-6-(p-tolysulfonyl)-b-cyclodextrin (1) and the intermediate mono-6-azido-6-deoxy-b-cyclodextrin (2) were
synthesized as reported previously (Melton & Slessor, 1971;
Zhong et al., 1998). Methylation of the latter with dimethylsulfate in concentrated aqueous sodium hydroxide
solution gave polymethylated 6-monoazido-RAMEB (3)
with 50% yield. 6-monoamino-RAMEB (4) was isolated
with a yield of 70% after purification.
Figure 2 depicts a schematic representation of the two
steps followed to construct the functionalized surface.
First, the substrate was functionalized with a monolayer
of 11-mercaptoundecanoı̈c acid, respectively (Au-MUA).
The acid functions were then activated into esters, to let
RAMEB-NH2 react via its amino group (Au-MUARAMEB). The esterification and the subsequent creation
of an amidic bound was carried out in one step after
24 h of incubation.
Both steps of the surface functionalization were characterized by PM-RAIRS. Figure 4 shows the PM-RAIRS
spectra recorded after functionalization of the gold
surface by MUA in (a) and after grafting of RAMEBNH2 in (b).
Table 1. Antifungal and antibiofilm activities of anidulafungin and thymol complexed or not to RAMEB
MIC (mg L1)* (mM)*
Free
Anidulafungin
Thymol
0.12
104
400
2.66
Antibiofilm activity (mg L1)*† (mM)*†
Complexed to RAMEB
±
±
±
±
0.07
6.1 9 105
115
0.77
0.12
104
200
1.33
±
±
±
±
0.07
6.1 9 105
153
1.01
Free
0.5
4.4 9 104
800
5.32
Complexed to RAMEB
±
±
±
±
0.25
2.2 9 104
231
1.54
0.25
4.4 9 104
800
5.32
±
±
±
±
0.14
1.2 9 104
0.00
0.00
MIC: Candida concentration: 5 9 103 cells mL1, 48-h incubation.
Antibiofilm activity: Candida concentration: 5 9 106 cells mL1, 1 h adhesion phase, 24 h biofilm.
*n = 3 (Different Candida cultures and complex preparations between replicates).
†
Corresponds to the concentration leading to 90% inhibition of biofilm growth.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
FEMS Immunol Med Microbiol 65 (2012) 257–269
265
Antibiofilm surfaces grafted with cyclodextrins
1118
1334
1598
Antimicrobial activities of gold samples
1104
1260
(b)
1465
1435
1619
1711
1743
2853
2925
1092
1048
1460
1380
1421
1550
1700
1652
2960
2853
1730
2960
PM-RAIRS Signal 0.02 a.u.
2879
2929
confirmed by the signature of the backbone of the molecules, mainly, the stretching of the C–O–C at 1118 and
1092 cm1, and the mC–O–CH3 at 1334 cm1. Eventually, the huge increase in the intensity of the stretching
bands in the CH2 and CH3 region (2800 and 3000 cm1)
also confirmed the successful grafting of the RAMEB onto
the MUA SAMs on gold surface.
Antifungal performance of gold samples
(a)
3000
28002000
1800
1600
1400
1200
1000
Wavenumber cm–1
Fig. 4. PM-RAIRS spectra of the two consecutive steps leading to the
immobilization of RAMEB: (a) Au–MUA; (b) covalent binding of
RAMEB-NH2.
Spectrum (a) is dominated by intense mC=O wide band
at 1711 and 1743 cm1, typical of carboxylic groups
(Bain et al., 1989; Tielens et al., 2008), confirming the
presence of the acid-terminated thiol. In addition, two
bands at 1619 and 1435 cm1, assigned respectively to
the antisymmetric and symmetric mCOO, suggest the
presence of deprotonated carboxylate end groups
(Bertilsson & Liedberg, 1993; Briand et al., 2006a, b).
Thus, acidic functions are present under protonated and
deprotonated chemical forms, both capable of reacting
with EDC during the esterification process. In addition,
the symmetric and antisymmetric stretching modes of the
CH2 groups of the back bone of the thiol molecule are
clearly visible at 2925 and 2853 cm1, respectively. Eventually, PM-RAIRS spectra present bands related to the
scissor mode of CH2 groups at 1455 cm1 and to the
stretching of C–OH of protonated COOH moieties at
1260 cm1. Finally, there are some signs of residual contaminations in the MUA self-assembled monolayers, with
the presence of a band at 2960 cm1 (usually assigned to
CH3 groups) and a sharp band at 1104 cm1 whose
assignment is not clear.
Spectrum (b) exhibits differences with respect to the
MUA layer, suggesting some reactions after 24 h of
immersion in EDC + RAMEB solution. In fact, the binding of RAMEB is indicated by the presence of new IR features in the 1500 and 1700 cm1 region. Intense peaks at
1652 and 1550 cm1 are assigned to the amide I and
amide II bands of the peptidic backbone. One can also
notice the presence of bands at 1700 and 1730 cm1 that
could be ascribed to intact MUA carboxylic acid end
groups not transformed into ester and to ester having not
fully reacted with the NH2 groups of RAMEB, respectively. More importantly, the grafting of RAMEB is also
FEMS Immunol Med Microbiol 65 (2012) 257–269
The results, presented on Fig. 5, showed that the viability
after adherence of C. albicans ATCC 3153 on the gold
samples was inhibited when the antifungal, ANF as well as
THY, was loaded onto the surface. Because the samples
were prepared in parallel, we postulated that the grafting
ratio of RAMEB on different samples was similar. Thus, it
was expected that the ANF-loaded surfaces displayed a
higher antifungal activity than THY-loaded substrates
according to the lower MIC of ANF (1.1 9 107 M) as
compared to THY (2.7 9 103 M). However, inhibition
of the adhesion of yeast cells appeared higher for the samples loaded with THY (75 ± 15%) than with ANF
(64 ± 8%). This could be related to the molecular size of
ANF, which is about seven times bigger than THY, so we
can hypothesize that the number of immobilized ANF
molecules was reduced as compared to THY. Moreover,
only a small part of ANF is supposed to be entrapped in
the CD so we can imagine that the antifungal could act on
yeast cells before they are definitely attached to the surface.
On the contrary, the THY, which is supposed to destabilize
the cell membrane (Braga et al., 2008; Xu et al., 2008;
Nostro et al., 2009), is completely included in the CD and
could be released only as the result of the contact with the
cell membrane when the yeast is attached.
The samples grafted with RAMEB but not loaded with
antifungal were also tested for their anti-Candida activity.
The results obtained from these assays, 4% of inhibition
with a high variability among the three assays, indicated
that the adhesion of C. albicans ATCC 3153 was not
really inhibited (Fig. 5).
The antifungal molecules (ANF and THY) were also
deposited on the Au-MUA-modified surfaces before
RAMEB functionalization as controls, and the inhibition
percentage of adherence of C. albicans ATCC 3153 was
evaluated. For both molecules, adherence was similar than
for the control Au-MUA (data not shown).
Mode of action
Two phenomena could explain the decrease in the number
of culturable cells attached to the functionalized surfaces.
ª 2012 Federation of European Microbiological Societies
Published by Blackwell Publishing Ltd. All rights reserved
266
InhibiƟon percentages of adherence of yeasts (%)
A. Gharbi et al.
100
Initial study
75
Repetition on the same gold surface after 1 month
64
75
55
50
25
6
4
3
0
Au-MUA-NH-RAMEB
Au-MUA-NH-RAMEB +
Anidulafungin
Au-MUA-NH-RAMEB + Thymol
Fig. 5. Inhibition percentages of adherence of culturable C. albicans ATCC 3153 on functionalized gold surfaces (compared to the control
Au-MUA). 106 Candida cells were deposited on the samples and allowed to adhere for 3 h. Error bars indicate standard deviation from three
independent CFU counts.
Either the loading of the antifungal provides antiadhesive
properties to the surfaces thus preventing the yeasts from
attaching onto the substrates, or the loaded antimicrobial
exerts its antifungal activity toward cells after their adhesion via a fungicidal or fungistatic mode of action. To
answer this question, confocal microscopy analyses were
carried out using BacLightTM staining. Briefly, the cellular
membrane of the yeasts which appeared red is damaged,
whereas green cells have maintained their integrity and are
considered as alive. Indeed, the red stain (PI) can only penetrate permeabilized cells, whereas the green one (Syto9)
can cross over intact cellular membranes. Images obtained
by confocal microscopy analyses are presented in Fig. 6.
Entire surfaces of two samples of each type of substrate
were analyzed. On the control surface, grafted with MUA
only, C. albicans ATCC 3153 cells appeared as small green
clusters corresponding to living yeasts (Fig. 6A). On the
surface functionalized with RAMEB loaded with ANF
(Fig. 6B), similar green clusters of cells were observed but
(a)
(b)
in lower amounts (about 56% as estimated by counting)
than on the control Au-MUA. This could indicate an
antiadherent mode of action of immobilized RAMEB-ANF
complexes against Candida cells.
On the surfaces functionalized with THY (Fig. 6C),
cells clusters appeared bigger and were mainly red, indicating that the membranes of the cells were permeabilized, probably dead. Cell density was evaluated on the
samples by counting the total average number of green
and red adherent yeast cells and was found to be similar
to the control MUA. Thus, this result could indicate a
fungicidal mode of action of the surfaces bearing immobilized RAMEB-THY inclusion complexes.
Activity of already used surfaces
To check the antifungal activity of already used modified
surfaces, microbial viability after adherence was assayed
after 1 month’s storage, using the protocol described
(c)
Fig. 6. Viability of C. albicans ATCC 3153, stained with BacLightTM kit, attached to gold surfaces grafted with MUA (a), grafted with RAMEB,
and loaded with ANF (b) or THY (c), observed by confocal microscopy (magnification 9 20). 106 Candida cells were deposited on the samples
and allowed to adhere for 3 h.
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FEMS Immunol Med Microbiol 65 (2012) 257–269
267
Antibiofilm surfaces grafted with cyclodextrins
above, on the same set of two samples (control Au-MUA
and with RAMEB-antifungal immobilized). Samples were
washed in pure water and stored at 4 °C before the
second assay. Results of the inhibition evaluated by CFU
counting of adhered C. albicans ATCC 3153 are presented
in Fig. 5. The inhibition of the growth of the yeasts
appeared slightly decreased on the THY-loaded surface
(55 ± 4%) as compared to the initial assay (75 ± 15%),
indicating that sufficient amount of terpen is still
included in immobilized RAMEB, whereas antifungal
activity was almost lost for the ANF-loaded samples. To
verify that this activity loss was related to a depletion of
the surface in ANF, same samples were washed again and
then reloaded with the echinocandin before a new antifungal assay. As expected, the inhibition of growth of the
yeasts was found to be 86% (n = 1, data not shown).
Thus, the loss of antifungal activity was certainly because
of the absence of sufficient amount of ANF on the surface, because the CDs were empty after the first assay,
and confirmed that the activity was effectively dependent
of the antifungal molecule.
Conclusion
To conclude, this study demonstrated that CDs could be
used to greatly improve the solubility of ANF, and maybe
of structurally related other echinocandins, into an aqueous medium.
The surfaces grafted with CDs and loaded with ANF and
THY displayed antifungal activity but not to a full extent.
That is why this system, which is still at its ‘Proof of the
concept’ step needs further development for more realistic
applications. These improvements imply the transposition
of the concept to materials which are usually used to manufacture indwelling medical devices (especially silicone and
polyurethane). The grafting yield of CDs on the surface
has to be determined, for example, by infrared imaging
and also the quantity of ANF or THY included into the
CDs. Another system should also be found to increase the
grafting yield of CD (e.g. 3D systems) for a better activity.
Nevertheless, we showed that the antagonistic activity
could be restored by re-loading of the CDs. This interesting property could be exploited during lock therapies.
Moreover, it can be imagined that this concept could be
adapted to every molecule that is able to form inclusion
complexes with fixed CDs to conceive systems which can
be used to deliver medicine.
Acknowledgements
This work was financially supported by Pfizer Inc. We
thank Anne Cantereau from ImageUP for confocal
microscopy analyses.
FEMS Immunol Med Microbiol 65 (2012) 257–269
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