FULL PAPER

DOI: 10.1002/adfm.200700793

Incorporation of a Hydrophobic Antibacterial Peptide

into Amphiphilic Polyelectrolyte Multilayers: A Bioinspired
Approach to Prepare Biocidal Thin Coatings**
By Aure´lie Guyomard, Emmanuelle De´, Thierry Jouenne, Jean-Jacques Malandain, Guy Muller, and Karine Glinel*

A non-water-soluble natural antibacterial peptide, gramicidin A, has been successfully incorporated into polyelectrolyte
assemblies to elaborate biocidal thin films. For this, we used a double strategy, the first step of which consists of complexing the
peptide by a non-denaturing anionic amphiphilic polysaccharide, namely a hydrophobically modified carboxymethylpullulan.
We demonstrate that the use of this amphiphilic anionic derivative allows to efficiently solubilize the peptide in aqueous
solution, without denaturation. The amount of peptide solubilized by the amphiphilic polysaccharide was optimized by
systematically varying the hydrophobicity and the molar mass of the CMP derivative. In a second step, the negatively charged
complex was layer-by-layer assembled with cationic poly(L-lysine) to form biofunctionalized thin films. The amount of peptide
incorporated in the multilayers was controlled by changing the number of deposited complex layers, and was quantified by UV
spectroscopy. The antibacterial activity of the resulting biofunctionalized films was evidenced against a gram-positive
bacterium, E. faecalis. We demonstrated that the biocidal activity resulted from a double mechanism: contact between
bacteria and the film surface, and release of the peptide into the solution surrounding the film. We also showed that the peptide
was not completely removed from the film after rinsing, which insured preservation of the biocidal activity of the film surface.

1. Introduction or heavy metals such as silver and tin derivatives[11–14]

Bacteria have a strong ability to attach to solid surfaces,
forming colonies and, subsequently, biofilms.[1,2] These
biofilms serve as reservoirs for the development of pathogens,
which cause infectious diseases.[3,4] In this context, the
development of coatings that impart antibacterial properties
to material surfaces has received continued attention for
several years. Different techniques, involving chemical graft-
ing, surface impregnation, Langmuir–Blodgett deposition, or
physical entrapment, have been explored to immobilize active
antimicrobial substances onto solid surfaces. Numerous
biocidal substances such as antibiotics,[5] quaternary ammo-
nium compounds,[6–8] phenol derivatives,[9] titanium oxide,[10]
[*] Dr. K. Glinel, Dr. A. Guyomard, Dr. E. Dé,
Dr. T. Jouenne, Dr. G. Muller
Laboratoire Polymères, Biopolymères, Membranes – CNRS
Université de Rouen
Bd. Maurice de Broglie, 76821 Mont-Saint-Aignan (France)
E-mail: karine.glinel@univ-rouen.fr
Dr. J.-J. Malandain
Groupe de Physique des Matériaux – CNRS
Université de Rouen
Av. de l’Université, 76801 Saint Etienne de Rouvray (France)
[**] This research was financially supported by ‘‘Réseau Normand Maté-
riaux Polymères, Plasturgie’’ and PNIR Biofilms from CNRS.
have thus been immobilized onto material surfaces to prepare
efficient sterile surfaces. However, the use of some of these
substances is restricted by their potential toxicity. Moreover,
the leaching of these substances into the environment tends to
increase the drug resistance of pathogen bacteria.
Beside these synthetic approaches, some living organisms
such as amphibians and fishes, which are surrounded by
pathogens, have developed an active system of defense to
prevent proliferation of bacteria on their skin; they secrete a
thin layer of mucus that contains antibacterial peptides and
proteins.[15–17] Although the exact mechanisms by which these
peptides exert antibacterial activity are still unclear, it has been
shown that they are able to penetrate into bacterial membranes
to form pores, which subsequently induce cell lysis.[18,19] By
comparison with conventional antibiotics, these natural
antibacterial peptides, which are also produced by plants,
insects, mammalians, and micro-organisms,[20–23] offer several
attractive advantages: they have been shown to have a broad
spectrum of antimicrobial activity, they rarely promote the rise
of bacterial resistance, and they act at a very low concentration.
Therefore, the development of antibacterial coatings using
these natural substances seems to be very attractive.
The layer-by-layer (LbL) deposition of polyelectrolytes
based on the alternate deposition of polycations and -anions
onto solid substrates is a simple and robust method to coat
various surfaces.[24,25] A wide variety of compounds, such as
proteins and peptides, or particles can be easily embedded in
the multilayer architecture to prepare functional films.[26–29]

758 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim Adv. Funct. Mater. 2008, 18, 758–765
A. Guyomard et al./Bioinspired Biocidal Thin Coatings
Scheme 1. Amphiphilic anionic polysaccharide derivatives used in this
study: R ¼ H, CH2COO
Naþ, or CH2C(O)OC10H21.
Antibacterial films incorporating silver particles[30–35] or
biocidal polycations[36] have been successfully prepared by
this technique. Recently, polyelectrolyte multilayers were also
functionalized with antifungal[37] and antimicrobial pep-
tides.[38,39] Experimentally, the peptides were simply
embedded in the polyelectrolyte self-assemblies by direct
electrostatic adsorption during film construction or through
precomplexation with the polyanion used for the LbL
assembly. Whatever the strategy used, they were restricted
to the use of highly charged and water-soluble antibacterial
peptides, which are not so frequently encountered. Moreover,
the electrostatic interaction existing between the peptides and
the polyelectrolyte matrix, may denature the peptide or reduce
its mobility, which could affect significantly the bactericidal
efficiency.
Recently, we reported on the use of hydrophobically
modified carboxymethylpullulans (HMCMP; Scheme 1),
which are amphiphilic anionic polysaccharides, to solubilize
hydrophobic biomacromolecules in aqueous solution.[40] These
amphiphilic derivatives were shown to form micelles around
hydrophobic proteins without denaturation.[41,42] Polyelectro-
lyte multilayers were also successfully built by complexing
these amphiphilic polyanions with various polycations.[43] The
resulting multilayered films were shown to contain hydro-
phobic domains in which hydrophobic compounds, such as
dyes, could be efficiently trapped. Here, we explore the use of
these hydrophobically modified polysaccharides to insert a
hydrophobic antibacterial peptide into polyelectrolyte films,
with the aim of designing a simplified artificial biocidal mucus.
Gramicidine A (Gram A), a non-water-soluble peptide acting
against Gram-positive micro-organisms, was used as a
hydrophobic model peptide.[44–46] We investigated the inser-
tion of this peptide into the amphiphilic multilayers and the
antibacterial activity of the resulting films against a Gram-
Scheme 2. Representation of the process used for the preparation of the peptide-functionalized LbL films. POE, respectively, close to the
Adv. Funct. Mater. 2008, 18, 758–765 ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
FULL PAPER
positive bacterium, Enterococcus faecalis, which is largely
involved in nosocomial infections.[47]
2. Results and Discussion
The strategy, which consists of inserting the hydrophobic
antibacterial peptide in the polyelectrolyte multilayers, is
described in Scheme 2: The peptide was first dispersed in an
aqueous solution of a hydrophobically modified CMP to form a
water-soluble complex via hydrophobic interaction. The
resulting anionic complex was then layer-by-layer assembled
with a polycation onto solid substrates to form peptide-
functionalized multilayer films. Gram A, of which the solubility
in aqueous solution does not exceed 0.3 mg L
1,[48] was chosen
as a model antibacterial hydrophobic peptide for this study.
2.1. Solubilization of the Hydrophobic Peptide by
Non-Denaturating Amphiphilic CMP’s
According to the process described in Scheme 2, the
efficiency of the Gram A insertion into the multilayered films
depends on the ability of the anionic amphiphilic polysacchar-
ide to trap the peptide in aqueous solution (step 1). To optimize
this step, different HMCMP derivatives bearing various
contents (from 2 to 18%) of decyl chains were tested. These
derivatives are denoted CMP-xC10, with x the grafting degree
defined as the number of decyl chains grafted per 100
anhydroglucose units. Experimentally, Gram A was gently
dispersed in aqueous solutions of CMP-xC10 (2 g L
1). The
amount of Gram A actually solubilized by the HMCMP was
determined by performing UV measurements at 280 nm on the
supernatant obtained after centrifugation of this suspension, as
described in the Experimental section. Figure 1 shows the
concentration of Gram A actually solubilized by CMP-xC10
derivatives versus the grafting degree x in decyl chains. The
data are given for amphiphilic derivatives of low (CMPL-xC10)
and high (CMP-xC10) molar mass. The ability of CMP-xC10
derivatives to solubilize the hydrophobic peptide increased
with the degree x in grafted decyl chains. Moreover, the
low-molar-mass CMPL-xC10 derivatives appeared to be more
efficient than the high-molar-mass ones, particularly regarding
the higher grafted derivatives. To get a better estimation of the
efficiency of CMP derivatives to solubilize the hydrophobic
peptide, we performed similar
tests in presence of sodium dode-
cyl sulfate (SDS) and n-octyl
poly(oxyethylene) (octyl-POE),
commercial surfactants that are
well-known to solubilize effi-
ciently hydrophobic biomacro-
molecules such as proteins or
peptides. The tests were per-
formed at concentrations of 2.3
and 4 g L
1 for SDS and octyl-
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Figure 1. Variation of the concentration of Gram A effectively solubilized

in CMP-xC10 (triangle) and CMPL-xC10 (circle) solutions vs. grafting degree
x in decyl chains. The lines are drawn as guide for the eye.

concentration of 2 g L
1 used for the CMP derivatives. Results

(Table 1) showed that the low-molar-mass CMPL-18C10
exhibited the same ability to solubilize Gram A as SDS and
a larger one when compared with octyl-POE. The ability of
CMP-xC10 derivatives to solubilize hydrophobic compounds is
related to their associating behavior in aqueous solution.
Indeed, amphiphilic chains interact via intra- and/or inter-
molecular hydrophobic interactions to form aggregates,[43] in
which hydrophobic compounds can be trapped.[41,42,49] As the
grafting degree in decyl chains x of CMP-xC10 derivatives
increases, their ability to form hydrophobic aggregates and Figure 2. CD spectra of CMPL-18C10 derivative in aqueous solution (A),
Gram A in methanol (B), CMPL-18C10Gram A complex in aqueous
consequently to trap hydrophobic compounds, is enhanced. solution (C), and (PLL/CMPL-18C10Gram A)16 film deposited on a slide
The higher ability of low-molar-mass derivatives to solubilize of fused silica (D). The dashed line is at 230 nm.
hydrophobic compounds can be explained by their higher
tendency to form intermolecular aggregates.[50] Similar results
were previously reported for the solubilization of membrane (Fig. 2A and B). The CD spectrum of Gram A displayed two
proteins.[51] characteristic intense bands centered around 210 nm and
The pore forming ability of Gram A in bacterial membranes 230 nm (Fig. 2B).[53–55] The negative band centered around
results from an uncommon process based on the formation of 230 nm also seen for CMPL-18C10Gram A complex (Fig. 2C)
an amino terminal-to-amino terminal helical dimer.[52] There- attests for the b-helix native conformation of Gram A. Thus,
fore, it is important to maintain this peptide in its native the presence of the amphiphilic derivative CMPL-18C10 does
conformation within the polyelectrolyte assembly to insure its not affect the conformation of the peptide; a necessary
bioactivity. Circular dichroism (CD) measurements were condition to keep its antibacterial activity.
performed on the water soluble CMPL-18C10Gram A
complex to control the peptide conformation (Fig. 2C). The
control CD spectra of CMPL-18C10 solubilized in water and of
2.2. Insertion of the Hydrophobic Peptide into
Gram A solubilized in methanol are provided for comparison
Polyelectrolyte Multilayers
Table 1. Solubilization of Gram A by different amphiphilic derivatives. Considering the higher ability of the CMPL-18C10 derivative
to solubilize Gram A while keeping its native conformation,
Amphiphilic derivative Concentration Concentration of this anionic derivative was used in combination with
of amphiphilic GramA in amphiphilic biocompatible poly(L-lysine) (PLL) to prepare functional
derivative [g L
1] solution [mg L
1]
multilayers according to step 2 of Scheme 2. The LbL assemb-
CMPL-18C10 2 169 lies of PLL and CMP-xC10 derivatives were already reported
SDS 1 205 elsewhere and were shown to provide stable films.[43]
Octyl-POE 4 62
The efficiency of the strategy (Scheme 2) used to incorporate

760 www.afm-journal.de ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim Adv. Funct. Mater. 2008, 18, 758–765
A. Guyomard et al./Bioinspired Biocidal Thin Coatings
Figure 3. UV spectrum of the (PLL/CMPL-18C10Gram A)16 film deposited
on fused silica slide. The absorbance band at 280 nm is specific of the
peptide, since the films do not have bands in this range.
Gram A peptide into the films was checked by performing UV
measurement on a 16-bilayer (PLL/CMPL-18C10Gram A)
sample deposited on a fused silica slide (Fig. 3). The presence
of an absorption band visible at 280 nm testified for the
presence of Gram A into the film.[55] Considering the thickness
of 355  16 nm determined by ellipsometry for the same film
deposited on a silicon wafer and the extinction molar
Figure 4. SEM images of the LbL films ended by (PLL/CMPL-18C10)3-PLL (A), (PLL/CMPL-18C10)3 (B), (PLL/CMPL-18C10Gram A)3-PLL (C), and (PLL/
CMPL-18C10Gram A)3 (D) exposed to E. faecalis suspension for 6 h.
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coefficient of Gram A of 22400 L mol
1 cm
1 experimentally
determined from aqueous solutions and taking into account
that the film was deposited on both sides of the fused silica
slide, the concentration of Gram A embedded into the film
could be estimated in the range of 10
4 mol cm
3 which is
higher than the minimum inhibitory concentration (MIC) of
Gram A (5.5  10
12 mol cm
3) reported against E. faecalis.[46]
In addition, CD measurements performed on the 16-bilayer
film confirmed that the LbL process did not affect the native
helical conformation of Gram A molecules (Fig. 2D). These
results demonstrate the efficiency of the strategy (Scheme 2)
used to incorporate the hydrophobic peptide within the films.
Moreover, the amount of Gram A embedded within the films
could be tuned by simply varying the number of deposited
biofunctionalized (CMPL-18C10Gram A) layers.
2.3. Antibacterial Assays
The antibacterial activity of Gram A-biofunctionalized films
was tested against the gram-positive E. faecalis bacterium. To
avoid any effect due to the substrate and to ensure the stability of
the films, the following deposition sequence was used to prepare
the samples used for antibacterial assays: (PEI/CMP-18C10)1-
(PLL/CMP-18C10)8-(PLL/CMPL-18C10Gram A)M.
While the first compartment (PEI/CMP-18C10)1 reinforces
the adhesion between the film and the substrate, the second
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one (PLL/CMP-18C10)8 ensures a sufficient thickness of the

film, the third one (PLL/CMPL-18C10Gram A)M with M
varying, providing the bioactivity of the multilayer. We used M
values ranging from 1 to 3 in order to investigate the influence
of the number of deposited Gram A-functionalized layers, as
well as the nature of the last deposited layer on the
antibacterial activity of the films.
In a first approach, the films were simply immersed in a
bacterial suspension for 6 h, rinsed, dried, and observed by
scanning electron microscopy (SEM). Microscopy images
displayed in Figure 4 show the surface of LbL samples ended
by a PLL or CMPL-18C10 layer, and containing or not Gram A in
the third deposited compartment. Bacterial colonies could be
observed on all samples that did not incorporate Gram A (Fig. 4A
and B). This result confirmed the non-toxicity of the non-
functionalized (PLL/CMPL-18C10) films. Very different images
were obtained when Gram A was incorporated within the films. Figure 5. Normalized OD550 of MMG solutions after incubation for 6 h
Indeed, Figure 4D reveals a total absence of bacteria on the with E. faecalis bacterium. Prior to incubation, the solutions were put in
contact for 1 h (A), 14 h (B), and 24 h (C) with films ended by (PLL/
surface of the Gram A-biofunctionalized sample ended by CMPL-18C10Gram A)2 (gray bars) or (PLL/CMPL-18C10Gram A)2-PLL
CMPL-18C10Gram A. Figure 4C shows the presence of swollen (black bars). Incubation was performed after the film removals. The
or lysed bacteria on the surface of Gram A-biofunctionalized normalization is performed with respect to the absorbance of a film-free
sample ended by PLL. These results demonstrated the biocidal bacterial growth test (control experiment). Higher OD550 correspond to
less active films.
surface activity of the Gram A-functionalized films. Considering
the mechanism that is proposed to explain the antibacterial
activity of the peptide towards the bacterial membrane,[45] these containing a low concentration of E. faecalis bacteria. The
data demonstrated that peptide molecules located close to the bacterial growth was estimated by measuring the OD550 of the
film surface can penetrate into the membrane of adherent bacterial suspension after 6 h of incubation at 37 8C. The data
bacteria, inducing the formation of pores and, consequently, the were compared to the value obtained in similar conditions
bacteria lysis. The presence of lysed bacteria on the PLL-ended without dipped film (control experiment) and are expressed as
films, compared to their absence on the CMPL-18C10Gram a percentage of growth. Results obtained for films ended by
A-ended films, suggested that PLL slightly promotes the cell PLL or CMPL-18C10 and containing zero, one, two or three
adhesion due to its positive charge. (PLL/CMPL-18C10Gram A) bilayers are shown in Figure 5.
To check whether Gram A is released from the films, we
monitored the growth of planktonic cells in minimal salt
medium (MMG). Therefore, the films were immersed in pure
MMG solutions for different durations, then removed. Next, a
low amount of E. faecalis cells was inoculated in these MMG
solutions. The bacterial growth was estimated by measuring
the OD550 of the bacterial suspension after 6 h of incubation at
37 8C. The data were compared to the value obtained in similar
conditions without prior dipping of a film in the MMG solution
(control experiment), and are expressed as a percentage of
growth. Results obtained for MMG solutions exposed to films
ended by PLL or CMPL-18C10 and containing two (PLL/
CMPL-18C10Gram A) bilayers are shown in Figure 5. Data
reveal that the inhibition of the bacterial growth increased with
the duration of the contact time between the MMG solution
and the biofunctionalized films. Moreover, the bacterial
growth was less inhibited in solutions exposed to PLL-ended
films. These results unambiguously attested for a slow diffusion
Figure 6. Normalized OD550 of bacterial suspensions after 6 h of incu-
of Gram A from the films to the surrounding solution. The bation in the presence of films ended by: (PLL/CMPL-18C10)3-PLL (A) and
influence of the number of biofunctionalized layers embedded (PLL/CMPL-18C10)3 (A’), (PLL/CMPL-18C10Gram A)1-PLL (B) and (PLL/
into the films was also tested. For this, we monitored the CMPL-18C10Gram A)1 (B’), (PLL/CMPL-18C10Gram A)2-PLL (C) and
growth of planktonic cells in the presence of films comprising (PLL/CMPL-18C10Gram A)2 (C’), and (PLL/CMPL-18C10Gram A)3-PLL
(D) and (PLL/CMPL-18C10Gram A)3 (D’). The normalization is performed
an increasing number of (PLL/CMPL-18C10Gram A) bilayers. with respect to the absorbance of a film-free bacterial growth test (control
In this case, the films were immersed in MMG solutions already experiment). The error bars indicate the standard deviation.

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A. Guyomard et al./Bioinspired Biocidal Thin Coatings
The presence of Gram A into the films induced a strong
inhibition of the bacterial growth, which is slightly more
pronounced for CMPL-18C10Gram A-ended films and which
increased with the number of Gram A-functionalized layers
embedded within the multilayers. In contrast, films which did
not contain Gram A do not affect the bacterial growth,
confirming their non toxicity. Together with the results
obtained in the first set of experiments, we can safely conclude
that the higher antibacterial activity observed with biofunc-
tionalized films containing a higher number of (PLL/
CMPL-18C10Gram A) bilayers is due to a higher amount of
peptide released in the solution.
Interestingly, experimental data obtained with the film
incorporating only one (PLL/CMP-18C10Gram A) bilayer
(Fig. 6B and B’), revealed that the presence of the Gram
A-functionalized bilayer strongly inhibited the bacterial
growth but not totally. If we assume that all the amount of
Gram A trapped into the film (of 1 cm2 surface) was released
into the surrounding medium (400 mL volume), a theoretical
Gram A concentration of about 1  10
9 mol cm
3 in solution
can be determined. This concentration is higher than the MIC
of Gram A against E. faecalis.[46] Consequently, the partial
inhibition of the bacterial growth that we observed, demon-
strates a partial release of the peptide from the film. These
results can be explained by a slow diffusion of Gram A to the
film/liquid interface. To check this assumption, a film ended by
two (PLL/CMP-18C10Gram A) bilayers was extensively
rinsed by immersion for 8 days in MMG solution (with
frequent renewal of the medium). Inhibition tests subsequently
performed (as described above) showed that this film did not
exhibit an antibacterial activity in solution. Moreover, SEM
observation of the film surface showed the absence of a biofilm
after 6 h of incubation (Fig. 7). Only few isolated or aggregated
cocci were observed on the sample surface. This suggests that
residual Gram A remained in the multilayer after rinsing and
continues to diffuse slowly to the external surface, preventing
the formation of biofilm on the film surface. This behavior is of
a particular interest for the preparation of biocidal coatings
showing a long time activity.
Figure 7. SEM image of a film ended by (PLL/CMPL-18C10Gram A)2
extensively rinsed by MMG solution and exposed to E. faecalis suspension
for 6 h.
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3. Conclusion
By developing a double strategy based on hydrophobic and
electrostatic interactions, we prepared thin polyelectrolyte
films incorporating an insoluble antibacterial peptide. These
films were shown to exhibit a strong antibacterial activity,
resulting from a double mechanism, i.e., a contact between
bacteria and the film surface and a release of the peptide into
the solution surrounding the film. The amount of released
peptide (and consequently the antibacterial activity of the
surrounding medium) could be tuned by varying the number of
biofunctionalized layers deposited during the film build-up.
Moreover, the peptide was not completely removed after
multilayers rinsing, which insures the preservation of the
biocidal activity of the film surface.
The strategy developed here is a promising approach to coat
various items or devices used in food, biomedical industry and
in medicine. Moreover, it could be easily adapted to other
hydrophobic or amphiphilic bioactive components, e.g.,
anti-cancer, antibiotic and hormones molecules to elaborate
bioactive coatings, which opens interesting possibilities for the
efficient preparation of smart bio-coatings.
4. Experimental
Materials: Branched poly(ethyleneimine) (PEI) (Mw ¼ 750000),
poly(L-lysine) (PLL) (Mw ¼ 62000), tris(hydroxymethyl)amino-
methane hydrochloride (tris-HCl), and Gramicidin A (Gram A)
(M ¼ 1811) from Bacillus brevis were purchased from Sigma–Aldrich.
Octyl-POE and SDS detergents were obtained from Bachem.
Carboxymethylpullulan (CMP) (Mw ¼ 235000) with a substitution
degree in carboxymethyl groups of 0.90 R 0.05 per anhydroglucose unit
was prepared from pullulan (Hayashibara Biochemical Laboratory,
Japan) according to a procedure already described. [56] Hydropho-
bically modified derivatives HMCMP were synthesized by grafting
decyl chains on carboxymethyl groups of the CMP through ester
linkages as reported elsewhere. [43, 49] Different hydrophobically
modified CMP derivatives CMP-xC10 were obtained by varying the
content (from 2 to 18%) of grafted alkyl chains. Additionally, low
molar mass derivatives noted CMPL-xC10 were prepared from
CMP-xC10 derivatives by ultrasound treatment as described elsewhere.
[50]
Solutions of Polyelectrolytes: The solutions of polyelectrolytes
were prepared by dissolving the polymers in 0.1 M Tris-HCl (pH 7.4) at
a concentration of 2 g LS1 and 1 g LS1 for CMP derivatives and PLL,
respectively. All solutions were filtered through a 8 mm Millipore
membrane before use.
Solubilization of Gramicidin A by Hydrophobically Modified
CMP Derivatives: A solution of Gram A (2 g LS1) in methanol was
prepared. 500 mL of this alcoholic solution were added under stirring in
2 mL of the CMP derivative solution prepared as described above. The
resulting suspension was stirred vigorously for 2 h at 30 -C to optimize
the Gram A solubilization and to remove the residual traces of
methanol. Then the insoluble part of Gram A was removed by
ultracentifugation at 70000 rpm for 1 h at 4 -C and the supernatant
containing the complex of CMP-xC10 and Gram A, noted
CMP-xC10Gram A, was recovered.
Preparation of Polyelectrolyte Films: The polyelectrolyte multi-
layers were built on one-side polished (100) silicon wafers (ACM,
France) cut into squares of 1 cm side and on SUPRASIL-type fused
silica plates (Hellma, France). The substrate was first cleaned by
treatment in a hot piranha solution (H2O2(35%)/H2SO4(98%) 1:1 v/v)
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for 20 min (caution: piranha solution is extremely corrosive) and then
thoroughly washed with pure Milli-Q water. The substrate was first
immersed in an aqueous solution of PEI (2 g LS1, pH 4) to get a positive
precursor layer covering the surface. Then multilayers were grown by
alternately dipping the substrate in aqueous solutions of CMP-xC10 or
CMPL-xC10 and PLL for 20 min each. Between each deposition step,
the substrate was rinsed to remove the excess polyelectrolyte by
dipping 10 times for few seconds in three beakers of pure Milli-Q water.
After the deposition of the required number of dipping cycles (N), the
substrate was blown dry with a stream of pure air. The same process
based on CMPL-xC10Gram A complex solution was used to prepare
peptide-functionalized films. A film obtained by dipping the substrate
N times in the solution of PLL and NR1 times in the solution of
CMP-xC10 is named (PEI/CMP-xC10)1-(PLL/CMP-xC10)N. More
complex peptide-functionalized films were also prepared by first
depositing N cycles of (PLL/CMP-xC10) followed by M cycles of (PLL/
CMPL-xC10Gram A).
UV-visible Spectroscopy: Gram A-functionalized films deposited
on fused silica slides and CMP-xC10Gram A complex solutions were
studied by UV spectroscopy. Experimentally, the quartz cuvette and
the fused silica slide were mounted in a Kontron UVIKON 860
spectrophotometer sample holder. The spectra were collected using a
quartz cuvette filled with buffer solution and an uncoated fused silica
slide as a reference, respectively.
Circular Dichroism Spectroscopy (CD): CD spectra of solutions of
CMPL-18C10, Gram A and CMPL-18C10Gram A complex were
recorded with a CD6 spectropolarimeter (Jobin–Yvon) routinely
calibrated with a standard solution of d-10 camphorsulfonic acid. The
measuring chamber was extensively purged with pure nitrogen gas
before the measurement to remove any trace of oxygen perturbing the
spectra. Scans were performed at a rate of 2 nm sS1 from 200 to 300 nm.
For each measurement, four spectra were recorded and averaged to
improve the signal/noise ratio. For film examinations, the fused silica
slides coated by the multilayers were mounted in a dedicated sample
holder. CD spectra of films and polyelectrolyte solutions were
systematically corrected by the spectra corresponding to uncoated
fused silica slide and buffer solution, respectively.
Ellipsometry: The thickness of the films grown on silicon wafers
was determined by a null ellipsometer from Multiskop instrument
(Optrel, Germany) at a fixed angle of 70 - and fixed wavelength of
6328 Å. The refractive index of the silicon and the polyelectrolyte films
were taken to be 3.882-j0.019 [57] and 1.5, respectively.
Bacterial Inhibition Assays: Enterococcus faecalis strain was kindly
provided by hospital in Rouen (CHU Charles Nicole, Rouen, France).
A 12h-preculture was performed aerobically in Columbia broth at
37 -C. Bacteria were recovered by centrifugation and resuspended in a
minimal salt medium (MMG) with the following composition (g LS1 in
distilled water): 10.5, K2HPO4; 3.5, KH2PO4; 0.5, NH4Cl; 0.05,
MgSO4  7H2O; 0.05, FeSO4  7H2O; 0.005, MnSO4  7H2O; 15, glucose.
A final bacterial concentration corresponding to an optical density at
550 nm (OD550) of 0.1 was used. 4 mL of this suspension were placed in
wells of a 24-well plate containing multilayer samples immersed in
400 mL of MMG. Peripheral wells of the plate were filled with pure
Milli-Q water to limit the evaporation effect. Additionally, two 24-well
plates which did not contain a multilayer sample were prepared for a
controlled test. After 6 h of incubation under gentle stirring (40 rpm) at
37 -C, the OD550 of the bacterial suspension was measured. The
percentage of bacterial growth was estimated by comparing the OD550
of the bacterial suspension in the presence and without multilayer
samples. Each experiment was performed twice at least to get a
standard deviation of the experimental data. Similar experiments were
performed by adding bacteria in MMG solutions exposed to multi-
layers films for different times prior to bacteria inoculation.
Scanning Electron Microscopy (SEM): The surface of the films
deposited on silicon wafers and exposed to the bacterial suspension for
a given incubation time was observed with a Leo 1530 field emission
scanning electron microscope using an acceleration voltage of 2 kV.
Experimentally, the sample was removed from the bacterial suspen-
764 www.afm-journal.de ß 2008 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
A. Guyomard et al. / Bioinspired Biocidal Thin Coatings
sion, rinsed by one dip in pure Milli-Q water, blown dried with stream
of pure air and then observed by SEM without deposition of additional
coating.
Received: July 18, 2007
Revised: September 25, 2007
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