Antibacterial poly(ethylene terephthalate) surfaces obtained from thymyl methacrylate polymerization

Article abstract of DOI:10.1002/pola.27647

Thymol, an antibacterial agent was used for the preparation of a methacrylic monomer. The conventional and atom transfer radical (ATRP) polymerizations of this monomer were studied using different conditions. Then, the functionalization of poly(ethylene t

Full text of DOI:10.1002/pola.27647

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Antibacterial Poly(Ethylene terephthalate) Surfaces Obtained From
Thymyl Methacrylate Polymerization
1
1
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2
ꢀ ꢀ
Sophie Bedel, Benedicte Lepoittevin, Ludovic Costa, Olivier Leroy,
1
3
3
3
ꢀ ^
Diana Dragoe, Jerome Bruzaud, Jean-Marie Herry, Morgan Guilbaud,
3
3
€
Marie-Noelle Bellon-Fontaine, Philippe Roger
1
ꢀ
ꢀ
ꢀ
Institut de Chimie Moleculaire et des Materiaux d’Orsay (ICMMO), UMR CNRS 8182, Universite Paris-Sud, Orsay,
F-91405, France
2
ꢀ
Laboratoire de Physique des Gaz et des Plasmas (LPGP), UMR CNRS 8578, Universite Paris-Sud, Orsay, F-91405, France
3
ꢀ
ꢀ
ꢀ
AgroParisTech INRA, Institut Micalis, Equipe Bioadhesion-Biofilms et Hygie`ne des Materiaux, 25 avenue de la Republique,
Massy, F-91300, France
Correspondence to: P. Roger (E-mail: philippe.roger@u-psud.fr)
Received 19 December 2014; accepted 7 March 2015; published online 00 Month 2015
DOI: 10.1002/pola.27647
ABSTRACT: Thymol, an antibacterial agent was used for the
preparation of a methacrylic monomer. The conventional and
atom transfer radical (ATRP) polymerizations of this monomer
were studied using different conditions. Then, the functionali-
zation of poly(ethylene terephthalate) (PET) films by “grafting
from” ATRP using this monomer was investigated. In this aim,
a three steps procedure was developed. The surfaces were first
treated by NH3 plasma treatment to incorporate primary amino
functions. Then, in a second step, ATRP initiator was grafted
by reaction with bromoisobutyryl bromide. Surface initiated
ATRP of thymyl methacrylate was performed in solution in the
reactions was confirmed by X-ray photoelectron spectroscopy.
Wetting properties and surface energy were found to vary sys-
tematically depending to the type of functionalization and
grafting. The poly(thymyl methacrylate)-grafted PET surfaces
exhibit resistance to bacterial adhesion toward Pseudomonas
aeruginosa, Listeria monocytogenes, and Staphylococcus aur-
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eus strains.
2015 Wiley Periodicals, Inc. J. Polym. Sci., Part
A: Polym. Chem. 2015, 00, 000–000
KEYWORDS: atom transfer radical polymerization (ATRP); living
radical polymerization (LRP); polyesters; radical polymerization;
surfaces
presence of
a sacrificial initiator. The efficiency of these
ple, surface modification with hydrated polymer chains (such
as poly(ethylene glycol), polysaccharides, phospholipids, and
polyvinyl pyrrolidone) provides a steric barrier that mini-
mizes non-specific interactions with organic matter and liv-
ing bacteria.6 Other studies report the use of silver
nanoparticles loaded on material surfaces for their bacteri-
cidal ability.7 Alternatively, many natural compounds possess
known activity against a range of Gram-positive and Gram-
negative bacteria. We have previously shown the use of poly-
meric derivates from gaiacol (obtained from gaiac) and myr-
tenol (essential oil) to obtain anti-adhesive and anti-biofilm
coating on glass slides.8,9
INTRODUCTION Microorganisms can colonize a wide variety
of surfaces including food packaging and medical devices,
this colonization leading to biofilm formation. Biofilms result
from the accumulation of organic molecules, microorganisms,
and metabolites and are ubiquitous on surfaces submerged
in aqueous environments.1 A reduction of surface contamina-
tion could be obtained through a well-adapted choice in the
materials to use or through modifying them by tailored sur-
face treatments.2 In the past years, there has been much
interest in developing methods to modify the surface proper-
ties of different polymers in order to obtain antibacterial
properties. Poly(ethylene terephthalate) (PET) is a semi-
aromatic and thermoplastic polymer commonly used in sev-
eral areas such as the textile industry, packaging, automo-
biles, membranes filtration, and the biomedical field.3,4 PET
has many applications due to its properties; however, bacte-
ria can easily colonize the surface of PET. Thus, various types
of modification methods have been developed to enhance
the antibacterial properties of organic surfaces.5 For exam-
Thymol (2-isopropyl-5-methylphenol), an essential oil found
in thyme and extracted from Thymus vulgaris, is used in den-
tistry, for example, for treatment of oral infection. Thymol
has strong bacteriostatic or bactericidal activity against a
wide range of bacteria.10 Very few studies report the use of
thymol for the preparation of polymeric materials.
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In 1994, Moszner et al.11 described the preparation of meth-
acrylic and p-styrenesulfonic acid esters of thymol and stud-
ied their free radical polymerization. These authors showed
an antibacterial activity of methacrylic polymer (water sus-
pension) against Streptococcus mutans probably caused by
enzymatic release of thymol. To our knowledge, this study
does not seem to be more detailed.
Plasma Processing
Surface modifications were performed in
a
microwave
plasma reactor as previously described.19 The plasma was
created and maintained in a quartz cylinder with a 12 cm
diameter and 40 cm high on top of a post-discharge chamber
where the substrates are located. The plasma was generated
using a Surfaguide excitation reactor connected to opposite
sides of two 2.45 GHz microwave generators. NH3 flux was
regulated at a total flow of 100 sccm (standard cubic centi-
meters per minute). Pressure was fixed at 47 mTorr. Sub-
strates were treated in the flowing afterglow of the
microwave plasma in the post-discharge chamber. Both gen-
erators were set at 500 W. The surfaces were exposed 60 s.
Then, the PET films were subjected to colorimetric titration.
The bicinchoninic acid method was used to determine the
concentration of primary amino groups.23
Polyesters of alternating maleic anhydride/styrene or vinyl
acetate copolymers with thymol (and eugenol, citronellol)
were prepared by esterification reaction.12 More recently,
phenolic derivatives as thymol were chemically incorpo-
rated into polyanhydrides as pendant groups via ester
linkages.13 These polymers were hydrolytically degraded
after 2 weeks releasing free phenolic antimicrobials in a
controlled manner.14 Some other recent studies report the
use of thymol in mixture with different polymers (encap-
sulation, coating, etc.) and the effect on the antimicrobial
properties.15–17
Monomer Synthesis
Thymyl (5.0 g, 3.30 3 1022 mol), anhydrous diethyl ether
(50 mL) and triethylamine (5.8 mL, 4.16 3 1022 mꢀol) were
introduced in a flask under argon atmosphere at 0 C. Then
methacryloyl chloride (4.9 mL, 4.16 3 1022 mol) was added
dropwise at low temperature and the reaction mixture was
stirred at room temperature during 20 h. Triethylammonium
salts were removed by filtration and washed with diethyl
ether. The filtrate was washed with an aqueous solution of
sodium hydroxide (1 mol L21) and saturated sodium chlo-
ride solution. The organic phase was rotary evaporated to
dryness to yield yellow syrup. The crude product was puri-
fied by flash chromatography using petroleum ether/ethyl
acetate (98/2 vol %) as mobile phase. The fractions were
then evaporated to afford the product as clear syrup (4.4 g,
65% yield).
Recently, we have published different methods to modify
PET surfaces as films and fibers. The techniques used were
plasma treatment,18,19 covalently grafting of biomolecules by
reductive amination,20 “grafting from” by controlled radical
polymerization,21 and grafting of UV-reactive molecule.22 In
this study, we have combined the use of plasma treatment
and grafting from polymerization to obtain poly(thymyl
methacrylate) brushes on PET surfaces presenting antibacte-
rial properties. Among the different controlled polymeriza-
tion technique, atom transfer radical polymerization (ATRP)
is especially attractive because it is a controlled process that
gives polymers with expected molar masses and low polydis-
persity index. In addition, ATRP has been widely used for
the synthesis of polymer brushes of various monomers from
(in)organic substrates.
1H NMR (360 MHz, CDCl3, d): 7.20 (d, 1H, Ar H), 7.02 (d, 1H,
Ar H), 6.84 (s, 1H, Ar H), 6.34 (s, 1H, CH@), 5.74 (t, 1H,
CH@), 2.96 (sept, 1H, (CH3)2CH), 2.30 (s,3H, CH3), 2.06 (s,
3H, CH3 methacrylic), 1.16 (d, 6H, (CH3)2CH) ppm.
The first part of this study demonstrates the ability of a
methacrylic monomer obtained from thymol essential oil to
be polymerized from conventional and ATRP polymeriza-
tions. In the second part, we study the ATRP grafting from
polymerization of this monomer from PET films previously
functionalized by plasma treatment and initiator grafting.
Antibacterial tests were carried out using different Gram-
positive and Gram-negative strains.
13C NMR (90 MHz, CDCl3, d) and HSQC (1H/13C): 166.2
(C@O), 148.4 (CAO), 137.2, 136.7 and 136.2 (C@CCH3,
CACH3 Ar and CACH(CH3)2 Ar), 127.2 (CH Ar), 127.1
(CH2@), 126.6 (CH Ar), 123.0 (CH Ar), 27.5 (CH isopropyl),
23.2 (23(CH3) isopropyl), 18.7 (CH3 methacrylic).
Immobilization of ATRP Initiator on PET Surface
This reaction was carried out in tubes under argon. PET
films, freshly plasma treated, were immersed in anhydrous
diethylether (10 mL). Then, triethylamine (1.5 mL) and bro-
moisobutyryl bromide (6.4 3 1023 mol, 0.8 mL) were added.
The reaction was allowed to proceed overnight with moder-
ate agitation. The PET films were purified by multiple wash-
ings with water, methanol, and acetone and dried in vacuum
at room temperature.
EXPERIMENTAL
Materials
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V
PET films Melinex OD with a thickness of 125 lm were
kindly provided by DuPont Teijin films and used without
cleaning. PET films were cut into pieces of 10 3 20 mm2.
Thymol, ethyl-2-bromoisobutyrate (99%), 2-bromoisobutyryl
bromide (98%), N,N,N0,N00,N00-pentamethyldiethylenetriamine
(PMDETA, 98%), 1,1,4,7,10,10-hexamethyltriethylenetetr-
amine (HMTETA, 97%) copper bromide (CuBr, 98%), tolu-
ene, and methanol were obtained from Aldrich and used
without purification. Diethylether and triethylamine were
distilled on calcium hydride before use. AIBN was recrystal-
lized from ethanol.
Conventional Radical Polymerization
Polymerizations were carried out under argon in toluene
with shaking. Aliquots of approximately 100 mL were with-
1
drawn to follow monomer conversion by H NMR and molar
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TABLE 1 Energy Characteristics (mJ m22) of Pure Liquids used
masses evolution by size exclusion chromatography (SEC). In
a typical experiment, thymyl methacrylate (0.5 g, 2.29 3
1023 mol), AIBN (3.8 mg, 2.29 3 1025 mol), and toluene
(1.8 mL) were used. At the end of the reaction, poly(thymyl
methacrylate) was isolated from the reaction medium by
precipitation in cold methanol, filtered, washed with cold
methanol, and dried under vacuum.
for Contact Angle Measurements
LW
AB
1
2
Liquid
dL
dL
dL
dL
dL
H2O
72.8
50.8
48.0
58.1
19.9
50.8
29.0
39.0
51.0
0
25.5
0
25.5
0
CH2I2
C2H6O
CH3NO
19.0
19.1
1.9
2.3
47.0
39.6
ATRP
Polymerizations were performed over
period of time using different (thymyl methacrylate)/(ethyl-
2-bromoisobutyrate)/(CuBr)/(PMDETA) molar ratio in
a predetermined
size of 400 mm was employed. The hemispherical analyzer
was operated in CAE (Constant Analyzer Energy) mode, with
a pass energy of 200 eV and a step of 1 eV for the acquisi-
tion of surveys spectra, and a pass energy of 50 eV and a
step of 0.1 eV for the acquisition of high resolution spectra.
A “dual beam” flood gun was used to neutralize the charge
build-up. The spectra obtained were treated by means of the
“Avantage” software provided by ThermoFisher. A Shirley
type background subtraction was used and the peak areas
were normalized using the Scofield sensitivity factors in the
calculation of elemental compositions. All binding energies
were referenced to C1s neutral carbon peak at 284.8 eV.
a
schlenk tube at 60 ꢀC. Before polymerizations, the solutions
were degassed (three freeze-pump thaw cycles) and back-
filled with argon. The reactions were stopped at the desired
conversions. In a typical experiment, the following amounts
were used: thymyl methacrylate (0.5 g, 2.29 3 1023 mol),
solution of ethyl-2-bromoisobutyrate and PMDETA in toluene
(0.5 mL, 4.5 3 1022 mol L21 for both initiator and ligand),
and CuBr (3.2 mg, 2.27 3 1025 mol). After polymerization,
the reaction medium was dissolved in CH2Cl2 and passed
through a column of neutral alumina to remove the copper
salts. Polymer was precipitated from cold methanol, filtered,
washed with cold methanol, and dried under vacuum.
For atomic force microscopy (AFM), tapping mode topogra-
phy and phase imaging was accomplished using a di Innova
AFM Bruker with NanoDrive v8.02 software. Tapping mode
images were acquired using silicon tips from Nanosensors
(PPP NCSTR) with a resonance frequency ranging between
76 and 263 kHz. Images were processed using WsXM soft-
ware, freely available on internet.
Grafting from polymerizations were performed in the same
manner by adding PET film in the schlenk. Very moderate
agitation was used during reaction. After polymerization,
PET films were washed thoroughly with toluene for several
hours to ensure a complete removal of the non-grafted poly-
mer chains prior to being dried with nitrogen and stored
under vacuum.
Determination of Energetic Characteristics of Surfaces
The approach of Good, Van Oss, and Chaudhury (acid–base
theory) was used to calculate the surface free energy.24,25
The surface energy di is seen as the sum of a Lifshitz-van
der Waals apolar component diLW and a Lewis acid–base
Molecules and Macromolecules Characterizations
1H NMR spectra were recorded at room temperature using a
Bruker 360 MHz spectrometer. CDCl3 was used as deuter-
ated solvent (10 mg/0.6 mL). The polymer molar masses
were determined using SEC with tetrahydrofuran as eluent
(1 mL min21) at room temperature. This apparatus was
equipped with a refractive index detector (Waters 410) and
two mixed-bed ViscoGELTM columns (7.8 3 300 mm, type
GMHH R-H) provided by Viscotek. The molar masses were
determined using a calibration curve based on polystyrene
standards. The hardware and software for data acquisition
and treatment were supplied from hs GmbH Ober-
Hilbersheim (Germany).
polar component diAB
:
di5diLW1diAB
(1)
The acid–base polar component diAB can be further subdi-
vided by using specific terms for an electron donor (di1) and
an electron acceptor (di2) subcomponent:
À
Á
1=2
diAB52 di1di2
(2)
The surface energy components of a surface (dS1, dS2, and
dSLW) were determined by performing contact angle measure-
ments with four liquids with known surface tension parame-
ters (dL1, dL2, and dLLW) (Table 1). Using the Young equation,
a relation between the measured contact angles, the liquid
surface energy, and the solid energy can be obtained.
Surfaces Characterizations
Contact angle measurements were carried out by the Young-
€
Laplace method using a DS100 Kruss goniometer with four
liquids of known surface properties, that is, high purity
water (Millipore, milliQ), diiodomethane, ethylene glycol and
formamide (supplied by Aldrich). The contact angle was
measured within 10 s of placing the drop (3 lL) on the
surfaces and an average of six measurements was reported.
1=2
1
dLð11 cosHÞ 52ððdSLWdL
Þ
1=21 ðdS1dL
Þ
1=21 ðdS2dL
Þ
Þ
LW
2
(3)
XPS measurements were performed on a K-Alpha spectrome-
ter from ThermoFisher, equipped with a monochromated X-
ray Source (Al Ka, 1486.6 eV). For all measurements a spot
Microbial Material
Pseudomonas aeruginosa PAO1 was graciously provided by
M. Klausen et al., Listeria monocytogenes (CIP 103574,
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bacteria was determined by enumeration of cultivable cell on
LB or TS Agar plates. Each experiment was conducted three
times. ANOVA variance analysis was performed using Stat-
graphics (Manugistic, Rockville, MD).
Biofilm Tests
After 3 h of bacterial adhesion, the solid surfaces were
rinsed with TSB in order to eliminate any non-adherent bac-
teria before being recovered with 10 mL TSB and incubated
for 72 h at 30 ꢀC. The observations were carried out by
using confocal laser scanning microscopy Leica SP2 AOBS
(Leica-Microsystems, Nanterre, France) after coloration with
acridine orange. All biofilms were scanned using a 633 with
a 1.3 N.A. (Leica HCX Apo) oil immersion objective lens with
a 488 nm argon laser. Emitted fluorescence was recorded
with the range 500–600 nm. Three stack of horizontal plane
image (512 3 512 pixels) with a z-step of 1 lm were
acquired for each contaminated solid surface at different
areas in the solid surface.
Three-dimensional projections of biofilms structure were
reconstructed using the Easy 3D function of the IMARIS 7.0
software.
SCHEME 1 Chemical pathways for: thymyl methacrylate mono-
mer synthesis (A), polymer synthesis by conventional free radi-
cal polymerization (B), and polymer synthesis by ATRP (C).
Collection de l’Institut Pasteur), and Staphylococcus aureus
were graciously provided by A. Horswill et al. Bacterial
strains were stored at 280 ꢀC. Adhesion tests were per-
formed on cells in the early stationary growth phase.
P. aeruginosa PAO1 was cultivated in LB medium (Luria Ber-
tani Broth: 10 g tryptone, 5 g yeast extract, 10 g NaCl) with
pH of 7.4. L. monocytogenes and S. aureus strains were culti-
vated in TSB medium (Trypcase Soy Broth, Biomerieux,
Lyon, France). Strains were cultivated with three consecutive
ꢀ
batch cultures at 37 C under 180 RPM shaking of 24, 8, and
16 h. Experiments were conducted using culture in station-
ary phase with a DO600nm of 1.2 (approximately 109 Colony
Forming Unit per mL (CFU mL21]).
Bacterial Adhesion Tests
Bacterial adhesion tests were performed by sedimentation.
Cells in stationary phase of growth were harvested and
washed three times by centrifugation at 7000 g for 10 min at
21
4 C and suspended again in a 1.5 3 10 mol L21 NaCl solu-
ꢀ
tion. Cells were adjusted to DO600nm 5 0.8 and concentration
was verified by enumeration of cultivable cell on LB or TS
Agar plates. Ten milliliter of cell suspension was allowed to
ꢀ
adhere for 3 h at 37 C to substrate samples in 55 mm Petri
dishes. After 3 h, non-adherent or weakly attached cells were
removed by six successive rinsing of substrate samples in a
1.5 3 1021 mol L21 NaCl solution. Adherent cells were
detached by ultrasonic treatment (Deltasonic, Meaux, France)
for 2 min at room temperature and vigorous shaking for 30 s
at room temperature. The number of viable and cultivable
FIGURE 1 1H NMR spectra of thymyl methacrylate (A) and pol-
y(thymyl methacrylate) (B). [Color figure can be viewed in the
online issue, which is available at wileyonlinelibrary.com.]
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of the monomer was confirmed by 1H and 13C NMR spec-
troscopy (Fig. 1). All integrations of 1H NMR spectrum
matched very well.
Conventional and Controlled Radical
Polymerizations Study
Conventional radical polymerization of thymyl methacrylate
was investigated in solution (1 mol L21 in toluene) using
AIBN as thermal initiator. Polymerization proceeds homoge-
neously and the polymers were isolated from the reaction
1
medium by precipitation in cold methanol. H NMR spectrum
of the polymer shows that the characteristic signals of vinyl
protons from the monomer at 5–6 ppm disappeared and by
the presence of broad peaks at 1–2.5 ppm corresponding to
ACH2ACH protons of the polymer backbone. All other peaks
of thymyl group were broader with nearly the same chemical
shifts as those of the monomer. Figure 2(A) shows the time-
monomer conversion curves observed in the temperature
ꢀ
range from 60 to 80 C with 1 mol % of AIBN. The polymer-
ization yield increases with time and temperature.
Influence of the temperature reaction on molar masses was
studied. Figure 2(B) shows the molar masses evolution of
the polymers formed under different polymerization condi-
tions. The number-average molar masses values were in the
range 1–3 3 104 g mol21 and decreased with increasing
temperature. Polydispersity indexes (Mw/Mn) are in the
range 2.5–3. These tendencies are similar to those for classi-
cal radical polymerizations.
FIGURE 2 Influence of temperature (60, 70, and 80 ꢀC) on con-
ventional radical polymerization of thymyl methacrylate (AIBN
1 mol %).(A) Evolution of monomer conversion according to
reaction time (B) Evolution of average number molar masses
according to monomer conversion. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.
com.]
To our knowledge, the ATRP of thymyl methacrylate has
never been reported. In this study, thymyl methacrylate
was polymerized by ATRP under various conditions and
the results are summarized in Table 2. Ethyl-2-
bromoisobutyrate was used as initiator. Polymerizations
were carried out in toluene using PMDETA/CuBr or
HMETA/CuBr as catalytic systems. Different ratio (mono-
mer)/(initiator) were used. Molar masses increase with
(monomer)/(initiator) ratio and chromatogram traces were
narrow and monomodal with polydispersity index below
1.5. A slight increase of dispersity was observed when
increasing the molar masses probably due to some termina-
tion and isopropyl transfer reactions, but the polymeriza-
tion was relatively well controlled.
RESULTS AND DISCUSSION
Preparation of Monomer
Several decades ago, the results of Moszner have shown the
antibacterial effect of poly(thymyl methacrylate).11 Unfortu-
nately, these preliminary results were not detailed anymore.
In our study, the thymyl methacrylate monomer was synthe-
sized in dry diethylether using the procedure described by
Moszner with slight modifications. The monomer is obtained
in one step by reaction of thymol with methacryloyl chloride
followed by flash chromatography purification (Scheme 1).
The reaction yield was equal to 65%. The chemical structure
TABLE 2 ATRP of Thymyl Methacrylate in Toluene Solution
[MM]/[I]/[C]/[L]a
Conv.b
Mn,th (g/mol)
Mn,exp (g/mol)
Mw/Mn
c
d
d
Entry
ꢀC
Time
Ligand
1
2
3
4
5
60
60
70
70
70
24 h
24 h
24 h
24 h
24 h
PMDETA
PMDETA
HMTETA
HMTETA
HMTETA
50/1/1/1
100/1/1/1
50/1/1/1
100/1/1/1
200/1/1/1
91%
85%
80%
83%
87%
9370
9530
1.34
1.31
1.31
1.41
1.49
17510
8240
13100
8480
17100
35850
13560
21480
a
c
[MM]/[I]/[C]/[L] molar concentration ratio of monomer/initiator/catalyst/
ligand.
Mn,th: theoritical average molar mass determined using monomer
conversion.
d
b
Estimated by 1H NMR.
Estimated by PS-calibrated SEC.
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SCHEME 2 Synthetic route for the grafting of ATRP initiators and thymyl methacrylate polymerization initiated from the PET
surfaces.
TABLE 3 Results of Surface-Initiated Styrene ATRP using [styrene]/[initiator]/[CuBr]/[PMDETA] Equal to 400/1/1/1 at 100 ꢀC.
Free PS Chains
Mn,exp (g mol21
Grafted PS Chains
Mn,exp (g mol21
Water contact
Angle
Conva
Mn,th (g mol21
)
)
Mw/Mn
)
Mw/Mn
1.18
b
98%
41,000
43,200
1.08
46,000
102ꢀ
a
b
Estimated by 1H NMR.
Mn,th: theoritical average molar mass determined using monomer
conversion.
Surface Polymerization
titration, contact angle, XPS, etc) have showed that NH3
plasma is effective in incorporating primary amino groups.
These conditions are used in this study. Primary amino
groups’ density was determined by colorimetric titration and
was equal to 1.5 –NH2/nm2, this value is high enough to
obtain polymer brushes on the surface.
The process of surface polymerization in three steps is
shown in Scheme 2: (step 1) primary amino groups were
generated on PET surface by ammonia plasma treatment,
(step 2) the amino groups were reacted with 2-
bromoisobutyryl bromide to obtain the alkyl halide-
functionalized surface, and (step 3) surface initiated atom
transfer polymerizations (ATRP) of thymyl methacrylate
were carried out.
Then, in a second step ATRP anchored initiators were intro-
duced by amidation reaction between primary amino groups
and 2-bromoisobutyryl bromine in toluene in the presence
of triethylamine.
Previously, we have studied the incorporation of primary
amino groups by two kinds of low-pressure microwave
plasma treatment: ammonia and a mixture of nitrogen and
hydrogen.19 Several plasma parameters were optimized in
our reactor and the different characterizations (colorimetric
For surface initiated polymerization, the initiator concentra-
tion on the PET surface is very low compared to that usually
used for bulk or solution polymerization. To quickly establish
equilibrium between dormant and active species during
polymerization, a sacrificial initiator was added. The final
molar masses of tethered chains on the surfaces are dictated
TABLE 4 Calculated Values of the Solid Surface Energy, the
Lifshitz-van der Waals Component, and the Electron Acceptor
and Electron Donor Parameters of the Acid–Base Component
of Native PET Surface and After Different Treatments
Surface Energetic
Characteristics (mJ m22
)
dS
dLW
d1
d2
PET
44.7
48.6
0.3
11.4
PET-NH2
PET-Br
53.4
38.5
40.5
45.7
36.6
40.0
0.4
0.1
0.1
33.4
11.8
0.5
FIGURE 3 Static water contact angles for native and modified
PET-poly(MT)
PET films.
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FIGURE 4 C1s, O1s, N1s, Br3d high resolution XPS spectra of native PET, PET-NH2, PET-Br, and PET-poly(MT) surfaces.
by the sacrificial initiator concentration (ethyl-2-bromoisobu-
tyrate) added. The determination of monomer conversion
and molar masses of polymer is done by analysis of free
polymers formed in solution using 1H NMR and SEC,
respectively.
check the polymerization control. Styrene polymerization
was performed in bulk at 100 ꢀC using classical ATRP
conditions. To better understand the growth characteristics
of the surface initiated polymerization, the chains
were cleaved from the PET surface using conditions
we have previously described.21 Results are reported in
Table 3.
Polymerizations were performed under very slow stirring
due to the presence of the PET film in the reaction
medium. After polymerizations, the PET films were sub-
jected to extensive washing to assure complete removal of
the non-grafted polymers eventually physisorbed on the
surfaces.
A good agreement between the theoretical molar mass and
the molar masses of the free and grafted polymers was
observed with narrow molar masses distribution in both
cases. The surface water contact angle obtained after styrene
polymerization is equal to 102ꢀ, value closed to those
reported in literature for PS surfaces. The results prove the
good control of polymerization with styrene as model
Preliminary, the polymerization of styrene, as model mono-
mer initiated from this surface was studied in order to
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has a molar mass equal to 12,400 g mol21 with a dispersity
around 1.6. Compared to dispersity value obtained in Table 2,
higher value was obtained probably due to the heterogeneity
of reaction medium with the presence of PET film.
TABLE 5 Surface Chemical Compositions (atomic %) of the
PET Surfaces Determined by XPS
Theoretical
Experimental Atomic
Ratio (%)
Atomic
The contact angle measurements are used to follow the dif-
ferent steps. It is a common method for characterizing the
hydrophilicity, hydrophobicity, and wetting properties of a
surface. In addition, by using polar and apolar liquids, one
can also determine the energetic characteristics of modified
surfaces. Results of water contact angle measurements and
surface energetic values are presented in Figure 3 and
Table 4, respectively.
Ratio (%)
Surfaces
PET
C1s
73
O1s
27
N1s
0
Br3d
0
C1s
O1s
29a
71a
–
PET-NH2
74
75
84
24
23
15
2
1
1
0
1
0
–
PET-Br
–
87b
–
13b
PET-poly(MT)
a
Calculated using the chemical formula of PET repeating unit: C10O4.
Calculated using the chemical formula of poly(MT) repeating unit:
b
The water contact angle of native PET is equal to 81ꢀ, in
agreement with value reported in literature. After NH3
plasma treatment, the water contact angle decreases to 41ꢀ
due to the presence of hydrophilic functional groups on the
surface. The contact angle increases after initiator grafting
(78ꢀ) confirming the disappearance of amine functions by
more hydrophobic amide and halogeno functions. These val-
ues are in good agreement with those we have previously
C14O2.
monomer. Then, the next step is devoted to surface-initiated
thymyl methacrylate ATRP.
Thymyl methacrylate polymerization is performed with CuBr/
HMTETA as catalytic system in toluene solution at 70 C dur-
ing 24 h. The free polymer obtained with sacrificial initiator
ꢀ
TABLE 6 C1s, O1s, N1s, and Br3d Peaks Assignments for Native PET Film and PET Films after Different Treatments
Surface
PET
Window
C1s
Center Peak (eV)
FWHM (eV)
Bond Candidates
Area (%)
284.7
286.3
288.7
531.8
533.1
284.6
286.2
288.6
530.9
531.9
533.2
398.4
399.6
284.9
286.6
289.0
532.1
533.6
68.7
1.2
1.1
0.9
1.3
1.4
1.1
1.7
1.2
1.3
1.4
1.6
1.4
1.9
1.4
1.4
1.1
1.5
1.5
2.1
2.1
2.3
1.5
1.8
1.3
1.8
1.5
2.6
C-C, C5C
68
17
13
51
49
63
24
13
10
39
51
91
9
C-O
C5O
O1s
C1s
C5O
C-O
PET-NH2
C-C, C5C
C-O, C-N
C5O
O1s
NH-C5O
C5O
C-O
N1s
C1s
C-NHx
C5N
PET-Br
C-C, C5C
73
16
11
55
45
–
C-O, C-N
C5O
O1s
C5O
C-O
Br3d
5/2
70.3
3/2
–
N1s
C1s
399.8
284.1
285.6
288.2
531.6
533.1
399.5
C-NHx, C5N, NH-C5O
–
PET-poly(MT)
C-C, C5C
72
18
10
68
32
–
C-O
C5O
O1s
N1s
C5O
C-O
C-NHx, C5N, NH-C5O
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This N1s region is fitted with two peaks assigned to amine
(primary and secondary) and imine as previously pub-
lished.19 The C1s is slightly modified with an increase of the
full width at half maximum of some components due to the
appearance of new chemical entities such as CAN, C@N and
AC@OANHA. The O1s region is fitted with three peaks
(530.9, 531.9, and 533.2 eV) assigned to NHAC@O, CAO/
CAN and OAC@O species.
After reaction of PET-NH2 surface with bromoisobutyryl bro-
mide, additional peak associated with the Br3d (Eb ꢁ 71 eV)
and Br3p (Eb ꢁ 184 eV) signals appear in the survey spec-
trum of PET-Br surfaces. This result indicates successful
reaction of bromoisobutyryl bromide with amino groups
localized on the surface.
After grafting from polymerization, the atomic ratio C/O is
equal to 84/15, close to the theoretical value 87/13 of the
thymyl methacrylate unit. This result indicates a good cover-
age of PET film with poly(thymyl methylmethacrylate). The
C1s-core level spectrum can be fitted with four peaks com-
ponents. These peaks with binding energy at 283.7, 284.5,
FIGURE 5 CFU counting of P. aeruginosa, L. monocytogenes,
and S. aureus strains on native PET and modified PET-pol-
y(MT). [Color figure can be viewed in the online issue, which is
available at wileyonlinelibrary.com.]
reported for PET aminolysis and initiator grafting.20 After
polymerization, the water contact angle was increased to
99ꢀ, indicating the hydrophobic nature of the surface.
From the Young-van Oss equation, it is possible to calculate
the dLW Lifshitz-van der Waals (non-polar) component, and
d1 and d2, the Lewis acid parameter and the Lewis base
parameter, respectively (Table 4). After plasma treatment,
the total surface energy (dS) increases from to 44.7 to 53.4
mJ m22 due to the increase of the electron donor component
(d2 varies from 11.4 to 33.4 mJ m22) coming from basic
amino groups. Reaction with bromoisobutyryl bromide yields
to a decrease of the electron donor component (11.8 mJ
m22) confirming the disappearance of primary amines func-
tion by more hydrophobic amide linkage and bromoalkyl
groups. After thymyl methacrylate polymerization, the elec-
tron donor component decrease to 0.5 mJ m22 due to a com-
plete coverage of polar groups coming from plasma
treatment by grafted polymer.
In order to quantify the chemical changes on the modified
PET surfaces, XPS analyses were performed after each step
(Fig. 4). Table
5 summarizes the atomic compositions
deduced from the XPS data.
FIGURE 6 Confocal laser scanning microscopy images after 3
days S. aureus culture of biofilm. [Color figure can be viewed
in the online issue, which is available at wileyonlinelibrary.
com.]
As expected, the analysis of native PET films shows two
peaks attributed to C1s and O1s with an atomic ratio C/O
equal to 73/27. After plasma treatment, a new peak appears
around 400 eV corresponding to N1s of nitrogen species.
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285.7, and 288.2 were assigned to CH aromatic, CH aliphatic,
CAO and C@O species, respectively. All peaks assignments
are summarized in Table 6 and confirmed the presence of
grafted polymer on the PET films.
the preparation of poly(thymyl methacrylate) brushes. Micro-
biological studies were performed using different bacterial
strains. Anti-adhesion tests showed that PET-poly(MT) is
highly effective against bio-adhesion of P. aeruginosa, L.
monocytogenes, and S. aureus. Moreover, results of anti-
biofilm test proved a strong anti-biofilm effect against S.
aureus.
Impact of Surface Treatments on Bacterial Adhesion
The bacterial adhesion tests were performed on modified
surfaces with suspensions of Gram-negative bacteria (P. aer-
uginosa) and Gram-positive bacteria (L. monocytogenes and
S. aureus). For the sake of comparison, the native PET sur-
face was studied in the same conditions. The results were
presented in Figure 5 as Colony Forming Units per cm2
(CFU cm22) versus bacteria strains (after an adhesion time
ACKNOWLEDGMENTS
The authors gratefully acknowledge the French National
Agency for Research (ANR CD2I SANBACT) for the financial
support of this work, and MIMA2 platform for microscopic
observations (http://www.jouy.inra.fr/mima2).
ꢀ
equal to 3 h at 37 C, adhered cells were detached from the
surfaces by sonication and the number of CFU was
counted).
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