Development of photoactivable glycerol-based coatings containing quercetin for antibacterial applications

Article abstract of DOI:10.1039/c5ra25267a

The development of new antibacterial coatings (against Escherichia coli and Staphylococcus aureus) using a natural dye, quercetin, according to a green chemistry process was investigated. Quercetin was used as both a photosensitizer and antibacterial agent. The synthesized material was developed according to a cationic photopolymerization process under light irradiation. The photosensitizing mechanism involving quercetin and an iodonium-based cationic photoinitiator was described for the first time according to steady state photolysis and fluorescence experiments. The resulting coatings showed excellent adhesion on a stainless steel plate as demonstrated by nanoindentation and scratch tests, with a high thermal stability up to 375 °C. Finally, a primary investigation was conducted to assess the antibacterial properties of the glycerol-derived coatings against Escherichia coli and Staphylococcus aureus under light illumination. Electron paramagnetic resonance spectroscopy confirmed the generation of reactive oxygen species, such as singlet oxygen, which is responsible for inhibiting bacteria proliferation.

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Development of photoactivable glycerol-based coatings
containing quercetin for antibacterial applications
Michael Condat,a Julien Babinota, Somia Toumanea, Jean-Pierre Malvalb, Inn-Kyu Kangc, Faustine
Spilleboutd, Pierre-Emmanuel Mazerane, Jacques Lalevéeb, Samir Abbad Andalloussif, Davy-Louis
Versacea*.
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The development of new antibacterial coatings (against Escherichia coli and Staphylococcus aureus) using a natural dye,
quercetin, according to a green chemistry process was investigated. Quercetin was used as both a photosensitizer and
antibacterial agent. The synthesized material was developed according to a cationic photopolymerization process under
light irradiation. The photosensitizing mechanism involving quercetin and an iodonium-based cationic photoinitiator was
described for the first time according to steady state photolysis and fluorescence experiments. The resulting coatings
showed excellent adhesion on stainless steel plate as demonstrated by nanoindentation and scratch tests, with a high
thermal stability up to 375°C. Finally, a primary investigation was conducted to assess the antibacterial properties of the
glycerol-derived coatings against Escherichia coli and Staphylococcus aureus under light illumination. Electron
Paramagnetic Resonance spectroscopy confirmed the generation of reactive oxygen species, such as singlet oxygen, which
is responsible for inhibiting bacteria proliferation.
2
.
To solve the problem of bacteria resistance, the
1. Introduction
development of new antimicrobial systems in the biomedical
industry for fighting infections has attracted considerable
attention. The main strategies are based on the decrease of
the capacity of bacteria to attach to a surface3-7, the lethal
contact8-10 which induces the biochemical death of bacteria,
the biocide leaching11-15 and finally the disinfection of surfaces
using coatings that produce reactive oxygen species16-19 (ROS)
upon light activation, also known as photodynamic inactivation
Contamination by microorganisms is of great concern in many
fields such as health care products, medical devices, hospital
surfaces/furniture, or surgery equipments. This problem is
associated with an annual mortality of many thousands of
people in the US and a constant increase of health-care costs1,
a.Université Paris-Est Créteil (UPEC), 61, Avenue Général de Gaulle, 94010 Créteil
cedex, Institut de Chimie et des Matériaux Paris-Est, Equipe Systèmes Polymères
Complexes, UMR CNRS 7182, 2-8 rue Henri Dunant–94320 Thiais, France. Fax:
+33 1 49 78 12 01; Tel: +33 1 49 78 12 28; E-mail: versace@icmpe.cnrs.fr.
b.Institut de Science des Matériaux de Mulhouse, IS2M-LRC 7228, 15 rue Starcky -
68057 Mulhouse, France.
(PDI). In this latter method, the light excitation of
a
photosensitizer (PS) generates ROS which subsequently cause
cell death. In this context, PSs do not have to penetrate the
bacterium or even come in contact with the cell to be
effective. Such antibacterial surfaces that work according to
the PDI principle could help to reduce the transmission of
c. Department of Polymer Science and Engineering, Kyungpook National University,
Daegu 702-701, South Korea
d.National Institute for Nanotechnology, Department of Chemistry, and Department
of Mechanical Engineering, University of Alberta, 11421 Saskatchewan Drive,
Edmonton, Alberta, T6G 2M9, Canada
multi-resistant microorganisms20-23
,
which is of great
importance in hospital hygiene, and can offer a constant
prevention of microorganism adhesion and proliferation on
any surface24. Some investigations have already been
performed essentially with porphyrins and phthalocyanine
derivatives25, 26, hydroxyethyl Michler’s ketone27, toluidine
e. Laboratoire Roberval, UMR CRNS-UTC 7337, Centre de Recherche de Royallieu,
Université de Technologie de Compiègne, 60205 Compiègne Cedex, France.
f. Unité Bioemco Equipe IBIOS, UMR 7618 CNRS - Université Paris-Est Créteil Val-de-
Marne, 61, Avenue Général de Gaulle, 94010 Créteil cedex.
Electronic Supplementary Information (ESI) available: Emission spectrum of the
lamp used during the different incubation times (Figure S1), evolution of the UV- blue28, eosin Y29, 30, methylene blue28, 31, 32 or rose bengal30, 33
visible spectra of quercetin alone and quercetin/Iod in ACN during irradiation
.
However, the use of natural dyes instead of the
aforementioned synthetic dyes is an interesting environmental
challenge. To our knowledge, there is no investigation on the
synthesis of antibacterial coatings using natural dyes which
could act as a “bacteria killer” according to the PDI process.
This lack of studies prompted us to develop new antibacterial
coatings based on a natural dye. Thus, the originality of this
(Figure S2), cyclic voltammetry of quercetin in ACN (Figure S3), evolution of the –
OH band from the polyether network during the 1200s of irradiation (Figure S4),
Influence of the incubation time (0, 2h, 6h), without illumination, on the growth of
A) E. coli and B) S. aureus with and without DMSO (Figure S5) and evolution of the
adhesion/proliferation of E. coli and S. aureus on the stainless steel substrate
(Figure S6). See DOI: 10.1039/x0xx00000x
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study relies on two points: i) the use of a natural flavonoid dye,
quercetin, which could be used as a reactive agent with dual
functionality, i.e an antibacterial promoter and
a
photosensitizer under light activation, and ii) the
photochemical studies of (quercetin/iodonium salt)
photoinitiating system (used for the initiation of the
polymerization and the synthesis of the coatings) which have
never been investigated yet.
Quercetin (3, 5, 7, 3’, 4’-pentahydroxyflavone) is one of the
most abundant flavonol-type flavonoids34-36 found in many
common fruits and vegetables (apple, grape, lemon, tomato,
onion, etc.), beverages (tea, red wine), olive oil, and propolis
from the bee hives. Quercetin has many health-promoting
effects37-39
, including anti-inflammatory and anti-allergic
effects, as well as the protection against cardiovascular health
and cancer risk. In addition, it has been reported that
quercetin enhances the antiviral activity39 owing to its strong
antioxidant action and can be used as a skin antioxidant
Fig. 1. 1H NMR spectra of A) triallyl glycerol and B) glycerol
triglycidyl ether.
protector against UV radiation35, 36, 40-42
.
This investigation presents a simple route to efficiently
synthesize an antibacterial coating derived from quercetin and
glycerol monomer, using environmental-friendly method
(photochemistry process), followed by a complete study of the
(quercetin/iodonium salt) photoinitiating system to perform
the synthesis of the quercetin-containing glycerol coatings.
The first part of this study focuses on the photochemical
properties of the photoinitiating system (quercetin/iodonium
salt) using steady state fluorescence and electrochemistry
experiments. In the second part, the thermal and mechanical
properties of the quercetin derivated coatings were
characterized by thermo gravimetric analysis, nanoindentation
and scratch tests. In a third part, the antibacterial properties of
the coatings against Escherichia coli and Staphylococcus aureus
were evaluated and correlated with the ability of the residual
quercetin embedded in the film to photogenerate singlet
oxygen.
2.2 Photophysical fingerprint of quercetin. Figure 2 displays the
normalized absorption and fluorescence spectra of quercetin in
acetonitrile. The low energy side of the absorption spectrum is
dominated by a distinctive band at 370 nm with moderate intensity
(єmax ~ 19000 M-1 cm-1).
2. Results and discussion
2.1 Synthesis of the glycerol triglycidyl ether (GTE). This monomer
was synthesized using a two-step procedure. In the first step,
glycerol was converted to triallyl glycerol by reacting it with allyl
bromide and sodium hydride in dry DMF via a SN2 reaction. After
purification, the structure of the molecule was confirmed by 1H
nuclear magnetic resonance (NMR) spectroscopy with the
appearance of allylic signals at 5.91, 5.27 and 5.16 ppm,
respectively (Figure 1A). The double bonds of triallyl glycerol were
subsequently epoxidized using m-CPBA in CH2Cl2. The reaction was
monitored by 1H NMR spectroscopy. Figure 1B shows the total
disappearance of the ethylenic signals along with the appearance of
signals related to epoxide moieties at 3.14, 2.78 and 2.59 ppm,
respectively. The 1H NMR spectrum of glycerol triglycidyl ether
appears more complex than that of triallyl glycerol. This is due to
the non-stereospecificity of m-CPBA epoxidation, which leads to the
formation of isomers.
Fig.2. Normalized absorption and fluorescence spectra of
quercetin in acetonitrile. Note: Raman scattering peaks of
acetonitrile are indicated by asterisks in the fluorescence
spectrum of quercetin.
The fluorescence spectrum shows a maximum located at 535 nm
leading to a significant Stokes shift of approximately 8350 cm-1.
Such a large Stokes shift is a clear indication of a significant
electronic change between ground and excited state. Moreover,
quercetin is a very low emissive chromophore that exhibits a
fluorescence quantum yield of less than 10-3 in acetonitrile.
44
Similarly to the 3-hydroxyflavone43,
and its 4’-substituted
derivatives43, the fluorescence of quercetin44 stems from the
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radiative deactivations of two relaxed excited species which are in
fast equilibrium at singlet excited state ( Scheme 1).
Fig. 3. Plots of the absorbance monitored at 370 nm as a function of
the irradiation time for the two solutions of quercetin (3.5 x 10-5 M
in acetonitrile non-degassed) without (A) and with (B) iodonium
salts ([Iod] = 8.6 x 10-4 mol/L). (λirr : 365 nm, I = 70 mW.cm-2)
Scheme 1. Schematic representation of the various radiative
deactivation pathways of quercetin at S1 state. (FC stands for: Frank
Condon state).
A
reaction between excited quercetin and iodonium salts is
therefore demonstrated. Interestingly, such a reaction leads to the
concomitant photogeneration of H+ which can be detected using an
acid indicator, such as Rhodamine B45, 46 (RhB). As shown in Figure
4A, the continuous irradiation of a solution of quercetin mixed with
iodonium salts and RhB leads to the progressive growth of an
absorption band in the 480-650 nm range corresponding to the acid
form of RhB (RhBH+). The photogeneration of H+ is observed only
when quercetin and iodonium salts are mixed together. This clearly
confirms the photosensitizing properties of quercetin. Moreover,
the amount of H+ detected during the irradiation of a solution that
was initially N2-saturated is globally equivalent to that of a non-
degassed solution, as illustrated in Figure 4B. It is clear that the
presence of dissolved oxygen in acetonitrile ([O2]diss ~ 10-3 M47) does
not have any inhibiting effects on the photoreaction between the
excited quercetin and the iodonium derivative. This suggests that
the photosensitizing process occurs mainly from the singlet excited
states of quercetin.
The first emitting species (blue emitting one) corresponds typically
to an internal charge transfer (ESICT) from catechol to pyrone
groups. Subsequent to this latter electronic relaxation, an internal
proton transfer process occurs from the 3-hydroxyl function to the
carbonyl oxygen atom, giving rise to the emission of an internal
proton transfer (ESIPT) tautomer. Such a new fluorescent species
exhibits a benzopyrylium-like configuration. It should be noted that
the fluorescence spectra of ESICT and ESIPT are strongly overlapped
in such manner that only one emission band is observed for
quercetin. However, we will see hereafter that the presence of
iodonium salts will lead to a specific quenching of the ESIPT species.
2.3 Photosensitizing properties of quercetin. Steady state
photolysis experiments in acetonitrile were first performed to
demonstrate the photosensitizing role of quercetin in the presence
of iodonium salts. Figure 3 displays the absorbance time profile at
370 nm of a solution of quercetin in the presence of iodonium salts.
The kinetics relative to a reference solution of quercetin without a
photoacid generator is also depicted to highlight the effects. In
absence of iodonium salts, the absorption spectrum of quercetin
remains constant upon irradiation whereas the presence of
photoacid generator clearly induces the rapid photobleaching of
quercetin. For instance, the intensity of the longest wavelength
absorption band of quercetin collapses by 97 % after 17 min
irradiation. Figure S2 shows the evolution of the UV-visible spectra
of quercetin alone and quercetin/Iod.
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0,40
0,35
0,30
0,25
1,5
1,0
0,5
0,0
0
100
200
300
400
time / s
A
300
400
500
600
λ
/ nm
0,40
0,35
0,30
0,25
Air-saturated
N2-saturated
Fig. 5. Evolution of the fluorescence spectrum of quercetin upon
gradual addition of Iod in acetonitrile (λex: 395 nm). Raman
scattering peaks of acetonitrile are indicated by asterisks.
According to the strong electron-accepting characteristics of
iodonium, it is proposed that the fluorescence quenching of
quercetin can be mainly ascribed to an efficient photoinduced
electron transfer (PeT) from the ESIPT state of quercetin to the
photoacid generator. The free energy associated to this proposed
mechanism can be estimated according to the Rehm–Weller
B
400
0
50
100
150
200
250
300
350
time / s
equation48
:
∆GeT = Eox – Ered – EESIPT + C
Fig. 4. A) Evolution of the absorption spectrum of quercetin (5 x 10-5
-
M in non-degassed ACN) in presence of Ph2I+, PF6 (1.5 x 10-3 M)
The following approximations were made: i) the Coulombic part of
the stabilization energy (C) is negligible, ii) the energy of ESIPT state
can be estimated by EESIPT ≈ ½ hc (νabs+νfluo) leading to a value of ca.
2.84 eV; and iii) Eox and Ered correspond to the oxidation potential of
the quercetin and the reduction potential of the diphenyliodonium
salt (Ered = -0.2 V vs. SCE47). The oxidation potential of quercetin in
acetonitrile was measured by cyclic voltammetry. According to the
cyclic voltammogram of the dye (see Figure S3), the first oxidation
wave, which is irreversible, exhibits a half-wave potential of about
1.1 V vs. SCE. As a consequence, ∆GeT has an estimated value of ca. -
1.54 eV in acetonitrile which is consistent with a largely exergonic
PeT process.
during irradiation at 365 nm (I = 10 mW. cm-2). RhB (1 x 10-5 M) is
added as acid indicator. Inset: Evolution of the absorbance of RhBH+
at 550 nm. B) Evolution of the absorbance of the protonated RhBH+
during irradiation of a non-degassed and a N2-saturated solution
This assumption is corroborated by the fluorescence quenching of
quercetin in the presence of increasing amounts of iodonium salts
(see Figure 5). The fluorescence spectrum clearly shows a decrease
in intensity. It should be emphasized that this quenching mainly
affects the ESIPT band which is located in the 530-680 nm range. As
a consequence, after addition of iodonium salts, the relative
contribution of ESIPT band to the total fluorescence spectrum drops
drastically in such manner that the low intensive ESICT band can be
detected in the 460-500 nm range. Noteworthy, the excitation
spectrum collected from this latter emission band matches the
absorption band of quercetin.
Therefore, quercetin, as a natural dye, constitutes an interesting
candidate that can promote acid photogeneration in the presence
of iodonium salts. This photosensitizing process will be addressed to
produce
quercetin-derivative
coatings
by
cationic
photopolymerisation.
2.4 Synthesis and characterization of the quercetin-derivative
coatings. The photochemical reactivity of the photosensitive
formulation
containing
glycerol
triglycidyl
ether
(GTE)/Quercetin/Iodonium was evaluated by Real-Time Fourier
Transform Infrared (RT-FTIR) spectroscopy by monitoring the
decrease of the epoxy group at 910 cm-1 upon irradiation (Figure
6A). The band associated with the epoxy group decreases
concomitantly with the growth of a new absorbance at 1080-1100
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cm-1 (Figure 6B). This latter band confirms the formation of the 2.5 Thermogravimetric analyses. Thermal stability of the coatings
polyether network, and leads to the appearance of a new band was studied by thermogravimetric analysis (TGA). The results are
between 3100 and 3600 cm-1 due to the -OH group of the polyether displayed in Figure 8. The coating reached 50% weight loss at
network (Figure S4). These results demonstrate the 375°C
photopolymerization of GTE. As shown in Figure 6A, the final epoxy
conversion is approximately 75% after 1200s of irradiation. The
coating is totally take-free. An optical image of the coating
deposited on a stainless steel plate is displayed on Figure 7.
.
Fig. 8. TGA thermogram of the photoinduced quercetin-derivative
coating.
2.6 Mechanical analyses of the coatings. The hardness of the
glycerol-derived coatings was first determined relative to
a
standard set of pencil leads. Considering that the pencil hardness is
always above 7H, it means that no macroscopic scratches were
observed up to a hardness of 7H. To highlight more precisely the
hardness of the coatings, further investigations, i.e.
nanoindentation and scratch tests, were performed.
2.6.1 Nanoindentation. The loading and unloading curves during
the nanoindentation tests were superimposed, showing that the
coatings exhibit a viscoelastic behavior at room temperature,
indicating that the polymer is in a rubber-like state at room
temperature. The elastic modulus and the hardness, as measured
by the Oliver and Pharr method49 was approximately 189 ± 18 MPa
(Mean ± Standard deviation) and 12 ± 1 MPa, respectively.
Fig. 6. A) Photopolymerization kinetic of GTE under light activation
and B) FTIR spectra of the coating during the 1200s of irradiation.
Xe lamp, intensity= 70 mW/cm2, film thickness = 100 μm.
2.6.2 Scratch tests. Regarding the scratch tests, a comparison of the
height profiles before and after scratch tests reveals the complete
recovery of the material, confirming the rubber-like behavior
(Figure 9). The lack of bulging on the height profile during and after
scratching indicates the apparition of brittle fracture or
delamination of the coating. Moreover, the optical images of the
sample after scratching show no residual imprint, confirming that
neither brittle fracture nor delamination occurs. Hence, there is a
good adhesion to the substrate and the good resistance of the
coatings to brittle fracture.
Fig. 7. Optical image of the stainless steel substrate (left) and the
quercetin-derivative coating deposited on
substrate.
a stainless steel
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900s of irradiation. Inset = Fluorescence spectrum of the embedded
quercetin into the polymer film after polymerization.
2.8. Antibacterial properties. Prior to investigate the antibacterial
effect of the quercetin derivative-coatings, the ability of quercetin
to inhibit bacterial proliferation in solution was investigated against
Gram-Negative (E. coli) and Gram-Positive (S. aureus) bacteria after
2h and 6h, with and without light illumination. Initially, 6.5 x 106
CFU/mL of each bacteria was introduced in solution at t = 0 h.
First, a reference study referring to the light irradiation against
the two bacteria strains was performed (Figure 11 and Figure S5).
The results show that the irradiation does not have any influence on
the proliferation of E. coli and S. aureus alone. The two strains
proliferate in the same way with or without irradiation. A second
reference study was performed in the presence of DMSO, because
quercetin is insoluble in water. According to these results, DMSO
does not affect the bacterial activity because the decrease in CFU is
not significant regardless of the stains used with or without light
illumination (Figure 11 and Figure S5).
Fig. 9. Height profile before, during and after scratch. A complete
recovery of the sample after scratch and the absence of bulge in the
profile during scratch evidence the rubber like behavior of the
coatings and its very good adhesion and resistance to brittle
fracture.
2.7. Fluorescence of the coatings. After irradiation of the quercetin
derived formulation (900s), unreactive quercetin molecules are left
inside the coating, as observed by UV-vis spectrometry (Figure 10A,
dot line) and its epifluorescence (Figure 10B). The remaining
fluorescence of quercetin can be used to generate reactive oxygen
species (ROS) able to eliminate bacteria, as it will be demonstrated
in the following section.
Fig. 10. A) Evolution of the UV-vis spectra of the quercetin derived
coating after 900s of irradiation. Solid line = before irradiation, dot
=
70 mW/cm2.
Fig. 11. Influence of the incubation time (0, 2h, 6h) and the
irradiation on the growth of A) E. coli and B) S. aureus. [Quercetin] =
500 mM.
line
=
after irradiation. Xe lamp, intensity
glass plates. B)
Formulation was sandwiched under
2
Epifluorescence of quercetin located into the polymer coating after
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The last interesting results are the effect of light irradiation on
the proliferation of bacteria. The antibacterial effect of quercetin
was then evaluated in DMSO, under light activation. The results
show that irradiated quercetin only leads to an antibacterial effect
with S. aureus. Indeed, the development of gram-negative bacteria
(E. coli) is neither inhibited by the illumination nor affected by the
presence of quercetin in the dark. On the contrary, the irradiated
quercetin solutions allow the total death of S. aureus after 2h of
incubation. The antibacterial effect against S. aureus remains
efficient even after 6h. The variation in quercetin killing efficacy at
different exposure durations was statistically significant (p value <
0.05 on comparing percentage survival). In the absence of
irradiation, more than half of bacteria are always alive after 6h of
incubation with S. aureus.
Some studies50 on the inactivation of bacteria demonstrate that
the photosensitizers do not need to penetrate the cell membrane
or even to come into contact to be effective. Indeed, if sufficient
quantities of singlet oxygen can be generated near the outer
membrane of the bacteria, it will lead to its damage. However, only
gram-positive bacteria (S. aureus) are affected by this oxygen
singlet process. Indeed, the lipopolysaccharide (LPS) coatings of the
cell wall of the gram-negative bacteria offers some protection from
the toxic effects of exogenous agents51, 52. In addition to possibly
forming a structural barrier to penetration, this outer membrane
may form a chemical trap for singlet oxygen; it is composed of
unsaturated fatty acids and proteins, which are compounds known
to react chemically with singlet oxygen53, 54
.
As a result, some strains that fail to produce a large portion of
the LPS displayed greater sensitivity to exogenous singlet oxygen
than the strains with this capability53. Most gram-positive bacteria
(S. aureus) lack this protective structure analogous to the gram
negative LPS and the outer membrane in which it is anchored. This
can explain the 99% inhibition of the proliferation of S. aureus after
2h of incubation. ROS and particularly singlet oxygen probably
diffuses readily through the relatively open structure of the
peptidoglycan layer of the S. aureus cell wall to react with the vital
target.
Fig. 12. A) ESR spectra obtained under light irradiation of quercetin
in tert-butylbenzene in aerated conditions after 240s. The increase
of the ESR spectrum is ascribed to TEMPO creation. Light intensity =
270 mW/cm2. B) Evolution of the generated TEMPO radicals in
solution during light activation.
In this study, Electron Paramagnetic Resonance (EPR)
According to these results, it is likely that quercetin, which was
spectroscopy was used to monitor the ROS generation ability of introduced and immobilized in the coatings, could generate reactive
quercetin upon photo-illumination (Figure 12). Amount of 2,2,6,6- oxygen species like singlet oxygen to inhibit the proliferation of
tetramethylpiperidine (TEMP), a diamagnetic molecule, was used to bacteria on stainless steel substrates. Mechanistic interpretations46,
53, 58
capture oxygen radical species by yielding a paramagnetic product,
have already demonstrated that the resulting reactive species,
the nitroxide radical TEMPO55-57. As shown in Figure 12, the EPR such as singlet oxygen could be generated by these immobilized
spectral signal of three lines of equal intensity, which is attributed photosensitizers on the material surface, followed by its diffusion,
to the TEMPO nitroxide radical, was observed when an oxygen- resulting in the damage of the bacteria envelope. Interestingly, the
saturated solution of quercetin was irradiated in the presence of diffusion of quercetin through the synthesized coatings was not
TEMP at room temperature. During illumination of quercetin observed until 6h of incubation. The ability of the quercetin
solution, the intensity of the EPR signal increases gradually, derivative-coatings (deposited on stainless steel substrates) to
indicating the formation of reactive oxygen species like singlet inhibit the bacteria adhesion/proliferation was investigated with
oxygen.
and without the light activation (Figure 13). A quantitative and well-
tried method in biological studies for quantifying the total biofilm
population was used, as reported by many antibacterial
investigations10, 59, 60
.
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Conclusions
This study examined the synthesis of antibacterial coatings
derived from a natural dye (quercetin) and a glycerol triglycidyl
ether monomer. The ability of quercetin to be used both as a
photosensitizer of iodonium salt and as an efficient antibacterial
agent (even incorporated into a coating) against S. aureus was
demonstrated for the first time. For this purpose, the
photochemical behavior of quercetin in the presence of iodonium
salts was described by steady state and fluorescence experiments,
demonstrating i) the efficiency of this system to be used as a
photoinitiating couple and ii) its ability to generate H+ photoacid for
the cationic photopolymerization. The resulting coatings
synthesized under light illumination showed very good adherence
properties to the stainless steel substrates and a high thermal
stability up to 375°C.
The synthesized coatings containing quercetin led, under light
activation, to an inhibition of S. aureus proliferation of 99% after 2h
and 6h of incubation. This new coating could be used successfully to
avoid bacteria proliferation and be deposited onto disposable
paramedical devices, such as clamp or scalpels, which can be used
one-time for a few hours.
3. Experimental
Materials. Glycerol (≥ 99%), sodium hydride (97%), 3-
chloroperbenzoic acid (≤ 77%, m-CPBA), quercetin (≥ 95%, Qr),
2,2,6,6-tetramethyl-1-piperidinyloxy (98%, TEMPO), 2,2,6,6-
tetramethylpiperidine (≥ 99%, TEMP), rhodamine B (≥ 95%, RhB)
and anhydrous DMF were purchased from Sigma-Aldrich. Iodonium
Fig. 13. Comparison of the antibacterial properties of quercetin-
derivative coatings against A) E. coli and B) S. aureus with and
without light activation after 2h and 6h of incubation.
4-(2-methylpropyl)phenyl-hexafluorophosphate
(Iod)
was
purchased from Badische Anilin und Soda Fabrik (BASF). Allyl
bromide (99%) was acquired from Alfa Aesar. Petroleum ether,
ethyl acetate (analytical grade) and dichloromethane (synthesis
grade) were obtained from Carlo Erba. All chemicals were used as
received. Table 2 lists the chemical structure of the compounds
used in this study.
In this case, 15 x 106 CFU/mL of each bacteria was introduced
and placed in contact with the coatings at t = 0h. According to
Figure 13, the antibacterial effect is clearly more efficient against S.
aureus than for E. coli. Upon light illumination, the number of CFU
S. aureus drastically decreases, whereas it increases significantly on
the stainless steel substrate (Figure S6). After 2h and 6h of
incubation, 99% inhibition of S. aureus adhesion was observed. On
the other hand, without illumination of the coatings, the
adhesion/proliferation of S. aureus remains important: the number
of CFU per cm2 increases from 1.5 x 106 (2h of incubation) to 107
after 6h (Figure 13). This later result demonstrates that light is
essential for leading to an antibacterial effect. For E. coli, the
antibacterial property of the coating is not efficient. Indeed,
whatever the conditions used (illumination or not), the number of
CFU increase after 2h and 6h of incubation. These results are in
accordance with the results obtained in solution.
This experiment highlights the efficiency of reactive oxygen
species, which are generated from the illumination of the
quercetin-derivative
coatings, to strongly
reduce
the
adherence/proliferation of S. aureus on stainless steel supports.
8 | J. Name., 2012, 00, 1-3
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Table 2. Chemical structure of the compounds used in this study.
View Article Online
DOI: 10.1039/C5RA25267A
ARTICLE
Photopolymerization procedure.
For
the
cationic
photopolymerization, 8 mg of Iod (4 wt % with respect to the epoxy
monomer), 5 mg of Qr (2.5 wt % with respect to the epoxy
monomer) were dissolved into epoxy monomer formulation (GTE,
200 mg) containing 100 μL of acetone. The kinetics of
photopolymerization were followed by real time Fourier transform
Compound
Glycerol
Function
Monomer
precursor
Structure
OH
HO
OH
OH
OH
Quercetin
Photosensitizer
and
infrared spectroscopy (RT-FTIR) using
a Thermo-Nicolet 6700
HO
O
instrument. The liquid samples were applied to a BaF2 chips by
means of calibrated wire-wound applicator. The thickness of the
UV-curable film was evaluated at 100 μm. The RT-FTIR analyses
were carried out under air conditions. Samples were irradiated at
room temperature, by means of a Lightningcure LC8-03 lamp from
Hamamatsu, equipped with a xenon lamp (200 W) coupled with a
flexible light guide. The end of the guide was placed at a distance of
4 cm. The maximum UV light intensity at the sample position was
found to be 70 mW/cm2. The photopolymerization was monitored
by the disappearance of the epoxy function of the GTE monomer at
910 cm-1. The decreases of epoxy function at 910 cm-1 and the
increase of polyether at 1080-1100 cm-1 demonstrated the
efficiency of the photopolymerization. Conversion rate was
calculated with the followed equation (Eq 1):
antibacterial
agent
OH
OH
O
I+
Iodonium 4-(2-
Cationic
methylpropyl)phenyl- photoinitiator
hexafluorophosphate
(Iod)
F
F
F
P
F
F
F
H
N
2,2,6,6-
Tetramethylpiperidine Singlet oxygen
(TEMP)
scavenger
O
N
2,2,6,6-Tetramethyl-1-
piperidinyloxy
(TEMPO)
Determination
of 1O2
Rhodamine B (RhB) Acid indicator
Epoxy conversion (%) = 100 x (A0 - At)/A0
(Eq 1)
O
OH
A0 represents the area at t = 0s and At represents the area at time t.
N+
N
O
Synthesis of the glycerol-derivative coatings. Prior to the
deposition of formulation on stainless steel plate, the latter was
intensively cleaned with ethanol and toluene. The photosensitized
formulation containing GTE, Iod and Qr (100 μL) were then
deposited and spin-coated (2000 rpm during 3s) on the dried
stainless steel plates. Both sides of stainless steel plate were
irradiated at a distance of 4 cm at room temperature using a
Lightningcure LC8-03 lamp from Hamamatsu, equipped with a
xenon lamp (200 W) coupled with a flexible light guide. The
irradiation time was fixed to 900 s per side with an intensity of 70
mW/cm2.
Cl
Synthesis of triallyl glycerol. Triallyl glycerol was synthesized using
a procedure reported elsewhere. Glycerol (5 g, 54.3 mmol) and NaH
(6.84 g, 285 mmol) were dissolved in 50 mL of anhydrous DMF at
0°C. After 10 min, allyl bromide (17 mL, 285 mmol) was added and
the solution was stirred for 3h at room temperature. The mixture
was then poured into water and extracted with CH2Cl2. The organic
phase was concentrated and the residue was purified on a silica gel
column (petroleum ether-EtOAc 1:1.5 as eluent). The evaporation
of the solvent and subsequent drying under high vacuum led to
8.272 g (yield = 72%) of triallyl glycerol. The product structure was
UV-Visible absorption and fluorescence measurements. The
absorption measurements were carried out using a Perkin-Elmer
Lambda 2 spectrometer. Steady-state fluorescence spectra were
collected from a FluoroMax-4 spectrofluorometer. All emission
spectra were spectrally corrected.
confirmed by 1H NMR. H NMR (CDCl3) δ (ppm) 5.91 (m, 3H), 5.27
1
(m, 3H), 5.16 (m, 3H), 4.15 (dt, J = 5.7, 1.4 Hz, 2H), 4 (dt, J = 5.6, 1.4
Hz, 4H), 3.69 (m, 1H), 3.54 (m, 4H).
Fluorescence microscopy. An inverted microscope IX73 from
Olympus equipped with a 75W Xe Lamp housing was used. The
excitation and emission light is filtered with a fluorescence mirror
unit (U-FUN from Olympus) associating a band pass filter centered
at 365 nm (BP360-370), a dichroic mirror (DM410) and a long pass
filter (BA420IF).
Synthesis of glycerol triglycidyl ether (GTE). Triallyl glycerol (5 g,
23.6 mmol) was dissolved in 50 mL of CH2Cl2. m-CPBA (24.42 g, 142
mmol), dissolved in 75 mL of CH2Cl2 was added dropwise over a one
hour and the solution was stirred overnight at room temperature.
After filtering the precipitate, the solution was concentrated to
approximately 50 mL and placed at -20°C. The precipitate was then
filtered and the operation was repeated 3 times until no more
precipitate appeared. Finally, the solution was passed through a
silica gel pad, evaporated and dried under vacuum, yielding 4.93 g
of glycerol triglycidyl ether (yield = 80%). The total conversion of
double bonds to epoxide was confirmed by 1H NMR spectroscopy.
1H NMR (CDCl3) δ (ppm) 3.90 (dt, J = 11.7, 2.7 Hz, 1H), 3.78 (dd, J =
11.6, 2.6 Hz, 2H), 3.71 (m, 1H), 3.59 (m, 5H), 3.39 (m, 2H), 3.14 (m,
3H), 2.78 (t, J = 4.6 Hz, 3H), 2.59 (m, 3H).
Cyclic Voltammetry. The oxidation potential (Eox) of quercetin was
measured by cyclic voltammetry61 using a computer-controlled
Radiometer Voltalab 6 potentiostat with a three-electrode single
compartment cell. The working electrode was a platinum disk. A
saturated calomel electrode (SCE) used as a reference was placed in
a separate compartment. Measurements were performed at 300 K,
in N2-degassed acetonitrile with a constant concentration (0.1 M) of
(n-Bu)4BF4. Ferrocene was used as the internal reference.
Pencil hardness. The hardness of the coating was determined
relative to a standard set of pencil leads. The pencil hardness
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Journal Name
measurement begins with the lowest pencil and continues up the conducted four times. The levels of adhesion are given as the
scale to determine the maximum hardness able to scratch the number of cells per square centimeter.
surface of the glycerol-derived coatings (Method: ASTMD3363-74, Antibacterial properties of quercetin in solution. The reference
2000). The surface hardness is determined by scratching the leads solution (bacteria/DMSO) and the quercetin solution in DMSO (160
across the coating at a controlled angle of 45º, the value given are µM) containing bacteria were placed on a slantwise rotating wheel
the lowest grade of pencil that could induce a scratch on the to avoid the sedimentation of bacteria, and incubated for 2h and 6h
coating surface. The pencil hardness was measured using a No. 553 at room temperature under illumination or not. The volume of
pencil hardness tester (Yasuda Seiki Seisakusho LTD.). Pencils were DMSO corresponds to 3% v/v of the total bacteria solution. The
supplied by Staedtler Mars Lumograph 100 (Germany).
process is the same as described in the previous paragraph. The
Nanoindentation and scratch tests. Nanoindentation and scratch suspensions were pooled and diluted serially. A 100 µL volume of
tests were carried out on the coatings deposited on a stainless steel the viable bacteria solution was introduced onto the surface of a
substrate using a Nano Indenter G200 (Agilent Technologies) with a Plat Count agar plate.
Berkovich tip (Micro Star Technologies). Twenty Nanoindentation Finally, the number of viable bacteria was determined by a counting
tests per sample were performed. Samples were loaded and of the CFUs, after overnight statically incubation of the agar plates
unloaded at constant strain rate (0.05 s-1) using the Continuous at 37°C. Each experiment was done four times.
Stiffness Measurement (CSM) method until an indentation depth of Statistical analysis. All values corresponding to the anti-adherence
1 µm was reached. The unloading stage was performed after a hold property of E. coli and S. aureus are expressed as mean ± standard
load plateau of 300s to exhibit the viscous behavior. Twenty scratch deviation. Statistical analysis was performed using Student’s t-test
tests were performed; face forward, with an increasing load from to calculate the significance level of the data. Differences were
0.1 to 100 mN for a scratching distance of 500 µm. The distance considered statistically significant at P < 0.05. Ten samples per
between two scratches was fixed to 500 µm.
group were evaluated.
Thermogravimetric analyses (TGA). 10 mg of the glycerol-derivative
films were introduced into aluminum pans and analyzed using a
Setaram Setsys Evolution 16 thermobalance by heating the samples
at a rate of 15 °C/min from 20 to 800 °C under argon atmosphere.
Antibacterial properties of the coatings. The initial adhesion assays
were performed using two strains of bacteria, namely E. coli
ATCC25922 and S. aureus ATCC6538 on the quercetin derivative-
coatings. Before the in vitro antibacterial tests, the bacterial strains
were grown aerobically overnight in Luria–Bertani broth at 37 °C
with stirring. Overnight cultures of E. coli and S. aureus grown in
Luria–Bertani broth were diluted to an optical density (OD 600 nm)
of 0.05 in sterile LB broth. At this point, the stainless steel supports
and stainless steel substrates with coatings (1.5 cm x 1.5 cm) were
immersed in the culture. The corresponding vials were placed on a
slantwise rotating wheel to avoid the sedimentation of bacteria,
incubated for different times (2h and 6h) and shaken at 150 rpm to
allow initial adhesion to occur (INFORS AG-CH 4103, Bottmingen-
Basel, Switzerland). During the incubation time, some samples were
illuminated with 4 lamps (intensity = 170 µmol/m2/s). The emission
spectrum of the lamp is given in Figure S1. After adhesion, the
samples were rinsed seven times with sterile saline solution (NaCl,
0.9% w/v) to remove any non-adherent cells.
Acknowledgements
We would like to thank CNRS institute and University of Paris-Est
Creteil (UPEC) for financial support, Léon Preira for the cutting
process of stainless steel substrates and Séna Hamadi for the TGA
analysis.
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TABLE of CONTENT
Synthesis of antibacterial coatings derived from glycerol and quercetin for the inhibition of bacteria proliferation.
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