Triazinyl porphyrin-based photoactive cotton fabrics: Preparation, characterization, and antibacterial activity

Article abstract of DOI:10.1021/bm200082d

In the present work, we report on the synthesis of cellulose cotton fibers bearing different types of photosensitizers with the aim to prepare new efficient polymeric materials for antimicrobial applications. Anionic, neutral, and cationic amino porphyrin

Full text of DOI:10.1021/bm200082d

ARTICLE
pubs.acs.org/Biomac
Triazinyl Porphyrin-Based Photoactive Cotton Fabrics: Preparation,
Characterization, and Antibacterial Activity
Cyril Ringot,† Vincent Sol,†,§,* Matthieu Barriꢀere,† Naïma Saad,† Philippe Bressollier,† Robert Granet,†
Pierre Couleaud,‡ Cꢁeline Frochot,‡,§ and Pierre Krausz†,§
†Laboratoire de Chimie des Substances Naturelles, Universitꢁe de Limoges, EA 1069, 123 avenue Albert Thomas, 87060 Limoges, France
‡Laboratoire Rꢁeactions et Gꢁenie des Procꢁedꢁes, UPR 3349, Nancy-Universitꢁe, CNRS, 1 rue Grandville, F-54001 Nancy, France
§GDR CNRS 3049, Mꢁedicaments PhotoactivablesꢀPhotochimiothꢁerapie (PHOTOMED), France
ABSTRACT: In the present work, we report on the synthesis of
cellulose cotton fibers bearing different types of photosensiti-
zers with the aim to prepare new efficient polymeric materials
for antimicrobial applications. Anionic, neutral, and cationic
amino porphyrins have been covalently grafted on cotton fabric,
without previous chemical modification of the cellulosic sup-
port, using a 1,3,5-triazine derivative as the linker. The obtained
porphyrin-grafted cotton fabrics were characterized by infrared
(ATR-FTIR), diffuse reflectance UVꢀvis (DRUV) spectro-
scopies, and thermogravimetric analysis (TGA) to confirm
the triazine linkage. Antimicrobial activity of porphyr-
inꢀcellulose materials was tested under visible light irradiation
against Staphylococcus aureus and Escherichia coli. The results
showed excellent activity on the Gram-positive bacterium, showing structureꢀactivity relationship, although no photodamage of the
Gram-negative microorganism was recorded. A mechanism of bacterial inactivation by photosensitive surfaces is proposed.
’ INTRODUCTION
(PACT).22ꢀ27 Although the cellular mechanism of the photo-
dynamic process is not yet fully understood, it is presently
admitted that phototoxicity primarily relies on the formation of
singlet oxygen (1O2) after illumination.28,29 This highly reactive
species is able to react with almost every cellular component,
bringing irreversible damages that ultimately lead to cell death.30
This strong reactivity, combined with a low specificity, gives rise
to a promising approach for the photoinactivation of micro-
organisms, such as bacteria. It has been shown that porphyrins
keep their antimicrobial properties when grafted to chitosan or
cellulose and that these modified polymers can be cast into
photobactericidal membranes or films.31ꢀ33 The first photoanti-
microbial porphyrinic textiles with covalent links have been
obtained by grafting protoporphyrin IX and zinc protoporphyrin
IX on nylon fiber.34,35
Nosocomial infections have become a major concern, com-
plicated by the emergence of multi-drug-resistant microbial
strains such as the so-called superbugs belonging to Staphylococ-
cus aureus and Escherichia coli bacterial species. Hospital equip-
ment, including polymeric materials and textiles, are potential
vectors for microbial dissemination. As a consequence, interest
has grown in the preparation of materials with antibacterial pro-
perties, and numerous research works on antimicrobial surfaces
have been published.1ꢀ11 In addition to their antimicrobial
properties, it would be desirable that these materials also possess
a pronounced duration of properties and hence could resist
repeated washing or exposure to cleaning products. Modifica-
tions by the means of coating, impregnating, or simple blending
often lead to short-lived properties. Irreversible modifications
resulting from covalent attachment of biocidal moieties are thus
highly preferable, assuming that the chemical reactions do not
affect the genuine properties of the starting material. Cotton
fabrics, which essentially consist of cellulose fibers, can be easily
modified by substitution of a relative small number of their
numerous ꢀOH nucleophilic groups. Among the solutions pro-
posed, antibacterial cellulosic surfaces or fabrics have been trans-
formed by grafting quaternary ammonium salts,12ꢀ14 antibiotics,15
N-halamines,16 chitosan,17ꢀ20 or guanidine polymer.21
In connection with our research program on PACT,32,33,36,37
we have recently presented the first elaboration of photobacter-
icidal cotton fabrics with porphyrinic moieties using “click chem-
istry” reaction as a covalent binding protocol.38 Nevertheless, this
strategy needs prior modification of both cellulose substrate and
photosensitizer. The present article describes a simpler and faster
method which avoids the modification of cellulose. So, cyanuric
chloride, a 1,3,5-triazine derivative, previously substituted by
During the last 20 years, photosensitizers (PS) such as
porphyrins have been intensively studied for their use as photo-
bactericidal agents in photodynamic antimicrobial chemotherapy
Received: January 18, 2011
Revised:
March 17, 2011
Published: March 25, 2011
r
2011 American Chemical Society
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photosensitizers, shows a very high reactivity with the hydroxyl
groups of cellulose.39 Three types of photosensitizers (neutral,
anionic, and cationic) have been used, and chemical attachment
of the porphyrinic macrocycle to the cellulosic surface was scruti-
nized by polymer analysis methods (ATR-FITR, DRUV, and
TGA). The study of the structureꢀactivity relationship of the
photosensitizers attached to the polymer support is reported, and
the photobiocidal activity of the modified cotton fabrics was
tested against E. coli and S. aureus.
at 2000ꢁ magnification. All samples were conditioned on disk (diameter
1 cm) and purified with acetone at 70 °C for 24 h and dried at 100 °C
for 15 min prior to SEM preparations.
Grafting Ratio. The molar grafting ratio (%) of each porphyrin-
grafted cotton fabric was calculated from the difference between the
initial porphyrin amount in the grafting reaction and the unreacted
porphyrin present at the end of the grafting reaction, assuming that this
difference represented the amount of porphyrin actually bound to each
cotton sample, according the following formula
ꢀ
ꢁ
A
Soret=εSoret ꢁ V ꢁ d
grafting ratio ð%Þ ¼ 1 ꢀ
ꢁ 100 ð1Þ
’ EXPERIMENTAL SECTION
ninitial
Materials. Cyanuric chloride (99%, Acros), diisopropyl ethylamine
(99%, Aldrich), and sodium carbonate (99%, SDS) were used as
received at the highest purity. Photosensitizers were synthesized follow-
ing a procedure available in the literature.39ꢀ42 Cotton fabrics (dimension
3.5 ꢁ 3.5 cm, without optical bleach) were purchased from Avelana,
France. Light source (LED model Luxꢁeon Star white Lambertian LXHL-
MW1D 5500K) for the photoinactivation system was obtained from
Dioptik, France. Cultures of Staphylococcus aureus (S2375) and Escher-
ichia coli (S2025) were obtained from Institut Pasteur, Paris. For the
antibacterial tests, glassware was autoclaved at 120 °C for 15 min. Un-
modified and porphyrinic cotton fabric samples were purified with
acetone at 70 °C for 24 h, dried at 100 °C for 15 min, and autoclaved
at 120 °C for 15 min before antibacterial assessment.
where ASoret is the Soret band absorbance of the porphyrinꢀtriazine
compound corresponding to the free photosensitizer in a solution
consisting, for each cotton sample, of the final reacting solution mixed
with the various washings operated after the grafting reaction; εSoret is
the Soret band molar absorption coefficient of the free photosensitizer;
V is the volume of prepared solution for obtaining an absorbance value
between 0 and 1; d is the dilution factor done for UVꢀvis measurement;
and ninitial is the initial amount of photosensitizer (mol) present before
initiating the grafting reaction.
Preparation of Neutral Photosensitive Cotton Fabric. Photo-
sensitizer 1 (Figure 2), TPP-NH2 (11 mg, 1 equiv), was solubilized in
THF (15 mL), then cyanuric chloride (4 mg, 1.2 equiv) and DIPEA
(4 μL, 1.2 equiv) were added.39 After 15 min at 0 °C, the mixture was
stirred at 25 °C for 15 min. Then cotton fabric (3.5 ꢁ 3.5 cm, 0.27 g),
previously soaked in 100 mL of 0.5 M NaOH during 24 h, was
introduced and grafting was carried out at 70 °C for 24 h under reflux
and stirring. Neutral cotton was collected and thoroughly washed with
THF (3 ꢁ 100 mL), CHCl3 (3 ꢁ 100 mL), and finally with hot DMF
(100 mL) at 120 °C for 24 h. Neutral cotton was obtained after drying at
100 °C for 1 h. The molar grafting ration was calculated using eq 1.
Grafting ratio, 57%. FTIR (cmꢀ1): 1726, 1660 (ꢀCONHꢀ stretching,
amido triazine), 880, 840 (CdC deformation, aromatic macrocycle).
DRUV (nm): 422, 518, 555, 594, 649.
Preparation of Anionic Photosensitive Cotton Fabric. For
photosensitizer 2 (Figure 2), TPPS-NH2 (10 mg, 1 equiv), water was
used as reaction solvent (15 mL). Cyanuric chloride (2.5 mg, 1.2 equiv),
dissolved in a minimum of THF, and 1.2 equiv of saturated aqueous
NaHCO3 (1 mL) were added, and reaction mixture was stirred at 0 °C
for 30 min then at 25 °C for 30 min. Cotton fabric (3.5 ꢁ 3.5 cm, 0.27 g),
previously soaked in 100 mL of 0.5 M NaOH during 24 h, was
introduced and grafting reaction was heated at 80 °C for 24 h. Then,
anionic cotton was washed with water (3 ꢁ 100 mL) and finally with hot
DMF (100 mL) at 120 °C for 24 h. Sample was obtained after drying at
100 °C for 1 h. The molar grafting ration was calculated using eq 1.
Grafting ratio, 73%. FTIR (cmꢀ1): 1730, 1655 (ꢀCONHꢀ stretching,
amido triazine), 880, 840 (CdC deformation, aromatic macrocycle).
DRUV (nm): 425, 521, 559, 594, 650.
Preparation of Cationic Photosensitive Cotton Fabric. For
elaboration of cationic porphyrinic cotton, the procedure was similar to
the preparation of neutral cotton. Ten milligrams (1 equiv) of photo-
sensitizer 3, trans-MePyþ-NH2, cyanuric chloride (3 mg, 1.2 equiv), and
DIPEA (3 μL, 1.2 equiv) were used. Grafting ratio, 54%. FTIR (cmꢀ1):
1636, 1562 (ꢀCONHꢀ stretching, amido triazine), 1352 (ꢀCdNþ-
stretching), 805 (CdC deformation, aromatic macrocycle). DRUV (nm):
425, 520, 557, 595, 655.
UVꢀVisible Spectroscopy (UVꢀvis). UVꢀvis spectra were
recorded on a Perkin-Elmer Lambda 25 double-beam spectrophotometer
using 10 mm quartz cells. Spectra were realized at adequate concentra-
tion (10ꢀ5ꢀ10ꢀ6 M).
Determination of Singlet Oxygen Quantum Yield, Φ (1O2).
Quantum yield of 1O2 production was determined by direct analysis of
1
the O2 near-infrared luminescence at 1270 nm. Excitation occurred
with a Xe arc, and light was separated in a SPEX 1680 spectrofluorom-
eter, 0.22 μm double monochromator. Detection at 1270 nm was
realized through a PTI S/N 1565 monochromator, and emission was
monitored by a liquid-nitrogen-cooled Ge detector model (EO-817L,
North Coast Scientific Co.). Absorbance of reference (ethanolic solu-
tion of Rose Bengal,29 Φ 1O2 = 0.68) and sample solutions (at 415 nm)
was set equal (between 0.2 and 0.5) by dilution.
Attenuated Total Reflectance Fourier Transform Infrared
Spectroscopy (ATR-FTIR). ATR-FTIR spectra of unmodified and
functionalized samples were recorded on Varian 800 FT-IR Scinitar
Series spectrometer using a single reflection, horizontal ATR accessory.
For the measurement of ATR-FTIR spectra, each sample piece was
placed under the ATR source and was then slowly pressed on the
sample. Each spectrum was collected in the range of 4000ꢀ400 cmꢀ1
,
and the baseline correction was applied for all spectra using Varian
Scinitar Series software.
Diffuse Reflectance UVꢀVis Spectroscopy (DRUV). DRUV
spectra of porphyrin-modified samples were obtained with a CARY 5000
Varian spectrometer using a 110 mm PTFE integrating sphere. Reflec-
tance spectra were recorded against Teflon standard reflectance spec-
trum. Each spectrum was recorded in the range of 350ꢀ750 nm.
Thermogravimetric Analysis (TGA). Thermogravimetric ana-
lyses of unmodified and modified samples were carried out using a
SETARAM series Setsys 2400 thermogravimetric analyzer under an air
atmosphere. Samples (25ꢀ30 mg) were heated from room temperature
to 500 °C at a rate of 5 °C minꢀ1. Calcined alumina was used as internal
standard.
Scanning Electron Microscopy (SEM). Surface morphology of
unmodified and functionalized cellulosic samples was observed by SEM
using a XL30 Philips scanning microscope. Dried cotton samples were
coated with a 17 nm goldꢀpalladium layer using SCD 050, BAL-TEC
coating unit accessory. Electron micrographs of the sample were recorded
Antibacterial Activity of Photosensitive Cotton. Growth
Conditions of Bacterial Cells. Gram-positive bacteria, S. aureus, and
Gram-negative bacteria, E. coli, were inoculated into liquid tryptic soy
(pancreatic casein extract 17 g/L, soy flour papaic digest 3 g/L, dextrose
2.5 g/L, NaCl 5 g/L, and K2HPO4 2.5 g/L) and incubated at 37 °C
overnight under aerobic conditions. The stock solution was further diluted
to give a working suspension of approximately 106ꢀ107 CFU/mL.
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Light Source and Exposure. We developed a derivative technique of a
normalized protocol (“ATCC Test Method 100-1999, Antibacterial
Finishes on Textile Materials: Assessment of”). Figure 1 shows a photo-
graph of the photodynamic inactivation device inside an incubator. A
LED (light-emitting diode) system for irradiation over the entire visible
spectrum (400ꢀ800 nm) was obtained with Luxꢁeon Star white Lam-
bertian LXHL-MW1D 5500K LED model (45 lm, 3.42 V, 350 mA). The
distance between the light source and the exposed sample surface was
adjusted to 10 cm (light intensity = 690 lx). Samples of photosensitive
textiles were exposed to this white light irradiation (0.16 mW/cm2)
under aerobic conditions at 37 °C in a humidified incubator for 24 h.
Light fluence dose was equivalent to 9.5 J/cm2.
was counted manually. The results, after adjustment by dilution factors,
were expressed as mean CFU per square textile.
Each test was done in triplicate and was conducted along with
necessary controls: a photosensitive sample was processed immediately
after bacterial impregnation (t = 0), another one was incubated at 37 C
for 24 h in the dark; unmodified cotton has also been processed in the
same conditions (24 h at 37 °C in the dark and under white light
irradiation). Finally, we checked Triton X-100 for its possible deleterious
effect on tested organisms; this extracting agent did not affect bacterial
viability when used at working concentrations (0.5% v/v for S. aureus
and 0.05% v/v for E. coli).
Photodynamic Treatment with Photoactive Cotton Surfaces. Sterile
photosensitive textiles (3.5 ꢁ 3.5 cm) were impregnated with 1 mL of
bacterial inoculum at a cell density of approximately 106 CFU/mL,
deposited on a sterile Petri dish, and then incubated at 37 °C for 24 h
under white light irradiation in wet atmosphere. Then, each sample was
removed and transferred into 20 mL of extraction solution: Triton
X-100, 0.5% (v/v) for S. aureus and 0.05% (v/v) for E. coli. After 15 min
of gentle stirring at room temperature, serial dilutions of these suspen-
sions were prepared. Aliquots (100 μL) of diluted samples were then
spread on tryptic soy agar plates. After incubation at 37 °C for 24ꢀ48 h,
plates were examined and the number of colony forming units (CFU)
’ RESULTS AND DISCUSSION
Synthesis of Porphyrin-Bound Cotton Fibers via Triazine
Linkage. According to our previous results, cyanuric chloride
was used for the covalent grafting of porphyrins to cellulose
fabrics. Functionalized para-aminophenylporphyrin derivatives
1ꢀ3 were used as nucleophilic agents to react with cyanuric
chloride; these photosensitizers were synthesized according to
classical methods as previously described.39ꢀ42 Structures of differ-
ent photosensitizers used, neutral aminoporphyrin 1 (TPP-NH2),
anionic sulfoned aminoporphyrin 2 (TPPS-NH2), and cationic
trans-pyridinium aminoporphyrin 3 (trans-MePyþ-NH2), are
illustrated in Figure 2.
In a first step, porphyrins were reacted with cyanuric chloride
at 0 °C, which allows substitution of a first triazine chlorine atom
with the amino function conducing to porphyrinꢀtriazine
derivatives 1-a, 2-a, and 3-a (Scheme 1). From these intermedi-
ates, a highly reactive and not isolated porphyrinꢀtriazine link
has been characterized after the complete substitution of chlorine
atoms with the use of piperidine (for neutral and cationic product)
and sodium sulfanilate (for anionic compound).39 Then, alkali-
treated cotton fabrics (3.5 ꢁ 3.5 cm surface) were introduced in
the porphyrinꢀtriazine reaction mixture, as described above (see
Experimental Section).
Grafting reactions were followed by washing cycles in order to
remove unbound photosensitizer. Neutral and cationic cotton
was intensively washed with chloroform and THF and anionic
cotton with water; these washes were followed by hot DMF (24 h,
120 °C). These cycles were repeated until washing solutions did
not show any trace of porphyrins (UV titration at 420 nm). After
drying at 100 °C, the resulting cotton samples gained a reddish-
brown tint (Figure 3).
Figure 1. Photodynamic inactivation device. Triplicate samples of
photosensitive surfaces (reddish) and native cotton (white) were placed
in sterile Petri dishes and subjected to light irradiation in a humidified
oven at 37 °C.
Figure 2. Chemical structure of anionic, neutral, and cationic photosensitizers used in this work.
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Scheme 1. Synthetic Route to Photoantimicrobial Cotton
Table 1. Grafting Yield Determination of Photosensitizers by
UVꢀVis Titration
amount of grafted
grafted
grafting yield
(%)
photosensitizer (μmol/mg of cotton
porphyrin
sample)
anionic
neutral
cationic
73
57
54
0.03
0.037
0.028
ATR-FTIR Spectroscopy. Grafted cottons have been analyzed
by ATR-FTIR spectroscopy with the aim to characterize the
covalent link between cotton fabric and porphyrinic macrocycles.
ATR-FTIR spectra of unmodified and modified cotton fibers are
presented in Figure 4. Concerning native cotton spectra, classical
spectral data have been found: 3340 cmꢀ1 (OH stretching),
1325, and 1045 cmꢀ1 (CꢀO stretching). Samples of modified
cotton display characteristic signals within the 1750ꢀ1550 cmꢀ1
zone (amide stretching); 1726 and 1660 cmꢀ1 (neutral cotton),
1730 and 1655 cmꢀ1 (anionic cotton), 1636 and 1562 cmꢀ1
(cationic cotton). Moreover, the presence of peaks in the
900ꢀ800 cmꢀ1 zone has been assigned to a deformation vibration
band of bound aromatic macrocycle (ꢀCdCꢀ), and the intense
1352 cmꢀ1 signal observed with cationic cotton corresponds to a
stretching vibration of the iminium form (ꢀCdNþ pyridinium).
The appearance of new bands in the 1750ꢀ1550 cmꢀ1 zone
attests to the presence of a bond corresponding to the amide
function. The presence of an amide group comes from the
substitution of the last chloride atom on the 1,3,5-triazine ring
by the hydroxyl group after treatment of native cotton with
sodium hydroxide solution. Introduction of hydroxyl groups
onto the triazine ring provides evidence for a tautomeric shift
toward the corresponding amide form (Figure 5).43 The pre-
sence of the cyclic amide form was confirmed by the absence of a
Figure 3. Photographs of (a) native cotton; (b) neutral cotton; (c)
anionic cotton; and (d) cationic cotton obtained after treatment.
Molar grafting yield for each amino porphyrin was realized by
UVꢀvis titration (eq 1). Results are presented in Table 1.
Significant grafting yields were obtained (54ꢀ73%), owing to
the proper nature of the cotton fiber and to the heterogeneous
reaction conditions. To our knowledge, these molar grafting values
between the cellulose surface and a photosensitizer like porphyr-
in were reported for the first time. There is no correlation between
the amount of grafted photosensitizer and the value of grafting yield
because the molar absorption coefficient, corresponding to the Soret
band, is different for each porphyrin. Amount of grafted porphyrin
on the order of 0.03 μmol/mg of cotton shows homogeneity of the
grafting system with amino porphyrins of different nature.
The covalent bond between the photosensitizer and cotton
surface was confirmed by attenuated total reflectance Fourier
transform infrared (ATR-FTIR), diffuse reflectance UVꢀvis
(DRUV) spectroscopies, and thermogravimetric analysis (TGA).
CꢀCl band between 600 and 800 cmꢀ1
.
Low intensity of characteristic signals in the ATR-FTIR spectra
has complicated the interpretation of the results; however, ATR-
FTIR analysis allowed us to better understand the chemical
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Figure 4. ATR-FTIR spectra (4000ꢀ600 cmꢀ1) of (a) unmodified cotton and (b) grafted cotton. A, neutral; B, anionic; and C, cationic cotton.
Figure 5. Triazine ring tautomeric shift in the porphyrinꢀcellulose complex.
Table 2. Electronic Diffuse Reflectance UVꢀVis Spectral
Data
products
Soret band maxima (nm)
Q bands (nm)
1a
421
422
416
425
424
425
517, 554, 591, 648
518, 555, 594, 649
514, 552, 590, 648
521, 559, 594, 650
523, 572, 595, 656
520, 557, 595, 655
neutral cotton
2b
anionic cotton
3a
cationic cotton
a UVꢀvis spectra in CHCl3. b UVꢀvis spectra in MeOH.
that the surface of support did not bring any distortion of the
conjugated plane of grafted porphyrins. However, the following
changes were observed in diffuse reflectance UVꢀvis spectra
upon chemical grafting: (i) the ratio of molar absorption
coefficients of the Soret to Q bands was reduced; and (ii) Soret
bands broadened. The latter was attributed to π-electron inter-
action with surface hydroxyl groups. Moreover, maximum of
absorption shows no change in time, providing evidence of the
stability of the grafted cotton surface and that photosensitizers
are not leached from the materials in the solid states.
DRUV analyses, where porphyrins are covalently attached
onto surface, have not been reported in the literature. Similar
observations have been reported by Rahiman et al. for porphyrins
encapsulated into mesoporous silica structures.44
Thermogravimetric Analysis. Thermogravimetric analysis
(TGA) was used to investigate the thermal properties of grafted
cotton. TGA thermograms of neutral, anionic, and cationic pho-
tosensitizers grafted on cotton fabric are presented in Figure 7 in
comparison with unmodified cotton. Initial weight loss was
attributed to loss of water of each sample (dehydration phenom-
enon) which follows the order neutral, cationic > anionic and
unmodified cotton. At higher temperatures, cotton displayed
better thermal stability than grafted cotton as significant weight
Figure 6. DRUV spectra (350ꢀ750 nm) of modified cotton. Red, blue,
and green curves refer to anionic, neutral, and cationic grafted cotton,
respectively. Black dotted line ( ) refers to anionic porphyrin 2
3 3 3
recorded at the concentration of 2 ꢁ 10ꢀ6 M in MeOH and the dash-
dotted line (ꢀ ꢀ ) refers to cationic porphyrin 3 at the concentration
3
3 3
of 2 ꢁ 10ꢀ6 M in CHCl3.
structure adopted by the triazine ring and to show the existence
of a covalent link between the photosensitizer and cellulosic cotton
surface.
DRUV Spectroscopy. In order to observe the presence of
porphyrins on the surface of cotton fabric, modified cotton was
also analyzed by DRUV spectroscopy and compared to reference
molecules as anionic and cationic compound (Figure 6). DRUV
analysis led to spectra similar to UVꢀvis porphyrin derivatives in
solution: Soret band near 420 nm and Q bands between 500 and
700 nm clearly show up (see Table 2 for electronic data).
Results showed the Soret band maxima at 422 nm for neutral
and 425 nm for anionic and cationic cottons, which were identical to
those of free TPP-NH2 1, TPPS-NH2 2, and trans-MePyþ-NH2
3, respectively. Moreover, the appearance of a Q band implies
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Figure 7. TGA thermograms of cotton fabric before and after grafting.
Figure 9. Bacterial counts (log10 CFU) of (A) S. aureus and (B) E. coli.
Antibacterial Activity of Photosensitive Cotton. Photody-
namic activity of modified cotton was evaluated in vitro against S.
aureus and E.coli, used as model Gram-positive and Gram-neg-
ative bacteria.
Figure 8. SEM photomicrographs (20 μm scale) of cotton fibers.
Unmodified cotton (a) and modified cotton; neutral (b), anionic (c),
and cationic (d) porphyrin-grafted cotton.
Experimental data, expressed as the number of CFU per cotton
square sample, are reported in Figure 9. Untreated control samples
(in the dark and under light irradiation) and control treated
samples (in the dark) allow bacterial growth of 4 log compared
with reference (t = 0, number of bacteria initially deposited on
textile square). These controls allowed us to assess that neither
the chemical modifications of cotton nor the light dose (9.5 J/
cm2) have any influence on bacterial growth or viability.
After 24 h exposure to light irradiation at a fluence dose of
9.5 J/cm2 and at a temperature of 37 °C, all modified surfaces
cause a photobactericidal effect in Gram-positive bacteria. Never-
theless, observed activities are different according to the kind of
modified cotton; in fact, percentages of bacterial growth inhibi-
tion are 37% for anionic cotton, 93.7% for neutral cotton, and
100% for cationic cotton. Electric charge of photosensitizers directly
influences photoinactivation efficacy, and these results confirm
the presence of a structureꢀactivity relationship on photoinacti-
vation of Gram-positive bacteria cells. These data are in accor-
dance with former results showing that bacterial cell photo-
inactivation by free base porphyrins was also strongly dependent
on the global electric charge of the photosensitizers whose pho-
tobactericidal powers had the same ranking: cationic > neutral >
anionic.47,48 For cationic cotton fabric, toxicity in the dark (80%)
is probably due to the presence of the quaternary ammonium
charge, known to disorganize bacterial cell walls without light
loss was observed at temperatures of 295, 280, 245, and 240 °C
for cotton, grafted cotton with photosensitizers 2, 1, and 3, re-
spectively. At much higher temperatures, grafted cotton samples
showed multistep weight loss due to decomposition of photo-
sensitizers, removal of linker groups, or degradation of polymeric
material or backbone itself. The lower thermal stability of grafted
cotton in comparison to parent cotton was due to faster decomposi-
tion of modified cotton fibers in relation to the strong compact pure
cotton. Similar results on loss of thermal stability of grafted cotton
(quaternary ammonium polymers or quaternary ammonium triazine
derivatives, for example) have also been reported recently.45,46
Moreover, TGA analyses show that the change of thermal properties
correlated with the covalently modified cotton surface.
Scanning Electron Microscopy. The surface of unmodified
cotton and porphyrin-grafted cotton was examined by scanning
electron microscopy (SEM). Typical scanning electron photo-
micrographs are presented in Figure 8. Photomicrographs did
not show any destructuration of cotton fibers (similar diameter
and structure). These observations indicated that porphyrin
grafting did not affect fiber morphology; no destruction was
observed. SEM results show that cotton fiber structures are
resistant to chemical treatment.
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Table 3. Photoinactivation of Triazinyl Cotton (%)
chemical modification of the cellulosic material which appears
very interesting for further industrial applications. These func-
tionalized cellulose samples displayed an antibacterial activity
against S. aureus, which was shown to depend on the nature of
photosensitizers. These surfaces could be efficiently used in
biomedical fields to prevent microbial infections.
photoinactivation (%)
porphyrin-grafted cotton
S. aureus
E. coli
neutral
anionic
cationic
93.7 ( 3.2
37 ( 1.1
100
0
0
0
’ AUTHOR INFORMATION
Corresponding Author
*Phone: þ33(0)-5-5545-7490. Fax: þ33(0)-5-5545-7202. E-mail:
Table 4. Singlet Oxygen Quantum Yield
vincent.sol@unilim.fr.
amino precursor
Φ (1O2)
TPPS-NH2 1
0.59
0.65
0.82
’ ACKNOWLEDGMENT
TPP-NH2 2
trans-MePyþ-NH2 3
We thank the “Conseil Rꢁegional du Limousin” for financial
support. The authors are indebted to Dr. Michel Guilloton for help
in writing the manuscript, and Pierre Carles (SPCTS-Limoges) for
MEB analysis.
irradiation.49 Concerning Gram-negative bacteria, the three materi-
als did not show any photoactivity. These results are summarized in
Table 3.
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