Control of Listeria innocua biofilms by biocompatible photodynamic antifouling chitosan based materials

Article abstract of DOI:10.1016/j.dyepig.2016.10.020

New materials obtained through the incorporation of meso-tetraarylporphyrins, bearing phenyl or pentafluorophenyl groups at the meso positions with or without acid groups, in chitosan films have been prepared and characterized in detail. The efficiency of these porphyrinic-chitosan films (PS-CF) to prevent Listeria innocua attachment and subsequent biofilm formation was evaluated under different irradiation protocols (dark, light, light + dark, dark + light). All tested porphyrinic-chitosan films were photostable and effective in reducing Listeria innocua attachment during a period of 24 h exposure to white light. Biofilm development was nearly completely inhibited in two of the PS-CF films (one containing the 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin and the other one of the thio-carboxylate porphyrins) after being exposed to white light for 24 h followed by incubation in the dark for 48 h. Experiments with the non-immobilized porphyrins against cell suspensions of the same Listeria innocua strain showed that, the photodynamic inactivation efficiency was dependent on porphyrin. The demonstrated antimicrobial activity of PS-CF films, associated with the easy recovery of these films, make them promising photodynamic antifouling materials.

Full text of DOI:10.1016/j.dyepig.2016.10.020

Accepted Manuscript
Control of Listeria innocua biofilms by biocompatible photodynamic antifouling
chitosan based materials
Kelly A.D.F. Castro, Nuno M.M. Moura, Ana Fernandes, Maria A.F. Faustino, Mário
M.Q. Simões, José A.S. Cavaleiro, Shirley Nakagaki, Adelaide Almeida, Ângela
Cunha, Armando J.D. Silvestre, Carmem S.R. Freire, Ricardo J.B. Pinto, Maria da
Graça P.M.S. Neves
PII:
S0143-7208(16)30752-5
10.1016/j.dyepig.2016.10.020
DYPI 5536
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 16 September 2016
Accepted Date: 13 October 2016
Please cite this article as: Castro KADF, Moura NMM, Fernandes A, Faustino MAF, Simões MMQ,
Cavaleiro JAS, Nakagaki S, Almeida A, Cunha E, Silvestre AJD, Freire CSR, Pinto RJB, Neves
MdGPMS, Control of Listeria innocua biofilms by biocompatible photodynamic antifouling chitosan
based materials, Dyes and Pigments (2016), doi: 10.1016/j.dyepig.2016.10.020.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Control of Listeria innocua biofilms by biocompatible photodynamic antifouling
chitosan based materials
Kelly A.D.F. Castro,a,c Nuno M.M. Moura,a Ana Fernandes,b Maria A.F. Faustino,a*
Mário M.Q. Simões,a José A.S. Cavaleiro,a Shirley Nakagaki,c Adelaide Almeida,b
Ângela Cunha,b* Armando J.D. Silvestre,d Carmem S.R. Freire,d* Ricardo J.B. Pinto,d
Maria da Graça P.M.S. Nevesa*
aQOPNA, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal
bCESAM, Department of Biology, University of Aveiro, 3810-193 Aveiro, Portugal
cLaboratório de Bioinorgânica e Catálise; Universidade Federal do Paraná,
Departamento de Química, Curitiba, Brazil
dCICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro,
3810-193 Aveiro, Portugal.
*Corresponding
authors:
e-mail
address:
gneves@ua.pt,
faustino@ua.pt,
acunha@ua.pt and cfreire@ua.pt.
Keywords: porphyrins, chitosan films, photodynamic inactivation, Listeria innocua,
biofilms
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Abstract
New materials obtained through the incorporation of meso-tetraarylporphyrins, bearing
phenyl or pentafluorophenyl groups at the meso positions with or without acid groups, in
chitosan films have been prepared and characterized in detail. The efficiency of these
porphyrinic-chitosan films (PS-CF) to prevent Listeria innocua attachment and
subsequent biofilm formation was evaluated under different irradiation protocols (dark,
light, light + dark, dark + light). All tested porphyrinic-chitosan films were photostable
and effective in reducing L. innocua attachment during a period of 24 h exposure to
white light. Biofilm development was nearly completely inhibited in two of the PS-CF
films (one containing the 5,10,15,20-tetrakis(pentafluorophenyl)porphyrin and the other
one of the thio-carboxylate porphyrins) after being exposed to white light for 24 h
followed by incubation in the dark for 48 h. Experiments with the non-immobilized
porphyrins against cell suspensions of the same of L. innocua strain showed that, the
photodynamic inactivation efficiency was dependent on porphyrin. The demonstrated
antimicrobial activity of PS-CF films, associated with the easy recovery of these films,
make them promising photodynamic antifouling materials.
1. Introduction
The unique physicochemical properties of porphyrins justify the wide range of
applications found for these molecules in different fields, including medicine [1]. These
macrocycles are considered promising photosensitizers (PS) for photodynamic therapy
(PDT) of tumors and some of them are already being used with success in clinical
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applications [2-7]. Porphyrins have also been successfully explored as PS for the
photodynamic inactivation (PDI) of microorganisms, namely virus, Gram-negative and
Gram-positive bacteria and fungi [8-16]. Both approaches (PDT and PDI) rely on the
combination of nontoxic dyes with harmless visible light to produce reactive oxygen
species (ROS), mainly singlet oxygen (1O2), that can selectively kill cancer cells [2] or
microorganisms [15]. This photodynamic action is largely dependent on the efficiency of
the PS to produce those ROS in the presence of light and molecular oxygen [17]. One
relevant application of PDI, is its use as an alternative approach to control microbial
biofilms which are typically more resistant to classical biocides than the corresponding
planktonic forms [15].
Listeria monocytogenes, a biofilm-forming bacterium, is the causative agent of
listeriosis, a severe food-borne disease that can lead to gastrointestinal and
neurological disorders, sepsis and abortion in humans. Because of the severity of the
symptoms and of the relatively high mortality, L. monocytogenes represents a growing
food safety issue. It is capable of forming dense biofilms tolerant to detergents and
disinfectants [18], and consequently difficult to eliminate. These biofilms are able to
survive for long periods in harsh conditions like refrigeration temperatures used to
preserve food [19].
Due to the risks of food spoilage and deterioration and the consequent impacts in
economy and human health, strategies to control and delay biofilm development, as
well as the inactivation of installed biofilms are being intensively searched [14].
The immobilization of a PS in different solid supports has been explored in order to
promote the recovery and reuse of the PS after PDI, in the perspective of making
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photodynamic antimicrobial approach economically viable and more environmentally
friendly. In this perspective, the use of non-toxic and biodegradable supports such as
chitosan is regarded as promising carriers for drug delivery and also for the design of
photodynamic antimicrobial materials [20-24]. Chitosan is a linear polysaccharide
composed of ß(1‰4) linked glucosamine units obtained by alkaline deacetylation of
chitin, present as a structural biopolymer in the exoskeletons of some crustaceans and
mollusks [25, 26]. This polysaccharide exhibits interesting properties such as film-
forming ability, biodegradability and an intrinsic antimicrobial activity associated with the
protonated amino groups, which is the base of its wide use in biomedical applications
[27-31]. The antimicrobial activity of chitosan based materials can be further enhanced
by the incorporation of bioactive materials/compounds like metal nanoparticles,
photosensitizers, phenolic compounds, antibiotics and tetrahydrocurcominoid
derivatives [19, 32-36]. Recently, the synergic action of chitosan films loaded with a
series of antibiotics and tetrahydrocurcuminoid derivatives was demonstrated in the
disruption of L. monocytogenes biofilms, or in the complete inactivation of Listeria
innocua [36].
Considering the adequate features of chitosan to immobilize photosensitizers
associated with its antimicrobial activity and film-forming ability, the principal aim of this
work was to develop efficient photosensitizing materials based on chitosan and
adequately substituted meso-tetraarylporphyrins (phenyl units with acid groups or
pentafluorophenyl units with or without acid groups) (Figure 1). The efficiency of the
new porphyrinic materials (PS-CF) to prevent Listeria innocua biofilm development was
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tested on the phases corresponding to cell adhesion and biofilm maturation. The
inactivation of planktonic cells with non-immobilized PS was also tested for comparison.
2. Experimental section
General information
All reagents were purchased from Aldrich, Sigma or Merck and were of analytical grade.
Chitosan was kindly provided by Mahtani Chitosan Pvt. Ltd. (India) and was purified by
dissolving the commercial sample in 1% (v/v) aqueous acetic acid solution, followed by
filtration to remove solid impurities, and then precipitated by neutralization with 0.5 M
aqueous NaOH up to a pH of 8.5. Solid chitosan was recovered by filtration, washed
with distilled water until neutral pH and air dried. Its viscosity-average molar mass,
obtained at 25 ºC from a 0.3 M CH3CO2H/ 0.2 M CH3CO2Na solution, using the
published Mark–Houwink constants [37], was 350 000 g mol-1. Its degree of
1
deacetylation (DDA), determined by H NMR, (in D2O containing 1% of CD3COOD),
was 97% [38].
1
Techniques used for the materials characterization: H and 19F NMR spectra were
recorded on a Bruker Avance 300 spectrometer at 300.13 and 282.38 MHz,
respectively. Deuterated dimethyl sulfoxide and deuterated chloroform were used as
solvent and TMS (
δ = 0 ppm) as the internal standard; chemical shifts are reported in
ppm ( ) and coupling constants (J) are given in Hz. Mass spectra were recorded on a
δ
4800 Proteomics Analyser mass spectrometer (MALDI TOF/TOF) and HRMS were
recorded on an APEXQe FT-ICR mass spectrometer (Bruker Daltonics, Billerica, MA).
Electronic spectra (UV-VIS) were obtained on a Cary-Varian or a Shimadzu UV-2501PC
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spectrophotometer, in the 200-800 nm range. The fluorescence emission spectra of P1-
4 were recorded in DMF in 1 × 1 cm quartz optical cells under normal atmospheric
conditions on a computer controlled Horiba Jobin Yvon Fluoro Max-3 spectrofluorimeter.
The widths of both excitation and emission slits were set at 2.0 nm. The fluorescence
quantum yield (ΦF) of P1-4 was calculated in DMF by comparison of the area below the
corrected emission spectra using [H2(TPP)] as standard (λexc at 420 nm, ΦF = 0.11 in
DMF) [39]. For the solids diffuse reflectance characterization an UV-VIS Jasco V560
spectrophotometer was used. Attenuated total reflectance Transmission Fourier
Transform Infrared (ATR-FTIR) spectra were registered on a FT Mattson 7000 galaxy
series spectrophotometer in the 400-4000 cm-1 range, the spectra were collected with a
resolution of 4 cm-1 and accumulation of 32 scans. Thermogravimetric (TG)
measurements were performed on a Thermal Analyst TGA-50, in nitrogen, with a
heating rate of 10 ºC min-1, from 25 to 800 ºC. For the X-ray diffraction (PXRD)
measurements, self-oriented solids were placed on neutral glass sample holders. The
measurements were performed in the reflection mode using X'Pert MPD Philips
diffractometer (Cu Kα1,2 X-radiation, λ1 = 1.540598 Å and λ2 = 1.544426 Å), equipped
with an X'Celerator detector and a flat-plate sample holder in a Bragg–Brentano para-
focusing optics configuration (40 kV, 50 mA). Intensity data were collected by the step
counting method (step 0.02), in continuous mode. The thickness of the films was
determined using a digimatic micrometer (model MDE-25TJ, Mitutoyo Corp., Tokyo,
Japan). Mean thicknesses were calculated from 20 measurements taken at different
locations of the films.
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Synthesis of porphyrin derivatives
Free base porphyrins P1, P2 and P4 (see structures in Figure 1) and 5,10,15,20-
tetraphenylporphyrin [H2(TPP)] were synthesized as described in the literature [40, 41].
Product purity was confirmed by thin layer chromatography and by 1H NMR
spectroscopy.
1
Porphyrin P1, mp > 300°C: H NMR (CDCl3): δ -2.92 (broad s, 2H, NH), 8.92 (s, 8H, β-
H). 19F NMR (CDCl3): δ - 160.0 (dd, J = 25.4 and 8.5 Hz, 8F, Ar-o-F), -174.7 (t, J = 21.2
Hz, 4F, Ar-p-F), -184.8 to -184.7 (m, 8F, Ar-m-F). UV-VIS (CH2Cl2) λmax (nm): 410, 504,
539, 582 and 638; MS (MALDI) (m/z): 975 [M]+•.
Porphyrin P2, mp > 300°C: δH ppm (DMSO-d6): -2.87 (s, 2H, NH), 7.82 (d, J = 8.4 Hz,
8H, S-C6H4-CO2H), 8.14 (d, J = 8.4 Hz, 8H, S-C6H4-CO2H) and 9.46 (broad, 8H, H-β).
19F NMR: δF (DMSO-d6, in ppm): δ -161.1 to 161.0 (dd, J = 11.3 and 25.4 Hz, 8F, m-F),
and -156.2 to -156.1 (dd, J = 11.3 and 25.4 Hz, 8F, o-F). UV-VIS (DMF) λmax (nm): 412,
504, 540, 580 and 646; HRMS (FAB+): [M+H]+: 1511.0752.
1
Porphyrin P4, mp > 300°C: H NMR (CDCl3): δ-2.94 (s, 2H, NH), 8.35 and 8.40 (AB
system, J = 8.3 Hz, 16H, Ar-o,m-H), 8.87 (s, 8H, β-H), and 13.3 (s, 4H, CO2H). UV-VIS
(DMF) λmax (nm): 418, 518; 552; 584; 630. MS (MALDI) (m/z): 790.2 [M]+•.
Synthesis of porphyrin P3 from the reaction of 3-mercaptobenzoic acid with
porphyrin P1: In a round-bottom flask, 0.24 mmol of 3-mercaptobenzoic acid were
dissolved in 20 mL of DMF with 1.0 mL of pyridine and kept under magnetic stirring at
room temperature for 30 min. After this time the porphyrin P1 (0.035 mmol) was added
and the reaction mixture was kept under magnetic stirring for 48 h. The reaction was
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monitored by TLC and the reaction was stopped when no P1 was detected. The solvent
was evaporated under reduced pressure and the crude material dissolved in
CH2Cl2:CH3OH was purified by column chromatography using silica as the stationary
phase and CH2Cl2:CH3OH (9:1 v/v) as eluent. Porphyrin P3 was obtained in 85% yield,
after crystallization from a mixture of CH2Cl2:CH3OH:hexane.
Porphyrin P3, mp > 300°C: δH ppm (DMSO-d6): -3.13 (s, 2H, NH), 7.72 to 7.67 (t, J =
7.8 Hz, 4H, S-C6H4-CO2H), 8.06 to 7.97 (m, 8H, S-C6H4-CO2H), 8.23 (s, 4H, S-C6H4-
CO2H) and 9.50 (s, 8H, H-β). 19F NMR: δF ppm (DMSO-d6) -161.2 to -161.1 (dd, J =
11.4 and 26.6 Hz, 8F, F-meta), and -156.7 to -156.6 (dd, J = 11.4 and 26.6 Hz, 8F, F-
ortho) (see ESI). UV-VIS (DMF) λmax, nm (log ε): 412 (5.14), 504 (4.07), 582 (3.57).
HRMS-ESI: calcd. for C72H29F16N4O8S4 [M-H]+• 1509.0764 found 1509.0657; calcd. for
C72H30F16N4O8S4 [M]+• 1510.0691 found 1510.0683; calcd. for C72H31F16N4O8S4 [M+H]+
1511.0725 found 1511.0687.
Immobilization of porphyrins in chitosan (Px-chitosan): The porphyrin-chitosan (Px-
chitosan where x = 1, 2, 3 or 4) solids were prepared by dissolving 150 mg of chitosan
in 5.0 mL of 1% (v:v) aqueous acetic acid. A solution of P1, P2, P3 or P4 (1 mmol/L) in
THF was added, and the mixture was stirred for 16 h, at 30 °C. The solvent was
evaporated under reduced pressure and the resulting solid was filtered and exhaustively
washed with DMF, methanol and THF. The supernatants resulting from the washing
process were analyzed by UV-VIS in order to quantify the Px that leaked from the
matrix during the washing process and the loading of the porphyrin in each solid was
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calculated. The light brown Px-chitosan solids obtained were dried in the oven at 55 °C
for 48 h.
Preparation of chitosan-porphyrin films (PSx-CF): The PSx-CF and chitosan films
(CF) were obtained by dispersing the unmodified chitosan (30 mg) or the Px-chitosan
samples (about 30 mg) in 5.0 mL of 1% (v:v) aqueous acetic acid. The suspension was
maintained at 30 °C under stirring until complete solubilization of solid chitosan (40 min)
or Px-chitosan (5 h). The obtained solutions were poured into molds (2.5 x 2.5 cm) and
allowed to dry at room temperature for 48 h (see ESI, Figure S3). The obtained films,
identified as PSx-CF were washed with DMF and air-dried. The PSx-CF films were
characterized by XRD (powder), TG/DTG, ATR-FTIR and UV-VIS.
Singlet oxygen generation and photostability studies: A 10 mM stock solution of
1,3-diphenylisobenzofuran (DPiBF, a singlet oxygen quencher) in DMF and 0.1 mM
stock solution of each porphyrin derivative in DMF were prepared. In a 1 x1 cm optic
glass cell (2 mL), a solution of DPiBF (50 µM) and 0.5 µM of each PS in DMF was
irradiated with a LED array at an irradiance of 9.0 mW.cm-2. A LED square array (5 x 5
LEDs light sources) with an emission peak centered at 654 nm and a bandwidth at half
maximum of ± 20 nm was constructed and used as light source. Irradiation was
conducted at room temperature under stirring. The breakdown of DPiBF, indicative of
singlet oxygen production, was monitored by measuring the decreasing of the
absorbance at 415 nm at irradiation intervals of 1 min.
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[H2(TPP)], an efficient singlet oxygen generator, was included as reference [42]. A
solution of DPiBF (50 µM), without added porphyrin, was used to evaluate DPiBF
photodecomposition.
The photostability of the porphyrin derivatives was evaluated by irradiation of a
solution of each porphyrin in DMF (2 µM) with white light (400-800 nm) from a
compatible optic fibre probe attached to a 250 W quartz/halogen lamp (LumaCare®
model LC122, USA) at an irradiance of 40 mW.cm-2. During the experiments, the
solutions were magnetically stirred and kept at room temperature. The stability of the
porphyrin derivatives was verified by visible absorption spectroscopy at regular intervals
up to 90 min.
PDI irradiation conditions: A white Back-Light LED-Setup (AG Photobiophysik,
Humboldt-Universität zu Berlin) delivering white light (400–800 nm) at an irradiance of
10 mW cm-2 was used to expose cells suspensions during 5, 10, 20, 30 or 40 min in
assays with non-immobilized porphyrins (P1-P4) and for 24 h in assays with PSx-CF
films. The light irradiance was measured with a Power Meter Coherent FieldMaxII-Top
combined with a Coherent PowerSens PS19Q energy sensor.
Photodynamic inactivation of L. innocua cell suspensions by non-immobilized
photosensitizers P2-P4: For the screening of the photosensitization potential of the
newly synthesized porphyrins, 500 µM stock solutions of porphyrins P2, P3 and P4
were prepared in DMSO and maintained at 4 °C. L. innocua was used as a low-risk
surrogate of the pathogenic L. monocytogenes. Fresh cultures in Tryptic Soy Broth
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(TSB) were prepared before each assay by inoculating an isolated colony obtained from
a culture in Tryptic Soy Agar (TSA) in 30 mL of liquid medium and incubating overnight
at 37 ºC with stirring (~150 rpm) to obtain cultures with an OD600 ≈ 0.8, corresponding to
a concentration of viable cells of ~109 CFU.mL-1.
Suspensions of L. innocua were prepared by 10-fold diluting the fresh culture in
phosphate buffered saline solution (PBS, pH= 7.1). Four-mL aliquots of bacterial
suspension were distributed in individual wells of 12-well plates and the appropriate
volume of the porphyrin derivative (P2, P3 or P4) (Figure 1) was added in order to attain
final concentrations of 2.0 and 5.0 µM. The suspensions were incubated for 15 min in
the dark to allow for the binding of the PS to bacterial cells, and subsequently exposed
to irradiation conditions, during a maximum period of 40 min. Dark and light controls
were included in the experiments. In the light control, the bacterial suspensions without
the porphyrin were exposed to the same irradiation protocol as the test suspensions. In
the dark control, the porphyrin was added at the highest tested concentration (5.0 µM)
and the experimental setup was protected from light with aluminum foil. Aliquots of 0.1
mL of test and control samples were aseptically collected at times corresponding to 0, 5,
10, 20, 30 and 40 min irradiation. Serial 10-fold dilutions were prepared in PBS and
aliquots of the highest dilutions were pour-plated in duplicate in Tryptic Soy Agar (TSA).
The cultures were incubated for 48 h at 37 ºC and colonies were counted in the plates
of the most suitable dilution. The concentration of survivors was calculated as the
average colony count corrected for the dilution factor. At least two independent assays
were performed.
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Photodynamic effect of PSx-CF films on different stages of L. innocua biofilms
development: The effect of PS1-CF to PS4-CF films on the development of L. innocua
biofilms was tested using 0.2 µmol of immobilized PS (about 6 mg of PSx-CF) and an
irradiation time of 24 h. Suspensions of L. innocua were prepared 10-fold diluting a
fresh culture in PBS. The PSx-CF films were washed with DMF and ethanol, and air-
dried before use. Two-mL aliquots of bacterial suspension were transferred to glass
vials and the sterilized chitosan-porphyrin films (PS1-CF, PS2-CF, PS3-CF or PS4-CF)
containing 0.2 µmol of incorporated PS were immersed in the cell suspensions. A glass
slide with dimensions equivalent to those of the PS-chitosan films was also included in
the experiment as a positive control for biofilm development. The vials were exposed to
different irradiation regimes: Dark (24 h), Light (24 h), Dark + Light (24 h + 24 h) or Light
+ Dark (24 h + 24 h). Experiments were conducted at 20 ºC. To create dark conditions,
vials were wrapped in aluminium foil during the experiment.
In the Dark + Light and Light + Dark regimes, the vials were swapped after 24 h-
incubation so that films that had been incubated in the dark during the attachment
phase were exposed to light and films that had been exposed to light during the
attachment phase were further incubated in the dark. In the Dark + Light regime, the
solid supports immersed in cell suspensions were kept in the dark for 24 h (non-
photosensitizing conditions during adhesion stage) followed by 24 h light exposure
(photosensitization during proliferation stage). In the Light + Dark regime, cell
attachment to films occurred under light exposure (photosensitizing conditions during
adhesion stage) and the proliferation of attached cells was allowed to proceed in the
dark (non-photosensitization conditions during maturation) for further 24 h. At least two
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independent assays were performed, each one with replicates for each experimental
condition.
For the determination of the concentration of cells in the biofilms at the beginning,
and at the end of the experiments (24 h or 48 h), PSx-chitosan and chitosan films were
recovered from the suspensions, twice washed by gentle immersion in 1.0 mL of PBS
for removal of loosely bound cells, transferred to a microtube containing 1.0 mL of PBS
and sonicated during 15 min (Silvercrest Ultrasonic Cleaner) in order to promote the
detachment of cells from the solid support (PSx-CF films or glass slide) to the liquid
phase. The cell suspension was serially diluted and pour-plated in Tryptic Soy Agar
(TSA), in duplicate. The cultures were incubated for 48 h at 37 ºC and colonies were
counted in the plates of the most suitable dilution. The concentration of viable cells was
calculated as the average colony count corrected for the dilution factor.
Photodynamic effect of PS3-CF films on L. innocua cells: In order to evaluate the
direct photodynamic inactivation of planktonic cells in the presence of PSx-CF films, the
most efficient material (PS3-CF) was used. Cell suspensions were prepared from fresh
liquid cultures, transferred to glass vials and PS3-CF films were immersed as previously
described. The vials containing the bacterial suspension and PS3-CF were exposed to
different light regimes: Dark (24 h), Light (24 h). A light control (bacterial suspension
without PS3-CF) was submitted to the same irradiation protocol.
In order to determine the concentration of viable planktonic cells at the beginning
and at the end of the experiment, all cell suspensions were serially diluted and pour-
plated in Tryptic Soy Agar (TSA), in duplicate. The cultures were incubated for 48 h at
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37 ºC and colonies were counted in the plates of the most suitable dilution. The
concentration of viable cells was calculated as the average colony count corrected for
the dilution factor.
In order to discriminate between two processes that could underlie photodynamic
inhibition of cell adhesion to the solid supports (direct cell inactivation or loosening of
the binding and detachment of viable cells to the suspension medium) the films used in
the Light (24 h) treatment were removed from the suspension, transferred to TSB (1/10
strength) and further incubated 4 h in the dark to allow the recovery of cells that might
have detached from the film. The concentration of viable cells was determined by
colony counting as previously described.
Statistical analysis: Statistical analysis was performed in OriginPro 8.5. The
significance of the PDI effect of each PS and of the irradiation time on bacterial cell
viability was assessed by an unvaried analysis of variance (ANOVA) model with the
Bonferroni post hoc test. Normal distributions were assessed by the Kolmogorov–
Smirnov test and homogeneity of variances was assessed by the Levene test. A value
of p < 0.05 was considered significant.
3. Results and Discussion
3.1. Synthesis and photophysical characterization of porphyrin derivatives
In the selection of porphyrins P2-P4 to be immobilized in chitosan (Figure 1) it was
considered that the presence of carboxylic groups in the porphyrin ring could improve it
electrostatic interaction with the chitosan amino groups [43]. P1 was assessed once it is
the chemical precursor of P2 and P3.
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5,10,15,20-tetrakis(pentafluorophenyl)porphyrin (P1) and 5,10,15,20-tetrakis(4-
carboxyphenyl)porphyrin (P4) were obtained directly by condensation of pyrrole with the
adequate aldehydes in acidic conditions according to the procedure reported in the
literature [40, 41]. Porphyrin P1 was used as precursor of P2 and P3. The thio-
carboxylate porphyrins P2 and P3 were obtained by nucleophilic aromatic substitution of
the p-fluorine atoms in (P1) with 4-mercaptobenzoic acid and 3-mercaptobenzoic acid,
respectively [41]. The structure of P3 was confirmed by using the usual spectroscopic
techniques and for the other porphyrins the analytical data were in accordance with
literature [40, 41].
Figure 1. Schematic representation of the photosensitizer P1-P4 and chitosan
structures.
The absorption spectra of the selected porphyrins P1-P4 in DMF (see ESI, Table
1S and Figure 2) evidence the typical features of free base meso-tetraarylporphyrins
due to π–π* transitions: a highly intense Soret band between 410 nm and 418 nm and
four less intense Q-bands in the range 504-648 nm of the visible spectrum. Upon
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excitation, the porphyrins showed the two expected fluorescence emission bands in the
red spectral region at ca 648 and 706 nm (Figure 2) [39].
P1
P2
P3
P4
P4
P3
P2
P1
B
A
600
620
640
660
680
700
720
740
350
400
450
500
550
600
650
700
Wavelength (nm)
Wavelength (nm)
Figure 2. Normalized electronic spectra A) absorption and B) fluorescence (λ excitation
= 420 nm) spectra of P1, P2, P3 and P4 in DMF.
3.2. Immobilization of porphyrins on chitosan
The immobilization of the porphyrins on chitosan was performed by adding
adequate amounts of porphyrins P1-P4, dissolved in THF, to an aqueous acetic acid
(1% v/v) of chitosan. The amount of each porphyrin immobilized on the chitosan matrix
was determined indirectly based on the amount of porphyrin in the supernatants
resulting from the washing process, determined by UV-VIS. Table 1 summarizes the
porphyrin percentage loading based on the amount used and the corresponding
concentration in the chitosan support.
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Table 1. Porphyrin incorporation on chitosan.
Loading (porphyrin per solid prepared)
% w/wd
% of
immobilizationb
16%
Solid
(mol g-1)a
0.50 x 10-5
3.15 x 10-5
2.96 x 10-5
3.08 x 10-5
P1-chitosan
P2- chitosan
P3- chitosan
P4- chitosan
[H2(TPP)]-
0.48
4.76
4.47
2.43
0
100%
96%
76%
0
-
chitosanc
a)
b)
Mol of porphyrin/mass (g) of chitosan; Immobilization percentage based on the mass of
porphyrin used; c) There was no interaction. dMass (g) of porphyrin per gram of chitosan.
In order to evaluate how the load efficiency was affected by substituents in
porphyrin meso-aryl groups (phenyl with acid groups or pentafluorophenyl with or
without acid groups) the immobilization protocol was also repeated for the non-
substituted free base 5,10,15,20-tetraphenylporphyrin [H2(TPP)].
The highest immobilization percentages were obtained for porphyrins P2 (100%)
and P3 (96%) followed by P4 (76%) and P1 (16%). In contrast, [H2(TPP)] was
completely removed after the washing process. This fact suggests that the electrostatic
interaction of the carboxylic groups present in the meso-substituents of the porphyrin
with the chitosan amino groups can have an important contribution for the
immobilization of porphyrins P2-P4. In fact, chitosan is considered an interesting
candidate to assemble macrocycles by electrostatic interactions, due to the amino
positive charge density at low pH values (pKa ~ 6.2) [27]. In the case of P1, the
interaction with the chitosan is probably facilitated by hydrogen bonding involving the
fluorine atoms, which could justify the incorporation of this neutral derivative when
compared with the non-substituted [H2(TPP)] macrocycle.
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3.3. Preparation and characterization of chitosan-porphyrin films (PSx-CF)
The chitosan-porphyrin films, designated as PS1-CF, PS2-CF, PS3-CF and PS4-
CF, were prepared by dissolution of the porphyrins and chitosan in a 1% acetic acid
solution (Figure S3, Supporting information) followed by casting in a ventilated oven at
30 ºC. All samples were able to filmify and the resulting films with thickness between
35.2 and 77.4 µm displayed a brownish color, compared to the fully transparent
chitosan film (CF) (Figure 3). The films were characterized by UV-VIS, powder XRD,
TG/DTG and ATR-FTIR.
CF
PS2-CF
PS4-CF
PS1-CF
PS3-CF
Figure 3. Digital photographs of chitosan and chitosan-porphyrin films; chitosan (CF),
PS2-chitosan (PS2-CF), PS4-chitosan (PS4-CF), PS1-chitosan (PS1-CF) and PS3-
chitosan (PS3-CF) (left to right).
The solid UV-VIS spectra of the chitosan-porphyrin films (Figure 4) show the typical
absorption bands for porphyrins (Supporting information, Table 1S and Figure 2). The
Soret bands of the films PS2-CF, PS3-CF and PS4-CF are broad and slightly red-
shifted (ca 3-9 nm) when compared with those of the corresponding non-immobilized
porphyrins in solid state (Figure S4, Supporting information); PS4-CF displayed another
broad band at c.a. 406 nm. The solid-state interactions lead to band broadening and
elevation of the base line [44]. Besides that, the broadening of the Soret and Q bands of
18

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the films PS-CF may be caused by small alteration of porphyrin molecular environment
due to the interaction with chitosan [45].
The four Q-bands are shifted (ca 4-16 nm) depending on the porphyrin (Figure S4,
Supporting Information). For PS1-CF the Soret band and Q-bands did not shift
significantly, and such behavior can be attributed to the mode of interaction with
chitosan.
Porphyrin P4 tends to aggregate in solution, depending on the solvent, and on the
pH in aqueous solutions [46]. Self-aggregation lead to drastic changes in the spectral
profile, namely decrease in absorption intensity, enlargement of bands, appearance of
new bands and elevation of the baseline, as shown in Figure 2 for P4 in DMF and in
Figure 4 for PS4-CF.
It is well known that porphyrins can suffer different types of aggregation
phenomena which can be related with intrinsic factors (e.g., presence or absence of a
central metal atom, nature of substituents) and extrinsic factors such as solubility, pH
and ionic strength of the solution, or even the nature of the solvent [47].
For the other PSx-CF (x= 1, 2 and 3) the broadening and the bathochromic shifts
of the Soret and Q bands observed seem to indicate that during the process of
immobilization a significant distortion in porphyrin conformation occurred, which can be
related with alterations in the aromatic arrangement, affecting the porphyrin ring
symmetry [48]. These red shifts can be associated with the destabilization of the frontier
orbitals HOMO in relation to LUMO due to physical and chemical interactions between
the porphyrin and the chitosan [49].
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406
433
422
522
557
596
652
510
422
412
PS4-CF
586
540
540
544
510
512
650
650
PS3-CF
PS2-CF
586
586
652
PS1-CF
CF
300
350
400
450
500
550
600
650
700
750
800
Wavelength (nm)
Figure 4. UV-VIS spectra of the solid samples: chitosan and PSx-CF films.
The ATR-FTIR spectra of CF and PSx-CF films (Figure S5, Supporting
information) clearly show the characteristic bands of chitosan [50], notably at 3364 and
3288 cm-1 (
ν-OH and
ν
-NH2); 2869 cm-1 ( NH); 1422 cm-1
ν
-CH asymmetric); 1590 cm-1 (
δ
(δ
OH e
δ
CH2 (scissoring)); 1375 cm-1 (
δ
CN); 1313 e 1256 cm-1 (
ν-CN); 1064 and 1030
cm-1 (
ν
-COC). However, the characteristic bands of porphyrins (Figure S6, Supporting
information) could not be detected, mainly due low amount of porphyrins incorporated in
the chitosan films (Table 1).
The XRD pattern of the CF films have been studied in the 2θ
range of 5o to 50o
and was compared with the XRD pattern of pure chitosan and of the corresponding
porphyrins (Figure 5 and Figure S7). The X-ray diffraction profile of CF shows peaks at
16.3o, 19.9o and 42.9o (2
θ) characteristic of semi-crystalline chitosan (Figure 5) [51].
The peaks at 19.9º (d = 0.445 nm) and 42.9º (d = 0.211 nm) characterized the presence
20

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of a crystalline phase, associated with the planes (200) and (241) respectively. The
peaks observed in 10-16º (2θ) can be associated with hydrated and anhydrous crystals
[51, 52].
The peak observed at 8.2º (2θ) for PS1-CF and PS3-CF is probably related with
the plane (020) of chitosan crystalline cell. The appearance of this peak can be due to a
rearrangement of chitosan chains during the porphyrin immobilization process. PS4-CF
revealed only a halo in the region of 17 and 30º for 2θ which suggests that the
amorphous phase is predominant. No significant alteration on CF crystallinity was
detected on PS2-CF.
PS4-CF
PS3-CF
PS2-CF
PS1-CF
CF
5
10
15
20
25
30
35
40
45
50
θ
2 (Degree)
Figure 5. X-Ray powder diffraction patterns for: chitosan and PSx-CF films.
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Interesting, the results of the XRD analysis allow concluding that the crystallinity
observed for each PS-CF is dependent on porphyrin crystallinity (Figure S7, Supporting
information).
The thermal stability of the new materials PS1-CF to PS4-CF was evaluated by
thermogravimetry and it was compared with the thermal stability of chitosan (Figure 6).
The thermogram of the CF film shows the expected two weight losses [53]. The first
one, below 100 ºC, is due to the desorption of physisorbed water and acetic acid and
the second thermal event at 298 ºC is associated with the polysaccharide degradation.
The TGA of the materials PS1-CF to PS4-CF exhibited two steps of decomposition
upon heating under N2 besides of the mass loss of around 10% centered below 100 ºC
that is related with the loss of the adsorbed water. The first one is related to the
degradation of the chitosan chains and appeared at 260 ºC for the PS1-CF, at 289 ºC
for the PS4-CF and at 304 ºC for the PS2-CF and PS3-CF. For all the four solids a third
mass loss event is also observed between 600-660 ºC that can be attributed to the
porphyrins decomposition.
In fact, the thermal analysis of the non-immobilized porphyrins showed that the
main mass loss occurred between 400 and 600 ºC (Figure S8, Supporting information).
In general, porphyrins display high thermal stability [54] and the thermal decomposition
can occur in several steps, depending on the peripheral substituents, followed by the
macrocycle break [55]. The decomposition of P2, P3 and P4 consists of two or three
main stages and of P1 only on one stage. Considering the main loss, the thermal
stability observed was P2 (590 ºC) ~ P3 (574 ºC) > P4 (411 and 601 ºC) > P1 (442 ºC)
22

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(Figure S8). These results suggest that the presence of the carboxylic group moieties in
the macrocycle improved its thermal stability.
According to the DTG curves of the PS-CF films, the second thermal event for
PS1-CF and PS4-CF occurs at lower temperatures than for chitosan. Probably in these
two films the insertion of the porphyrin macrocycle was responsible by weakening the
interconnection between the chitosan polysaccharide chains turning these materials
less thermostables [45].
However, chitosan seems to slightly protect the porphyrinic macrocycle as it is
evidenced by the displacement of the third mass loss (attributed to the porphyrin
decomposition) for temperatures higher than the ones observed for the non-
immobilized porphyrins.
The thermal stability observed for PS-CF seems to be influenced not only by the
stability of the immobilized porphyrin but also by the different interactions between the
porphyrin and the chitosan (Figure 6 and S8).
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CF
100
0.0
80
60
40
20
0
-1.0x10-3
-2.0x10-3
-3.0x10-3
-4.0x10-3
-5.0x10-3
100
200
300
400
500
600
700
800
Temperature (ºC)
100
80
60
40
20
0
100
80
60
40
20
0
PS1-CF
PS2-CF
0.0
0.0
-5.0x10-4
-1.0x10-3
-1.5x10-3
-2.0x10-3
-2.5x10-3
-3.0x10-3
-5.0x10-4
-1.0x10-3
-1.5x10-3
-2.0x10-3
100
200
300
400
500
600
700
800
100
200
300
400
500
600
700
800
Temperature (ºC)
Temperature (ºC)
0.0002
0.0000
-0.0002
-0.0004
-0.0006
-0.0008
-0.0010
100
PS3-CF
PS4-CF
100
80
60
40
20
0
0.0
80
60
40
20
0
-5.0x10-4
-1.0x10-3
-1.5x10-3
-2.0x10-3
100
200
300
400
500
600
700
800
100
200
300
400
500
600
700
800
Temperature (ºC)
Temperature (ºC)
Figure 6. Thermal analyses TG/DTG curves of materials PS1-CF to PS4-CF and CF.
3.4. Singlet oxygen generation and photostability studies
The photodynamic efficiency of a PS is dependent on its ability to generate reactive
oxygen species (ROS), mainly singlet oxygen (1O2), and also of its photostability upon
irradiation. The efficiency of the new PS-CF films and of the non-immobilized porphyrins
P1-P4 in the generation of 1O2 was qualitatively assessed by monitoring the
decomposition of 1,3-diphenylisobenzofuran (DPiBF) during irradiation with red light
24

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(654 ± 20 nm). The first order decay observed in DPiBF absorption band at 415 nm
results from its reaction with 1O2 in a [4 + 2] cycloaddition process affording a colourless
o-dibenzoylbenzene.
1.2
DPiBF
H2(TPP)
1.0
P1
P2
P3
0.8
P4
CF
PS1-CF
0.6
PS2-CF
PS3-CF
PS4-CF
0.4
0.2
0.0
0
200
400
600
800
Time (s)
Figure 7. Time dependent photodecomposition of DPiBF (50 µM) photosensitized by
P1-P4 and PS1-CF-PS4-CF in DMF upon irradiation with red light (654 ± 20 nm) with or
without PS (0.5 µM). [H2(TPP)] and CF were used as reference.
The results summarized in Figure 7 show that the DPiBF decomposition is highly
enhanced in the presence of the PS1-CF, PS2-CF and PS3-CF films or in the presence
of corresponding non-immobilized porphyrins P1, P2 and P3. The low efficiency of P4 in
1
generating O2 can be related with its strong tendency to aggregate in solution (as
previously discussed), but this feature is attenuated after its incorporation in the
1
1
chitosan, since PS4-CF film is able to generate moderately O2. The most efficient O2
producer using the PS-CF film was PS1-CF, followed by PS2-CF
≈
PS3-CF and finally
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ACCEPTED MANUSCRIPT
PS4-CF. This is coherent with the efficacy of the non-incorporated porphyrins in
solution: P1 > P2
≈ P3 >> P4. In these studies [H2(TPP)] was used as reference due to
its high efficiency to generate 1O2 [43].
All the PS-CF films were photostable once the UV-VIS and TGA analysis obtained
before and after the PDI biological assays were similar (Figure S9 and S10, Supporting
information). These results are in accordance with the photostability found for
porphyrins P1 to P4 in PBS solution under the same PDI irradiation conditions. Only P4
suffered a decay (<50%) during the irradiation period (Figure S11, Supporting
information) which can be associated with some decomposition and the tendency to
aggregate.
3.5. Photodynamic inactivation of Listeria innocua with the non-immobilized
porphyrins
Considering that the ability of the new PS-CF materials to avoid biofilm formation
was likely determined by the ability of the corresponding porphyrins to photosensitize
Listeria innocua, the inactivation kinetics of cells suspensions was initially screened.
The experiments were performed in the presence of the non-immobilized porphyrins P2-
P4 at concentrations of 2.0 and 5.0 µM, under white light (400-800 nm) (Figure S12,
Supporting information). P1 was not used in this set of experiments due to its insolubility
in PBS the suspension medium used in the PDI experiments. The viability of L. innocua
was not affected neither by light alone (light control in which bacterial suspension were
irradiated with white light in the absence of PS) nor by the direct effect of any of the
tested PS (dark control in which bacterial suspension were exposed to the PS in the
absence of light). These results indicate that the reduction in the concentration of viable
26

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cells bacterial was due to the photodynamic effect of the PS. Photosensitizers P2-P4
showed significant differences in terms of inactivation efficiency (ANOVA – p < 0.05)
(Figure 8). P3 at a concentration of 5.0 µM was the most effective PS (Figure 8)
causing a reduction of ca 6 log in the concentration of viable cells, after 10 min (6 J/cm2)
of irradiation. At this concentration, P3 was able to completely inactivate L. innocua
(concentration of viable cells below the quantification limit) after 20 min of irradiation. At
a concentration of 2.0 µM, 30 min of irradiation are required for complete inactivation
(Figure S12). P2 was the next most effective PS (Figure 8), causing an inactivation of
ca 3 log after 20 min of irradiation, at a concentration of 5.0 µM. With this PS, the
reduction in L. innocua viability reached the detection limit after 30 min of irradiation,
with either 2.0 or 5.0 µM. With P4 (Figure 8) the maximum bacterial reduction was ca 4
log, achieved after 40 min of irradiation at concentration of 5.0 µM and complete
inactivation was not attained under the experimental conditions used. These results can
1
be explained by the limited efficacy of P4 to generate cytotoxic O2 and consequently
reducing its PDI efficiency [8]. Figure 8 shows a comparison of the inactivation kinetics
with the different porphyrins, used at a concentration of 5.0 µM. The difference
observed for each PS can be justified by a balance between the efficiency to produce
1O2 (P2
∼ P3 >> P4) and the affinity of PS to the bacteria that it is dependent on
porphyrin structure [56]. The efficiency of P2 and P3 is comparable to the one reported
for the inactivation of several strains of Listeria monocytogenes by a Na-chlorophyllin
PS and ALA (5-aminolevulinic acid) produced endogeneous porphyrins [57, 58].
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Figure 8. Variation of the concentration of viable cells of Listeria innocua in suspension,
after irradiation with white light at an irradiance of 10 mW/cm2 in the presence of P2-P4
at a concentration of 5.0 µM. Each value represents the average of at least two
independent experiments. Deviations to CFU/mL were not greater than ± 2%.
All the porphyrin derivatives tested (P2, P3 and P4) caused reductions higher than 3
log in L. innocua suspensions under the experimental conditions tested, and can,
therefore, be considered effective antibacterial agents of the planktonic form of this
species according to the American Society for Microbiology (for a new approach to be
considered as "antimicrobial" or "antibacterial" a minimum inactivation of 3 log must be
obtained) although P3 was effective within a shorter irradiation time (20 min) [59].
3.6. Photodynamic effect of PSx-CF films on L. innocua biofilms development
The effect of the new materials PS1-CF - PS4-CF on L. innocua biofilms was
evaluated at two different biological stages of biofilm development - adhesion and
maturation [60].
28