A nonionic porphyrin as a noninterfering DNA antibacterial agent

Article abstract of DOI:10.1111/j.1751-1097.2011.00984.x

The increasing interest in clinical bacterial photodynamic inactivation has led to the search for photosensitizers with higher bactericidal efficiency and less side effects on the surrounding tissues. We present a novel nonionic porphyrin, the 5,10,15-tri

Full text of DOI:10.1111/j.1751-1097.2011.00984.x

Photochemistry and Photobiology, 2011, 87: 1395–1404
A Nonionic Porphyrin as a Noninterfering DNA Antibacterial Agent
1
Sonia Mendes , Fabio Camacho , Tito Silva , Cecılia R. C. Calado , Armenio Coimbra Serra* ,
1
1
1
2
´
Antonio M. d’A. Rocha Gonsalves and Monica Roxo-Rosa*
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2
1
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1
´
Faculdade de Engenharia, Universidade Catolica Portuguesa, Rio de Mouro, Portugal
Chymiotechnon, Departamento de Quımica, Universidade de Coimbra, Coimbra, Portugal
2
´
Received 3 May 2011, accepted 1 August 2011, DOI: 10.1111/j.1751-1097.2011.00984.x
biomolecules in their immediate vicinity leading to severe
cell damage (1).
ABSTRACT
The increasing interest in clinical bacterial photodynamic
inactivation has led to the search for photosensitizers with
higher bactericidal efficiency and less side effects on the
surrounding tissues. We present a novel nonionic porphyrin,
the 5,10,15-tris(2,6-dichlorophenyl)-20-[4-N-(6-amino-hexyl)sul-
fonamido)phenyl]-porphyrin (ACS769F4) with substantial
improvements in the efficiency of nonionic sensitizers. This
porphyrin causes eradication of both Escherichia coli and
Staphylococcus aureus by the photodynamic effect but in higher
concentrations compared with 5,10,15,20-tetrakis (4-N,N,N-
trimethylammoniumphenyl)-porphyrin p-tosylate (TTAP4+), a
known bactericidal tetracationic porphyrin. More important,
under such conditions, ACS769F4 proved to be harmless to two
mammalian cells lines (human embryonic and baby hamster
kidney), causing no reduction in their viability or negative impact
on their cytoskeleton, despite its accumulation in cellular
structures. On the contrary, TTAP4+ is shown to accumulate
in the nucleus of mammalian cells, in association to DNA,
causing chromatin condensation after exposure to light. Fur-
thermore, dark incubation with TTAP4+ was shown to have a
deleterious effect on the microtubule network. Based on its
bactericidal efficiency, also observed without exposure to light,
and on the low tendency to be harmful or genotoxic to
mammalian cells, ACS769F4 should be looked at as an
interesting photosensitizer to be evaluated for clinical purposes.
In many countries, PDT has regulatory approval for
treatment of several malignant and nonmalignant diseases.
The application of PDT in in vitro inactivation of viruses (2),
bacteria (3), fungi (4) and protozoa (5) has also been shown to
be effective. The interest in PDT bactericidal effect increased in
the last decade strongly reinforced by the worldwide rise in
antibiotic resistance (3). Data from multiple treatments suggest
that bacterial photodynamic inactivation (PDI) is unlikely to
induce bacterial resistance (3,6) and that its efficiency is
independent of the pattern of antibiotic resistance of the strain
(7). With its broad spectrum of action (8), PDI has shown
promising results in treating animal models (9) and human
infections. Indeed, PDI seems efficient in eradicating Helico-
bacter pylori from its natural niche, the human stomach, in a
small group of infected patients (10). It was also shown to be a
better approach than the standard endodontic treatment in the
elimination of bacterial biofilms in root canals (11). Even
better results were obtained by combining these two therapies
(11), a procedure that is already in clinical trials (12).
Gram-positive strains are generally sensitive to PDI (8). On
the contrary, Gram-negative bacteria, with their additional
lipid membrane, located externally to the peptidoglycan
network, strongly impairing the electrostatic attraction to
negatively and noncharged sensitizers, are not efficiently
inactivated (8). This can be overcome using permeabilization
agents or cationic porphyrins (13–17), as for example
5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)-por-
phyrin p-tosylate (TTAP4+), a frequently used and active
sensitizer established to eradicate Escherichia coli (14,18).
Recently, research has focused on the study of structural
features of sensitizers that somehow potentiate their antimi-
crobial effects. In fact, properties such as the number and type
of charge, its distribution over the molecule and the presence
of long hydrocarbon chains, have an influence on the
hydrophilicity of the sensitizer and therefore, on its cellular
distribution and its effectiveness (19). For treatment of
infections and notwithstanding the bactericidal potential of
sensitizers, their interaction with surrounding healthy tissues
should be considered. Therefore, the intracellular distribution
of sensitizers in mammalian cells should be evaluated. It is
known that accumulation of a sensitizer in the nucleus of cells
increases the risk of DNA damage, mutations and carcino-
genesis, while its accumulation in mitochondria is likely to
INTRODUCTION
The use of light as a therapeutic tool, known in ancient
Greece, Egypt and India and forgotten for centuries, was
rediscovered in the early 20th century. The clinical method
now known as photodynamic therapy (PDT), is based on
using a nontoxic photosensitive molecule that once activated
by visible light, triggers a destructive action in biological
systems due to the generation of cytotoxic oxygen species of
the radical type (Type I reactions) or singlet oxygen (1O2)
(Type II reactions) (1). Due to their short lifetimes and
diffusion paths, these highly reactive species interact with
*Corresponding author email: armenio.serra@gmail.com (Arme
mroxorosa@fe.lisboa.ucp.pt (Monica Roxo-Rosa)
´
´
nio Serra);
ꢀ 2011 The Authors
Photochemistry and Photobiology ꢀ 2011 The American Society of Photobiology 0031-8655/11
1395

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1396 Sonia Mendes et al.
Both sensitizers were stored protected from light, at room temper-
ature, as 500 lM stock solutions in 2.5% (vol ⁄ vol) dimethylformamide
(DMF) (Sigma-Aldrich) and diluted in ultrapure water for the
following experiments.
Light source and irradiation setup. The light source was a 300 W
halogen lamp (Halolineꢂ lamp; OSRAM, Germany) (emission wave-
length 350–950 nm), positioned 42 cm above samples, delivering a
fluence rate of 9.35 mW cm)2 (X97 radiometer; Gigahertz-Optik).
Room temperature was insured by using a thermostatic circulatory
system (MultiTempTM III; GE Healthcare, UK) and by installing the
irradiation setup inside a safety cabinet for bacterial and mammalian
cell experiments, respectively.
Sensitizers’ photobleaching. UV–Visible absorption spectra (350–
650 nm) of 15 lM sensitizer solutions, before and during irradiation,
were recorded and processed with a multi detection microplate reader
(SynergyTM 2; BioTek Instruments, Inc., Vermont) and the software
GEN5TM (BioTek Instruments, Inc.). Photobleaching kinetics were
evaluated by following the absorbance (A) decrease at the Soret band
wavelength (ca 420 nm) throughout irradiation. Photobleaching rate
constants (k) were obtained by a linear least squares fit of the
following semilogarithmic plot: natural logarithm of the ratio
between the optical density (O.D.) (at 422 nm for ACS769F4 and
412 nm for TTAP4+) at the beginning of the experiment (t = 0 min)
(A0) and the O.D. at a given time (A) [ln(A0 ⁄ A)] versus irradiation
time (t, min).
Cell culture conditions. E. coli (ATCC 25922) and S. aureus (ATCC
25923) bacterial cells were grown aerobically at 37ꢁC, 220 rpm, in a
complex growth medium (glucose 0.05% [wt ⁄ vol], yeast extract 0.2%
[wt ⁄ vol], bactotryptone 0.1% [wt ⁄ vol], phosphate buffer at pH 7.0, all
reagents purchased from Difco). BHK-21 (baby hamster kidney-21)
and HEK (human embryonic kidney) cell lines were grown at 37ꢁC
with 5% CO2 and 99% humidity in Dulbecco’s modified Eagle’s
medium (DMEM ⁄ F12) (Invitrogen, Life Technologies, Carlsbad, CA)
supplemented with 10% (vol ⁄ vol) fetal bovine serum (Invitrogen) and
1% (vol ⁄ vol) penicillin and streptomycin (Invitrogen).
induce apoptosis. Moreover, their accumulation in lysosomes
or endosomes leads to permeabilization of these organelles,
with the consequent release of the sensitizer to the cytosol,
where it can sensitize tubulin to photodamage (20).
The present study focuses on the evaluation of the
a
bactericidal efficiency of
novel neutral porphyrin,
5,10,15-tris(2,6-dichlorophenyl)-20-[4-N-(6-amino-hexyl)sulfo-
namido phenyl] porphyrin (ACS769F4), in inactivation of
Staphylococcus aureus and E. coli. Furthermore, the potential
impact of this molecule on human tissue was assessed by
characterizing the events that occur in mammalian cells treated
with ACS769F4 in the dark and after photosensitization.
These results were compared with those obtained using
TTAP4+
.
MATERIALS AND METHODS
Synthesis and characterization of ACS769F4 porphyrin. At room
temperature, 20 mL of chlorosulfonic acid were added to 650 mg of
a mixture of porphyrins 1 and 2, in a ratio of 13–72% prepared as
previously described (21) (Fig. 1). After stirring for 2 h, the chloro-
sulfonyl derivative 3 and the unreacted porphyrin 1 were precipitated
by pouring the solution carefully over ice. The precipitate was filtered,
dried, dissolved with dichloromethane and the solution dried with
sodium sulfate (Na2SO4). The solution was concentrated to 30 mL and
1,6-hexanediamine (4 g) and pyridine (10 mL) were added. The
mixture was stirred overnight at 30ꢁC, filtered, washed with water,
dried with Na2SO4 and chromatographed on silica gel type 60 (particle
size of 0.035–0.070 lm; Acros Organics). The porphyrin fraction was
eluted using dichlorometane with increasing amounts of ethanol up to
40%. Evaporation of the solvent gave 120 mg of pure ACS769F4
(Fig. 1), as a purple powder. 1H NMR (300 MHz, CDCl3) dH = 1.4–
1.7 (m), 3.3 (bs), 7.9 (m), 8.4 (m), 8.8 (m) (300 MHz Bruker-AMX
spectrometer, J values are given in Hz), MS (ESI): m ⁄ z = 999
Phototoxicity assays on bacteria. Fresh bacterial cell cultures were
grown up to a cell density of 108 cells mL)1 (0.6 O.D. at 600 nm for
E. coli or 0.8 for S. aureus), washed twice with cold PBS and
resuspended in PBS at the same cell density. Cells were then incubated
in the dark with different concentrations (1, 5, 10 and 15 lM) of
ACS769F4 or TTAP4+, for 30 min at 37ꢁC and immediately irradiated
for 15 min (light dose 8.4 J cm)2). Control experiments: without
irradiation in the presence of sensitizers; with and without irradiation
in the absence of sensitizers; with DMF excluding porphyrins. Cell
samples were then serially diluted 10-fold in PBS, in order to obtain
dilutions of 10)1–10)6 times of the initial concentration and 50 lL of
([M + 1]+
, 100%) (Finnigan Advantage spectrometer), C50H38
Cl6N6O6S.2H2O: Calcd. C, 58.0; H, 4.09; N, 8.11. Found: C, 58.3;
H, 3.73; N, 7.63% (EA1108-CHNS-0 elemental analyser; Fisons
Instruments). UV–Vis spectra (CH2Cl2), kma (nm): 418, 512, 587.5,
´
x
648 (U-2001 spectrophotometer; Hitachi) (see Figures S1–S4 for
characterization).
TTAP4+ was purchased from Sigma-Aldrich (Sigma-Aldrich Com-
pany, St. Louis, MO) (molecular mass: 1531.9 Da).
R1
OHC
Cl
Cl
Cl
R1=R2=
porphyrin 1:
NH
N
Cl
AcOH/PhNO2
+
R1
R2
N
H
Cl
CHO
N
HN
R2=
R1=
porphyrin 2:
Cl
R1
HClSO3
R1
R1
NH
N
N
NH
N
Cl
Cl
R1
NH2(CH2)6NH2
R2
R1
R2
R1=
R2=
HN
N
porphyrin 3:
HN
SO2Cl
R1
R1
Cl
Cl
SO2NH(CH2)6NH2
R1=
R2=
ACS 769F4:
Figure 1. Sequence of reactions for the synthesis of the porphyrin ACS769F4.

Photochemistry and Photobiology, 2011, 87 1397
each dilution were spread on agar plates (in triplicates) and incubated
for 16 h at 37ꢁC. Bacteria viability was then monitored by counting the
CFUs and determining the survival fraction compared to the untreated
control. Effective killing was taken to be >99.9% which corresponds
to a reduction of 3 log10 in the survival fraction.
Phototoxicity assays on mammalian cells. BHK-21 and HEK cells
grown in 24 multiwell plates (Nalge Nunc, Roskilde, Denmark) until
60–70% confluence and rinsed twice with cold PBS, were incubated
with different concentrations up to 20 lM of ACS769F4 or TTAP4+
for 30 min, in the dark, at 37ꢁC. Cells were then irradiated for 40 min
(light dose 22.4 J cm)2). Control experiments: without irradiation in
the presence of sensitizers; irradiation in the absence of sensitizers;
DMF excluding porphyrins. Cell viability was assessed, immediately
after light exposure, by a standard MTT assay. For normalization the
value of 100 corresponds to cell viability without treatment with
sensitizers and without light exposure.
reaction of that mixture and, therefore, the production of the
chlorosulphonyl derivative 3. Finally, the reaction of porphy-
rin 3 with 1,6-hexanediamine originated the nonsymmetric,
noncharged, hydrophobic porphyrin, termed as ACS769F4.
The absorption spectra of ACS769F4 in DMF (Fig. 2A),
showed a typical porphyrinic Soret band at 422 nm and the
additional low intensity Q bands (ca 515, 550, 590 and 650 nm)
(22). In agreement with the literature (14), similar values were
observed for TTAP4+ (Soret band at 412 nm) (Fig. 2B). The
photobleaching experiment (Fig. 2) was carried out using the
irradiation setup described above (fluence rate of 9.35
mW cm)2) and was analyzed by following the decrease of
absorbance at the Soret band. Within the analyzed spectral
region (350–650 nm), no other photosensitive molecule was
Cellular localization. Cells from fresh cultures grown up to an O.D.
(at 600 nm) of 2.0 for E. coli and of 4.0 for S. aureus were washed twice
with cold PBS and were incubated with 10 lM of ACS769F4 or 5 lM
TTAP4+ in PBS, for 1 h in the dark. Then 4 lL of each cellular
suspension were spread on a slide, dried and fixed by passing the slide
through a flame.
BHK-21 cells grown on eight well chamber slides (Nalge Nunc),
until 60–70% confluence were dark incubated with 10 lM of
ACS769F4 or TTAP4+ for 5 min, 1 h and 12 h at 37ꢁC. After two
washes with cold PBS, cells were fixed for 30 min at 4ꢁC in 4%
(vol ⁄ vol) formaldehyde (Sigma-Aldrich) and 3.7% (wt ⁄ vol) sucrose
(Merck, Darmstadt, Germany) solution, in PBS.
Both bacterial and BHK-21 cell slides were mounted in Vectashield
(Vector Laboratories, Burlingame, CA), containing DAPI (4,6-diami-
no-2-phenylindole) (Sigma-Aldrich) for nucleic acid staining whenever
necessary. Sensitizers’ fluorescence was observed and recorded on an
Axiovert 40CFL fluorescence microscope (Carl Zeiss, Jena, Germany)
equipped with the dual-band filter set-05 (excitation 395–440 nm;
detection 470–850 nm; Carl Zeiss) and with an Axiocam MRc5 (Carl
Zeiss) camera. Images were processed with the software AxioVision
Rel. 4.6.3 (Carl Zeiss).
Immunocytochemistry analysis. BHK-21 cells grown on eight well
chamber slides were dark incubated with 10 lM of ACS769F4 or 10 lM
of TTAP4+ for 1 h at 37ꢁC. Cells were rinsed and fixed as above. After
two washes with PBS, cells were permeabilized for 15 min with 0.2%
(vol ⁄ vol) TritonX-100 (Sigma-Aldrich) in PBS at RT, then washed
three more times with PBS and blocked with 1% (wt ⁄ vol) bovine
serum albumin (BSA) in PBS for 30 min prior to incubation for 90 min
at RT with the organelles’ specific monoclonal antibodies (mAb)
(1:1000 diluted in 0.5% [wt ⁄ vol] BSA in PBS). Primary mAbs:
anti-Rab7 (clone Rab7-117) mAb (Sigma-Aldrich); anti-Golgi 58K
protein ⁄ formimino-transferase cyclodeaminase (clone 58K-9) mAb
(Sigma-Aldrich); anti-a-tubulin (clone DM1A) mAb (Sigma-Aldrich);
antiactin (clone AC40) mAb (Sigma-Aldrich). Cells were then washed
three times with PBS and incubated with the fluorescein isothiocya-
nate-conjugated antimouse IgG (Sigma-Aldrich) diluted at 1:100 for
30 min at RT and washed twice again. Immunofluorescence was
observed as above, now using the filter set-10 (excitation 450–490 nm;
detection 515–565 nm; Carl Zeiss) for detection of fluorescein alone or
the filter set-05 for simultaneous detection of sensitizer and fluorescein.
Statistics. Results are expressed as means
standard deviations
(SD) of n observations. Differences were tested by Student’s t-test,
being considered as statistically significant when P < 0.05. Each
experiment was performed at least in triplicate.
RESULTS
Synthesis, properties and photobleaching of ACS769F4
A nonionic porphyrin derivative was synthesized by a three-
step method based on our chlorosulfonation methodology
(21). The one-pot mixed aldehyde–pyrrole condensation with
nitrobenzene as oxidant, originated a mixture of the symmetric
porphyrin 1 and the nonsymmetric porphyrin 2 (Fig. 1). The
phenyl ring showed a higher reactivity than the 2,6-dichlor-
ophenyl ring, allowing the controlled chlorosulphonation
Figure 2. Photobleaching assays. UV–Visible absorption spectra vari-
) of (A)
ations upon exposure to visible light (9.35 mW cm)2
ACS769F4 and (B) TTAP4+ in DMF. (C) First-order plots for the
first hour of photobleaching of ACS769F4 ( ) and TTAP4+ (d).
Symbols and error bars are means
for three experiments.
SD of the values at each point,

´
1398 Sonia Mendes et al.
produced by photodegradation of ACS769F4 or TTAP4+, as
no additional absorption peak was formed during irradiation
(Fig 2A,B). In agreement with the literature (14), photo-
bleaching reactions of both porphyrins showed first-order
kinetics in the initial hour of exposure to light (Fig. 2C).
Photo-degradation rate decay constants (k [min)1]) and half-
lifetimes (t1 ⁄ 2 [min]), for ACS769F4 and TTAP4+, were
determined from their photobleaching first-order plots
(Fig. 2C). Accordingly, a lower photodegradation rate decay
constant (kACS769F4 = 0.8 · 10)2 min)1) and, consequently, a
higher half-lifetime (t1 ⁄ 2 ACS769F4 = 86.6 min) were observed
that a minimum of 15 min of light exposure (8.4 J cm)2) is
required to cause a significant reduction in bacteria survival.
Viability of bacteria was not affected by this light dose
without prior treatment with sensitizers or by incubation
with solvent alone. A significantly (P < 0.05) higher PDI
effect was observed for TTAP4+ since dark incubation with
a 1 lM solution of this sensitizer, followed by irradiation,
caused a 4.6 log10 reduction in the viability of both E. coli
and S. aureus. In contrast, 15 lM of ACS769F4 were
required to reach 1.6 log10 and 3.0 log10 of photoinactivation
of E. coli and S. aureus, respectively (Fig. 3), indicating a
higher sensitivity of the latter strain to PDI treatment with
this novel noncharged porphyrin.
Interesting was the performance of ACS769F4 in our
experiments. They revealed that light exposure has only a
small additional effect on the toxicity, since the survival curves
of both bacterial strains incubated in the dark with this
sensitizer were similar to those obtained from PDI experi-
ments. Indeed, dark incubation with 15 lM of ACS769F4
caused a reduction in the viability of S. aureus and E. coli of
2.3 log10 and 1.4 log10, respectively. This effect was not
observed for TTAP4+, a statistically significant (P < 0.05)
difference having been registered between the survival curves
of dark incubation and PDI treatment with this sensitizer at
concentrations above 1 lM. Moreover, dark incubation with
ACS769F4 at concentrations below 15 lM was shown to have
a significantly (P < 0.05) greater bactericidal effect than the
dark incubation with TTAP4+ at the same concentrations.
Indeed, in the absence of light, TTAP4+ caused less than 0.5
log10 reductions in the number of survivors of S. aureus and
E. coli (Fig. 3).
for ACS769F4, compared with TTAP4+ (k
= 1.17 ·
TTAP4+
10)2 min)1 and t1 ⁄ 2 TTAP4+ = 59.2 min). The broader shape
of the Soret band of ACS769F4 is indicative of some self-
aggregation (14). The presence of chlorine atoms in the
structure of ACS769F4 allows the increase of the singlet
1
quantum yield of O2 by the heavy atom effect (23) and the
stability of the porphyrin. Although different environmental
conditions are expected in biological systems (24) these are
important properties for a photosensitizer.
Photoinactivation and dark-inactivation of bacteria
In order to determine and compare the antibacterial effects
of both porphyrins, suspensions of E. coli (ATCC 25922
strain) and of S. aureus (ATCC 25923 strain), in PBS, were
treated with different concentrations of ACS769F4 and
TTAP4+, from 1 to 15 lM. After dark incubation for
30 min at 37ꢁC, cells were exposed to light for 15 min
(8.4 J cm)2) and 10-fold serial dilutions were then plated.
Bacteria viability was assessed by counting the number of
CFUs formed after 16 h at 37ꢁC in the dark (Fig. 3). The
effect of the light dose on the survival of bacteria after PDI
treatment was evaluated (data not shown), becoming clear
As mentioned before, the cellular distribution of a sensitizer
and, consequently, its interaction with surrounding biomole-
cules influences its toxicity during PDI (8). Therefore, to better
understand the bactericidal effect of each sensitizer, we
assessed their distribution within the bacterial cells by fluores-
cence microscopy. This was performed taking advantage of the
red fluorescence (detected with the filter set-05) emitted by
ACS769F4 and TTAP4+, upon excitation at 412 and 422 nm,
respectively.
After 1 h incubation of E. coli and S. aureus with
ACS769F4, red fluorescence was detected only in aggregates,
apparently located externally to the cells (Fig. 4A,E, respec-
tively). Indeed, red fluorescence did not colocalize at each
bacterial cell but, instead, appeared to be concentrated over
the cells. Corroborating this observation, in such conditions,
bacterial cells stained green (more visible in the panel E of
Fig. 4), which may result from a reflection of the red
fluorescence of the externally located sensitizer, as no green
bacterial autofluorescence was detected. On the other hand,
our data suggests that TTAP4+ is internalized by both Gram-
negative and Gram-positive bacteria (Fig. 4C,G, respectively).
Control images showed no red autofluorescence in the
bacterial cells. From these studies and in addition to those
previously described (15,19,25), we believe that the strong
phototoxicity of TTAP4+ results from its intracellular local-
ization. On the other hand, the significant toxicity observed for
ACS769F4 may be justified by the presence of a long alkyl
chain with a terminal amino group which can be protonated at
cellular medium. This structure can interact with the lipophylic
cell wall and cause some disturbance that allows bacteria
Figure 3. Dose-dependent toxicity of sensitizers in prokaryotic cells,
Escherichia coli (A) and Staphylococcus aureus (B). (D) dark experiment;
(L) irradiated experiment (see experimental). Symbols and error bars are
means
SD of the values at each point for three experiments.

Photochemistry and Photobiology, 2011, 87 1399
destruction with an activity similar to that observed for
sphingosine (26).
30 min in the dark with different concentrations of both
sensitizers, in a range from 1 to 20 lM, cells were irradiated for
15 or 40 min (light dose of 8.4 and 22.4 J cm)2, respectively)
or, alternatively, kept in the dark for the same time. Cell
viability was immediately assessed by the MTT assay, in order
to understand the magnitude of toxicity of these treatments.
Dark incubation for 70 min caused no reduction in the
viability of mammalian cells, even at concentrations of
porphyrin as high as 20 lM, indicating a lower susceptibility
of either HEK (Fig. 5A) or BHK-21 (Fig. 5B) cells, to both
sensitizers. Moreover, in the subsequent 96 h after treatment,
mammalian cells continued to proliferate in a similar manner
to nontreated cells (data not shown), proving their viability.
With the same light dose used in bacteria experiments
(8.4 J cm)2) we did not observe any phototoxicity to the two
Mammalian cell sensitivity to ACS769F4 and TTAP4+
An important issue to be considered for clinical use is the
potential toxicity of an antibacterial agent against the healthy
surrounding tissues. Therefore, the phototoxicity of ACS
769F4 and TTAP4+, their uptake, their intracellular dis-
tribution and their impact on the cytoskeleton of mammalian
cells were evaluated using the HEK and BHK-21 cell lines.
These cellular models, widely used in cellular biology research,
are easily maintained in culture and give reproducible results,
being appropriate for the present study. After incubation for
lines of mammalian cells. Higher doses of light (22.4 J cm)2
)
were required to cause some phototoxicity to mammalian
cellular models. The two tested porphyrins seem to affect the
two line cells in similar ways (Fig. 5). For HEK we observed a
little more susceptibility for TTAP4+ than for ACS769F4 and
the contrary was observed in the case of BHK-21 cells. Control
experiments showed that irradiation in the absence of sensi-
tizers, and incubation with solvent, were harmless to mam-
malian cells.
Despite the relatively low toxicity observed of both sensitiz-
ers, ACS769F4 and TTAP4+, for mammalian cells, it was
important to assess where these molecules were internalized and
a)
TTAP (D)
ACS769F4 (D)
ACS 769 (L)
TTAP (L)
100
75
50
25
0
0
5
10
15
20
uM
b)
TTAP (D)
ACS 769F4 (D)
ACS 769F4 (L)
TTAP (L)
100
75
50
25
0
Figure 4. Evaluation of the external ⁄ internal accumulation of sensi-
tizers in bacterial cells. Bacterial cells collected from a fresh culture,
grown up until stationary phase, were incubated with 10 lM of
ACS769F4 (red staining) or 5 lM of TTAP4+ (red staining), for 1 h in
the dark. (A, B) Escherichia coli with ACS769F4; (C, D) E. coli with
TTAP4+; (E, F) Staphylococcus aureus with ACS769F4; (G, H)
S. aureus with TTAP4+. Panels B, D, F and H correspond to the same
image of panels A, C, E and G, respectively, observed by light
microscopy. Bar: 20 lm.
0
5
10
uM
15
20
Figure 5. Dose-dependent toxicity of sensitizers in mammalian HEK
(A) and BHK-21 (B) cell lines. (D) dark experiment; (L) irradiated
experiment (see experimental). Symbols and error bars are
means
SD of the values at each point for six experiments.

´
1400 Sonia Mendes et al.
Figure 7. Intracellular distribution of TTAP4+ in mammalian cells (A)
BHK-21 cells (control without sensitizer). (B), (C) and (D), cells
incubated with 10 lM of TTAP4+ in the dark at 37ꢁC for 5 min, 1 h
and 12 h, respectively. (E) and (F), dark incubation with the sensitizer
Figure 6. Intracellular distribution of ACF769F4 in mammalian cells.
(A) BHK-21 cells (control without sensitizer). (B), (C) and (D), cells
incubated with 10 lM of ACS769F4 in the dark at 37ꢁC for 5 min, 1 h
and 12 h, respectively. (E) and (G), dark incubation with the sensitizer
for 1 h followed by immunodetection of the rab7 protein (1:1000 anti-
Rab7 mAb) for lysosomes staining and of the 58K proteins ⁄ formimi-
no-transferase cyclodeaminase (1:1000 anti-Golgi mAb) for Golgi
staining, respectively. Images taken with the filter set-05: ACS769F4 in
red (B, C, D, E and G); DAPI in blue, for nuclei staining (A, B, C and
D); and fluorescein in white blue (E and G). White arrows show
colocalization of ACS769F4 and both rab7 (E) and Golgi proteins (G).
(F) and (H) correspond to the same image of (E) and (G), respectively,
but taken with the filter set-10 (fluorescein in green). White bar: 50 lm.
for 1 h followed by 40 min of irradiation (22.4 J cm)2
) or by
immunodetection of microtubules’ tubulin (1:1000 antitubulin mAb),
respectively. Images taken with the filter set-05: TTAP4+ in red (B, C,
D, E and F); DAPI in blue, for nuclei staining (A); and fluorescein in
white blue (F). White bar: 50 lm.
that was even more pronounced after longer periods of
incubation (Fig. 7C,D). TTAP4+ was found to be directly
associated with nuclear DNA, colocalizing with condensed
chromosomes during mitosis (Fig. 7F). Its unusual cellular
distribution pattern and its affinity for DNA raise doubts
about the genotoxicity of TTAP4+. Consistent with this, it
became clear that irradiation in the presence of this sensitizer
caused condensation of chromatin (Fig. 7E).
As the cellular integrity is dependent on the cytoskeleton
organization, the toxicity of sensitizers was further assessed
evaluating by immunodetection of tubulin and actin filaments
the fate of microtubule and microfilament networks of BHK-
21 cells. Our results indicated that incubation with 10 lM of
ACS769F4 in the dark for 1 h did not disturb the microtubule
(Fig. 8B) or the microfilament (Fig. 8H) networks, compared
to controls (Fig. 8A,G, respectively). Furthermore, no altera-
tions in the microtubule network were observed for cells
incubated with 10 lM of ACS769F4 and then irradiated for
40 min (22.4 J cm)2) (Fig. 8E in comparison to D). The
microfilament network was disturbed by light exposure alone
(Fig. 8J), becoming more concentrated around the nucleus,
but this was not enhanced by incubation with 10 lM of
ACS769F4 (Fig. 8K). We concluded that, in contrast to other
sensitizers, that once released from lysosomes or endosomes
cause cytoskeleton’s photodamage (20), ACS769F4 was harm-
less to tubulin and actin networks.
On the other hand, BHK-21 cells incubated in the dark with
10 lM of TTAP4+ (Fig. 8C in comparison to A) showed some
level of disturbance in their microtubule network, which
became less regular and less sharp, although at a level that did
not compromise the mitotic spindle formation (Fig. 7F). A
greater level of depolymerization was achieved after 40 min of
exposure to light (22.4 J cm)2) (Fig. 8F in comparison to D),
which clearly shows that TTAP4+ disturbs the integrity of the
cell. However, no significant alterations were observed in the
microfilament network after incubation with 10 lM of
TTAP4+, either in the dark (Fig. 8I) or after 40 min of
irradiation (Fig. 8L) when compared to respective controls
(Fig. 8G,J, respectively).
accumulated. Figure 6 shows BHK-21 cells, which do not
exhibit autofluorescence (Fig. 6A), incubated with ACS769F4
at different time points. After 5 min of incubation, ACS769F4
was detected as dotted red fluorescence throughout the cell
cytoplasm (Fig. 6B). Internalization of ACS769F4 should occur
quite rapidly as, at this time point, we were not able to detect
cytoplasmic membrane staining. No fluorescence was found in
the nucleus of the cells, lowering the probabilities of genotox-
icity. Longer incubation led to intracellular accumulation of
additional ACS769F4 (Fig. 6C,D). Cell membrane permeabili-
zation with TritonX-100 led to the loss of most intracellular
ACS769F4, together with the cytosolic fraction (data not
shown), suggesting that this sensitizer was mainly distributed
through the cytosol. Additionally, ACS769F4 was found to be
accumulated in vesicle-like structures, some of which colocal-
ized with protein Rab-7, a lysosomal marker (white arrows in
Fig. 6E) and with proteins from the Golgi complex (white
arrows in Fig. 6G). These observations lead us to think that
ACS769F4 may follow the pathway described for other sensi-
tizers (20), which are internalized by endocytosis and accumu-
late in early and late endosomes and finally in lysosomes for
degradation. Alternatively, once in the early endosomes,
ACS769F4 may follow the classical secretion pathway through
the Golgi apparatus. Further work should be done in order to
clarify whether those spots of ACS769F4 that remained
intracellularly after treatment with TritonX-100, and that did
not colocalize with lysosomes or Golgi, were accumulated
within endosomes. As demonstrated before (Fig. 4), due to its
hydrophobicity, ACS769F4 form aggregates that may interfere
with the ability to cross membranes. Therefore, it is expected
that ACS769F4 should be internalized by pinocytosis and ⁄ or
endocytosis, a feature that is not shared by bacteria.
In contrast, 5 min of incubation with TTAP4+ revealed a
faint fluorescence in the cytoplasm of the cell and its
preferential accumulation in the nucleus (Fig. 7B), an effect

Photochemistry and Photobiology, 2011, 87 1401
and the more hydrophilic ones (tri and tetracationic), are
internalized by endocytosis, being accumulated afterward in
lysosomes (29). The charged porphyrins acting as lipophylic
cations, are targeted to the mitochondria, encouraged by the
transmembrane potential of this organelle (29–31). In both
cases, apoptotic cell death is induced upon exposure to light.
Sensitizers in general do not accumulate in the nucleus of the
mammalian cells and, when that happens, the risk of DNA
damage, mutations and carcinogenesis increases (20,27).
However, cationic sensitizers are known to have a high affinity
for nucleic acids, showing an enhanced ability to induce DNA
photodamage (32), inhibition of telomerase (33) and impair-
ment of the topoisomerases activities (32–34). Therefore, the
use of cationic porphyrins, which accumulate in the nucleus of
mammalian cells, has as the biggest problem for the surround-
ing tissues the possibility of developing genotoxic effects.
In the search for an efficient bactericidal sensitizer, harmless to
mammalian cells, a novel nonionic porphyrin, ACS769F4 was
synthesized. Its structure was modeled to have chlorine atoms
in an appropriate place in order to improve the quantum yield
of 1O2 (21), and a long alkyl chain to favor the penetration into
biological membranes as shown by others (8,17). All studies
were carried out in comparison with TTAP4+, a cationic
porphyrin known for its efficiency in eradicating E. coli cellular
suspensions (14,15). Both ACS769F4 and TTAP4+ showed the
typical absorption spectrum, with a Soret band at 422 and
412 nm, respectively. The broader shape of the Soret band of
ACS769F4 is indicative of self aggregation (14), an undesirable
property that may lead to a lower photosensitizing efficiency,
since only monomeric species are appreciably photoactive (35).
The noncationic porphyrin ACS769F4 showed a lower decay
Figure 8. Impact of sensitizers in the cell cytoskeleton. BHK-21 cells
incubated with 10 lM of ACS769F4 or of TTAP4+ for 1 h in the dark
at 37ꢁC were irradiated for 40 min (22.4 J cm)2) or were kept in the
dark for control. Microtubules (panels A, B, C, D, E and F) and
microfilaments (panels G, H, I, J, K and L) were immunostained with
antitubulin mAb (1:1000) and with antiactin mAb (1:1000), respec-
tively. (A, G) control without sensitizers, in the dark; (B, H)
ACS769F4, in the dark; (C, I) TTAP4+, in the dark; (D, J) control
without sensitizers, with light exposure; (E, K) ACS769F4, with light
exposure; (F, L) TTAP4+, with light exposure. Images taken with the
filter set-10 (fluorescein in green). White bar: 50 lm.
rate constant and, therefore, a longer half-life (kACS769F4
0.8 · 10)2 min)1 and t1 ⁄ 2
=
= 86.6 min) during the
ACS769F4
first hour of the photobleaching reaction compared with those of
TTAP4+ (k = 1.17 · 10)2 min)1 and t1 ⁄ 2 TTAP4+
=
TTAP4+
59.2 min). As is fairly well known, the photophysical proper-
ties measured in homogenous solution may differ from those in
a biological environment (15,24). Indeed, porphyrins with
similar photophysical properties, presented different antimi-
crobial phototoxicity based on differences in their cellular
distribution and intrinsic photoactivity (13). In our case,
TTAP4+ was found to be more photoactive, since the
treatment with just 1 lM of this sensitizer, followed by light
exposure (8.4 J cm)2), was sufficient for 99.999% bacterial
killing (reduction of 4.6 log10) of both E. coli and S. aureus. By
using higher doses of ACS769F4 (15 lM for S. aureus and
25 lM for E. coli), it was also possible to cause a 3 log10
reduction of bacteria after PDI treatment, achieving the
recommended killing efficiency of 99.9% (36). In agreement
with published data (13,16,28), S. aureus presented a higher
photosusceptibility to both porphyrins than E. coli. However,
significant improvements were achieved with ACS769F4 com-
pared to other nonionic porphyrins described in the literature,
since it presented an efficient PDI effect against Gram-negative
bacteria, even without changing the permeability of the outer
membrane of these (13,17,25). More studies are needed to
assess whether administration of ACS769F4 with polycationic
liposomes (37) or with bacterial outer membrane disrupting
agents (i.e. CaCl2, EDTA or polymixin B) (8) potentiates the
PDI effect of this sensitizer. Another important consideration
is that, while we were working with high cell density cultures
DISCUSSION
Antimicrobial PDT is a very promising alternative to anti-
biotics to eradicate bacteria in local infections (8). In the
absence of structural features that turn sensitizers specific to
bacteria, the challenge of antimicrobial PDT is to find
molecules that efficiently eradicate bacteria without damage
the host cells. The photophysical properties of sensitizers such
1
as photostability and O2 yields are crucial for the PDT event
but intracellular distribution of molecules is also very impor-
tant (13) due to the restricted radius of action and the
extremely short lifetimes of the high-energy species produced
(27). The cellular distribution of a sensitizer is determined by
its structure and charge which influence the sensitizer hydro-
philicity and aggregation state (19). Cationic porphyrins are
among the most efficient sensitizers for antimicrobial PDT.
The electrostatic interaction between these sensitizers and the
negative charges on the outer surface of bacterial cells, allows
their penetration to the inner plasma membrane of bacteria,
most likely through the self-promoted uptake pathway,
thereby enhancing their PDI effect (8,13–17,19,28). Differences
in the mechanism of interaction of porphyrins with bacteria
and mammalian cells are not difficult to accept given the
structural differences between the cell walls of the former and
the cytoplasmic membranes of the latter. It is thought that
both amphiphilic cationic porphyrins (mono and dicationic)

´
1402 Sonia Mendes et al.
(108 cells mL)1), some authors claim that PDI becomes more
effective as the cell density decreases (38), suggesting that
(photo)toxicity of both ACS769F4 and TTAP4+ could be
enhanced in bacterial cultures of lower density.
The accumulation of porphyrins was analyzed by fluores-
cence microscopy and showed that ACS769F4 and TTAP4+
accumulate in different compartments of the cell in
a
time-dependent manner. In both cell lines ACS769F4 showed
spotty fluorescence signals throughout the cytoplasm, part
colocalizing with lysosomes and Golgi apparatus and most
with the cytosolic fraction. The hydrophobicity properties of
monomeric ACS769F4 enhanced by its long alkyl chain, favor
the affinity toward lipid membranes. Therefore, after diffusion
through the cytoplasmic membrane, ACS769F4 may bind to
cytosolic proteins, insuring its solubilization. On the other
hand, aggregated forms of ACS769F4 are expected to be
internalized by endocytosis (20), justifying their accumulation
in lysosomes (41). Sensitizers with this pattern of intracellular
distribution have been described as being unable to photoin-
duce nuclear DNA damage (42). Moreover, we found that
despite this intracellular distribution and in contrast to other
cytosolic dyes (20), ACS769F4 caused no visible deleterious
effect on the cytoskeleton of mammalian cells, even upon
irradiation.
A different situation was found for TTAP4+ which rapidly
accumulates in the nucleus of mammalian cells and associates
with DNA, probably by electrostatic attraction. This finding
supports the suggested TTAP4+ uptake by bacterial cells.
A similar situation was described for meso-tetrakis(N-methyl-
4-pyridiniumyl)-porphyrin (TMPyP) (43), known intercalating
agent that present a nuclease-like activity, even in the absence
of light and oxygen (44). The structural features that allow
TMPyP and TTAP4+ to concentrate in the nucleus are not
fully known but the molecule charge must be determinant. It
has been suggested that after endocytosis, TMPyP localizes
temporarily in lysosomes, causing a faint staining of the
cytoplasm, as observed for TTAP4+, being then rapidly
relocalized to the nucleus. There is evidence that its translo-
cation to the nucleus is mediated by nuclear proteins, following
a nuclear transport pathway (45). It is still unknown whether
the TTAP4+ ⁄ DNA association happens by intercalation,
outside groove binding, or outside binding with porphyrin
self-stacking, mechanisms described for other cationic por-
phyrins (19). Given the similarities between the TTAP4+ and
TMPyP in terms of their structure and cellular distribution, it
is expected that the mechanisms used by one or the other are
also similar. Per se this is a huge drawback for the use of
TTAP4+ in antimicrobial PDT, as even working in conditions
that apparently do not undermine the viability of mammalian
cells, i.e. 1 lM, followed for 15 min of light exposure, there is
no guarantee on safety in terms of genotoxic effects. In fact, we
found that dark incubation with 10 lM of TTAP4+ causes a
deleterious effect on the microtubule network. Moreover, after
irradiation, TTAP4+ remained in the nucleus leading to
chromatin condensation and changes in the cytoskeleton of
the cell. More plausible is the possibility of induction of
apoptosis in response to DNA damage caused by TTAP4+, an
event that will necessarily influence the organization of the
cytoskeleton (46). For this porphyrin the higher reduction of
viability observed in the case of BHK cells may be due to this
effect. Highlighting the similarities between TTAP4+ and
TMPyP, it was shown that after irradiation, TMPyP-contain-
ing cells lose their mitochondrial transmembrane potential,
inducing apoptosis (43).
Interestingly, we found that with our experimental condi-
tions, ACS769F4 also behaves as an effective bactericidal
agent in the absence of light. Indeed, the PDI-toxicity dose-
dependent curve of ACS769F4, in either E. coli or S. aureus,
was slightly sharper than its dark-toxicity dose-dependent
curve. Therefore, dark incubation of S. aureus and E. coli with
15 lM of ACS769F4 lead to a reduction of 2.3 log10 (99.5%)
and of 1.4 log10 (93%) in the number of survivors, respectively.
This fact is more relevant if we consider that the tested
mammalian cell lines (HEK and BHK-21 cells) showed a very
low dark-sensitivity for ACS769F4, even at concentrations as
high as 20 lM. Also this is suggestive that the killing efficiency
of this bactericidal agent can be improved by increasing the
amount of light delivered. Dark toxicity was already described
for some cationic porphyrin derivatives (39), however, to the
best of our knowledge, side effects resulting for their use in
mammalian cells are not known. More studies should be
conducted to determine the dark-susceptibility to ACS769F4
in methicillin resistant S. aureus strains as, being the major
cause of healthcare-associated infections, new methods to
control and prevent their spread are urgently required (40).
Our data from fluorescence microscopy analyses suggested
an influx of TTAP4+ into bacterial cells. Further supporting
this observation, Caminos and coworkers reported the ability
of TTAP4+ to tightly bind to E. coli cells (15). This was
determined by measuring the fluorescence of the sensitizer in
cell lysates, obtained from bacteria previously incubated with
TTAP4+ in the dark and washed immediately before lyses (15).
In addition, Salmon-Divon et al. reported that the bactericidal
effect of tetracationic porphyrins on E. coli is primarily
dependent on genomic DNA photodamage (25). The suggested
uptake of TTAP4+ by bacterial cells may be justified by the
electrostatic attraction to DNA (19). On the other hand, our
data suggests that ACS769F4 forms aggregates on the external
surface of the bacteria. As described for other sensitizers (19),
this aggregation is probably due to its hydrophobicity and may
potentiate the affinity of ACS769F4 for lipid membranes,
while not allowing it to cross them. In fact, we are convinced
that the long alkyl chain of the ACS769F4 acts as a
hydrophobic arm, which penetrates deeply into the bacterial
wall potentiating the PDI effect of the monomeric forms of
this sensitizer.
Both prokaryotic and mammalian cells are known to be
susceptible to photodynamic treatment and this is an impor-
tant subject if a clinical application is expected. The HEK and
BHK-21 mammalian cell lines showed a very low dark-
sensitivity for either ACS769F4 or TTAP4+. Indeed, we found
that cells treated in the dark for 1 h with 20 lM of any of those
sensitizers, continued to proliferate in a similar manner to
nontreated cells, during the subsequent 96 h (data not shown).
At this concentration, exposure to light pledged the mamma-
lian cell viability. Nonetheless, it took a higher dose of light
(i.e. 22.4 J cm)2), than that used in bactericidal conditions
(8.4 J cm)2), to cause a significant reduction in their viability.
In this case TTAP4+ proved to be more damaging than
ACS769F4 for HEK cells.

Photochemistry and Photobiology, 2011, 87 1403
7. Tang, H. M., M. R. Hamblin and C. M. Yow (2007) A compar-
ative in vitro photoinactivation study of clinical isolates of mul-
tidrug-resistant pathogens. J. Infect. Chemother. 13, 87–91.
8. Jori, G., C. Fabris, M. Soncin, S. Ferro, O. Coppellotti, D. Dei,
L. Fantetti, G. Chiti and G. Roncucci (2006) Photody-
namic therapy in the treatment of microbial infections: Basic
principles and perspective applications. Lasers Surg. Med. 38,
468–481.
9. Hamblin, M. R., D. A. O’Donnell, N. Murthy, C. H. Contag and
T. Hasan (2002) Rapid control of wound infections by targeted
photodynamic therapy monitored by in vivo bioluminescence
imaging. Photochem. Photobiol. 75, 51–57.
10. Lembo, A. J., R. A. Ganz, S. Sheth, D. Cave, C. Kelly, P. Levin,
P. T. Kazlas, P. C. Baldwin III, W. R. Lindmark, J. R. McGrath
and M. R. Hamblin (2009) Treatment of Helicobacter pylori
infection with intra-gastric violet light phototherapy: A pilot
clinical trial. Lasers Surg. Med. 41, 337–344.
In conclusion, ACS769F4 is a novel nonionic porphyrin
sensitizer which showed evident in vitro ability to inactivate
E. coli and S. aureus. The results showed clear differences in
sensitivity of prokaryotic and mammalian cells, in terms of
concentration of the sensitizer, and in terms of the light dose
required to cause cell death. These results justify their
disclosure and make ACS769F4 a very promising sensitizer
to be considered for clinical use as an antimicrobial agent. This
is even more relevant taking into account the comparison with
TTAP4+ which was shown to localize in the nucleus of the
tested mammalian cells with interactions with DNA, a
situation with awful perspectives for a molecule intended to
be applied in humans.
11. Garcez, A. S., M. S. Ribeiro, G. P. Tegos, S. C. Nunez, A. O.
Jorge and M. R. Hamblin (2007) Antimicrobial photodynamic
therapy combined with conventional endodontic treatment to
eliminate root canal biofilm infection. Lasers Surg. Med. 39, 59–
66.
12. Garcez, A. S., S. C. Nunez, M. R. Hamblin and M. S. Ribeiro
(2008) Antimicrobial effects of photodynamic therapy on
patients with necrotic pulps and periapical lesion. J. Endod. 34,
138–142.
13. Merchat, M., G. Bertolini, P. Giacomini, A. Villanueva and G.
Jori (1996) Meso-substituted cationic porphyrins as efficient
photosensitizers of gram-positive and gram-negative bacteria.
J. Photochem. Photobiol. B 32, 153–157.
Acknowledgements—The authors wish to thank Professor F. Mendes
(Nuclear and Technological Institute, Portugal) for the valuable
advices for the immunocytochemistry assays, Professor M. D. Amaral
(Centre of Human Genetics, National Institute of Health, Portugal)
for the BHK-21 and HEK cell lines, Mr. Benjamin for the photometer
(Konica Minolta AutoMeter IV F) used to measure the light dose of
our irradiation setup at the beginning of the experiments, and finally
Professor L. Clarke (Faculty of Sciences, University of Lisbon,
Portugal) for revising the manuscript. This work was supported by
‘‘Cancer Light Assisted Receeding Oncologic’’ (CLARO) project grant
˜
(Ageˆ ncia de Inovac¸ ao, Portugal). Both SM and FC were recipients of
research fellowships from CLARO’s grant (Portugal).
14. Caminos, D. A. and E. N. Durantini (2006) Photodynamic inac-
tivation of Escherichia coli immobilized on agar surfaces by a
tricationic porphyrin. Bioorg. Med. Chem. 14, 4253–4259.
15. Caminos, D. A., M. B. Spesia, P. Pons and E. N. Durantini (2008)
Mechanisms of Escherichia coli photodynamic inactivation by an
amphiphilic tricationic porphyrin and 5,10,15,20-tetra(4-N,N,N-
trimethylammoniumphenyl) porphyrin. Photochem. Photobiol.
Sci. 7, 1071–1078.
16. Hamblin, M. R., D. A. O’Donnell, N. Murthy, K. Rajagopalan,
N. Michaud, M. E. Sherwood and T. Hasan (2002) Polycationic
photosensitizer conjugates: Effects of chain length and Gram
classification on the photodynamic inactivation of bacteria.
J. Antimicrob. Chemother. 49, 941–951.
17. Alves, E., L. Costa, C. M. Carvalho, J. P. Tome, M. A. Faustino,
M. G. Neves, A. C. Tome, J. A. Cavaleiro, A. Cunha and A.
Almeida (2009) Charge effect on the photoinactivation of Gram-
negative and Gram-positive bacteria by cationic meso-substituted
porphyrins. BMC Microbiol. 9, 70.
18. Caminos, D. A., M. B. Spesia and E. N. Durantini (2006)
Photodynamic inactivation of Escherichia coli by novel
meso-substituted porphyrins by 4-(3-N,N,N-trimethylammonium-
propoxy)phenyl and 4-(trifluoromethyl)phenyl groups. Photo-
chem. Photobiol. Sci. 5, 56–65.
SUPPORTING INFORMATION
Additional Supporting Information may be found in the online
version of this article:
Figure S1. NMR spectrum of ACS 769F4 (*TFA sinal).
Figure S2. IR spectrum of ACS 769F4: NH band at
3429 cm)1
; ; sulfonamide at
CH saturated at 2929 cm)1
1159 cm)1; porphyrin macocycle at 1557, 1428 and 803 cm)1
Figure S3. ESI spectrum of ACS 769F4.
.
Figure S4. Visible spectrum of ACS 769F4 (CH2Cl2 ⁄
MeOH).
Please note: Wiley-Blackwell is not responsible for the
content or functionality of any supporting information
supplied by the authors. Any queries (other than missing
material) should be directed to the corresponding author for
the article.
19. Lang, K., J. Mosinger and D. M. Wagnerova (2004) Photophys-
´
REFERENCES
ical properties of porphyrinoid sensitizers non-covalently bound
to host molecules; models for photodynamic therapy. Coord.
Chem. Rev. 248, 321–350.
1. Plaetzer, K., B. Krammer, J. Berlanda, F. Berr and T. Kiesslich
(2009) Photophysics and photochemistry of photodynamic ther-
apy: Fundamental aspects. Lasers Med. Sci. 24, 259–268.
2. Tao, J. N., S. M. Duan and J. Li (2007) Experimental studies on
treatment of HSV infections with photodynamic therapy using
5-aminolevulinic acid. Zhonghua Shi Yan He Lin Chuang Bing Du
Xue Za Zhi 21, 79–82.
3. Hamblin, M. R. and T. Hasan (2004) Photodynamic therapy: A
new antimicrobial approach to infectious disease? Photochem.
Photobiol. Sci. 3, 436–450.
4. Fuchs, B. B., G. P. Tegos, M. R. Hamblin and E. Mylonakis
(2007) Susceptibility of Cryptococcus neoformans to photodynamic
inactivation is associated with cell wall integrity. Antimicrob.
Agents Chemother. 51, 2929–2936.
5. Wagner, S. J., A. Skripchenko, J. Salata and L. J. Cardo (2007)
Photoinactivation of Trypanosoma cruzi in red cell suspensions
with thiopyrylium. Transfus. Apher. Sci. 37, 23–25.
20. Dougherty, T. J., C. J. Gomer, B. W. Henderson, G. Jori, D.
Kessel, M. Korbelik, J. Moan and Q. Peng (1998) Photodynamic
therapy. J. Natl Cancer Inst. 90, 889–905.
21. Ribeiro, S. M., A. C. Serra and A. M. d. Rocha-Golsaves (2007)
Covalently immobilized porphyrins as photooxidation catalysts.
Tetrahedron 63, 7885–7891.
22. Barnett, G. H., M. F. Hudson and K. M. Smith (1975) Con-
cerning meso-tetraphenylporphyrin purification. J. Chem. Soc. 14,
1401–1403.
23. Azenha, E. G., A. C. Serra, M. Pineiro, M. M. Pereira, J. Seixas de
Melo, L. G. Arnaut, S. J. Formosinho and A. M. d’ A. Gonsalves
(2002) Heavy-atom effects on metalloporphyrins and polyhalo-
genated porphyrins. Chem. Phys. 280, 177–190.
24. Bezdetnaya, L., N. Zeghari, I. Belitchenko, M. Barberi-Heyob,
J. L. Merlin, A. Potapenko and F. Guillemin (1996) Spectroscopic
and biological testing of photobleaching of porphyrins in solu-
tions. Photochem. Photobiol. 64, 382–386.
6. Maisch, T. (2007) Anti-microbial photodynamic therapy: Useful
in the future? Lasers Med. Sci. 22, 83–91.

´
1404 Sonia Mendes et al.
25. Salmon-Divon, M., Y. Nitzan and Z. Malik (2004) Mechanistic
aspects of Escherichia coli photodynamic inactivation by cationic
tetra-meso(N-methylpyridyl)porphine. Photochem. Photobiol. Sci.
3, 423–429.
26. Possemiers, S., J. Van Camp, S. Bolca and W. Verstraete (2005)
Characterization of the bactericidal effect of dietary sphingosine
and its activity under intestinal conditions. Int. J. Food Microbiol.
105, 59–70.
and photosensitising ability in relation to mTHPC and poly-
haematoporphyrin. J. Photochem. Photobiol. B 45, 28–35.
36. Boyce, J. M. and D. Pittet (2002) Guideline for hand hygiene in
health-care settings. Recommendations of the Healthcare Infec-
tion Control Practices Advisory Committee and the HI-
PAC ⁄ SHEA ⁄ APIC ⁄ IDSA Hand Hygiene Task Force. Am.
J. Infect. Control 30, S1–S46.
37. Ferro, S., F. Ricchelli, D. Monti, G. Mancini and G. Jori (2007)
Efficient photoinactivation of methicillin-resistant Staphylococcus
aureus by a novel porphyrin incorporated into a poly-cationic
liposome. Int. J. Biochem. Cell Biol. 39, 1026–1034.
27. Sobolev, A. S., D. A. Jans and A. A. Rosenkranz (2000) Targeted
intracellular delivery of photosensitizers. Prog. Biophys. Mol. Biol.
73, 51–90.
28. Maisch, T., C. Bosl, R. M. Szeimies, N. Lehn and C. Abels (2005)
Photodynamic effects of novel XF porphyrin derivatives on pro-
karyotic and eukaryotic cells. Antimicrob. Agents Chemother. 49,
1542–1552.
29. Mroz, P., J. Bhaumik, D. K. Dogutan, Z. Aly, Z. Kamal, L.
Khalid, H. L. Kee, D. F. Bocian, D. Holten, J. S. Lindsey and M.
R. Hamblin (2009) Imidazole metalloporphyrins as photosensi-
tizers for photodynamic therapy: Role of molecular charge,
central metal and hydroxyl radical production. Cancer Lett. 282,
63–76.
30. Lei, W., J. Xie, Y. Hou, G. Jiang, H. Zhang, P. Wang, X. Wang
and B. Zhang (2010) Mitochondria-targeting properties and
photodynamic activities of porphyrin derivatives bearing cationic
pendant. J. Photochem. Photobiol. B 98, 167–171.
31. Ngen, E. J., P. Rajaputra and Y. You (2009) Evaluation of
delocalized lipophilic cationic dyes as delivery vehicles for
photosensitizers to mitochondria. Bioorg. Med. Chem. 17, 6631–
6640.
38. Demidova, T. N. and M. R. Hamblin (2005) Effect of cell-pho-
tosensitizer binding and cell density on microbial photoinactiva-
tion. Antimicrob. Agents Chemother. 49, 2329–2335.
39. Ooi, N., K. Miller, C. Randall, W. Rhys-Williams, W. Love and I.
Chopra (2010) XF-70 and XF-73, novel antibacterial agents active
against slow-growing and non-dividing cultures of Staphylococcus
aureus including biofilms. J. Antimicrob. Chemother. 65, 72–78.
40. Kurlenda, J., M. Grinholc, K. Jasek and G. Wegrzyn (2007)
RAPD typing of methicillin-resistant Staphylococcus aureus: A
7-year experience in a Polish hospital. Med. Sci. Monit. 13, MT13–
MT18.
41. Bonneau, S., P. Morliere and D. Brault (2004) Dynamics of
interactions of photosensitizers with lipoproteins and membrane-
models: Correlation with cellular incorporation and subcellular
distribution. Biochem. Pharmacol. 68, 1443–1452.
42. Egyeki, M., K. Toth, W. Waldeck, P. Schmezer, J. Langowski and
G. Csik (2006) DNA damaging capability of hematoporphyrin
towards DNAs of various accessibilities. J. Photochem. Photobiol.
B 84, 119–127.
43. Hatz, S., J. D. Lambert and P. R. Ogilby (2007) Measuring the
lifetime of singlet oxygen in a single cell: Addressing the issue of
cell viability. Photochem. Photobiol. Sci. 6, 1106–1116.
44. Meunier, B. (1992) Metalloporphyrins as versatile catalysts for
oxidation reactions and oxidative DNA cleavage. Chem. Rev. 92,
1411–1456.
32. Munson, B. R. and R. J. Fiel (1992) DNA intercalation and
photosensitization by cationic meso substituted porphyrins.
Nucleic Acids Res. 20, 1315–1319.
33. Dixon, I. M., F. Lopez, J. P. Esteve, A. M. Tejera, M. A. Blasco,
G. Pratviel and B. Meunier (2005) Porphyrin derivatives for
telomere binding and telomerase inhibition. Chembiochem. 6, 123–
132.
34. Shuai, L., S. Wang, L. Zhang, B. Fu and X. Zhou (2009) Cationic
porphyrins and analogues as new DNA topoisomerase I and II
inhibitors. Chem. Biodivers. 6, 827–837.
45. Patito, I. A., C. Rothmann and Z. Malik (2001) Nuclear transport
of photosensitizers during photosensitization and oxidative stress.
Biol. Cell 93, 285–291.
35. Ball, D. J., S. R. Wood, D. I. Vernon, J. Griffiths, T. M. Dubb-
elman and S. B. Brown (1998) The characterisation of three
substituted zinc phthalocyanines of differing charge for use in
photodynamic therapy. A comparative study of their aggregation
46. Bursch, W., A. Ellinger, C. Gerner, U. Frohwein and R. Schulte-
Hermann (2000) Programmed cell death (PCD). Apoptosis,
autophagic PCD, or others? Ann. NY Acad. Sci. 926, 1–12.