Antimicrobial photodynamic efficiency of novel cationic porphyrins towards periodontal gram-positive and gram-negative pathogenic bacteria

Article abstract of DOI:10.1111/php.12198

The Gram-negative Aggregatibacter actinomycetemcomitans and Fusobacterium nucleatum are major causative agents of aggressive periodontal disease. Due to increase in the number of antibiotic-resistant bacteria, antimicrobial Photodynamic therapy (aPDT) see

Full text of DOI:10.1111/php.12198

Photochemistry and Photobiology, 2014, 90: 628–640
Antimicrobial Photodynamic Efficiency of Novel Cationic Porphyrins
towards Periodontal Gram-positive and Gram-negative Pathogenic
Bacteria
1
2
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3
Chandra Sekhar Prasanth , Suneesh C. Karunakaran , Albish K. Paul , Vesselin Kussovski* ,
4
2
5
4
6
Vanya Mantareva , Danaboyina Ramaiah* , Leslie Selvaraj , Ivan Angelov , Latchezar Avramov ,
Krishnankutty Nandakumar and Narayanan Subhash*
Biophotonics Laboratory, Centre for Earth Science Studies, Trivandrum, India
Photosciences and Photonics, Chemical Sciences and Technology Division, CSIR- National Institute for Interdisciplinary
Science and Technology, Trivandrum, India
7
§1
1
2
3
4
5
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7
The Stephan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria
Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia, Bulgaria
Microbiology Division, Doctors Diagnostic Research Centre, Trivandrum, India
Institute of Electronics, Bulgarian Academy of Sciences, Sofia, Bulgaria
Department of Periodontics, Azeezia Dental College, Kollam, India
Received 8 July 2013, accepted 9 October 2013, DOI: 10.1111/php.12198
ABSTRACT
INTRODUCTION
The Gram-negative Aggregatibacter actinomycetemcomitans
and Fusobacterium nucleatum are major causative agents of
aggressive periodontal disease. Due to increase in the number
of antibiotic-resistant bacteria, antimicrobial Photodynamic
therapy (aPDT) seems to be a plausible alternative. In this
work, photosensitization was performed on Gram-positive
and Gram-negative bacteria in pure culture using new-age
cationic porphyrins, namely mesoimidazolium-substituted
porphyrin derivative (ImP) and pyridinium-substituted por-
phyrin derivative (PyP). The photophysical properties of both
the sensitizers including absorption, fluorescence emission,
quantum yields of the triplet excited states and singlet oxygen
generation efficiencies were evaluated in the context of aPDT
application. The studied porphyrins exhibited high ability to
accumulate into bacterial cells with complete penetration into
early stage biofilms. As compared with ImP, PyP was found
to be more effective for photoinactivation of bacterial strains
associated with periodontitis, without any signs of dark toxic-
ity, owing to its high photocytotoxicity.
The photodestruction study of Oscar Raab (1) on Paramecia
predates the first anticancer reports. Tappeiner and Jessionek (2)
performed the first photodynamic therapy (PDT) on skin cancer
using eosin as photosensitizer. Photodynamic antimicrobial
agents assumed significance in the beginning of twenty-first cen-
tury due to the growing resistance of pathogenic microorganisms
against the conventional therapy with antibiotics (3). Although a
broad spectrum of antibiotics are available, various pathogenic
microorganisms are gaining resistance as a result of chromo-
somal mutation, inductive expression of latent chromosomal
genes, exchange of genetic material via transformation, bacterio-
phage transduction and plasmid conjugation (4). Thus, there is
an emergent need to develop and evolve alternate methodologies
against the pathogenic microbes.
Photodynamic therapy is a promising and effective alternative
to conventional therapeutic methods (5–10). In PDT, the photosen-
sitizer (PS) gets activated by light of suitable wavelength and this
in turn reacts with molecular oxygen in ground state to produce
excited singlet state oxygen (11,12), which is very reactive and
has the ability to oxidize bio-organic molecules (13). Thus, antimi-
crobial PDT (aPDT) has significant advantages over other thera-
peutic modalities of treatment owing to its ability to get attached
directly to the membranes of pathogenic cells and the possibility
for accurate delivery of light to the affected tissue (14).
Abbreviations: ImP, 5,10,15,20-Tetrakis[4-(1-methyl-1H-imi-
dazol-3-ium)phenyl]porphyrin tetrabromide; PyP, 5,10,15,20-
Tetrakis[4-(8-pyridiniooctyloxy)phenyl]porphyrin tetrabromide;
aPDT, antimicrobial photodynamic therapy; PDT, photodynamic
therapy; PS, photosensitizer; ROS, reactive oxygen species; DPBF,
The cell wall of Gram-negative bacteria is more extensive and
complex than that of Gram-positive species (3). Anionic and neu-
tral photosensitizers bind efficiently on Gram-positive bacteria to
induce photoinactivation by irradiation in the visible light region
with a suitable wavelength, whereas Gram-negative bacteria
appear resistant to the same treatment. Growth inhibition of certain
Gram-negatives by porphyrin photosensitization occurs only in the
presence of cell membrane disorganizing substances, such as
EDTA, nitrilotriacetic acid and sodium hexametaphosphate (15).
Recent studies (16–20) reveal that different chemical classes of
1
,3-diphenylisobenzofuran; m-THPP, meta-tetrahydroxyporphy-
rin; CSLM, confocal scanning fluorescence microscopy; HpD,
hematoporphyrin derivative ; EDTA, Ethylenediaminetetraa-
cetic acid; TLC, Thin layer chromatography.
§
Current address: Vinvish Technologies Ltd., Thejaswini Bldg., Technopark,
Trivandrum 695581, India
*
Corresponding authors email: subhashnarayanan@gmail.com (Narayanan Sub-
hash); veselinkusovski@yahoo.com (Vesselin Kussovski) and rama@niist.res.in
Danaboyina Ramaiah)
2013 The American Society of Photobiology
(
©
6
28

Photochemistry and Photobiology, 2014, 90 629
positively charged PSs, including porphyrins and phthalocyanines,
are effective as photodynamic sensitizers against Gram-positive
and Gram-negative bacteria. Photosensitizers with an overall cat-
ionic charge like meso-substituted cationic porphyrins can effi-
ciently kill Gram-negative bacteria in the presence of visible light
by binding via ionic attraction to carboxylated or sulfonated resi-
dues in the various strata of the bacterial cell wall.
The new-age tetrapyrrolic macrocyclic compounds like por-
phyrins (15) have been studied as useful sensitizers in aPDT due
to their favorable spectral properties and negligible dark toxici-
ties. Among the various porphyrins, cationic porphyrins are more
promising for the use as sensitizers in aPDT (20) as, positively
charged photosensitizers seem to move across to the outer mem-
brane via a self-promoted uptake pathway, in a mechanism
involving interaction between divalent cations of the compound
with adjacent bacterial lipopolysaccharide (21). The photochemi-
cal property of the favorable triplet quantum yields and singlet
oxygen generation efficiency of the new generation porphyrins,
which are chemically pure compounds as compared with hemato-
porphyrin derivatives, also make them potential sensitizers in
aPDT.
MATERIALS AND METHODS
Materials. The chemicals and reagents used in the study were purchased
from S. D. Fine Chemicals, India; Sigma-Aldrich; Merck Chemicals,
Germany and were used as such without further purification. 1,3-Diph-
enylisobenzofuran (DPBF) was recrystallized using methanol and acetone
mixture (1:3) and b-carotene from a mixture (1:1) of ethanol and chloro-
form. The solvents were dried and purified before use. The aldehyde used
for the synthesis of porphyrins was synthesized by following the previ-
ously reported procedure (27).
General experimental techniques. The equipment and procedures for
melting point determination and spectral recordings are described else-
where (28–32). A 500 MHz Bruker advanced spectrometer (Model:
1
13
DPX) was used to record both H and C NMR spectra. A Shimadzu
Biotech Axima CFR plus instrument equipped with a nitrogen laser in
the linear mode was used to perform the MALDI-TOF MS analysis using
2
,5-dihydroxybenzoic acid (DHB) as the matrix. The electronic absorp-
tion spectra were recorded on a Shimadzu UV–VIS–NIR spectrophotom-
eter. A SPEX-Fluorolog F112X spectrofluorimeter was used to record
fluorescence spectra. Triplet studies involving the transients were carried
out using a nanosecond laser flash photolysis Nd:YAG laser system
(
5
OCR-12 Series Quanta Ray Laser) with an output energy of 110 mJ at
32 nm by employing a laser kinetic spectrometer (Applied Photophysics
model LKS-20). The analyzing and laser beams were fixed at right angles
to each other. Quantum yields of fluorescence were measured by relative
methods using optically dilute solutions. Tetraphenylporphyrin
Aggregatibacter actinomycetemcomitans is a microaerophilic
Gram-negative pathogenic bacterium that colonizes the human
oral cavity. It is the causative agent for localized aggressive peri-
odontitis, a disease characterized by rapid destruction of tooth-
supporting tissues. This bacterium secretes leukotoxin which
helps to evade the host immune response during infection
(
Φ
F
= 0.11) was employed as standard for the porphyrin derivatives. An
Elico pH meter was used for pH measurements. Sodium hydroxide,
NH OH or HCl were used to vary the pH of solutions. All experiments
4
were carried out at room temperature (25°C ꢀ 1°C), unless otherwise
specified. Each experiment was carried out in triplicate and the data are
presented as a mean ꢀ standard deviation (SD). The difference between
two means was compared by a two-tailed unpaired Student’s t-test. The
values of P < 0.05 were considered as significant.
(
22,23). Fusobacterium nucleatum is an anaerobic Gram-negative
bacteria found in the normal flora of subgingival plaque. It is
one of the most common oral species isolated from extra-oral
infections, such as blood, brain, lung, liver, joints, abdominal,
obstetrical and gynecological infections and abscesses. Recent
reports reveal that this bacterium causes liver abscess in patients
suffering from recurrent periodontal disease (24). Enterococcus
faecalis is a Gram-positive pathogenic bacteria usually found in
the tooth caries of patients that survives and grows within the
dentinal tubules and reinfects an obturated root canal. This bacte-
rium has been associated with a wide range of human infections
comprising endocarditis, urinary tract infections, persistent
endodontic infection and biomaterial centered infections in
humans. Furthermore, it produces biofilms on anatomic sites that
are highly resistant to conventional treatment strategies (25,26).
Targeted local killing of periodontopathogenic bacteria using
aPDT has shown potential to be an alternative to the systemic
application of antibacterial drugs used in the treatment of peri-
odontal diseases. Development of new PSs and their clinical
application for a variety of oral infections are required to
improve the therapeutic outcome of aPDT, thereby minimizing
the side effects, such as dark toxicity, mutagenesis and intensive
coloration. In many clinical cases, aPDT is applied as an adjunc-
tive treatment procedure to the conventional treatment modalities
of curettage and antibiotics. In this context, development of opti-
mal dosages and treatment parameters for these new PSs could
possibly help in replacing the conventional methods of treatment.
This study explores the potential of newly synthesized new-
age cationic porphyrins, namely mesoimidazolium-substituted
porphyrin derivative (ImP) and pyridinium-substituted porphyrin
derivative (PyP) for application in aPDT toward the Gram-posi-
tive bacterium E. faecalis and two Gram-negative bacterial
species A. actinomycetemcomitans and F. nucleatum, which are
typical for oral cavity infections.
Synthesis of porphyrin derivatives. Synthesis of 5,10,15,20-tetrakis
(4-(8-bromooctyloxyphenyl)porphyrin: Synthesis of 5,10,15,20-Tetrakis
(
4
(
4-(8-bromooctyloxy phenyl) porphyrin involved the condensation of
-(8-Bromooctyloxy)benzaldehyde (1 g, 3.2 mmol) and distilled pyrrole
0.25 mL, 3.2 mmol) in dry dichloromethane (400 mL) taken in a 1 L
round-bottomed flask kept under argon atmosphere in presence of trifluo-
roacetic acid (1.3 mmol). The reaction mixture was stirred under argon
atmosphere for 2 h at 30°C. 2, 3-dichloro-5, 6-dicyanobenzoquinone
(
3
DDQ) (4.8 mmol) was added, and the reaction mixture was stirred at
0°C for 2 h. The reaction progress was monitored by TLC. The reaction
mixture was then filtered through an alumina column using dichlorome-
thane as eluent. The solvent was removed under reduced pressure to get
a purple solid, which was chromatographed over silica gel using dichlo-
romethane as the eluent to produce 4-(8-bromooctyloxy phenyl) porphy-
1
rin. Yield: (18%). mp > 300°C; H NMR (500 MHz, CDCl
3
, 30°C,
),
), 3.459 (t, 8H,
), 4.243 (t, 8H, J = 6.0–6.5 Hz, –CH ), 7.254 (d,
H, J = 9 Hz, –Ar–H, Phenyl), 8.094 (d, J = 8 Hz, 8H, –Ar–H, Phenyl),
TMS): d (ppm) ꢁ2.743 (s, 2H, -Py NH), 1.448–1.534 (m, 24H, –CH
.607–1.653 (m, 8H, –CH ), 1.898–1.993 (m, 16H, –CH
J = 6.5–7.0 Hz, –CH
2
1
2
2
2
2
8
8
1
3
3
.860 (s, 8H, –Ar–H, pyrrole); C NMR (125 MHz, CD OD, 30°C,
TMS): d (ppm) 26.17, 28.19, 28.80, 29.34, 29.47, 32.87, 34.08, 68.21,
12.71, 119.84, 134.49, 135.62, 158.93; IR (KBr): mmax: 635.58, 740.08,
805.29, 840.98, 967.32, 1176.60, 1244.11, 1283.65, 1250.19, 1465.93,
1
ꢁ
1
1509.32, 1606.73, 2361.88, 1852.77, 2930.89, 3396.76 cm ; MALDI-
TOF m+/z = Calcd for 90Br 1443.17, Found: 1444.82.
M + 1) .
Synthesis of 5,10,15,20-tetrakis[4-(8-pyridiniooctyloxy)phenyl]porphy-
76
C H
4 4 4
N O :
(
rin tetrabromide (PyP): 4-(8-Bromooctyloxy phenyl) porphyrin (200 mg,
0.18 mmol) was dissolved in 2 mL of dry pyridine and heated at 100°C
for 8 h. Excess pyridine was removed under reduced pressure. The resi-
due obtained was dissolved in water, filtered and the saturated solution of
NH PF was added to precipitate the PF salt of the porphyrin derivative.
4 6 6
The PF6 salt was then dissolved in acetonitrile and a saturated solution
of tetrabutylammonium bromide was added to give PyP. Yield: (60%).
1
mp > 300°C; H NMR (500 MHz, DMSO-d
6
, 30°C, TMS): d (ppm)
ꢁ
2.882 (s, 2H, –Py NH), 1.379–1.460 (m, 26H), 1.59 (s, 8H), 1.925 (s,
8
8
8
H), 1.993 (t, 8H, J = 6.5–7 Hz), 2.090 (s, 2H), 4.278 (s, 8H), 4.658 (t,
H, J = 7–7.5 Hz), 7.375 (d, 8H, J = 8 Hz), 8.118 (d, 8H, J = 7.5 Hz),
.197 (t, 8H, J = 6.5 Hz), 8.630–8.661 (t, 4H, J = 7.5–8 Hz), 8.869 (s,
1
3
8H), 9.172 (s, 8H); C NMR (125 MHz, MeOD, 30°C, TMS): d (ppm)

6
30 Chandra Sekhar Prasanth et al.
1
1
3.91, 20.71, 24.8, 27.11, 30.02–30.44, 32.44, 59.53, 63.04, 69.25,
02.88, 113.95, 121.26, 129.45, 135.28, 136.71, 145.84, 146.79, 160.59;
irradiation time and F is the absorption correction factor, which is given
OD
ꢁ
by F = 1–10
(OD is the optical density at the irradiation wavelength).
IR (KBr): υmax: 684.73, 802.39, 966.34, 1174.65, 1244.09, 1502.55,
1
z = Calcd for C96
ꢁ
1
mporFref
ref
604.77, 1743.65, 2922.16, 3051.39, 3390.86 cm ; MALDI-TOF m/
1
por
1
ꢁ
+
/ð O Þ ¼ /ð O2Þ
ð2Þ
H
110 24
F N
8
O
4
P
4
2019.94, Found: 1875.68 (M-2PF
6
) .
2
mrefFpor
Synthesis of 5,10,15,20-tetrakis[4-(1-methyl-1H-imidazol-3-ium)phe-
nyl]porphyrin tetrabromide (ImP): 4-(Bromomethyl phenyl) porphyrin
Bacterial strains and growth conditions: A. actinomycetemcomitans
strain 8324 from the collection DSMZ Germany); E. faecalis (Bulgarian
National Bank for industrial microorganisms) and F. nucleatum (ATCC
5586) were used in this study. A. actinomycetemcomitans were incu-
(
200 mg, 0.2 mmol) was dissolved in 2 mL of dry 1-methyl-1H-imidaz-
(
ole and heated at 100°C for 8 h. The precipitated product was filtered.
The residue obtained was dissolved in water, filtered and saturated solu-
2
tion of NH
derivative. The PF
tion of tetrabutylammonium bromide was added to give ImP. Yield:
4
PF
6
was added to precipitate the PF
6
salt of the porphyrin
2
bated in microaerophilic conditions (5% CO ) at 37°C for 48 h on solid
6
salt was dissolved in acetonitrile and a saturated solu-
medium of Trypticaseâ Soy agar (supplemented with 0.5% yeast extract).
Before aPDT experiments, the bacterial suspensions were diluted to the
required cell densities. A. actinomycetemcomitans strains were subcul-
tured in GC agar base (BD 228950), which was supplemented by dried
bovine hemoglobin (BD 212392) and IsoVitaleX (BD 211876). The sub-
cultures were incubated under microaerophilic conditions, at 37°C for
1
(
(
56%). mp > 300°C; H NMR (500 MHz, DMSO-d
ppm) -2.971 (s, 2H, –Py NH), 3.994 (s, 12H, –N–CH ), 5.821(s, 8H, Ar–
), 7.848 (d, 8H, J = 8 Hz, –Ar–H), 7.902 (s, 4H), 8.089 (s, 4H),
6
, 30°C, TMS): d
2
CH
2
8
.269 (d, 8H, J = 8 Hz, –Ar–H), 8.819 (s, 8H, –Ar–H), 9.490 (s, 4H);
C NMR (125 MHz, MeOD, 30°C, TMS) d (ppm) 37.75, 54.60, 121.45,
24.58–124.62, 126.18-126.22, 129.05, 135.77, 136.94, 138.94, 144.41;
1
3
4
8 h. F. nucleatum was maintained on anaerobic blood agar plates, fol-
1
lowing growth at 37°C in an atmosphere of N /CO /H generated using
2
2
2
IR (KBr): υmax: 621.08, 738.74, 756.1, 802.39, 966.34, 1161.15, 1336.67,
anaerobic gas packs (HiMedia, India). The culture plates were kept in
anaerobic jar containing the gas pack.
ꢁ
1
1
409.66, 1564.27, 1680, 3089.96, 3142.04, 3325.28 cm ); MALDI-TOF
ꢁ
+
m/z = Calcd for C64
H12Br
4
N
12: 1314.84; Found 1234.92 (M–Br ) .
) of PyP
and ImP were determined employing an earlier described method of energy
5
Uptake study: The cellular suspensions with different densities of 10 ,
Calculation of triplet quantum yields: The triplet yields (U
T
6
7
8
ꢁ1
1
(
0 , 10 and 10 CFU mL
were incubated with ImP and PyP
8.0 lM) for 15 min at 22°C by gentle stirring and covered with alumi-
2+
3
transfer to b-carotene, using Ru(bpy)
ments, optically matched solutions of Ru(bpy)
were mixed with a known volume of b-carotene solution (end concentra-
as the reference. For these experi-
num foil. The supernatants were removed and stored for fluorescence
measurements. The experimental setup for the PS uptake evaluation in
bacteria consisted of a red light-emitting diode (LED) (637 nm) as excita-
tion source and a spectrometer for acquiring spectral emissions and are
described elsewhere (33).
The cells were washed with phosphate-buffered saline (PBS) in tripli-
cate and then resuspended in aqueous 2% sodium dodecyl sulfate (SDS).
The extraction was continued for 30 min by mild stirring with the sam-
ples kept in the dark. These were then centrifuged, and the collected
extracts were examined by fluorescence analysis. The results are pre-
sented as number of dye molecules per bacterium cell by processing the
obtained values of fluorescence intensity with reference to the calibration
curves taken for the studied porphyrin in the extraction solvent.
2+
3
and PyP/ImP at 532 nm
ꢁ
4
tion of b-carotene was ca. 2.0 9 10 M). The transient absorbance (DA)
of the b-carotene triplet, formed by the energy transfer from Ru(bpy)
2
3
+
or
the porphyrin triplet, was monitored at 515 nm. Comparison of plateau
absorbance (DA) following the completion of sensitized triplet formation,
properly corrected for the decay of the donor triplets in competition with
energy transfer to b-carotene, enabled us to estimate U
T
of PyP/ImP triplet
excited state based on the following equation (Eq. (1)).
DApor
ref
por
obs
ref
ref
0
K
ðK ꢁ K
obs
Þ
por
T
/
¼ /T
ð1Þ
por
por
por
DArefðKobs ꢁ K0 ÞKobs
In vitro antimicrobial photodynamic therapy: The efficiency of the
cationic porphyrins at different concentrations (2, 5, 10, 11 and 22 lM)
was evaluated through quantification of the colonies of bacteria under
laboratory conditions. Samples of microbial suspensions were incubated
for 20 min in the dark with 10 lL photosensitizer taken from a stock
solution to final concentrations between 2 and 22 lM of PyP/ImP. The
incubation was carried out at room temperature and by gentle stirring.
Bacterial suspensions of 2 mL were prepared from bacterial cultures,
which were then diluted 10-fold in phosphate-buffered saline (pH = 7.2)
wherein, superscripts por and ref designate the various porphryins and
2
+
Ru(bpy)
the growth of b-carotene triplet and K
of the donor triplet, in the absence of b-carotene, observed in solutions
3
, respectively; Kobs, is the pseudofirst-order rate constant for
0
is the rate constant for the decay
2
+
containing Ru(bpy)
used for sensitization.
3
or porphyrin at the same optical density (OD) as
Calculation of singlet oxygen quantum yields: Irradiation was carried
out with light from a 200 W Hg lamp (Oriel model 3767) fitted on an
optical bench (Oriel model 11200) with a grating monochromator (Oriel
model 77250). The intensity of light was maintained constant throughout
the irradiation by measuring the output using a photodiode detection
system (Oriel model 7072). Reinecke’s salt actinometry provided the cali-
bration check. Quantum yields for singlet oxygen generation in oxygen-
saturated methanol were determined by monitoring the photooxidation of
6
ꢁ1
to a concentration of ~10 CFU mL . In all the experiments, 1 mL of
bacterial suspension was aseptically distributed in sterilized glass test
tubes and the PS was added from the stock solution to achieve final con-
centrations. After the addition of appropriate volume of porphyrin, test
tubes (total volume of 50 mL) were incubated for 15 min at 30°C. The
tubes were stirred every 5 min of incubation, covered with aluminum foil
to avoid accidental light exposure. Light and dark control experiments
were carried out simultaneously. In the light controls, the bacterial sus-
pension without PS was exposed to light irradiation. In the dark controls,
the PS was added to the beaker containing the bacterial suspension,
which was covered with aluminum foil to protect from light exposure.
The controls also followed the preirradiation incubation protocol. This
photosensitization procedure was used for each of the PS tested and for
the bacterial strains under investigation and the procedure was repeated
for each porphyrin in triplicate.
1
,3-diphenylisobenzofuran (DPBF) sensitized by PyP/ImP. DPBF is a
convenient acceptor because it absorbs in the region of dye transparency
and rapidly scavenges singlet oxygen to give colorless products. This
reaction occurs with little or no physical quenching. Singlet oxygen
quantum yields were measured at low dye concentrations (optical density
was 0.05 at irradiation wavelengths above 495 nm) to minimize the pos-
sibility of singlet oxygen quenching by the dyes. The concentration of
DPBF was between 2 and 2.8 10 M, and irradiation was carried out at
low conversion (5–10%) of DPBF such that its concentration may be
assumed as fixed at the initial value. The photooxidation of DPBF was
monitored between 0 and 50 s. No thermal recovery of DPBF (from a
possible decomposition of endoperoxide product) was observed under
ꢁ4
After incubation, an aliquot (200 lL) from the treated suspension was
placed in a standard 96-well polystyrene microtiter plate, where the irra-
diation was performed. The samples were exposed to laser light (635 nm,
ꢁ2
1 W CW, CNI Laser, China) for 20 min at an intensity of 50 mW cm
,
these experimental conditions. The quantum yield of singlet oxygen gen-
measured with power meter (Ophir, Israel, Model: PD-300–30W). Four
samples groups with microbial cells were collected: (1) light control (LC)
—without photosensitizer, but illuminated; (2) dark control (DC)—with
photosensitizer, but no light (dark toxicity); (3) control (C)—only bacte-
rial suspension (no photosensitizer, no light) and (4) aPDT treated group.
Following irradiation, 0.1 mL samples were taken and serially diluted
(10-fold) with PBS. Aliquots (0.025 mL) were spread over respective
agar plates. The number of colonies (CFU) formed on each plate was
1
eration [Φ( O
2
)] was calculated by a relative method using optically
matched solutions and comparing the quantum yield of photooxidation of
DPBF sensitized by the dye of interest to the quantum yield of meta-tet-
1
rahydroxyporphyrin (m-THPP), (Φ O
2
= 0.46) sensitized DPBF photoox-
idation as the reference. The following Eq. (2) was used, where
superscripts ‘por’ and ‘ref’ designate PyP/ImP and m-THPP,
1
respectively, [Φ( O
2
)] is the quantum yield of singlet oxygen, m is the
slope of the plot of change in absorbance of DPBF (at 410 nm) with the
2
counted following 48 h incubation at 37°C in 5% CO .

Photochemistry and Photobiology, 2014, 90 631
A standard volume (20 lL) of undiluted and serially diluted (up to
0 cells mL by 10-fold dilutions) irradiated samples and controls were
plated. After 96 h of incubation at 37°C in the dark, the number of colo-
nies was counted. The dark control Petri plates were kept in darkness
immediately after plating and during the incubation period. The assays
for each concentration of porphyrins and for each bacterial strain were
done in duplicate and averaged.
by oxidation with DDQ yielded 18% of the corresponding
bromo-substituted porphyrin, which on quarternization with pyri-
dine at 100°C gave PyP with 60% of yield. Similarly, the imi-
dazolium-substituted porphyrin derivative ImP, was synthesized
starting with 4-(bromomethyl)benzaldehyde, which on reaction
with pyrrole in chloroform in the presence of boron trifluoride
diethyl ether, followed by oxidation using p-chloranil yielded
3
ꢁ1
1
Biofilm assay and evaluation: Biofilm assay was performed on cover-
slips covered with gelatin, which were placed in commercial presterilized
polystyrene flat bottomed 12-well cell culture test plates (Switzerland).
3
0% of the corresponding bromo-substituted porphyrin (35),
7
ꢁ1
which on quarternization, with 1-methyl imidazole at 100°C gave
ImP with 56% of yield. All the starting materials as well as the
porphyrin derivatives were purified through recrystallization,
counter anion exchange and characterized on the basis of various
analytical and spectral evidences.
Standard bacterial suspension (1 mL, 10 CFU mL ) of A. actinomyce-
temcomitans and E. faecalis was prepared after serial dilutions and was
placed onto the surface of the coverslip in each well of the plate. The
incubation was carried out between 30 min and 1.5 h at 37°C to promote
cellular adherence to the gelatin surface. After the initial adhesion phase,
the cell suspensions were aspirated and the coverslips were gently
washed with PBS to remove loosely adherent cells. In the biofilm phase
formation, an addition of 4 mL Tryptic soy broth (supplemented with
Photophysical properties of photosensitizers
0
.5% yeast extract) (Difco Lab, MD) was placed in each well. The plates
were incubated for 48 h at 37°C to form the biofilm.
The biofilms of A. actinomycetemcomitans and E. faecalis cells were
developed on the coverslips covered with gelatin for 48 h incubation
The pyridinium porphyrin derivative PyP shows the characteris-
tic porphyrin absorption with the Soret band at 419 nm and
four Q bands at 523, 561, 592 and 654 nm, whereas the fluo-
rescence spectrum exhibited two emission maxima at 665 and
(
37°C). The biofilms were examined with confocal laser scanning micro-
scope (CLSM) of Leica Microsystems (Model: Leica TCS SPE). The
images were processed via the Leica LAS AF software provided with the
CLSM. An oil immersion 639 objective (NA = 1.23) was used. The
images of biofilm slices with thickness of 0.150 lm were scanned by the
fluorescence using 635 nm laser as excitation light. The red fluorescence of
PyP and ImP bound to the cells of biofilm was imaged at excitation with a
laser at 635 nm (or 408 nm) and the fluorescence emission was recorded in
the region 660–720 nm. The green autofluorescence emission of the bacte-
rial cells organized as biofilm was imaged in the 500–580 nm wavelength
range with excitation at 488 nm. Slices of 0.150 lm were scanned through
the whole biofilm to picture the typical far-red fluorescence emission of the
porphyrin dye. The transmission channel and laser light at 635 nm was
also used to determine the exact thickness of biofilms in comparison to the
porphyrin penetration depth into biomass.
7
27 nm in water (Fig. 1a). On the other hand, the imidazolium-
substituted porphyrin (ImP) exhibited the Soret absorption band
at 413 nm with four Q bands at 516, 553, 582 and 635 nm,
whereas its emission spectrum showed two emission peaks cen-
tered at 649 and 707 nm (Fig. 1b). Fluorescence quantum yields
of the porphyrins PyP and ImP were found to be 0.13 and
0
F
.07 using tetraphenylporphyrin as the standard (Φ = 0.11), in
toluene (36).
Nanosecond laser flash photolysis employing a 532 nm laser
pulse was carried out to understand the transient species involved
in these derivatives. The transient absorption spectrum of the
pyridinium derivative PyP in methanol is shown in Fig. 2a. The
absorption maximum of the transient species was found to be
centered at 450 nm, with bleach at 410 nm, where the compound
has significant ground state absorption. The inset of Fig. 2a
shows the decay profile at 450 nm from which the lifetime of
the transient species was calculated as 2 ls. The quenching
experiments were carried out to characterize the transient species
formed as triplet excited state of PyP based on its insignificant
formation in presence of dissolved oxygen (37). Laser excitation
of ImP at 532 nm also showed similar observations, with triplet
RESULTS
Synthesis of porphyrin derivatives
The novel water soluble cationic porphyrins (Chart 1) with
pyridinium (PyP) and N-methyl imidazolium (ImP) were synthe-
sized through Lindsey’s method (34). The synthesis of the pyr-
idyl-substituted porphyrin PyP was achieved through the
condensation of 4-(8-bromooctyloxy)benzaldehyde with pyrrole
in dichloromethane in presence of trifluoroacetic acid, followed
N
N
N
O
4
N
4Br
N
NH
N
NH
N
N
N
4
O
O
N
HN
N
HN
4
N
N
4Br
O
N
N
4
N
PyP
ImP
Chart 1. Chemical structures of cationic porphyrin derivatives PyP and ImP.

6
32 Chandra Sekhar Prasanth et al.
Figure 1. UV-Vis absorption and fluorescence spectra (shown as inset) of the cationic porphyrin derivatives (a) PyP and (b) ImP in water. kex
,
430 nm.
Figure 2. Triplet absorption spectra after laser excitation of the cationic porphyrins (a) PyP and (b) ImP (20 lM) with transient decay (inset) at 450 nm
for PyP and 440 nm for ImP in argon saturated and oxygen saturated methanol. (c) Absorption spectra of diphenylisobenzofuran (DPBF, 60 lM) at var-
ious irradiation time intervals in the presence of PyP (10 lM). Inset shows the expanded portion of absorption changes in DPBF at 410 nm. (d) Changes
in absorbance of diphenylisobenzofuran (DPBF) at 410 nm against irradiation time in the presence of various porphyrin derivatives PyP (■), ImP (▲)
and meta-tetrahydroxyporphyrin (m-THPP) (●) as standard.
excited state absorption maximum at 440 nm and a lifetime of
.2 ls in methanol (Fig. 2b). The quantum yields of triplet
excited states (Φ ) were estimated using triplet–triplet energy
using 1,3-diphenylisobenzofuran (DPBF) as the singlet oxygen
scavenger (39–41). To calculate yields, the sensitizer along with
DPBF was irradiated using a xenon lamp with a long pass (LP)
filter (495 nm) at different time intervals between 0 and 45 s.
The DPBF absorption at 410 nm was monitored and the decrease
in absorbance were compared with the standard, tetra(meta-
hydroxyphenyl)porphyrin (m-THPP), under identical conditions.
The absorbance changes of diphenylisobenzofuran (DPBF) at
various time intervals in the presence of the cationic porphyrin
derivative PyP is shown in Fig. 2c. The singlet oxygen quantum
yields were calculated from the slope of the graph obtained by
plotting the change in absorbance of DPBF against irradiation
1
T
2
+
transfer method to b-carotene and [Ru(bpy)3] as the standard
38). The values in methanol are
= 0.62 ꢀ 0.03 and
.56 ꢀ 0.02, for PyP and ImP, respectively.
(
Φ
T
0
Photosensitized singlet oxygen generation
As, singlet oxygen is considered to be the main cytotoxic agent
1
in photodynamic therapy, the quantum yields [Φ ( O )] of singlet
2
oxygen generation efficiencies of PyP and ImP were determined

Photochemistry and Photobiology, 2014, 90 633
Table 1. Photophysical parameters of ImP and PyP.
ꢁ
1
Mꢁ1
1
Porphyrin
ImP
k
abs (nm)
ɛ (cm
)
Φ
F
Φ
T
s
Τ
(ls)
Φ ( O
2
)
5
413
35
419
54
2.45 9 10
0.079
0.103
0.56 ꢀ 0.02
0.62 ꢀ 0.03
1.2 ꢀ 0.01
2 ꢀ 0.01
0.52 ꢀ 0.02
0.60 ꢀ 0.03
3
6
2.2 9 105
PyP
2.3 9 10
3
6
4.59 9 10
1
k
abs: Absorption maximum Φ
F
: quantum yield of fluorescence; Φ
T
: quantum yield of triplet excited state; s
Τ
: triplet excited state lifetime; Φ( O
2
): singlet
oxygen generation efficiency.
1
time (Fig. 2d) and are found to be Φ( O ) = 0.60 ꢀ 0.03 for
(Fig. 4). The bacterial cells were visualized by autofluorescence
using laser excitation, kex: 488 nm and emission between kem
520–580. The red porphyrin (ImP) fluorescence emission was
obtained at kex: 635 nm and kem: 650–720 nm). Figure 5 shows
the z-profile of the biofilm grown for 2 days, with thickness of
2
PyP and 0.52 ꢀ 0.02 for ImP, in methanol. The detailed photo-
physical properties of the porphyrin derivatives ImP and PyP
are shown in Table 1.
:
9
–11 lm as seen by autofluorescence. A complete penetration
Uptake of porphyrins by bacteria and biofilms
into biomass was observed for porphyrin PyP as well as for
ImP.
The biofilms E. faecalis formed at 72 h were analyzed by
CLSM from autofluorescence (kex: 488 nm; kem: 520–580 nm)
The uptake of the porphyrins ImP and PyP in incubated bacte-
rial cells (E. faecalis and A. actinomycetemcomitans) was evalu-
ated after chemical extraction procedure by fluorescence
measurements of the supernatants from extracted cell samples.
Nevertheless, both the porphyrins have low fluorescence with
quantum yields as measured in methanol (Table 1). The estima-
tion of the uptake capacity for 8 lM ImP and PyP toward these
microbial species was determined from the fluorescence emission
spectra recorded in the spectral range 660–750 nm with excita-
tion at 637 nm from a LED. The results are presented in Fig. 3
as bound molecules of ImP and PyP, calculated for one bacterial
cell.
and porphyrin fluorescence (k : 635 nm; kem: 650–720 nm) and
ex
the overlap of both images to visualize the accumulation for a
slice of the whole biofilm (Fig. 6). The biofilms with thicknesses
between 4 and 8 lm were evaluated by the scans of 0.150 mm
slices for detection of autofluorescence from cell (k : 488 nm;
ex
kem: 500–580 nm) as well as by porphyrin fluorescence. The
overlap of both images for E. faecalis incubated with ImP and
PyP is indicative of the localization of porphyrins (Fig. 6).
The bacterial biofilm formed at 48 h was evaluated for its
thickness and the penetration depth of the porphyrins into the
biomass with a CLSM. The images present the slice (0.150 mm)
of a scan for detection of A. actinomycetemcomitans biofilm
Photostability of porphyrins during PDT irradiation
The photostability of the porphyrins as photosensitizers for PDT
was studied at different intervals of light exposure (Fig. 7a,b).
Figure 3. Uptake of PyP and ImP in Enterococcus faecalis and Aggregatibacter actinomycetemcomitans in dependence on the cell density of bacterial
suspension. Inset: the percentage of photosensitizer uptake versus cell density at 8 lM PyP and ImP.

6
34 Chandra Sekhar Prasanth et al.
a)
(
(b)
(c)
Figure 4. The images showing Aggregatibacter actinomycetemcomitans bacterial biofilm with ImP porphyrin. (a) green-colored autofluorescence,
ex: 488 nm; kem: 500–600 nm, (b) the red-colored porphyrin fluorescence, kex: 635 nm; kem: 650–720 nm and (c) the overlap of both (a and b).
k
Objective: x63
concentrations of PyP and ImP toward the representative strains
associated with dental pathology. It can be seen that laser irradia-
ꢁ
2
tion at power density of 50 mW cm
6
and light dose of
ꢁ
2
0 J cm without photosensitizer did not affect the viability of
the studied bacteria, viz. F. nucleatum, E. faecalis and A. actino-
mycetemcomitans. The dark control groups treated at a concentra-
tion of 22 lM of PyP, without laser irradiation, showed lack of
dark toxicity on all tested bacterial species (Fig. 8). In contrast,
the porphyrin ImP showed dark toxicity for all concentrations
studied (2–22 lM) on the Gram-positive aerobic strain E. faecalis
(Fig. 9a). The optimal inactivation of E. faecalis cells was
obtained at a very low concentration of 2 lM of ImP, even
though the dark toxicity at this concentration was negligible.
Figure 8a presents the effect of PyP on the Gram-positive aer-
obic species E. faecalis. Figures 8b,c show the effect of PyP on
the Gram-negative microaerophillic species A. actinomycetem-
comitans and the Gram-negative anaerobe F. nucleatum, respec-
tively. At a concentration as low as 5 lM of porphyrin PyP, a
complete photoinactivation was induced. The photodynamic
effect toward the three tested bacterial species was high for 5 lM
porphyrin PyP, whereas at 2 lM, the less effective process of
inducing photodamage to all of the studied microbial species
was observed.
Figure 9a presents the photodynamic effect of ImP on the
Gram-positive aerobic species E. faecalis. Figures 9b,c show the
effect on two Gram-negative microaerophillic species A. actino-
mycetemcomitans and F. nucleatum, respectively. The phorpyrin
ImP was also able to induce high PDT effect at a concentration
as low as 5 lM. The photodynamic damage to all the three
bacterial species was observed at 2 lM of ImP. However, both
the porphyrins were less effective in inducing photodamage to
the membranes of the Gram-negative bacterial species, with
exception of the Gram-positive bacterial strain.
Figure 5. Confocal laser scanning microscope image of Aggregatibacter
actinomycetemcomitans incubated with PyP. The z-profile of the biofilm
was 9–11 lm in thickness and is seen on the right and bottom. Objec-
tive: x 63.
The variations in the fluorescence intensity of the studied por-
phyrins characterize the changes in the amount of dye in the irra-
diated volume. The reason for this change can be attributed to
the photobleaching of the porphyrins ImP and PyP at therapeutic
light application. The spectra were recorded repeatedly in inter-
vals of 6 min up to 1 h. As observed from Fig. 7a,b ImP is
more stable than PyP.
DISCUSSION
In vitro antimicrobial PDT study on pathogenic bacteria
Photophysical properties of Porphyrin derivatives
PyP and ImP
The photodynamic efficiency of the cationic porphyrins PyP and
ImP as antimicrobials were studied (Figs. 8 and 9). The results
are presented as viable bacteria in CFUs per mL for various
Porphyrins are known to have optical absorption peaks in the visi-
ble region and high triplet state quantum yields (9,43). These

Photochemistry and Photobiology, 2014, 90 635
(
a)
(b)
Figure 6. Confocal laser scanning microscope images of Enterococcus faecalis biofilms incubated with ImP (a) and PyP (b). The z-profile of the
biofilms shows the thicknesses between 4 and 8 lm as seen on the right and bottom position inside the images. Objective: x63.
Figure 7. Fluorescence spectra of ImP (a) and PyP (b) obtained for equal time of irradiation with a red light, which suggest the photostability of the
studied porphyrins during PDT.
characteristics make them suitable for various applications, such
as fluorescence probes in the optical diagnostics of malignancies
as well as the photosensitizers in PDT of solid tumors. Moreover,
for noninvasive photodiagnostic and therapeutic medicinal appli-
cations, the photosensitizer should have absorption bands roughly
between 630 and 850 nm due to low human tissue permeability to
light below 600 nm and the water absorptivity above 900 nm.
The longest absorption peak for PyP in the red spectral region is
at 654 nm and for ImP, it is at 635 nm. The absorption in the red
and far-red spectra is an important parameter as the endogenous
tissue chromophores such as hemoglobin, cytochrome and mela-
nin, have absorption between 280 and 600 nm. The wavelength
range from 630 to 850 nm is known as the phototherapeutic win-
dow in which the light penetrates into biological tissues with
lower absorption loss, thereby enabling treatment of deeper tissue
structures (44). The cationic porphyrin derivatives, PyP and ImP
showed typical porphyrin absorption corresponding to p–p* tran-
sition, called the Soret band, as well as at bands corresponding to
the p–p* transition, known as the Q bands. Upon excitation with
light of appropriate wavelength, these porphyrins get excited to
the first excited state and subsequently, cross over to the triplet
excited state. The involvement of triplet excited states in the por-
phyrin system was confirmed by nanosecond laser flash photoly-
sis, which showed the presence of transient absorption at 450 and
440 nm for PyP and ImP, respectively. The ready quenching of
the transient absorption by dissolved oxygen and b-carotene fur-
ther substantiates the formation of triplet excited states for both
the porphyrin derivatives. It is known that b-carotene possess low
energy level triplet, which cannot be excited using 532 nm laser
pulse. Nevertheless, excitation of porphyrin and b-carotene solu-
tions with 532 nm laser pulse results in the characteristic transient
absorption of the b-carotene at 515 nm. This observation of b-car-
otene triplet at 515 nm, accompanied by the loss of the transient
absorption of porphyrin triplet, is due to energy transfer from the
triplet of the porphyrin to the triplet of the b-carotene. In other
words, the formation of transient absorption at 515 nm, upon
excitation at 532 nm indicates that this transient absorption arises
from the triplet excited state of the porphyrin molecules.

6
36 Chandra Sekhar Prasanth et al.
Figure 8. Photodynamic efficiency of cationic porphyrin PyP for different concentration and irradiation with light emitting diode 637 nm
ꢁ2
(
60 mWcm ) on the Gram-positive bacterial strain Enterococcus faecalis (a), the Gram-negative microaerophilic bacterial strain Aggregatibacter
actinomycetemcomitans (b) and strictly anaerobic bacterial strain Fusobacterium nucleatum (c).
Figure 9. Photodynamic efficiency of cationic porphyrin ImP for different concentration and irradiation with light emitting diode at 637 nm with a
ꢁ
2
power density of 60 mWcm on (a) the Gram-positive bacterial strain Enterococcus faecalis, (b) the Gram-negative microaerophilic bacterial strain
Aggregatibacter actinomycetemcomitans and (c) the strictly anaerobic bacterial strain Fusobacterium nucleatum.
The evidence for involvement of singlet oxygen was obtained
by monitoring changes in the absorbance of the singlet oxygen
scavenger, DPBF. Both the photosensitizers on excitation get
excited to the first excited singlet state. Due to the high intersystem
crossing efficiency, the excited state molecule intersystem crosses
to the first excited triplet state. The triplet state of the neutral

Photochemistry and Photobiology, 2014, 90 637
porphyrin derivatives and their metal complexes transfers its
energy to the triplet molecular oxygen generating the singlet oxy-
gen. The singlet oxygen thus produced, being highly reactive,
readily undergoes reaction with DPBF resulting in the formation
of the endoperoxide. Therefore, the observed decrease in the
absorption band intensity of the DPBF on irradiation using light of
wavelength >495 nm, in the presence of the porphyrin derivatives,
is due to the formation of the corresponding endoperoxide. The
high quantum yield for singlet oxygen generation by the macrocy-
cles shows their efficiency in intersystem crossing to the triplet
excited state as well as the efficient energy transfer to the ground
state triplet oxygen. Furthermore, the triplet energy level of the
macrocycle is expected to lie close to or higher than
thickness between 9 and 11 lm for A. actinomycetemcomitans
(Fig. 5) and between 4 and 8 lm for E. faecalis (Fig. 6). In the
vertical section image of A. actinomycetemcomitans biofilm, it
can be seen that the cationic porphyrins were unable to penetrate
areas of high cellular density, which could be attributed to the
intact anaerobic interior clusters formed inside the biofilm
(Fig. 5). However, in the case of E. faecalis biofilms, both the
porphyrins were more uniformly distributed as a result of its
loose structure (Fig. 6).
Photostability of porphyrins during PDT irradiation
It can be seen from Fig. 1a that PyP has characteristic absorption
peak at 419 nm and the fluorescence spectrum has emission
maxima at 665 and 727 nm. In comparison, the ImP chromo-
phore (Fig. 1b) has characteristic absorption peak at 413 nm
whereas the fluorescence spectrum shows emission maxima at
ꢁ
1
2
3 kcal mol (~1 eV), which is required for energy transfer to
molecular oxygen.
Uptake and localization of porphyrins in pathogens
6
49 and 707 nm. The Soret bands of both the photosensitizers
The uptake and localization studies of porphyrins for the plank-
tonic cultured bacteria and the undisturbed, artificially grown
biofilms were evaluated by fluorescence measurements of the cel-
lular extracts and by direct visualization with CLSM. The results
obtained suggest that both cationic porphyrins, due to their posi-
tive charges, get easily attached to the cellular wall. This favors
a better target for the generated singlet oxygen, such as mem-
branes of pathogenic microorganisms. The phenomenon of an
inverse uptake which happens with an increase in cell density
were utilized for their excitation around 405 nm. The emission
spectra of both the porphyrins were studied and utilized for
in situ fluorescence monitoring of photosensitizer dosimetry dur-
ing aPDT (Fig. 7a,b). The rate of dissociation of PyP (Fig. 7b)
suggests that it undergoes swift photobleaching and not much of
the photosensitizer is left after 20 min of irradiation. On the other
hand, ImP remains stable during the first 20 min PDT irradiation
(Fig. 7a). This, in fact, is a good indication for using PyP in clin-
ical trials as it would be possible to lower the treatment time and
high efficiency can be attained at low concentrations. In PDT, in
situ quantification of PS concentration prior to initiation of ther-
apy is important. The current results indicate that the fluorescence
signal intensity would have value in measurement and dosimetry
during PDT, as reported by various groups (51–54).
5
8
ꢁ1
from 10 to 10 CFU mL
was observed for both ImP and
PyP. These results follow the uptake behavior of our recent
observation on differently charged zinc phthalocyanines (33,42).
The binding process occurs mainly by noncovalent, electrostatic
mechanism of interactions between porphyrin and cell membrane
(
45). The phenomenon of an inverse reliance on the number of the
photosensitizing dye molecules attached to a cell on the cell den-
sity of the suspension was firstly reported by Demidova and Ham-
blin for E. coli (46). The cationic dyes are more easily taken up in
the bacterial cells due to the nature of the membrane bilayers. This
phenomenon is a result of improved charge distribution and high
drug-cell tolerance, which is essential for the Gram-negative bacte-
rial strains. However, there are reports on the possibility of bacteria
developing resistance to aPDT due to selective survival of strains
with increased multidrug resistance pumps (MDR) expression lev-
els (47) and further studies are necessary for evaluating chances of
developing resistance (48) against tetrapyrrolic macrocyclic por-
phyrin compounds.
Cationic porphyrins as photosensitizers against
pathogenic bacteria
New-age cationic porphyrins PyP and ImP showed promising
photodynamic efficiency against the Gram-positive and the
Gram-negative bacterial strains associated with oral infections.
The microaerophilic Gram-negative bacterium A. actinomycetem-
comitans and anaerobic Gram-negative bacterium F. nucleatum
are very common periodontal pathogens. E. faecalis represents
dental pathogenic microorganisms, which are associated with dis-
eases like apical periodontitis, dental caries, etc. The photody-
namic activity of the proposed porphyrins PyP and ImP was
optimal on E. faecalis, which as a Gram-positive strain is more
susceptible to the applied therapy (Figs. 8a and 9a). At a concen-
tration as low as 2 lM, the cationic porphyrin ImP was able to
achieve complete inactivation of E. faecalis whereas 5 lM of PyP
was needed to completely inactivate the Gram-positive strain.
The Gram-negative strains F. nucleatum and A. actinomycetem-
comitans are well known as highly resistant against conventional
therapeutic techniques (33). However, the results obtained show
that it was able to inactivate the bacteria completely at a concen-
tration of 5 lM of porphyrins PyP and ImP (Figs. 8b,c and 9b,c).
Cationic photosensitizers are capable of binding to various
sections of the bacterial cell wall through ionic interaction to sul-
fonate or carboxylate residues. This is due to electrostatic force
(attraction) between the cationic PS and anionic (carboxylate,
The CLSM images showed that both the porphyrins (PyP and
ImP) penetrate through the whole biomass of the 48 h biofilm
(
Figs. 4–6). The structure as well as properties of biofilm orga-
nized cells differs from their planktonic counterparts (cells in
suspension). Figures 4a–c show the images of the biofilm slice
(
0.15 lm), where the green color represents the autofluorescence
of A. actinomycetemcomitans bacterial biofilm. It is reported that
the green fluorescent protein (GFP) can be used as a biomarker
to identify the bacterial cells by CLSM (49). Figure 4b shows
the red porphyrin fluorescence emission obtained in the 650–
7
20 nm range with laser excitation at 635 nm. The images show
heterogeneous structure in all the biofilms studied. Fluid-filled
channels and voids are clearly visible in the overlapped image
(
Fig. 4c) as was previously reported (50). The vertical sections
ꢁ
of CLSM images of the biofilms were taken to determine its
COO ) groups in the cell exterior. Therefore, selective adsorp-
thickness and architecture. The sections were found to have
tion to the cell membrane can takes place (3). Merchat et al.

6
38 Chandra Sekhar Prasanth et al.
(
18), in a study on cationic porphyrins as photosensitizers using
successful photoinactivation was 10 lM; however, a major draw-
back was that the dark toxicity on Gram-positive strain is
unknown (67). Alves et al. (57), in a recent study reported that
meso-substituted cationic porphyrins similar to the one used in
this study do not induce any dark toxicity on Gram-positive and
Gram-negative strains. The high efficiency of the new-age por-
phyrins observed in this study compares favorably with the clini-
cally approved and vastly used Hematoporphyrin derivative
(HpD) for PDT applications.
In our study, the both porphyrins PyP and ImP are cationic
in nature due to positive charges of four substituents. In a PDT
study of four types of meso-substituted cationic porphyrin deriva-
tives, Lazzeri et al. (55), observed higher photoinactivation of
cells for tricationic porphyrins. Alves et al. (57), in a study on
the effect of charge in meso-substituted cationic porphyrins on
Gram-negative and Gram-positive bacteria reported that cationic
porphyrins having three and four charges are highly efficient
photosensitizers against both the bacterial strains. The relatively
good results obtained in this study can also be attributed to the
number of positive charges of these compounds. Further in vivo
investigations, including clinical trials, are needed to evaluate the
advantages of PyP and ImP over the photosensitizers being used
at present for periodontal treatment.
Enterococcus seriolicida as Gram-positive strain and Vibrio
anguillarum as Gram-negative strain, observed the ability of
these compounds to inactivate the Gram-negative bacteria. In
another study (55), the photodynamic property of four deriva-
tives of meso-substituted cationic porphyrin derivatives were
studied on E. coli, which is a Gram-negative bacterium. Recent
studies with cationic porphyrins (56,57) have shown efficient
destruction of both Gram-positive and Gram-negative bacteria
with aPDT. Ergaieg and Seux (58) have carried out a compara-
tive study on the photoinactivation efficacy of meso-substituted
cationic porphyrin on Gram-positive Enterococcus hirae and
Gram-negative E. coli, which showed that cationic porphyrin has
higher photodynamic efficiency than rose bengal and methylene
blue. Alves et al.(46), studied the efficiency of seven cationic
porphyrins differing in meso-substituent groups on E. coli and E.
faecalis species. The control groups showed no dark toxicity or
light toxicity for both the strains and two of the seven cationic
porphyrin derivatives showed good efficiency in killing both the
strains. Ergaig et al.(56) in a study on the mechanism involved
in the phototoxicity of meso-substituted cationic porphyrin deriv-
atives found that both Type I and Type II mechanisms are
responsible for the photodynamic activity of these sensitizers. In
a recent study by Nagahara et al. (59), it was reported that a
new photosensitizer indocyanine green-loaded nanospheres could
photodynamically inactivate Porphyromonus gingivalis, a Gram-
negative periodontal pathogen. In this work, the adherence of the
photosensitizer was evaluated but the photo physical properties
were not studied in detail. Very recently, Pereira et al. (60) have
evaluated photodynamic inactivation (61) using already known
photosensitizers, erythrosine (ER) and Rose Bengal (RB), on the
viability of bacterial biofilms to oral infections Streptococcus
mutans and Streptococcus sanguinis.
The resistance of bacteria against the conventional therapy
with antibiotics (62) necessitates discovery of new antibiotics and
new treatment modalities. However, PDT appears more promising
as compared with other biocides therapies because the develop-
ment of bacterial resistance is not a concern. According to WHO,
resistance of bacteria to antibiotics is one of the greatest threats to
human health and the theme of World Health Day 2011 was on
combating of antimicrobial resistance (63). In this context, efforts
are required to find new photosensitizers with adequate structural
features for efficient antimicrobial photoinactivation. Furthermore,
several clinical and animal model studies are ongoing (64–66) to
evaluate the in vivo efficiency of photosensitizers with known
photophysical properties. The cationic porphyrins used in this
study appear effective against both the Gram-positive and the
Gram-negative strains with more than 99.9% reduction on cell
survival after light exposure. PyP showed no dark toxicity in both
the strains studied and was effective in killing of these microbes.
In comparison, ImP, which had no dark toxicity, showed photo-
toxicity against Gram-positive strain E. Faecalis (Fig. 9a) and
against both Gram-negative strains even at higher concentrations
of 22 lM (Figs. 9b,c). This is in agreement with previous reports
that the Gram-positive bacteria are more sensitive to photosensiti-
zation (67). The other photosensitizer PyP showed no dark toxic-
ity in both Gram-positive and Gram-negative strains (Figs. 7a, 8a
and 9a). This makes PyP a novel and promising photosensitizer
for future antibacterial applications. The hydrophilic tricationic
porphyrins bearing a trifluoromethyl group have shown promising
results. The concentration of the PS used in this study for
CONCLUSION
Two novel cationic porphyrins bearing pyridinium (PyP) and
imidazolium (ImP) substituents were synthesized and investi-
gated for their utility as PDT sensitizers for inactivation of the
Gram-negative and the Gram-positive periodontal pathogenic
bacteria associated with dental infections. The synthesized por-
phyrin derivatives exhibited favorable photophysical properties
including intensive, red absorption and fluorescence emission,
and relatively high quantum yields for the triplet excited states
and singlet oxygen generation efficiency. The uptake study on
Gram-positive aerobe E. faecalis and microaerophilic Gram-neg-
ative A. actinomicetemcomitans in suspension showed high abil-
ity of both the porphyrins for accumulation in bacterial cells.
The complete penetration of PyP and ImP into biofilms with
high photodynamic efficacy suggests their promising as applica-
tion as photosensitizers for targeting oral pathogens such as E.
faecalis, A. actinomicetemcomitans and F. nucleatum. An
advanced antimicrobial PDT efficiency was observed for PyP
porphyrin.
Acknowledgements—This project was carried out as part of an Indo-
Bulgarian collaborative project (INT/BULGARIA/B-64/06) funded by the
Department of Science and Technology, Government of India and the
Bulgarian Ministry of Education and Science. CSP is thankful to the
research committee of CESS for his research fellowship grants. The
authors declare no competing financial interest.
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