Synthesis, photophysical studies and anticancer activity of a new halogenated water-soluble porphyrin

Article abstract of DOI:10.1111/j.1751-1097.2007.00073.x

A water-soluble halogenated porphyrin, namely 5,10,15,20-tetrakis(2-chloro- 3-sulfophenyl)porphyrin (TCPPSO₃H), was prepared and evaluated as sensitizer for photodynamic therapy (PDT). Photophysical properties of TCPPSO₃H, such as high photostability, long triplet lifetime and high singlet oxygen quantum yield suggest high effectiveness of this class of halogenated porphyrins in PDT. TCPPSO₃H is non-toxic in the dark and causes a significant photodynamic effect examined against MCF7 (human breast carcinoma), SKMEL 188 (human melanoma) and S91(mouse melanoma) cell lines upon red light irradiation (cutoff < 600 nm) at low light doses. Time-dependent cellular uptake of TCPPSO₃H reached plateau at 120 min and was the highest for S91, 20% lower for MCF7 and 70% lower for SKMEL 188. Our results show that this halogenated water-soluble porphyrin is an efficient photosensitizer and reveal the potential of this class of compounds as PDT agents.

Full text of DOI:10.1111/j.1751-1097.2007.00073.x

Photochemistry and Photobiology, 2007, 83: 897–903
Synthesis, Photophysical Studies and Anticancer Activity
of a New Halogenated Water-Soluble Porphyrin
1
Janusz M. D a˛ browski , Mariette M. Pereira* , Luis G. Arnaut* , Carlos J. P. Monteiro ,
,2
3
3
3
3
1
2
1
Andreia F. Peixoto , Andrzej Karocki , Krystyna Urba n´ ska and Gra z_ yna Stochel*
1
Faculty of Chemistry, Jagiellonian University, Krakow, Poland
Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Krakow, Poland
Chemistry Department, University of Coimbra, Coimbra, Portugal
2
3
Received 10 October 2006; accepted 10 January 2007; DOI: 10.1111/j.1751-1097.2007.00073.x
ABSTRACT
A water-soluble halogenated porphyrin, namely 5,10,15,20-
tetrakis(2-chloro-3-sulfophenyl)porphyrin (TCPPSO H), was
prepared and evaluated as sensitizer for photodynamic therapy
PDT). Photophysical properties of TCPPSO H, such as high
photostability, long triplet lifetime and high singlet oxygen
quantum yield suggest high effectiveness of this class of halogen-
Porphyrins and their analogs are the most frequently studied
photosensitizers for PDT (19–21). They have a significant
absorption in red, low toxicity in the dark and a long triplet state
lifetime. This property is critical because the triplet state of the
photosensitizer is a precursor of the cytotoxic species and a high
quantum yield of this species can only be achieved through a
3
(
3
ated porphyrins in PDT. TCPPSO
causes a significant photodynamic effect examined against MCF7
human breast carcinoma), SKMEL 188 (human melanoma)
and S91(mouse melanoma) cell lines upon red light irradiation
cutoff < 600 nm) at low light doses. Time-dependent cellular
uptake of TCPPSO H reached plateau at 120 min and was the
highest for S91, 20% lower for MCF7 and 70% lower for SKMEL
88. Our results show that this halogenated water-soluble
3
H is non-toxic in the dark and
long-lived precursor (22). The photocytotoxicity is due to the
1
generation of free radicals or singlet molecular oxygen (the D
g
(
electronic state of molecular oxygen) upon irradiation of the
photosensitizer. The desirable photophysical properties already
mentioned must be combined with the controlled solubility of
the sensitizer in aqueous media (23,24).
In this work we describe the synthesis of the new water-
soluble 5,10,15,20-tetrakis(2-chloro-3-sulfophenyl)porphyrin
(
3
1
porphyrin is an efficient photosensitizer and reveal the potential
of this class of compounds as PDT agents.
3
(TCPPSO H) as well as its physicochemical and biological
properties. The selection of this photosensitizer was motiva-
ted by earlier work that demonstrated the efficacy of halogen
atoms in the ortho position of phenyl rings in the meso
positions to promote intersystems crossing and increase
singlet oxygen quantum yields (25). Cytotoxic and photocy-
totoxic effects were evaluated in human and mouse melan-
oma and human breast carcinoma cells, using the S91 (mouse
melanoma), SKMEL 188 (human melanoma) and MCF7
INTRODUCTION
There is a general interest in the development of more efficient
anticancer drugs with diminished side effects and better
selectivity. Photodynamic therapy (PDT) is gaining acceptance
as a complementary or alternative to traditional cancer
treatment because light can be employed to selectively activate
drugs that otherwise have no effect in the tumor (1–11). PDT
has been used to treat many different kinds of cancers (4,7–10)
and more recently has also been tested for other clinical
applications such as anti-bacteria and anti-virus treatments
(
human breast carcinoma) cell lines. Our studies reveal that
TCPPSO H has a small toxicity in the dark and is photo-
3
active in aqueous solutions. Its photodynamic effect is
ꢀ
compared with that of Photofrin and it is concluded that
it has a greater potential to generate singlet oxygen in vitro.
Considering the stability of the dihydro and tetrahydro
analogs of this porphyrin (17,18), our results indicate that this
family of halogenated water-soluble sensitizers is efficient for
PDT.
(
12,13). The most commonly used photosensitizer is Photof-
ꢀ
rin , a preparation of hematoporphyrin monomer, dimer,
oligomers and their dehydration products (14). Its complex
composition, the tendency to aggregate and the hyperphoto-
sensitivity of skin induced by this sensitizer for several weeks,
have been perceived as limitations of Photofrin (15). This
allied to the relatively low singlet oxygen quantum yield of the
ꢀ
MATERIALS AND METHODS
1
Apparatus. H NMR spectra were recorded on a 300 MHz Brucker-
aggregates in water, which drops to F = 0.11 in the case of
D
AMX; Mass spectra were obtained through the MS service of the
University of Swansea. The transient absorption spectra were measured
with an Applied Photophysics Laser Flash Photolysis Spectrometer,
model LKS.60, using a Continuum Surelite I Nd:YAG laser source.
Chemicals. All solvents used in the synthesis of the photosensitizer
were purified by standard methods before use. The chloroform used in
the preparative thin layer chromatography was neutralized with
neutral active alumina. Solutions were prepared with double distilled
water, either equilibrated with air or bubbled with argon at room
the hematoporphyrin derivative dimer in water (16), have
challenged researchers to synthesize new photosensitizers with
improved photodynamic action (1,2,9,17–20).
*
Corresponding author emails: stochel@chemia.uj.edu.pl (Gra z_ yna Stochel) and
mmpereira@qui.uc.pt (Mariette Pereira)
ꢁ
2007 The Authors. JournalCompilation. The AmericanSocietyofPhotobiology 0031-8655/07
8
97

8
98 Janusz M. D a˛ browski et al.
temperature. Media for cell culture (RPMI, F10) and phosphate-
buffered saline (PBS) were purchased from Biomed (Lublin, Poland).
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT),
penicillin and streptomycin are from Sigma Chemical Co.
Cell lines. The MCF7 (human breast carcinoma), SKMEL (human
melanoma) and S91 ⁄ I3 (mouse melanoma) cells were used in cytotox-
icity and photocytotoxicity experiments. MCF7 cells were cultured as
monolayers supplemented with 10% fetal calf serum (FCS),
solution. A continuous water extraction was carried out, with stirring,
until neutralization. The chloroform solution was then washed with
sodium hydrogenocarbonate and dried with MgSO4. After solvent
evaporation the chlorosulfonated porphyrins were isolated, yielding
70% and 75%, respectively.
5,10,15,20-Tetrakis(4-chlorosulfonylphenyl)porphyrin (TPPSO Cl)
2
NMR (300 MHz, CDCl3): d = 8.90 (s, 8H), 8.21 (d, 8H,
1
H
J = 7,5 Hz), 7.51 (d, 8H, J = 7,5 Hz), )2.70 (s, 2H); UV-visible
2
melanoma cells SKMEL-188 were grown in F10 medium supplemen-
5 U mL) of penicillin and 25 lg mL of streptomycin. Human
1
)1
spectrum (toluene); k
⁄ nm(ꢀ ⁄ mol cm ): 417 (3.8 · 10 ); 512
)1
)1
5
max
4
3 3 3
(1.9 · 10 ); 546 (3.61 · 10 ); 588 (6.48 · 10 ); 644 (4.4 · 10 );
)
1
)1
+
ted with 10% FCS, 100 U mL of penicillin and 100 lg mL of
streptomycin. I3 subline of Cloudman S91 melanoma cells were
cultured in RPMI 1640 medium supplemented with 100 U mL of
penicillin, 100 lg mL streptomycin and 5% FCS. All cell lines were
cultivated as monolayer in petri dishes of 60 mm diameter and
44 26 4 4 8 4.
MS(FAB)[M + H] : m ⁄ z = 1007; C H Cl N O S : calculated C
52.40, H 2.60, N 5.60, found C 52.30, H 2.90, N 5.20.
5,10,15,20-Tetrakis(2-chloro-3-chlorosulfonylphenyl)porphyrin
)1
)
1
1
(TCPPSO Cl) H NMR (300 MHz, CDCl ): d = 8.85 (m, 4H), 8.70
2
3
(s, 8H), 8.47 (m, 4H), 8.14 (m, 4H), )2.7 (s, 2H); UV-visible spectrum
)1 )1 5 4
incubated at 37ꢂC in a humid atmosphere containing 5% CO
Porphyrin synthesis. 5,10,15,20-Tetrakisarylporphyrins were pre-
2
.
(toluene); kmax ⁄ nm(ꢀ ⁄ mol cm ): 423 (2.27 · 10 ); 513 (1.40 · 10 );
3 3 2 +
545 (2.05 · 10 ); 589 (4.22 · 10 ); 645 (5.40 · 10 ); MS(FAB)[M ]:
44 22
m ⁄ z = 1146; C H Cl N O S 2H O: calculated C 44.69, H 2.22, N
pared according to the nitrobenzene method described in the literature,
(26,27) and involved the condensation of pyrrole (0.7 mL, 10 mmol)
8
4
8
4.
2
4.74, S 10.8, found C 44.82, H 2.77, N 4.24, S 10.66.
and the desired aromatic aldehyde (10 mmol) in the presence of acetic
acid (75 mL, 1.3 mol) and nitrobenzene (50 mL, 490 mmol) preheated
to 120ꢂC. The temperature was maintained for 1 h and then the
solution was cooled to room temperature to give the porphyrin crystals
General hydrolysis method. The hydrolysis of the chlorosulfonated
porphyrins (170 mg) was performed by suspending them in water
(150 mL) during 12 h at 80ꢂC to give the corresponding 5,10,15,
20-tetrakis (4-sulfophenyl) porphyrin (TPPSO H) and 5,10,15,20-
3
which were filtered off and dried.
5
CDCl
tetrakis(2-chloro-3-sulfophenyl)porphyrin (TCPPSO H), after water
3
1
,10,15,20-Tetrakisphenylporphyrin (TPP) H NMR (300 MHz,
evaporation, with 90 and 93%, respectively.
5,10,15,20-Tetrakis(4-sulfophenyl)porphyrin
1
3
): d = 8.80 (s, 8H), 8.17 (m, 8H), 7.68 (m, 12H), )2.82 (s, 2H);
(TPPSO H)
3
H
+
MS (FAB) [M + H] : m ⁄ z = 615; UV-visible spectrum (toluene);
NMR (300 MHz, D O): d = 8.75 (s, 8H), 8.30 (d, 8H, J = 7.7 Hz),
2
max ⁄ nm (ꢀ ⁄ mol cm)1 ): 418 (2.72 · 10 ); 514 (1.81 · 10 ); 548
)
1
5
4
)
k
(
7.20 (d, 8H, J = 7.7 Hz), MS(electrospray)[M ) H] : m ⁄ z = 933;
4
4
3
)1
)1
1.24 · 10 ); 592 (1.02 · 10 ); 650 (4.90 · 10 ); C44 N
C 86.00, H 4.90, N 9.10, found C 85.50, H 5.20, N 9.30. Yield: 20%.
H
30
4
: calculated
UV-visible spectrum (H O ⁄ PBS);
k
⁄ nm(ꢀ ⁄ mol cm
): 413
3
2
max
5
3
3
(2.00 · 10 ); 518 (5.80 · 10 ); 578 (2.40 · 10 ); 554 (3.10 · 10 ); 634
1
3
5
,10,15,20-Tetrakis(2-chloro-phenyl)porphyrin (TCPP) H NMR
300 MHz, CDCl ): d = 8.78 (s, 8H), 8.53–8.37 (m, 4H), 7.94–7.86 (m,
2H), )2.2 (s, 2H); MS (FAB): m ⁄ z = 751 (M ); UV-visible spectrum
(1.10 · 10 ).
5,10,15,20-Tetrakis(2-chloro-3-sulfophenyl)porphyrin (TCPPSO H)
(
1
3
3
+
1
H NMR (300 MHz, CD OD): d = 8.94 (s, 8H), 8.44 (8H, bs), 8.09
(4H, bs), 7.97 (4H, bs), MS(FAB)[M ]: m ⁄ z = 1075. UV-visible
spectrum (H O ⁄ PBS); k
3
)
1
)1
5
4
+
(toluene); kmax ⁄ nm(ꢀ ⁄ mol cm ): 418 (3.14 · 10 ); 512 (2.13 · 10 );
3
3
3
)1
)1
5
5
41 (4.99 · 10 ); 589 (6.49 · 10 ); 655 (4.09 · 10 ); C44
H26Cl
4
N
4
:
⁄ nm(e ⁄ mol cm ): 413 (1.60 · 10 ); 513
2
max
3
3 3 2
calculated C 70.23, H 3.48, N 7.45, found C 69.94, H 3.49, N 7.52.
Yield: 9%.
(7.03 · 10 ); 549 (1.20 · 10 ); 581 (2.50 · 10 ); 633 (5.04 · 10 ).
Light source dosimetry. All irradiation experiments were performed
using a microprocessor HOP 250 halogen lamp (Optel, Opole, Poland).
The light intensity could be modulated manually. The light source was
equipped with a set of by-pass filters covering various spectral regions.
The cutoff type filter < 600 nm was used to test the photodynamic
General chlorosulfonation method. Chlorosulfonation of the por-
phyrins was carried out according to a method developed by some of
us (28). The required porphyrin (150 mg) and chlorosulfonic acid
(
8 mL) were stirred at room temperature (TPP) and 50ꢂC (TCPP)
during 1 h. After this period, chloroform (200 mL) was added to the
effect of TCPPSO H toward S91, SKMEL-188 and MCF7 cells. In
3
3
Scheme 1. Schemes of synthesis of water-soluble 5,10,15,20-tetrakis(2-chloro-3-sulfophenyl)porphyrin (TCPPSO H).

Photochemistry and Photobiology, 2007, 83 899
each experiment the emission beam was directed to the irradiation site
with the optical fiber which ended 1 cm away from the culture plate.
The emission spectrum of the halogen lamp was recorded using a
spectroradiometer IL2000 (Spectrocube). This set-up supports the light
with fluence rate of 26 mW cm in the range 600–710 nm. In this
spectral range most of the photosensitizers currently used in PDT or
tested clinically have one of their absorption maxima. The photosen-
The cell survival was expressed by the absorbance changes of the
formazan salt, and survival rate was given as the percent ratio of viable
treated cells vs. the number of viable untreated cells. Number of cells
was determined from linear regression of a calibration curve. Addi-
tionally the long term survival test was performed to determine the
)
2
3
potential cytotoxicity of TCPPSO H against SKMEL 188, S91 and
MCF7 cells. The cells were incubated with photosensitizer at different
)4
)7
sitizer TCPPSO
3
H exhibits an absorption band at 633 nm, with a
concentrations (10 –10 M) for 90 min at 37ꢂC. After that the cells
were washed twice with PBS and incubated in growth medium at 37ꢂC
for 48 h. The cytotoxicity in the dark was monitored by counting the
width at half-maximum ca. 20 nm. We estimate that only one-fifth of
the available light flux is actually used by our photosensitizer.
UV ⁄VIS absorption spectra and photodegradation experiments. UV ⁄ VIS
absorption spectra were recorded in quartz cuvettes with Shimadzu
number of untreated cells and TCPPSO H treated cells.
3
Time-dependent cellular uptake. SKMEL 188, S91 and MCF7 cells
4
2100 spectrophotometer or with Carry 50 Biospectrophotometer
were seeded on 96-well plates at 1 · 10 cells per well and exposed to
(Varian, Mulgrave, USA). In order to check the possible photodeg-
3
20 lM concentrations of TCPPSO H in PBS for various time
radation during biological experiments the irradiation was carried out
using the halogen lamp described above. Solutions containing por-
phyrin dissolved in the phosphate buffer (pH 7.4) were irradiated in
petri dishes for various time intervals. The absorption spectra were
recorded before the irradiation and immediately after set irradiation
times. Time of irradiation was between 5 and 30 min.
Fluorescence quantum yield measurements. Fluorescence studies
were carried out using a SPEX Fluorolog 3-22 spectrofluorimeter,
equipped with a 300 W Xenon lamp. The fluorescence spectra of
intervals, from 10 min up to 180 min to probe time-dependent drug
accumulation. At the end of the incubation interval, the cells were
washed three times with PBS and solubilized in 100 lL of 0.25%
Triton X-100 in PBS. The retention of cell-associated porphyrin was
detected by fluorescence measurement of the accumulated porphyrin
with the ELISA reader using excitation ⁄ emission wavelengths of 405
and 635 nm, respectively.
Cell photosensitization. Cells were prepared as described above. On
)
5
the basis of cytotoxicity assays, a 2 · 10 M concentration of
TCPPSO H was selected for the photosensitization assays. The cells
were incubated for 1.5 h at 37ꢂC and then irradiated at a 26 mW cm
TCPPSO
3
H was measured in water buffer solutions (KH
2
PO
4
⁄ NaOH,
3
)
2
pH 7.4) with excitation wavelength at 413 nm (A413 = 0.025). The
water buffer solutions placed in 1 cm quartz cuvettes were air-
equilibrated before making the fluorescence measurements.
)
2
fluence rate and in doses ranging from 0.1 to 8.7 J cm . We recall that
TCPPSO H uses only approximately one-fifth of the available fluence
3
Triplet state lifetime measurements. The triplet state lifetimes were
measured with the Laser Flash Photolysis Spectrometer described
rate. The MTT test was performed immediately after irradiation. The
values were obtained from three independent experiments and
expressed as percent of cell survival with reference to control cells,
which were manipulated in the same manner but without incubation
with photosensitizer and without illumination.
above. The kinetic curves of the transient absorption of TCPPSO
the range 270–750 nm were registered after the laser excitation
k = 355 nm, Emax = 100 mJ ⁄ pulse and full width at half maximum,
3
H in
(
FWHM = 6 ns). At least ten kinetic runs were registered under each
set of conditions and their average was recorded. The measurements
were made at 20ꢂC. All porphyrin solutions were freshly prepared
before the measurements. The stability of the samples was confirmed
by comparing their absorption spectra before and after each flash
photolysis experiment. The samples showed less than 5% degradation
after 1000 laser flashes at 355 nm.
For the long-term phototoxicity test the S91 cells were incubated
with TCPPSO H (20 lM) for 90 min at 37ꢂC, then the photosensitizer
3
solution was substituted by 100 lL of PBS and cells were irradiated at
)
2
a light dose of 6.2 J cm . After irradiation the PBS solution was
replaced with culture medium. 24 h after irradiation the MTT test was
performed and photographs of the treated and treated S91 cells were
taken using a Nikon Eclipse TS-100 F inverted microscope equipped
with Nikon digital camera Coolpix 8400 (under magnification 40·).
The photolysis experiments run in the presence of oxygen involved
the saturation of the solutions with oxygen, by flushing with this gas at
2
0ꢂC. For the experiments in the absence of oxygen, the solutions were
purged with argon until no change in the decay rate was observed, and
were kept under argon during the measurements.
RESULTS AND DISCUSSION
Quantum yield of singlet molecular oxygen formation. Singlet molecu-
lar oxygen phosphorescence at 1270 nm was detected at room tempera-
ture following laser excitation of aerated D O solutions containing the
2
sensitizer at the concentration necessary to produce an absorbance of 0.2
in 1 cm quartz cuvette at the wavelength of excitation. The excitation of
the samples at 355 nm (the third harmonic of a Nd-YAG laser Spectra-
Physics Quanta-Ray GRC-130) was used as usual, but the flash
photolysis equipment was modified to allow for luminescence detection
in the 800–1400 nm region. The emission was filtered by a Melles Griot
dielectric mirror reflecting more than 99.5% of the incident light in the
Photophysical and photochemical properties of TCPPSO
The ground state absorption spectra of the 5,10,15,20-tetrakis
(2-chloro-3-sulfophenyl)porphyrin (TCPPSO H), recorded at
room temperature in phosphate buffer water solutions (PBS,
pH = 7.4) are presented in Fig. 1.
3
H
3
610–860 nm range, and by a Scotch RG665 filter. The infrared emission
transmitted by these filters was resolved by a monochromator with a
6
00-line grating mounted in the place of the usual UV ⁄ VIS grading. The
wavelength of 1270 nm was selected for detection in a Hamamatsu
R5509-42 photomultiplier, cooled to 193 K in a liquid nitrogen chamber
(
Products for Research model PC176TSCE005) (22).
Cytotoxicity and cell survival assay. SKMEL 188, S91 and MCF7
4
cells were seeded into a 96-well culture plate at 1 · 10 cells in a culture
medium. After 24 h, the cells were incubated with TCPPSO H
3
)
4
)7
photosensitizer of different concentrations (10 –10 M) for 90 min
at 37ꢂC. The photosensitizer solution was then substituted by 100 lL
of fresh medium and 20 lL (final concentration 0.5 mg mL ) of
MTT. Reduction of MTT to insoluble formazan dye by cellular
microsomal enzymes is an indicator of cellular metabolism efficiency
)
1
3
and was used as a measure of cell response to TCPPSO H. Cells were
incubated with MTT for 3 h. Then the culture medium was replaced
with DMSO-methanol solution (1:1) to dissolve the blue formazan
crystals. The 96-well culture plate was shaken for 0.5 min at room
temperature and immediately read for optical density at 560 nm using
an ELISA reader (GENios Plus; Tecan Trading AG, Switzerland).
Figure 1. UV-VIS absorption spectra of TCPPSO
3
H in aqueous
solution, room temperature. Inset shows photodegradation of TCP-
2
+
2+
3
PSO H in phosphate-buffered saline with Ca and Mg , pH = 7.4.

9
00 Janusz M. D a˛ browski et al.
Table 1. The molar absorption coefficients of TCPPSO
phosphate buffer solutions (pH = 7.4).
3
H in aqueous
3
Table 2. Photophysical properties of TCPPSO H in aqueous solu-
tions: Triplet lifetimes in aqueous solutions, with respective oxygen
quenching rate constants, singlet oxygen and fluorescence quantum
yields.
TCPPSO
(0,0)
549
3
H
1
B(0,0)
Q
y
(0,1)
Q
y
Q
x
(0,1)
x
Q (0,0)
Conditions
s
T
⁄ ls
k
q
[M) s)1
]
u
D
u
F
9
k
´
max
(nm)
413
513
581
633
Oxygen saturated
Air equilibrated
0.50
3.7
1.4 · 10
9.3 · 10
–
0.74
–
ꢀ
(M cm)1) 1.60 · 10 7.03 · 10 1.20 · 10 2.50 · 10 5.04 · 10
)
1
5
3
3
3
2
8
8 · 10
)3
The spectra show the five bands characteristic of porphy-
monitoring wavelength and are typical of triplet states
transferring the energy quantitatively to molecular oxygen
(30). The triplet lifetimes are presented in Table 2. In argon-
saturated aqueous solution, the decays remain monoexponen-
tial (inset in Fig. 3) and lifetimes increase to the millisecond
rins. The typical etio-type spectra of meso-phenyl porphyrins
changes to phyllo-type spectra (29) after the chlorosulfonation
reaction of o-halogenated porphyrins as was previously
observed (23). The longest-wavelength absorption band at
6
33 nm is crucial for PDT process, because only red light has
sufficient tissue penetration ability. Using measured absorb-
ance for various concentrations of TCPPSO H, the molar
3
range. Transient absorption spectrum of TCPPSO H in
deoxygenated water is shown in Fig. 3. The strong negative
absorbance change is due to the bleaching of the ground-state
absorption and the clearly defined pseudoisosbestic points
reflect the fact that all molecules return to this ground state,
that is, photodegradation is negligible. The rate constant of
energy transfer from the triplet state of the photosensitizer to
3
absorption coefficients were determined from Beer’s law.
Absorption bands and corresponding molar absorption coef-
3
ficients of TCPPSO H are presented in Table 1.
The photostability of TCPPSO H was studied using a
3
halogen lamp (HOP 250) equipped with the 600 nm cutoff
filter. The conditions of this experiment were similar to those
used during biological studies: the same light source and solvent
molecular oxygen (k ) can be calculated from the triplet
q
o
lifetimes in the absence, sT , and in the presence of oxygen, sT,
using the relationship:
(
phosphate buffer, pH 7.4). However, the maximum irradiation
ꢀ
ꢁ
1
1
1
time was 30 min, while the irradiation of cells with photosen-
sitizer was just 5 min. As seen in Fig. 1, the photodegradation of
kq ¼
ꢀ s0
ꢁ
sT
½O2ꢂ
T
3
TCPPSO H in these conditions was very limited.
and the oxygen concentration in oxygen-saturated and air-
)3
The fluorescence excitation spectrum in Fig. 2 is the mirror
image of the absorption spectra and confirms the purity and
non-aggregation of the samples. Earlier studies with other
halogenated water-soluble porphyrins had established that the
presence of bulky ortho-chloro groups reduce the tendency of
porphyrins to aggregate (23). The fluorescence quantum yield
)4
equilibrated water, [O ] = 1.39 · 10 and 2.9 · 10 M (30),
2
o
o
respectively. In fact, s
irrelevant to calculate the value of k reported in Table 2. Spin
T T T
>> s and the actual value of s is
q
statistics require that k should be 1 ⁄ 9 of the diffusion rate
constant, kdiff. The value of kdiff was estimated from the
fluorescence intensity of 5,10,15,20-tetrakis(4-sulfophe-
q
3 F
of TCPPSO H (u = 0.0082, air-equilibrated) was measured
according to published procedures (30) and calculated relat-
ively to that of TPPSO H (uF = 0.054, degassed).
nyl)porphyrin (TPPSO H) in the absence and in the presence
3
of oxygen, its lifetime in the absence of oxygen and the oxygen
3
The kinetics of the transient absorption changes (DA) of the
,10,15,20-tetrakis(2-chloro-3-chlorosulfophenyl)porphyrin in
5
solution were determined by laser flash photolysis. The spectra
were constructed from fitting data from the sets of the decay
curves, collected with 10-nm intervals. The transient decay
times in the presence of oxygen are independent of the
3
Figure 3. Transient absorption spectra of TCPPSO H in water,
recorded in the absence of oxygen, 20ꢂC, kexc = 355 nm. Inset: decay
Figure 2. Emission spectra of TCPPSO
temperature,kexc = 413 nm.
3
H in aqueous solution, room
curve of the triplet state signal of the TCPPSO
oxygen.
3
H in the absence of

Photochemistry and Photobiology, 2007, 83 901
(Fig. 4, inset). The potential toxicity of TCPPSO H in the dark,
0
concentration in air-saturated water. Using s
S
= 9.0 ns (31),
3
1
0
)1
)1, in good agreement with
literature values for similar systems (32). The values of kq
reported in Table 2 indeed approach 1 ⁄ 9 kdiff, as in related
compounds (23).
we calculate kdiff = 3 · 10
M
s
S91, SKMEL-188 and MCF7 cell lines (data not shown)
confirmed that S-91 was the most sensitive cell line. In all
subsequent PDT experiments the sublethal concentration of
)5
2 · 10 M was used to induce cell photodamage.
The singlet oxygen quantum yield was obtained from its
phosphorescence at 1270 nm using the method described in
Materials and Methods. The literature value for the singlet
Time-dependent cellular uptake
molecular oxygen quantum yield obtained with TPPSO H in
deuterated water is 0.64 at pH 7.4 (33). Extrapolating to time-
zero the decays of the singlet molecular oxygen emissions
3
The time dependent uptake was investigated in all tested cell
)5
3
lines exposed to 2 · 10 M of TCPPSO H solutions. The
uptake of tested compound occurred rapidly in the first 2 h,
after which it reached a plateau (Fig. 5). As can be seen the
3
measured with the reference and TCPPSO H, respectively, at a
given laser intensity, we obtained a relation between emission
intensities that is identical to the relation between the singlet
molecular oxygen quantum yields. The singlet molecular
3
uptake of TCPPSO H was the highest for S91, 20% lower for
MCF7 and 70% lower for SKMEL 188.
oxygen quantum yield was calculated relatively to TPPSO
(
3
H
Cell photosensitization
0.64) and is presented in Table 2. The observed improvement
was probably the result of lack of aggregation and of the
internal heavy atom effect. It is well known that only
monomeric forms of photosensitizer exhibit high yield of
singlet oxygen generation. The photophysical and photochem-
Cells treated with photosensitizer were irradiated with various
)2
light doses. The highest used light dose (8.7 J cm ) was
sufficient to inhibit cell metabolism in almost all cells (Fig. 4).
S91 cell line turned out to be the most sensitive (Table 3).
This cell line exhibits a low pigment content (34). Melanin, a
natural pigment, not only absorbs and scatters incident light,
but it also effectively scavenges reactive oxygen species such as
superoxide anions, hydroxyl radicals and singlet oxygen, and
3
ical properties of TPPSO H are comparable to those of other
halogenated water-soluble porphyrins (23).
Cytotoxicity assay
3
The cytotoxicity of TCPPSO H against S91 melanoma cells was
tested in the dark using the cell surviving techniques: MTT assay
or long term survival assay described in Materials and Methods.
Concentration–response experiments were performed to estab-
lish the optimal sublethal dose of the photosensitizer. Significant
)
4
cytotoxicity was observed only at a concentration of 2 · 10
M
3
Figure 5. Time-dependent uptake of TCPPSO H at 20 lM by S91,
SKMEL and 188 MCF7 cells as measured by fluorescence intensity
using excitation ⁄ emission wavelengths of 405 and 635 nm, respect-
ively.
Table 3. LD20, LD50 and LD90 values for TCPPSO
3
H (20 lM)
measured on S91, SKMEL 188 and MCF7 cells irradiated with red
.
)
2
light at the flounce rate of 26 mW cm
Light dose (J cm)
SKMEL 188
2
)
Figure 4. Surviving fraction of three different cell lines: S91 (d),
SKMEL 188 (m) and MCF 7 ( ) determined by MTT assay. Cells
Cell line
S91
MCF 7
were treated with TCPPSO
irradiated with the red light (fluence rate = 26 mW cm ). Inset shows
cytotoxicity of TCPPSO H tested in the dark against S91 cells (d). The
cells were incubated with TCPPSO H for 1.5 h, and MTT assays were
3
H at the concentration of 20 lM and
)2
LD20
LD50
LD90
0.1
1.0
4.0
0.9
2.3
5.4
1.1
2.8
6.3
3
3
then performed. The data shown are the means ± SD of three
independent experiments.

9
02 Janusz M. D a˛ browski et al.
Acknowledgements—This work has been supported in part by the
Polish Ministry of Science and Higher Education (Grant No.
1
˜
283 ⁄ T09 ⁄ 1005 ⁄ 29), Funda c¸ ao para a Ci eˆ ncia e Tecnologia and
FEDER (Project No. POCTI ⁄ QUI ⁄ 555505 ⁄ 2004).
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