Photodynamic studies of metallo 5,10,15,20-tetrakis(4-methoxyphenyl) porphyrin: Photochemical characterization and biological consequences in a human carcinoma cell line

Article abstract of DOI:10.1562/0031-8655(2001)074<0014:PSOMTM>2.0.CO;2

The photodynamic activities of the free-base 5,10,15,20-tetrakis(4-methoxyphenyl)porphyrin (TMP) and their metal complexes with zinc(II) (ZnTMP), copper(II) (CuTMP) and cadmium(II) (CdTMP) have been compared in two systems: reverse micelle of n-heptane/so

Full text of DOI:10.1562/0031-8655(2001)074<0014:PSOMTM>2.0.CO;2

Photodynamic Studies of Metallo 5,10,15,20-Tetrakis(4-methoxyphenyl)
porphyrin: Photochemical Characterization and Biological Consequences in a
Human Carcinoma Cell Line
Author(s): M. E. Milanesio, M. G. Alvarez, E. I. Yslas, C. D. Borsarelli, J. J. Silber, V. Rivarola, and E.
N. Durantini
Source: Photochemistry and Photobiology, 74(1):14-21.
Published By: American Society for Photobiology
DOI: http://dx.doi.org/10.1562/0031-8655(2001)074<0014:PSOMTM>2.0.CO;2
URL: http://www.bioone.org/doi/full/10.1562/0031-8655%282001%29074%3C0014%3APSOMTM
%3E2.0.CO%3B2
BioOne (www.bioone.org) is a nonprofit, online aggregation of core research in the biological, ecological, and
environmental sciences. BioOne provides a sustainable online platform for over 170 journals and books published
by nonprofit societies, associations, museums, institutions, and presses.
Your use of this PDF, the BioOne Web site, and all posted and associated content indicates your acceptance of
BioOne’s Terms of Use, available at www.bioone.org/page/terms_of_use.
Usage of BioOne content is strictly limited to personal, educational, and non-commercial use. Commercial inquiries
or rights and permissions requests should be directed to the individual publisher as copyright holder.
BioOne sees sustainable scholarly publishing as an inherently collaborative enterprise connecting authors, nonprofit publishers, academic institutions, research
libraries, and research funders in the common goal of maximizing access to critical research.

Photochemistry and Photobiology, 2001, 74(1): 14–21
Photodynamic Studies of Metallo 5,10,15,20-Tetrakis(4-methoxyphenyl)
porphyrin: Photochemical Characterization and Biological Consequences
in a Human Carcinoma Cell Line¶
M. E. Milanesio,1 M. G. Alvarez,2 E. I. Yslas,2 C. D. Borsarelli,3 J. J. Silber,1 V. Rivarola2, and
E. N. Durantini*1
1
2
Departamento de Qu´ımica y F´ısica and Biolog´ıa Molecular, Universidad Nacional de R´ıo Cuarto, R´ıo Cuarto, Argentina
and
3Instituto de Ciencias Qu´ımicas, Universidad Nacional de Santiago del Estero, Santiago del Estero, Argentina
Received 3 October 2000; accepted 8 April 2001
ABSTRACT
ing applications of porphyrins in medicine is in the detection
and cure of tumors (2,3). Photodynamic therapy (PDT)† is
an early new technique for treating cancer. After adminis-
tration of a photosensitizer, which is selectively retained by
tumor cells, the subsequent irradiation with visible light in
the presence of oxygen specifically inactivates neoplastic
cells (4,5). Recently, several porphyrin derivatives with po-
tential use in the treatment of tumors have been synthesized
(6–10). Basically two types of reactions can occur after pho-
toactivation of the photosensitizer. One involves the gener-
ation of free radicals (type-I photochemical reaction) and the
The photodynamic activities of the free-base 5,10,15,20-
tetrakis(4-methoxyphenyl)porphyrin (TMP) and their
metal complexes with zinc(II) (ZnTMP), copper(II)
(CuTMP) and cadmium(II) (CdTMP) have been com-
pared in two systems: reverse micelle of n-heptane/sodi-
um bis(2-ethylhexyl)sulfosuccinate/water bearing pho-
tooxidizable substrates and Hep-2 human larynx carci-
noma cell line. The quantum yields of singlet molecular
oxygen, O2(1
CdTMP in tetrahydrofuran, were determined yielding
values of 0.65, 0.73 and 0.73, respectively, while O2(1
⌬g), production (⌽⌬) of TMP, ZnTMP and
other the production of singlet molecular oxygen, O2(1
⌬ )
g
⌬
)
g
(type II) as the main species responsible for cell inactivation
(5). Evidence favors the role of the type-II process in cells,
although the photodynamic process of the sensitizers on neo-
plastic tissues is still not well understood (11). The mecha-
nism of PDT effects may involve a direct tumor cell injury
and also an indirect cell killing via microcirculatory changes
resulting in reduced blood flow in the tumor (5,12,13).
Photosensitizers are deemed to have specific chemical and
biological properties (2). Two of the photochemical requi-
sites are a high absorption coefficient in the visible region
of the spectrum, mainly in the phototherapeutic window
(600–1000 nm) and a long lifetime of the triplet excited state
formation was not detected for CuTMP. In the reverse
micellar system, the amino acid -tryptophan (Trp) was
used as biological substrate to analyze the O2(1
g)-medi-
L
⌬
ated photooxidation. The observed rate constants for Trp
photooxidation (kobsTrp) were proportional to the sensitiz-
Ϫ
Ϫ1
er quantum yield of O2(1 107 s 1 M
⌬g). A value of ϳ2 ؋
was found for the second-order rate constant of Trp
(krTry) in this system. The response of Hep-2 cells to cy-
totoxicity photoinduced by these agents in a biological
medium was studied. The Hep-2 cultures were treated
with 1
exposed to visible light. The cell survival at different light
exposure levels was dependent on ⌬. Under these con-
ditions, the cytotoxic effect increases in the order: Cu-
TMP TMP ZnTMP CdTMP, correlating with the
production of O2(1
g). A similar behavior was observed
in both the chemical and biological media indicating that
the O2(1
g) mediation appears to be mainly responsible
␮M of porphyrin for 24 h at 37؇C and the cells
to efficiently produce O2(1
⌬g) (3). Studies on the triplet state
⌽
carried out with a series of 5,10,15,20-tetrakis (methoxy-
phenyl) porphyrins have shown that these synthetic porphy-
rins are effective photosensitizers which can be used as mod-
el compounds to investigate the theoretical and instrumental
aspects of PDT (14,15). The spectroscopic properties and
K
Ͻ
ϳ
⌬
⌬
quantum yield of O2(1
⌬g) production (⌽⌬) of porphyrins can
for the cell inactivation.
INTRODUCTION
The tetrapyrrolic macrocycles play highly diverse roles in
biological systems (1). One of the more recent and promis-
†Abbreviations: AOT, sodium bis(2-ethylhexyl)sulfosuccinate;
CdTMP, cadmium 5,10,15,20-tetra(4-methoxyphenyl)porphyrin;
CuTMP, copper 5,10,15,20-tetra(4-methoxyphenyl)porphyrin;
DMA, 9,10-dimethylanthracene; ⑀, extinction coefficient; FCS, fe-
tal calf serum; 1HNMR, proton nuclear magnetic resonance;
HPLC, high-performance liquid chromatography; LDL, low-den-
sity lipoprotein; PBS, phosphate-buffered saline; PDT, photody-
namic therapy; SDS, sodium dodecyl sulfate; TB, tryptan blue;
TLC, thin-layer chromatography; TMP, 5,10,15,20-tetra(4-meth-
¶Posted on the website on 18 April 2001.
*To whom correspondence should be addressed at: Departamento
de Qu´ımica y F´ısica, Universidad Nacional de R´ıo Cuarto, Agen-
cia Postal Nro. 3, 5800 R´ıo Cuarto, Argentina. Fax: 54-358-
4676233; e-mail: edurantini@exa.unrc.edu.ar
oxyphenyl)porphyrin; TPP, tetraphenylporphyrin; Trp,
L-trypto-
᭧
2001 American Society for Photobiology 0031-8655/00
$5.00ϩ0.00
phan; ZnTMP, zinc 5,10,15,20-tetra(4-methoxyphenyl)porphyrin.
14

Photochemistry and Photobiology, 2001, 74(1) 15
panol/n-hexane, flow 0.5). Uniplate Silica gel GHLF 250 m thin-
layer chromatography (TLC) plates from Analtech (Newark, DE)
and silica gel 200–400 mesh for column chromatography from Al-
drich (Milwaukee, WI) were used.
All the chemicals from Aldrich were used without further purifi-
cation. Sodium dodecyl sulfate (SDS) from Merck (Deutschland,
Germany) was used as received. D,L-␣-Dipalmitoyl phosphatidyleth-
␮
be significantly changed by forming complexes with metals
(16–19).
The aim of this study was to compare the photodynamic
effect of metal-free 5,10,15,20-tetrakis(4-methoxyphenyl)
porphyrin (TMP), zinc (ZnTMP), copper (CuTMP) and cad-
mium (CdTMP), first in a simple biological model formed
by reverse micelles bearing a photooxidizable amino acid
and then in a more complex system of a human carcinoma
cell line. Tetrapyrrolic macrocycle–bearing methoxy substit-
uents in the meso-phenyl groups appear to present adequate
photophysical properties to be used as a sensitizer model
(14) and the increase in polarity with respect to tetraphen-
ylporphyrin (TPP), keeping its lipophilic character, could fa-
vor the interaction with biological media (20). In most cases,
the formation of metalloporphyrin complexes cause changes
in the free-base photophysical properties (21). Usually the
presence of Zn(II)- and Cd(II)-forming complexes with por-
anolamine from Sigma (St. Louis, MO) was used in liposome prep-
aration. Solvents from Merck (GR grade) were distilled and stored
˚
over 4 A molecule sieves. Tetrahydrofuran was distilled from lithi-
um aluminium hydride under an argon atmosphere. AOT from Sig-
ma was dried under vacuum and used without further purification.
Ultrapure water was obtained from Labonco equipment model
90901-01.
Porphyrins. TMP was synthesized by the condensation of 4-meth-
oxybenzaldehyde and pyrrole, using the Lindsey method (28). A
solution of 4-methoxybenzaldehyde (0.152 mL, 1.5 mmol) and pyr-
role (0.10 mL, 1.5 mmol) in 100 mL of dichloromethane was treated
with trifluoroacetic acid (35
␮L, 0.45 mmol) for 60 min at room
temperature in argon atmosphere. Then 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (0.34 g, 1.5 mmol) was added and the mixture
was stirred for an additional 60 min, open to the atmosphere. The
solvent was removed under reduced pressure and flash column chro-
matography (silica gel, dichloromethane) afforded 98 mg (35%) of
pure TMP. TLC (silica gel, dichloromethane) Rf 0.46. HPLC tR 7.4
phyrins produces an increase in the O2(1
⌬g) generation, while
the complex with Cu(II) presents a lack of photodynamic
activity. Thus, the Cu(II) complex can be used to analyze
the influence of type-I photoreaction in the biological system
studied (16,17,21).
The absorption and emission spectroscopic properties of
the porphyrins were first studied in a set of pure solvents
1
min. HNMR (CDCl3, tetramethylsilane) ␦ Ϫ2.76 (brs, 2H, pyrrole
N–H), 4.08 (s, 12H, Ar-OCH3), 7.26 (d, 8H, J
Ar 3,5-H), 8.10 (d, 8H, J 8.5 Hz, 10,15,20-Ar 2,6-H), 8.8–8.90
(s, 8H, pyrrole). MS [m/z] 734.3 (Mϩ) (734.2895 calculated for
C48H38N4O4). Absorption spectrum (dichloromethane, nm) (ex-
tinction coefficient [
], dm3 molϪ cmϪ1) 422 (463 200), 518
ϭ 8.5 Hz, 5,10,15,20-
ϭ
␭
max
1
and in various heterogeneous media. The O2(1
⌬
g) quantum
⌬) of the porphyrins were evaluated in a solution
of tetrahydrofuran.
⑀
yields (
⌽
(14 130), 556 (9750), 593 (4340), 650 (4820).
For the preparation of ZnTMP a solution of TMP (20 mg, 0.027
mmol) in 7 mL of dichloromethane was treated with 3 mL of a
saturated solution of zinc(II) acetate in methanol. The mixture was
stirred for 30 min at room temperature. Solvents were evaporated
under reduced pressure and flash chromatography (silica gel, dichlo-
romethane) afforded 20 mg (94%) of pure ZnTMP. TLC (silica gel,
dichloromethane) Rf 0.41. HPLC tR 7.9 min. MS [m/z] 797.7 (Mϩ)
Reverse micelles of n-heptane/sodium bis(2-ethylhex-
yl)sulfosuccinate (AOT)/water bearing photooxidizable bio-
logical substrate
mediation of O2(1
amino acid is subject to O2(1
L
-tryptophan (Trp) were used to study the
g) in the photooxidation mechanism. This
g) damage and it has been used
⌬
⌬
(797.666 calculated for C48H36N4O4Zn). Absorption spectrum
(dichloromethane, nm) (
(13 510), 598 (6940).
␭
to analyze the sensitizer activity toward biological substrates
in different systems (22–24). On the other hand, microhet-
erogeneous systems such as micelles and reverse micelles
are frequently used as simplified models for biological mem-
branes and enzyme pockets where various photodynamic ef-
fects can take place (25–27).
The response of Hep-2 human larynx carcinoma cell line
to cytotoxicity photoinduced by the porphyrins was evalu-
ated in the biological system. The survival of the irradiated
cells, previously treated with the porphyrin, was dependent
on both light exposure levels and sensitizer photochemical
properties. The effect in both the media shows a correlation
max
1
⑀
, dm3 molϪ cmϪ1) 425 (406 800), 558
CuTMP was prepared by using a solution of TMP (20 mg, 0.027
mmol), copper(II) acetate monohydrate (156 mg, 0.78 mmol) and
glacial acetic acid (5 mL) in 3 mL of chloroform which was refluxed
for 2 h in argon atmosphere. The solvents were removed under vac-
uum. Flash column chromatography (silica gel, dichloromethane/
methanol 0.7%) afforded 16 mg (75%) of pure CuTMP. TLC (silica
gel, dichloromethane) Rf 0.17; HPLC tR 9.2 min. MS [m/z] 795.8
(Mϩ) (795.8202 calculated for C48H36N4O4Cu). Absorption spectrum
1
␭
max
(dichloromethane, nm) (
⑀
, dm3 molϪ cmϪ1) 418 (325 000), 541
(17 540), 578 (3480).
For the preparation of CdTMP TMP (20 mg, 0.027 mmol) in
pyridine (8 mL) and saturated potassium hydroxide in methanol (4
mL) were treated with cadmium acetate dihydrate (18 mg, 0.067
mmol). After a 2 h reflux the solution was cooled and treated with
water (30 mL). The organic phase was extracted with three portions
of dichloromethane (15 mL each) and the solvents removed under
vacuum to give 14 mg (61%) of pure CdTMP. MS [m/z] 844.7 (Mϩ)
between the O2(1
ity upon photooxidizable substrates and Hep-2 cells. There-
fore, the intermediacy of O2(1
g) appears to be mainly re-
⌬g) production and the photodynamic activ-
⌬
sponsible for the photocytotoxic activities of these porphy-
rins. The results contribute to the understanding of the pho-
todynamic process induced by these agents and the
sensitivity of Hep-2 cells to this photodamage.
(844.6857 calculated for C48H36N4O4Cd). Absorption spectrum
␭
max
1
(dichloromethane, nm) (
(15 020), 616 (15 870).
⑀
, dm3 molϪ cmϪ1) 436 (367 700), 572
Irradiation. The light source used was a Kodak slide projector
equipped with a 150 W lamp. The light was filtered through a glass
filter and a 3 cm water layer to absorb heat. In biological studies, a
wavelength range between 350 and 800 nm was selected by optical
filters (29), while a range of 455–800 nm (GG455 cut-off filter) was
used in the micellar system. The light intensity at the treatment site
MATERIALS AND METHODS
General. UV–visible and fluorescence spectra were recorded on a
Shimadzu UV-2401PC spectrometer and on a Spex FluoroMax fluo-
rometer, respectively. Proton nuclear magnetic resonance (1HNMR)
spectra was recorded on a Varian Gemini spectrometer at 300 MHz.
Mass spectra were taken with a Varian Matt 312 operating in EI
mode at 70 eV. The high-performance liquid chromatography
(HPLC) chromatograms were performed in a Varian 5000 liquid
chromatograph equipped with a Varian 2550 UV–visible variable-
wavelength detector and Varian MicroPak SI-5 column (3% 2-pro-
2
was 18.0 and 15.5 mW cmϪ (Radiometer Laser Mate-Q, Coherent,
Hilton, Australia).
Spectroscopic studies. Absorption spectra were recorded at 25.0
Ϯ
0.5
Њ
C using 1 cm path length cells. Wavelength maxima (
␭
)
max
were measured by taking the midpoint between the two positions of
the spectrum where the absorbance of the band is equal to 0.9 Amax

16 M. E. Milanesio et al.
(30). The fluorescence quantum yield (
␾
F) of porphyrins was cal-
Table 1. The Soret band absorbance peak of porphyrins in differ-
culated by a comparison of the area below the corrected emission
spectrum in tetrahydrofuran with that of TPP as a fluorescence stan-
ent media
dard, exciting at
in tetrahydrofuran was calculated by comparison with the fluores-
cence spectrum in toluene using 0.11 (32) and taking into
account the refractive index of the solvents (31). Singlet molecular
oxygen O2(1
g) sensitization was measured by using time-resolved
␭
ϭ 550 nm (31). A value of ␾ ϭ 0.10 for TPP
e
x
F
Soret band
␭
(nm)
max
Medium
n-Heptane
Tetrahydrofuran
Dichloromethane
N,N-Dimethylformamide
AOT reverse micelles*
FCS suspension†
TMP ZnTMP CuTMP CdTMP
␾
ϭ
F
⌬
417.8
419.9
421.2
421.9
417.7
407.0
419.7
425.4
422.3
428.0
420.7
408.8
415.2
417.7
418.0
418.9
415.3
408.5
432.0
434.5
437.8
439.7
432.4
408.2
phosphorescence detection method. A Q-switched Nd:YAG laser
(Spectron SL400, Warwickshire, England) was used as the excitation
source operating at 532 nm (20 ns halfwidth) in order to excite the
porphyrins. The emitted radiation (mainly 1270 nm) was detected
at right angles with an amplified Judson J16/8Sp germanium detector
after passing it through appropriate filters. The output of the detector
was coupled to a digital oscilloscope (Hewlett–Packard HP-
54504A). About 20 shots are usually needed for averaging decay
times in order to get a good signal to noise ratio. No change, indi-
cating significant photodegradation of the sensitizer, was observed
in the porphyrin absorption spectrum after these experiments. The
averaged signals were analyzed as single exponential decays by us-
ing the calculation software (Microcal Origin 4.1, Northampton,
*n-heptane/AOT (0.1 M)/water (W
†10% vol/vol in PBS.
ϭ 10).
␮
L of porphyrin, incorporated into liposomes, was added to the cul-
ture flask bearing 5 mL of the medium. The cells were treated with
1 ␮M of porphyrin for 24 h in dark condition, the medium containing
MA). TPP in tetrahydrofuran was used as the standard (
⌽
ϭ
0.62)
⌬
(33). The absorbances of the sample and reference were matched at
the irradiation wavelength. Measurements of these solutions under
the photosensitizer was later discarded. Cells were washed three
times with the medium and kept in 5 mL of the same. The dishes
were exposed to visible light for different time intervals. After each
irradiation time the viability of the cells was estimated by micros-
copy with trypan blue (TB) exclusion test using a Neubauer chamber
counter (29). The same procedure without irradiation was carried
out to determine dark toxicity. The uptake of porphyrin by Hep-2
cells was determined by treating the cells with 2.0 mL of tetrahy-
drofuran. The mixture was sonicated for 15 min and centrifuged at
9000 rpm for 30 min. The concentration of the porphyrin in the
supernatant was measured by HPLC using the condition described
above. The pick area values obtained from each sample refer to the
total number of cells contained in the suspension. The concentration
of the porphyrin in this sample was estimated by comparison with
a calibration curve obtained with standard solutions of the porphyrin
the same conditions afforded
⌽ for the porphyrins studied. This
⌬
was obtained by a direct comparison of the slopes in the linear
region of the plots of phosphorescence amplitude extrapolate to zero
vs the laser total energy (34).
Studies in reverse micelles. A stock solution of AOT 0.1 M was
prepared by weighing and dilution in n-heptane. The addition of
water to the corresponding solution was performed using a calibrated
microsyringe. The amount of water present in the system was ex-
pressed as the molar ratio between water and the AOT present in
the reverse micelle (W
ϭ [H2O]/[AOT]). In all experiments, W ϭ
10 was used. The appropriate amount of stock solution (2 mL) was
transferred into a quartz cuvette of 1 cm optical path and appropriate
amounts of TMP, 9,10-dimethylanthracene (DMA) and Trp in stock
in tetrahydrofuran ([porphyrin]
ϳ 0.1–5 ␮M). For CdTMP, which
solution of AOT (0.1 M, W
ϭ 10) were added. The mixtures were
decomposes on silica gel column, the uptake was determined by
adding 1.0 mL of 4% SDS to 1 mL of the cellular suspension. The
mixture was incubated for a further 15 min (in the dark and at room
temperature) and centrifuged at 9000 rpm for 30 min. The concen-
tration of CdTMP in the supernatant was measured by spectrofluor-
imetry in a solution of 2% SDS in PBS. The fluorescence values
obtained from each sample referred to the total number of cells
contained in the suspension. The concentration of the porphyrin in
this sample was estimated by comparison with a calibration curve
obtained with standard solutions of CdTMP in 2% SDS ([CdTMP]
sonicated to obtain a perfectly clear micellar system. The photoox-
idation of Trp was evaluated using DMA as the actinometer under
the same experimental conditions. The kinetics of DMA and Trp
photooxidation in n-heptane/AOT/water reverse micelles were stud-
ied by following the decrease of the absorbance (A) at
␭
ϭ 378
max
nm and the fluorescence intensity (F) at ␭ ϭ 350 nm. The Trp fluo-
rescence was excited by 290 nm light (24). A control experiment
showed that under these experimental conditions the fluorescence
intensity is linearly correlated with Trp concentration. The observed
rate constants (kobs) were obtained by a linear least squares fit of the
semilogarithmic plot of ln F0/F or ln A0/A vs time. All the experi-
ϳ
0.1–5 ␮M). Four culture flasks were used for each incubation
time. Every experiment was compared with a culture control without
porphyrin.
ments were performed at 25.0
Ϯ 0.5ЊC. The pooled standard devi-
ation of the kinetic data, using different prepared samples, was less
than 5%.
Liposome preparation. The incorporation of the porphyrins into
the phospholipid bilayer of the D,L-␣-dipalmitoyl phosphatidyletha-
RESULTS AND DISCUSSION
nolamine was achieved by a modification of the ethanol injection
procedure of Kremer et al. (35). Typically, 2 mL of a solution bear-
ing 9.60 mM of phospholipid, 1.91 mM of cholesterol and 0.27 mM
of porphyrin in ethanol–tetrahydrofuran binary mixture (1:1, vol/vol)
Spectroscopic studies
The absorption spectrum of TMP, ZnTMP, CuTMP and
CdTMP in dichloromethane shows the typical Soret and Q-
bands, characteristic of a free base and the corresponding
matalloporphyrins (see ‘‘Materials and Methods’’) (18). In
addition the wavelength of the Soret band absorption max-
imum of these porphyrins was studied in different media.
The results are presented in Table 1. A sharp absorption
band was obtained in every case indicating that there is no
aggregation of the porphyrin in the environment in which
the photosensitizer is localized (19,37,38). The solvato-
chromic effect on the location of the Soret absorption band
in pure solvents shows a slight blueshift upon solubilization
in a low-polarity medium.
was injected into 10 mL of phosphate-buffered saline (PBS) at 80
Њ
C.
1
The injection was performed at a speed of 50
␮
L minϪ with mag-
netic stirring (36). The final volume was reduced to 10 mL by evap-
oration of organic solvents.
Cell culture. The Hep-2 human larynx carcinoma cell line (Aso-
ciacio´n Banco Argentino de Ce´lulas, ABAC, Instituto Nacinal de
Enfermedades Virales Humanas, Pergamino, Argentina) was main-
tained frozen in liquid nitrogen. The cells were grown as a mono-
layer employing Dulbecco modified Eagle medium containing 10%
fetal calf serum (FCS) and 50
␮g gentamycin as antibiotic. The cells
were incubated at 37 C in a humidified 5% CO2 atmosphere and the
Њ
medium was changed daily. The cell line was routinely checked for
the absence of mycoplasma contamination.
Cell photosensitization studies and quantification. An appropriate
ϳ1 ϫ
106 cells) was inoculated in 25 cm2 culture
In AOT reverse micelles an insignificant shift with respect
to n-heptane was observed, indicating no strong association
number of cells (
flasks and incubated to obtain nearly confluent cell layers. Then 100

Photochemistry and Photobiology, 2001, 74(1) 17
Table 2. Fluorescence emission maxima of porphyrins in different
media
␭
em
(nm)
Solvent
n-Heptane
Dichloromethane
Tetrahydrofuran
AOT*
TMP
657
657
658
657
655
ZnTMP
CdTMP
721
722
722
721
720
593
643
655
659
643
662
621
665
667
668
665
664
605
608
593
616
622
624
621
620
FCS†
*n-heptane/AOT (0.1 M)/water (W
ϭ
10).
†10% vol/vol in PBS.
Figure 1. Effect on the Soret band maximum absorption of TMP
), ZnTMP ( ), CuTMP ( ) and CdTMP ( ) produced by the
addition of FCS in PBS.
(᭡
᭢
●
Ⅲ
Singlet oxygen production and photooxidation in AOT
reverse micelles
In the presence of air, the emission signal of the O2(1
⌬ )
g
formed after laser excitation of the porphyrin solution in
tetrahydrofuran showed a first-order exponential decay. Typ-
ical results for ZnTMP are shown in Fig. 2. The initial
with the polar micellar interface (27,30). On the other hand,
these porphyrins are not soluble in saline solution. However,
when a fixed amount of precipitated porphyrin (1 nmol) in
PBS was treated with FCS, which is used in the biological
medium to incubate the cell line (see ‘‘Materials and Meth-
O2(1
⌬g) emission intensities (I0) were calculated from ex-
trapolation to zero time (determined by the laser shot). These
initial signal intensities were plotted vs laser total energy
(between 20 and 1 mJ; Fig. 2, inset). The quantum yield of
ods’’), a Soret band appears at
ϳ408 nm. An increase in the
O2(1
⌬
g) production (
plots for the porphyrins compared with the corresponding
slope obtained for the reference (TPP). The values of in
tetrahydrofuran are shown in Table 3. The results for
follow the order CdTMP ZnTMP TMP, while singlet
molecular oxygen (1
g) was not detected when CuTMP was
used as the photosensitizer. The values of ⌬ are comparable
⌽⌬) was calculated from the slope of the
amount of FCS added produces a narrowing and enhance-
ment in the intensity of the Soret band. Linear plots of the
Soret band absorption with the amount of FCS in PBS were
obtained under these conditions for all the porphyrins stud-
ied (Fig. 1). In a similar experiment using serum albumin
no significant disaggregating was observed by absorption or
fluorescence. These results indicate that FCS produces a
monomerization of the porphyrin which is preferentially
bound to the hydrophobic environment of the lipoproteins in
the serum (39). The selective transport by lipoproteins can
contribute to enhance the specificity of tumor targeting by
the photosensitizer, since mainly the low-density lipoprotein
(LDL) develops a very active interaction with neoplastic
cells (36,40).
⌽
⌬
⌽
⌬
ϳ
Ͼ
⌬
⌽
to those reported before for similar porphyrin derivatives
(14–16,17).
The aerobic irradiation of n-heptane/AOT (0.1 M)/water
(W
ϭ 10) reverse micelles with visible light containing the
photosensitizer was carried out in the presence of Trp. A
time-dependent decrease in the amino acid concentration
was observed following a decrease in its fluorescence emis-
sion at 350 nm. The photoprocess follows first-order kinetics
with respect to Trp concentration as shown below using dif-
ferent [Trp] (Fig. 3A). The photoexcitation of the porphyrins
in the AOT micellar system was also performed in the pres-
The steady-state fluorescence emission spectra of these
porphyrins were analyzed in different media. No detectable
fluorescence bands were observed for CuTMP, while TMP,
ZnTMP and CdTMP present two maxima, as shown in Table
2. The same values of
␭
em
were obtained on exciting the
sample at the wavelength of maximum absorption of the
Soret and the Q-bands. The two bands are characteristic of
similar porphyrins and they have been assigned to Q(0–0)
and Q(0–1) transitions (16). A small Stokes shift is expected
for TPP derivatives indicating that the spectroscopic energy
is nearly identical to the relaxed energy of the singlet state
(16). As can be observed, the
␭
em
values are not very dif-
ferent in these media and no changes in the fluorescence
profile were observed, showing that the same species (mono-
mer) was responsible for the fluorescence emission under
these conditions. The fluorescence quantum yield (
porphyrins were calculated by the steady-state comparative
method using TPP as a reference (31,32). The values of
0.14 0.01, 0.049 0.004 and 0.018 0.003 were
obtained in tetrahydrofuran for TMP, ZnTMP and CdTMP,
respectively. These results are consistent with that obtained
for similar metalloporphyrins (16).
␾F) of the
␾
F
ϭ
Ϯ
Ϯ
Ϯ
Figure 2. Decay traces of O2(1
⌬g) emission sensitized by ZnTMP
in tetrahydrofuran at different laser energies: (A) 0.84; (B) 0.60; (C)
0.40; (D) 0.20; and (E) 0.08 mJ. Inset shows a linear plot between
the initial O2(1
⌬g) emission intensities (I0) vs laser total energies (E).

18 M. E. Milanesio et al.
Table 3. Kinetic parameters for the photooxidation reaction of DMA and Trp in n-heptane/AOT (0.1 M)/water (W
ϭ
10) reverse micelles
Porphy-
rin
(sϪ1)†
kobs (sϪ1)†
krTrp (sϪ MϪ1)‡
DMA
Trp
1
⌽ *
⌬
kobs
4
4
5
5
5
5
TMP
0.65
0.73
—
(3.2
(3.8
Ϯ
Ϯ
0.2)
0.3)
—
ϫ
ϫ
10Ϫ
(8.5
(10.1
(0.6
Ϯ
Ϯ
Ϯ
Ϯ
0.3)
0.4)
0.1)
0.4)
ϫ
ϫ
ϫ
ϫ
10Ϫ
10Ϫ
10Ϫ
10Ϫ
(1.9
(1.9
Ϯ
Ϯ
0.1)
0.1)
—
ϫ
ϫ
107
107
ZnTMP
CuTMP
CdTMP
10Ϫ
§
4
0.73
(3.9
Ϯ
0.2)
ϫ
10Ϫ
(9.8
(1.8
Ϯ
0.1)
ϫ
107
*
⌽ in tetrahydrofuran.
⌬
†Spectral irradiated area 0.20 kK (kK
‡Calculated considering the mechanism described by Rodgers and Lee (25) and Borsarelli et al. (27) (Scheme 1).
§[Trp] 15 M.
ϭ kilo-Kaiser ϭ
103 cmϪ1; wavelength range 455–800 nm).
ϭ
␮
ence of DMA. It is known that this substrate quenches
O2(1
g) exclusively by chemical reaction (27). Similar be-
range of Trp concentrations shows that TMP, ZnTMP and
CdTMP appear to perform their photosensitizing actions via
the intermediacy of O2(1
⌬g). On the other hand, alternative
type-I photoreaction pathways involving a direct interaction
between the excited photosensitizer and the substrate are
concentration dependent. This can occur when CuTMP is
used as the sensitizer (19).
In this type of microheterogeneous system, Trp is exclu-
sively solubilized in the polar side of the interface and it is
oxidized by O2(1
⌬g) (25,41). Taking into account the mech-
⌬
havior to Trp was observed for DMA by following a de-
crease in its absorbance (Fig. 3B). From the plots in Fig. 3,
the values of the observed rate constant (kobs) were calculated
for both substrates. The results are shown in Table 3. As
can be observed, the increase in kobs is proportional to the
sensitizer quantum yield of O2(1
⌬g). Moreover, for TMP,
ZnTMP and CdTMP, the observed rate constant for Trp
(kobsTrp) does not change when the Trp concentration is var-
ied within the 5–15
action was only detected using highest Trp concentration.
The independence of kobs
␮
M interval, while for CuTMP the re-
anism described before for the photooxidation reactions in
AOT reverse micelles (25,27), the kinetic sequences shown
in Scheme 1 can be proposed, where subscripts f and b in-
dicate the organic phase and the micellar pseudophase, re-
Trp
photooxidation over a wide
3
spectively. S, 1S and S represent the ground, excited singlet
and triplet state of the sensitizer, respectively,
resent O2(1
g) and the ground state of molecular oxygen,
O2( g)¯, respectively.
⌬ and ⌺ rep-
⌬
⌺
To evaluate the reaction rate constant of Trp photooxi-
dation (krTrp) in this system, DMA was used as the actinom-
eter under the same experimental conditions. From the ratio
of the first-order slopes between the Trp and the actinometer
(Eq. 8), the values of krTrp were calculated in the micellar
system using TMP, ZnTMP and CdTMP as sensitizers.
Trp DMA
k
/k
ϭ
kTr rp
[⌬
b]/kDr MA
[⌬
] ϭ
f
kTr rpK/kDr MA
(8)
obs obs
0.11 (25) and krDMA
ϭ
0.8 ϫ
107 s
Ϫ
1
The values of K
ϭ
Ϫ
M
1 (27) reported earlier were used in these calculations. The
results for krTrp are shown in Table 3. As can be observed
very close values were obtained on photoexciting any of these
porphyrins. The mean value (krTrp
ϭ
1.9
ϭ
ϫ
2.3
107 s M ) is
ϫ
Ϫ
1
Ϫ1
close to the value reported before (krTrp
107 s 1 M )
Ϫ
Ϫ1
Figure 3. First-order plots for the photooxidation of: (A) Trp (5
M); and (B) DMA (35 M) photosensitized by TMP ( ), ZnTMP
) and CdTMP ( ) in AOT reverse micelles.
in a similar AOT reverse micellar system using rose bengal
as the sensitizer at W 22.2 (25). However, both results for
␮
␮
᭡
(Ⅲ
●
ϭ

Photochemistry and Photobiology, 2001, 74(1) 19
Table 4. Porphyrin uptake and cell inactivation parameters
Porphy-
rin
Uptake (
␮
mol/106 cells) t50 (min)*
D50 (J cmϪ2)†
3
TMP
(2.5
(2.1
(2.1
(1.7
Ϯ
Ϯ
Ϯ
Ϯ
0.1)
0.1)
0.1)
0.1)
ϫ
ϫ
ϫ
ϫ
10Ϫ
10Ϫ
10Ϫ
10Ϫ
‡
‡
‡
§
42
28
Ϯ
Ϯ
3
2
45
30
Ϯ
Ϯ
3
2
3
3
3
ZnTMP
CuTMP
CdTMP
—
Ϯ
—
Ϯ
29
2
31
2
*Irradiation time at which 50% of cells were inactivated under this
experimental condition.
†Light exposure level required to inactivate 50% of cell population.
‡Determined by HPLC.
§Determined by fluorescence.
Figure 4. Inactivation of the cells incubated with TMP (
) CuTMP and CdTMP ( ) and exposed to different irradiation
times with visible light; ( ) corresponding controls in dark condi-
tions; [sensitizer] 1 M, time 24 h. Values represent mean stan-
dard deviation of three separate experiments.
᭡), ZnTMP
(Ⅲ
●
▫
krTrp are lower than the value reported in water (krTrp
ϭ
6.1
107 s M ) (25) indicating that Trp at these W is not able
to quench O2(1
g) in the water pool of AOT reverse micelles
ϫ
␮
Ϯ
Ϫ1
Ϫ1
⌬
with the same effectiveness as in bulk water.
Studies in vitro on Hep-2 cells
(D50) were calculated. The results are shown in Table 4.
Under these conditions, the cytotoxic effect increases in the
Since these porphyrins are water-insoluble, they were added
to the cell cultures from a liposomal solution of
L
,
D
-
␣
-dipal-
order: CuTMP
K
TMP
Ͻ
ZnTMP
ϳ
CdTMP, correlating
mitoyl phosphatidylethanolamine bearing 20 mol% of choles-
terol. The use of phospholipid liposomes as vehicles to trans-
port hydrophobic photosensitizers could constitute an advan-
tage for its use in PDT, since liposomes optimizes the release
of lipophilic sensitizer to LDL (36). The Hep-2 cells were
with the sensitizer production of O2(1
⌬g). This behavior is
not always the case in cellular systems, where the biological
microenvironment of the porphyrins can induce significant
changes in the photophysics of the sensitizer established in
the homogeneous solution (43,44).
treated with 1
␮
M of the sensitizer for 24 h at 37
Њ
C. Similar
In conclusion, these studies provide information on the
photodynamic activity of lipophilic metalloporphyrin deriv-
atives. Although many other factors can contribute to pho-
todamage in cellular systems, a higher damage of Hep-2
cells could be obtained by forming complexes of porphyrin
values of porphyrin uptake were obtained for ZnTMP and
CuTMP (see Table 4), while a slightly higher value was found
for TMP and lower for CdTMP. It was reported before that
cellular uptake of porphyrins increase with lipophilicity,
which could be explained by a reduction in the membrane
permeability barrier toward lipophilic compounds (42).
Cell toxicity induced by the porphyrins was first analyzed
in dark condition and no toxicity in terms of cell survival
(measured by microscopy in the presence of TB) was de-
tected at any evaluated time (Fig. 4, control). Similar results
were found for cell cultures not treated with the sensitizer
and irradiated with visible light for 30 min. Therefore, the
cell mortality obtained after irradiation of the cultures treated
with the porphyrin is due to the photosensitization effect of
the agent produced by visible light.
The survival curves obtained after the combined treatment
of cells with the sensitizer and visible light are shown in Fig.
4. The irradiation system used in these studies is described
in ‘‘Materials and Methods.’’ As expected, Hep-2 cell in-
activation depends on the light exposure level employed. Af-
ter an irradiation of 32.4 J cm , the survival fractions were
0.61, 0.48 and 0.49 for cell line treated with TMP, ZnTMP
and CdTMP, respectively, while a very small effect (
was found for CuTMP. The results show higher cell mor-
tality with an increase of the O2(1
g) production by the por-
with metal which increase the O2(1
their metallic complexes with Zn(II) and Cd(II) produce
O2(1
g) with considerable quantum yield, while the genera-
tion of O2(1
g) by Cu(II) complex was negligible. When
⌬g) generation. TMP and
⌬
⌬
TMP, ZnTMP and CdTMP were used as photosensitizers in
the reverse micellar system, the photooxidation rates of
DMA were proportional to the corresponding
⌽⌬. Photoox-
idation of DMA was not observed with CuTMP. When Trp
was used as a model of the biological substrate, it was rap-
idly photodegraded by the photodynamic activity of TMP,
ZnTMP or CdTMP. Depending on the amino acid concen-
tration it was also slowly decomposed in the presence of
CuTMP. In biological medium, the relative photosensitizing
effectiveness of the porphyrins expressed as the photoinac-
Ϫ
tivation rate (1/D50) were 0.022, 0.033 and 0.032 J 1 cm2 for
TMP, ZnTMP and CdTMP, respectively, while under these
conditions CuTMP produced comparatively small photo-
damage on Hep-2 cells. These observations are consistent
with the reported lack of type-II activity for CuTMP, which
is presumed to undergo a type-I photodynamic response
(19,21). A similar behavior in both the media indicated that
the photodynamic effect of these porphyrins correlate with
Ϫ
2
ϳ0.94)
⌬
phyrins. The cell inactivation curves show approximately ex-
ponential decay (Fig. 4) for TMP, ZnTMP and CdTMP.
From these, the irradiation time required to inactivate 50%
of cell population (t50) was obtained (Table 4). Taking into
account the light intensity at the treatment site (see ‘‘Mate-
rials and Methods’’) the corresponding light exposure levels
the O2(1
erable
which involves mainly intermediacy of O2(1
⌬
⌽
g) production. Therefore, porphyrins with consid-
⌬ appear to act on Hep-2 cells through a mechanism
g), although, in
⌬
minor extension a type-I photoreaction process could be oc-
curring in this biological medium.

20 M. E. Milanesio et al.
Acknowledgements Authors are grateful to Consejo Nacional de
Investigaciones Cient´ıficas y Te´cnicas (CONICET) of Argentina,
Fundacio´n Antorchas, Agencia Nacional de Promocio´n Cient´ıfica y
Tecnolo´gica and SECYT of the Universidad Nacional de R´ıo Cuarto
for financial support. C.D.B., J.J.S., V.R. and E.N.D. are Researchers
at CONICET. M.G.A. thanks CONICET for a research fellowship.
iminium salts delivered by a lipid emulsion. Photochem. Pho-
tobiol. 68, 561–568.
20. Faustino, M. A. F., M. G. P. M. S. Neves, J. A. S. Cavaleiro,
M. Neumann, H.-D. Brauer and G. Jori (2000) Part 2. meso-
Tetraphenylporphyrin dimmer derivatives as potential photosen-
sitizers in photodynamic therapy. Photochem. Photobiol. 72,
217–225.
21. Ali, H. and J. E. van Lier (1999) Metal complexes as photo-
and radiosensitizers. Chem. Rev. 99, 2379–2450.
REFERENCES
22. Kimel, S., B. J. Tromberg, W. G. Roberts and M. W. Berns
(1989) Singlet oxygen generation of porphyrins, chlorines, and
phthalocyanines. Photochem. Photobiol. 50, 175–183.
23. Wefers, H. (1987) Singlet oxygen in biological systems. Bioelec-
trochemistry and bioenergetics. J. Electroanal. Chem. 232, 91–104.
24. Segalla, A., C. Milanesi, G. Jori, H.-G. Capraro, U. Isele and
K. Schieweck (1994) CGP 55398, a liposomal Ge(IV) phtha-
locyanine bearing two axially ligated cholesterol moieties: a
new potential agent for photodynamic therapy of tumours. Br.
J. Cancer 69, 817–825.
1. Milgrom, L. R. and F. O’Neill (1993) The chemistry of natural
products. In Porphyrins, Chap. 8 (Edited by R. H. Thomson),
pp. 329–376. Blackie, London.
2. Grossweiner, L. I. (1994) The science of phototherapy. In Pho-
todynamic Therapy, Chap. 8, pp. 139–155. CRC Press, London.
3. McCaughan, J. S. Jr (1999) Photodynamic therapy. A review.
Drugs Aging 15, 49–68.
4. Dougherty, T. J. (1993) Photodynamic therapy. Photochem.
Photobiol. 58, 895–900.
5. Penning, L. C. and T. M. Dubbelman (1994) Fundamentals of
photodynamic therapy: cellular and biochemical aspects.
AntiCancer Drugs 5, 139–146.
25. Rodgers, M. A. J. and P. C. Lee (1984) Singlet molecular ox-
ygen in micellar system. 2. Quenching behavior in AOT reverse
micelles. J. Phys. Chem. 88, 3480–3484.
6. Hombrecher, H. K., S. Ohm and D. Koli (1996) Synthesis of gal-
actopyranosyl substituted porphyrins. Tetrahedron 52, 5441–5448.
7. Gaud, O., R. Granet, M. Kaouadji, P. Krausz, J. C. Blais and
G. Bolbach (1996) Synthe`se et analyse structurale de nouvelles
me´so-arylporphyrines glycosyle´es en vue de l’application en
photothe´rapie des cancers. Can. J. Chem. 74, 481–499.
8. Driaf, K., R. Granet, P. Krausz, M. Kaouadji, F. Thomasson, A.
J. Chulia, B. Verneuil, M. Spiro, J.-C. Blais and G. Bolbach
(1996) Synthesis of glycosylated cationic porphyrin as potential
agents in photodynamic therapy. Can. J. Chem. 74, 1550–1563.
9. Sol, V., J. C. Blais, V. Carre´, R. Granet, M. Guilloton, M. Spiro
and P. J. Krausz (1999) Synthesis, spectroscopy, and photocy-
totoxicity of glycosylated amino acid porphyrin derivatives as
promising molecules for cancer phototheraphy. J. Org. Chem.
64, 4431–4444.
10. Durantini, E. N. (2000) Synthesis of meso-nitrophenylporphyr-
ins covalently linked to a polyphenylene chain bearing methoxy
groups. J. Phorphirins Phthalocyanines 4, 233–242.
11. Jori, G., L. Schindl, A. Schindl and L. Polo (1996) Novel ap-
proaches towards a detailed control of the mechanism and ef-
ficiency of photosensitized process in vivo. J. Photochem. Pho-
tobiol. A: Chem. 102, 101–107.
12. Star, W. M., H. P. C. Marijnissen, A. E. Berg-Blok, J. A. C.
Versteeg, K. A. P. Franken and H. S. Reinhold (1986) Destruc-
tion of rat mammary tumor and normal tissue microcirculation
by hematoporphyrin derivative observed in vivo in sandwich
observation chambers. Cancer Res. 46, 2532–2540.
13. Moan, J., Q. Peng, R. Sorensen, V. Iani and J. M. Nesland
(1998) The biophysical foundations of photodynamic therapy.
Endoscopy 30, 387–391.
14. Katona, Z., A. Grofcsik, P. Baranyai, I. Bitter, G. Grabner, M.
Kubinyi and T. Vidoczy (1998) Triplet state spectroscopic stud-
ies on some 5,10,15,20-tetrakis(methoxyphenyl) porphyrins. J.
Mol. Struct. 450, 41–45.
26. Gandini, S. C. M., V. E. Yushmanov, I. E. Borissevitch and M.
Tabak (1999) Interaction of tetra (4-sulfonatophenyl) porphyrin
with ion surfactants: aggregation and location in micelles. Lang-
muir 15, 6233–6243.
27. Borsarelli, C. D., E. N. Durantini and N. A. Garcia (1996) Sin-
glet molecular oxygen-mediated photooxidation of nitrophenolic
compounds in water-in-oil microemulsions. J. Chem. Soc. Per-
kin Trans. 2 2009–2013.
28. Lindsey, S. J., I. C. Schreiman, H. C. Hsu, P. C. Kearney and
A. M. Marguerettaz (1987) Rothemund and Adler–Longo re-
actions revisited: synthesis of tetraphenylporphyrins under equi-
librium conditions. J. Org. Chem. 52, 827–836.
29. Alvarez, M. G., M. La Penna, E. I. Yslas, V. Rivarola and E.
N. Durantini (2000) Photodamaging effects of porphyrin in a
human carcinoma cell line. Chem. Educator 5, 24–26.
30. Correa, N. M., E. D. Durantini and J. J. Silber (1998) Binding
of nitrodiphenylamines to reverse micelles of AOT in n-hexane
and carbon tetrachloride: solvent and substituent effects. J. Col-
loid Interface Sci. 208, 96–103.
31. Demas, J. N. and G. A. Crosby (1971) The measurement of pho-
toluminescence quantum yields. J. Phys. Chem. 75, 991–1024.
32. Tatman, D., P. A. Liddel, T. A. Moore, D. Gust and A. L. Moore
(1998) Carotenohematoporphyrins as tumor-imaging dyes. Syn-
thesis and in vitro photophysical characterization. Photochem.
Photobiol. 68, 459–466.
33. Schmidt, R. and E. Afshari (1990) Effect of solvents on the
phosphorescence rate constant of singlet molecular oxygen
1
(
⌬
g). J. Phys. Chem. 94, 4377–4378.
34. Valduga, G., S. Nonell, E. Reddi, G. Jori and S. E. Braslavsky
(1988) The production of singlet molecular oxygen by zinc(II)
phthalocyanine in ethanol and in unilamellar vesicles. Chemical
quenching and phosphorescence studies. Photochem. Photobiol.
48, 1–5.
35. Kremer, J. M. H., M. W. J. van der Esker, C. Pathmanoharan and
P. H. Wiersema (1977) Vesicles of variable diameter prepared by
a modified injection method. Biochemistry 16, 3932–3935.
36. Ginevra, F., S. Biffanti, A. Pagnan, R. Biolo, E. Reddi and G.
Jori (1990) Delivery of the photosensitizer zinc(II)-phthalocya-
nine to serum proteins by different liposomes: studies in vitro
and in vivo. Cancer Lett. 49, 59–65.
37. Kostenich, G., T. Babushkina, A. Lavi, Y. Langzam, Z. Malik,
A. Orenstein and B. Ehrenberg (1998) Photosensitization by the
near-IR-absorbing photosensitizer lutetium texaphyrin: spectro-
scopic, in vitro and in vivo studies. J. Porphyrins Phthalocya-
nines 2, 383–390.
38. Hadjur, C., N. Lange, J. Rebstein, P. Monnier, H. van den Bergh
and G. J. Wagnie`res (1998) Spectroscopic studies of photo-
bleaching and photoproduct formation of meta(tetrahydroxy-
phenyl) chlorin (m-THPC) used in photodynamic therapy. The
production of singlet oxygen by m-THPC. J. Photochem. Pho-
tobiol. B: Biol. 45, 170–178.
15. Alvarez, M. G., E. I. Yslas, V. Rivarola, G. Mori, M. La Penna,
J. J. Silber and E. N. Durantini (2000) Photodynamic effect of
5,10,15,20-tetrakis(4-methoxyphenyl) porphine (TMP) on Hep-
2 cell lines. Molecules 5, 379–380.
16. Pineiro, M., A. L. Carvalho, M. M. Pereira, A. M. d’A. Rocha
Gonsalves, L. G. Arnaut and S. J. Formosinho (1998) Photoa-
coustic measurements of porphyrin triplet-state quantum yields
and singlet-oxygen efficiencies. Chem. Eur. J. 4, 2299–2307.
17. Mosinger, J., K. Losinska, T. Abrhamova, S. Veiserova, Z.
Micka, I. Newcova and B. Mosinger (2000) Determination of
singlet oxygen production and antibacterial effect of nonpolar por-
phyrins in heterogeneous systems. Anal. Lett. 33, 1091–1104.
18. Bonnett, R., S. Ioannou, A. G. James, C. W. Pitt and M. M. Z.
Soe (1993) Synthesis and film-forming properties of metal com-
plexes of octadecyl ethers of 5,10,15,20-tetrakis(4-hydroxy-
phenyl) porphyrin. J. Mater. Chem. 3, 793–799.
19. Garbo, G. M., V. H. Fingar, T. J. Wieman, E. B. Noakes III, P.
S. Haydon, P. B. Cerrito, D. H. Kessel and A. R. Morgan (1998)
In vivo and in vitro photodynamic studies with benzochlorin
39. Belitchenko, I., V. Melnikova, L. Bezdetnaya, H. Rezzoug, J.

Photochemistry and Photobiology, 2001, 74(1) 21
L. Merlin, A. Potapenko and F. Guillemin (1988) Characteriza-
tion of photodegradation of meta-tetra (hydroxyphenyl) chlorin
(mTHPC) in solution: biological consecuences in human tumor
cells. Photochem. Photobiol. 67, 584–590.
42. Edrei, R., V. Gottfried, J. E. van Lier and S. Kimel (1998)
Sulfonated phthalocyanines: photophysical properties, in vitro
cell uptake and structure–activity relationships. J. Porphyrins
Phthalocyanines 2, 191–199.
43. Aveline, B. M. and R. W. Redmond (1998) Exclusive free rad-
ical mechanisms of cellular photosensitization. Photochem. Pho-
tobiol. 68, 266–275.
44. Aveline, B. M. and R. W. Redmond (1999) Can cellular pho-
totoxicity be accurately predicted on the basis of sensitizer pho-
tophysics? Photochem. Photobiol. 69, 306–316.
40. Jori, G. and E. Reddi (1993) The role of lipoproteins in the
delivery of tumour-targeting photosensitizers. Int. J. Biochem.
25, 1369–1375.
41. Lissi, E. A., M. V. Encinas, S. A. Bertolotti, J. J. Cosa and C.
M. Previtali (1990) Fluorescence quenching of indolic com-
pounds in reverse micelles of AOT. Photochem. Photobiol. 51,
53–58.