The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205

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The International Journal of Biochemistry

& Cell Biology
journal homepage: www.elsevier.com/locate/biocel

Cellular inactivation and antitumor efficacy of a new zinc

phthalocyanine with potential use in
photodynamic therapy
Natalia B. Rumie Vittar a,1 , Cesar G. Prucca a,1 , Cristian Strassert b ,
Josefina Awruch b , Viviana A. Rivarola a,∗
a
Departamento de Biologı́a Molecular, Facultad de Ciencias Exactas, Fı́sico-Quı́micas y Naturales,
Universidad Nacional de Rı́o Cuarto, Agencia Postal Nro 3, 5800 Rı́o Cuarto, Córdoba, Argentina
b
Departamento de Quı́mica Orgánica, Facultad de Farmacia y Bioquimica, Universidad de Buenos Aires,
Junı́n 956, 1113 Buenos Aires, Argentina

a r t i c l e i n f o a b s t r a c t

Article history: The aim of the present study was to evaluate the photodynamic efficacy of a
Received 26 August 2007 novel phthalocyanine derivate 2,3,9,10,16,17,23,24-octakis[(N,N-dimethylamino) ethylsul-
Received in revised form 18 February 2008
fanyl]phthalocyaninatozinc(II) (referred here as S1) using MCF-7c3 human breast cancer
Accepted 27 February 2008
cells and the LM2 adenocarcinoma subcutaneously implanted in Balb/c mice as experi-
Available online 6 March 2008
mental models.
The S1-l-␣-dimyristoyl-phosphatidylcholine liposome was selected as the best delivery
Keywords:
Photodynamic Therapy system because it showed greater internalization into cells (35 nmol/106 cells), relative to
Photosensitizer other liposomes. After 3 h incubation S1 was partially localized in lysosomes, the compart-
Apoptosis ment that represented its primary photodamage site. The S1 treated cultures also revealed
Necrosis a degree of mitochondrial morphology alteration. Indeed, S1 leads to photokilling of the
Cancer cells with different efficacies indicating that cell photoinactivation was dependent on both
the phthalocyanine concentration and the light dose applied. Analyses of morphology and
nuclear condensation level indicated that some of the cells exposed to photodynamic ther-
apy were undergoing apoptosis within 8 h after treatment.
To assess the in vivo effectiveness of S1, animals bearing tumors were treated with
0.2 mg/kg S1 followed 24 h later by 108 J cm−2 light at 600–800 nm and 60 mW cm−2 ,while
other animals served as controls (no treatment, light alone, or S1 alone). All S1 treated
tumors and none of the controls exhibited complete or partial responses, and these
responses continued for the entire observation period of 12 days. Evaluation of tumor size
showed that the treatment effectively delayed tumor growth. Light microscopy investi-
gations of irradiated tumor specimens showed that S1 causes an early direct damage of
malignant cells, largely via processes leading to random necrotic pathways.
© 2008 Elsevier Ltd. All rights reserved.

Abbreviations: PDT, photodynamic therapy; Pc, phthalocyanine; PS, photosensitizer; DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; DMPC, l-␣-
dimyristoyl-phosphatidylcholine; DPPEc, d,l-␣-dipalmitoyl phosphatidylethanolamine containing cholesterol; PBS, phosphate-buffered saline; SDS,
sodium dodecyl sulfate; DMF, dimethylformamide; THF, tetrahydrofuran; HO258, Hoechst33258; MTT, 3-(4,5-dimethylthiazolil-2)-2,5
diphenyltetrazolium bromide; H&E, hematoxylin-eosin.
∗ Corresponding author. Tel.: +54 358 4676437; fax: +54 358 4676232.
E-mail address: vrivarola@exa.unrc.edu.ar (V.A. Rivarola).
1
Both these authors contributed equally to this work.

1357-2725/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biocel.2008.02.024
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205
1. Introduction
Photodynamic therapy (PDT) involves the combination
of non-toxic dyes known as photosensitizers (PSs) and
harmless visible light which in the presence of oxygen leads
to the generation of reactive oxygen species that can dam-
age cellular constituents leading to cell death. The use of
PDT as a cancer therapy is particularly attractive because of
its intrinsic dual selectivity. The PS localizes in the malig-
nant tissue and the light is also spatially focused on the
lesion (Castano, Demidova, & Hamblin, 2005a,b). Provided
that the PS is non-toxic, only the irradiated areas will be
affected, even if the PS does bind to normal tissues.
Precisely why cells die when subjected to the PDT-
generated reactive oxygen species (ROS) has been the
subject of intense investigation in recent years. The dis-
covery of programmed cell death or apoptosis (Samali,
Gorman, & Cotter, 1996) has revolutionized the field of cyto-
toxic therapies in general (Dixon, Soriano, Lush, Borner,
& Figg, 1997) and PDT in particular (Oleinick, Morris, &
Belichenko, 2002).
Agarwal et al. were the first to report apoptosis after
PDT with chloroaluminium phthalocyanine in mouse lym-
phoma L5178Y cells. The crucial factors in determining the
type of cell death, e.g. apoptosis or necrosis following PDT
are: cell type, the subcellular localization of the PS, and
the light dose applied to activate it locally (Agarwal et
al., 1991; Alvarez, Prucca, Milanesio, Durantini, & Rivarola,
2006; Plaetzer, Kiesslich, Krammer, & Hammerl, 2002).
Phthalocyanines (Pcs) are a new generation of PSs for
PDT. This group of compounds has several advantages
over the sensitizers currently used, which are based on
hematoporphyrin (Gomes, Cruz, Lopes, Carvalho, & Duarte,
1999). These azoporphyrins derivatives have stronger
absorbances at longer wavelengths than do porphyrins and
tend to have improved photophysical and photochemical
properties. Their chemical synthesis and structure per-
mit the addition of substituents to both the central metal
ion and the periphery of the hydrophobic Pc macrocyle,
altering the physical properties of these PSs such as pho-
tostability, long lifetimes of the photoexcited triplet state,
and high molar absorption in the red region of the visible
spectrum.
In complex environments, such as cells and tissues, the
subcellular localization of the PS is important for effective
photochemistry to occur. Therefore, the cellular structure
close to both a high concentration of sensitizer and a high
oxygen concentration will be preferentially damaged upon
illumination. However, many efficient PSs are hydropho-
bic in nature and lose their efficacy when brought into
the aqueous phase, due to the formation of photochem-
ically inactive aggregates. Amphiphilic carriers, such as
micelles (Röder et al., 1996) and liposomes (Röder, Hanke,
Oelckers, Hackbarth, & Symietz, 2000) are currently used
to bring insoluble or hardly soluble dyes into water and
to avoid aggregation. In these systems, the PS is located
in the hydrophobic core of the microheterogeneous host.
Naturally, suitable carriers should facilitate dye transport
without substantially affecting photochemical activity.
Many PSs, which are currently used in clinical and exper-
imental treatment of cancer localize extranuclearly in cells:
2193
in mitochondria, in the plasma membrane, in endoplasmic
reticulum (ER), Golgi apparatus and lysosomes. It has been
proposed that intracellular localization is dependent upon
the chemical properties of the PS and is cell-line depen-
dent. Localization of PSs in cytoplasmic organelles during
photochemical therapy plays a major role in cell damage.
For example, the PSs lutetium texaphyrin (LuTex) and N-
aspartyl chlorine e6 (NPe6) cause an almost immediate
disruption of lysosomes upon irradiation (Dougherty et al.,
1998; Agarwal et al., 1991; Oleinick et al., 2002; Woodburn
et al., 1997). In contrast, irradiation of cells preloaded with
the PSs 9-capronyloxy-tetrakis(methoxyethyl) porphycene
(CPO), hexyl pheophorbide (HPPH) and phthalocyanine
(Pc4) has no effect on lysosomes. Instead, one sees a rapid
loss of mitochondrial membrane potential and release of
Cytochrome c (Chiu & Oleinick, 2001; Dougherty et al.,
1998; Kessel, Luo, Deng, & Chang, 1997; Kessel, Caruso,
& Reiners, 2000; Kessel, Luo, Mathieu, & Reiners, 2000;
Oleinick et al., 2002). These effects most likely reflect the
site of sensitizer accumulation (Trivedi, Wang, Nieminen,
Oleinick, & Izatt, 2000; Woodburn et al., 1997) because
the ROS formed upon irradiation have a limited ability to
migrate from the site(s) of formation (Trivedi et al., 2000).
Apoptosis is an intrinsic physiological event, which
can also be triggered by external stimuli like oxidative
stress attributable to photosensitization (Castano et al.,
2005a). During apoptosis the cells shrink, the nuclear chro-
matin becomes pyknotic and condenses against the nuclear
membrane, and eventually the cytoplasm and the nucleus
break up into apoptotic bodies. Although the cytoplasmic
organelles remain intact, DNA is digested at internucleo-
somal sites, giving rise to fragments that are multiples of
180–200 bp (Almeida, Manadas, Carvalho, & Duarte, 2004).
An apoptotic response was observed in a variety of tumor
cell lines after photosensitization with different porphyrin
and Pc derivatives (He, Skies, Thomsen, Chung, & Jacques,
1994; Kessel & Luo, 1999). However, the ROS that are pro-
duced during PDT have been shown to destroy tumors by
multifactorial mechanisms (Castano et al., 2005a,b). PDT
has a direct effect on cancer cells, producing cell death by
necrosis, apoptosis and autophagy (Oleinick et al., 2002;
Kessel et al., 2006). For most sensitizers in clinical and pre-
clinical use, three primary mechanisms of PDT-mediated
tumor destruction in vivo have been proposed: cellular,
vascular, and immunologic.
The aim of the present study is to evaluate the photo-
dynamic efficacy of a novel sulfur linked octaalkylamino
substituted zinc(II) phthalocyanine for suitable PDT appli-
cations. A preliminary study of zinc 1,4,8,11,15,18,22,25-
octaalkylphthalocyanines has proven that these com-
pounds are effective PSs with an unexpected mode of PDT
action (Cook, Chambrier, Cracknell, Mayes, & Russell, 1995).
In this work, we assessed the different Pc S1 delivery
methods with the purpose of disaggregating the PS and cor-
relating such parameters with time courses for cell uptake
using MCF-7c3 human breast cancer cells as an experimen-
tal model. To further elucidate the cellular mechanism of
action, we examined intracellular localization, lysosomal
and mitochondrial changes, with the goal of determining
the primary photodamage site, which reflects the localiza-
tion of the sensitizer at that time of light irradiation. In
2194 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205
addition, we extended our studies to determine whether
necrosis or apoptosis was the prevailing mechanism of
cell death after treatment. In order to optimize the effi-
ciency of this approach, it was essential for us to define the
pharmacological properties of the photosensitizing agent
in a suitable animal model (LM2 adenocarcinoma subcu-
taneously implanted Balb/c mice) and tumor regression of
the neoplastic lesion.
2. Material and methods
2.1. Cells and culture conditions
The human breast cancer MCF-7 (WS8) cell line trans-
fected with the pBabepuro retroviral vector encoding
human procaspase-3 cDNA (here referred to as MCF-7c3
cells) was derived by Dr. C.J. Froelich (Northwestern Uni-
versity, Evanston, IL). The cells were cultured in RPMI
1640 media supplemented with 1% l-glutamine, 80 ␮g/ml
gentamicine and 10% (v/v) fetal bovine serum (FBS). The
cultures were maintained at 37 ◦ C in a humidified atmo-
sphere of 5% CO2 –95% air.
2.2. Photosensitizer
Instrumentation. The 1 H NMR of S1 was recorded on
a Bruker AM 500 and HR-MALDI-TOF mass spectra was
measured with a Mod 4700 Applied Biosystems spec-
trometer. Electronic absorption spectra were determined
with a Shimadzu UV-3101 PC spectrophotometer. Fluo-
rescence spectra were monitored with a QuantaMaster
Model QM-1 PTI spectrofluorometer. Infrared spectra were
performed with a PerkinElmer Spectrum One FT-IR spec-
trometer.
Synthesis of 2,3,9,10,16,17,23,24-octakis[(N,N-dimethyl-
amino)ethylsulfanyl]phthlocyaninatozinc(II) (referred
here as S1). This compound was readily prepared by the
cyclotetramerisation of 1,2-dicyano-4,5-di[(N,N-dimethyl-
amino)ethylsulfanyl]benzene, sinthesized by a method
similar to that described in the literature (Gürsoy, Cihan,
Kocak, & Bekaroglu, 2001).
S1 was obtained as described in the above reference
with some minor modifications (Strassert, 2006).
A mixture of 1,2-dicyano-4,5-di[(N,N-dimethylamino)
ethylsulfanyl]benzene (0.100 g, 0.30 mmol), anhydrous zinc
acetate (0.100 g, 0.50 mmol), and DBU (1 ml, 6.7 mmol)
in anhydrous butanol (10 ml) was stirred and heated at
130 ◦ C under argon during 2 h. The mixture was cooled
down, evaporated in vacuo and 5 mL of methylene chlo-
ride was added, and centrifuged to eliminate the excess of
zinc acetate. The methylene chloride solution was washed
with water (3 × 5 mL) and evaporated in vacuo. The green
solid residue was dissolved in a small volume of toluene
and filtered through an aluminium oxide column packed
and pre-washed with toluene. After washing with toluene
and methylene chloride, the title compound was eluted
with methanol-methylene chloride (9:1). After evapora-
tion in vacuo the dye was recrystallized dissolving in a
small volume of toluene–methylene chloride (9:1) follow-
ing by the addition of hexane. Yield 0.050 g (50%). IR (KBr):
2936; 2858; 2818; 2774; 2572; 2295; 1719; 1623; 1593;
1482; 1459; 1405; 1371; 1333; 1295; 1261; 1202; 1168;
1150; 1087; 1012; 944; 896; 871; 849; 802; 780; 743;
730; 699; 547; 491 cm−1 . 1 H NMR (500 MHz, DMSO-d6 ):
ı = 2.42 (br, 48 H, CH3 ); 3.20 (br, 16 H, CH2 N); 3.71 (br, 16
H, SCH2 ); 8.29 (br, 8 H, ArH); HRMS-MALDI-TOF: m/z [M+ ]
calcd for C64 H88 N16 S8 Zn 1400.4435; found [M+ ] 1400.4635
and [M+ + H] 1401.4579. UV/Vis (DMF): max (␧) = 705 nm
(2.0 × 105 M−1 cm−1 ).
2.3. Liposome preparation
For use on cells, S1 was dissolved in tetrahydrofuran
(THF) at concentrations indicated in the experiments. It
was then entrapped in liposomes according to Kremer’s
injection method (Kremer, Esker, Pathmamanoharan, &
Wiersema, 1977). For liposomes of d,l-␣-dipalmitoyl
phosphatidylethanolamine containing cholesterol
(DPPEc), sensitizer (1 ␮mol), phospholipid (33 ␮mol)
and cholesterol (7 ␮mol) were dissolved in ethanol-THF
binary mixture (400 ␮l, 1:1 (v/v)). For l-␣-dimyristoyl-
phosphatidylcholine liposomes (DMPC), the phospholipid
(SIGMA) was dissolved in ethanol at a final concentration
of 3 × 10−3 M. About 400 ␮l of the ethanolic solution was
then taken up and mixed with 80 ␮l of the photosensitizer
solution. For both liposomes, the resulting solution was
quickly injected through a Hamilton syringe into 5 ml
of vigorously stirred phosphate-buffered saline (PBS)
at 80 ◦ C (for DPPEc) or 35 ◦ C (DMPC) depending of the
phospholipids employed. The injection was performed at
a speed of 50 ␮l/min with magnetic stirring and the final
volume was reduced to 5 ml by evaporation of organic
solvent. After adding the phthalocyanine stock solution,
samples were incubated for at least 20 min to allow dye
incorporation into the liposomes. The preparation was
used within a week after preparation.
2.4. Spectroscopic studies
The fluorescence emission spectra of S1 in solutions and
in cell suspensions were measured using a SPEX Fluoro-
Max spectrofluorometer. The sensitizer fluorescence was
collected at the maxima for the S1 excitation and emission
at 630 and 713 nm, respectively. The fluorescence signal
was analyzed using the software provided by the manu-
facturer. All the measurements were performed at room
temperature.
2.5. Quantification of photosensitizer uptake
Cells were incubated with 1 ␮M of either S1-DPPEc
or S1-DMPC during a time lapse of 3, 6, 18 and 24 h at
37 ◦ C in the dark. After incubation the cell pellets were
collected by scrapping and washed, then dispersed in
2 ml of sodium dodecyl sulfate (SDS, in 1:10 water dilu-
tion, v/v). The extraction of sensitizer material from the
cells was facilitated by agitation. Drug levels in cell pellet
were assessed by fluorescence (excitation 630 nm, emis-
sion 713 nm) and each value obtained was related to
the total number of cells contained in the suspension.
Sensitizer concentration in the samples was calculated
from standard calibration curves obtained with known
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205
concentrations of S1 treated with SDS. The kinetics of
uptake (concentration of drug/106 cells vs time) was cal-
culated.
2.6. Photodynamic treatment and viability determination
One day before the experiment, MCF-7c3 cells
(2 × 105 cells per dish) were seeded into 35 mm tissue cul-
ture plates in 2 ml RPMI containing 10% FBS, and incubated
overnight (37 ◦ C, 5% CO2 ). On the day of the experiment, the
Pc S1 entrapped in DMPC liposomes dispersed into culture
medium supplemented with 4% FBS, was added to cells.
Culture dishes were placed for 18 h at 37 ◦ C in a cell culture
incubator. In preparation for irradiation of cells, the dye
containing medium was removed and replaced with RPMI
dye-free (10% FBS). For all illuminations, the light source
used was a slide projector with a 150 W halogen lamp
(Reflecta Classic AF-IR) equipped with a water filter with
5 cm used to attenuated IR radiation and a cut-off filter
for remove emission wavelengths shorter than 480 nm.
The light intensity at the treatment site was 31 mW cm−2
(Radiometer Laser Mate-Q, Coherent). All irradiations
were performed at room temperature. After incubation
for 24 additional hours, cell viability was then determined
by quantization of the cleavage of the tetrazolium salt
MTT (3-(4,5-dimethylthiazolil-2)-2,5-diphenyltetrazolium
bromide (SIGMA) by mitochondrial deshydrogenases,
as recommended by the manufacturer (Denizot & Lang,
1986). Optical density of the resulting solution of formazan
salt was read at 540 nm, after subtraction of the blank.
Irradiation of the cells in the absence of Pc did not result in
alteration of cell viability (data not shown). Control cells
were treated in the same way as above without irradiation.
Results are presented as percentage of survival, taking
control as 100%.
2.7. Subcellular localization and fluorescence staining
procedures
Tumor MCF-7c3 cells grown on coverslips were incu-
bated with 1 ␮M of Pc for 3 h at 37 ◦ C. Then the subcellular
distribution of S1-DMPC was visualized under fluorescence
microscopy using a DBP 406/23 + 530/45, DFT 435 + 570,
DBP 467/30 + 618/75 filter (Carl Zeiss, Germany).
All fluorescent dyes used in the fluorimetric studies
were purchased from Molecular Probes (Eugene, OR, USA).
Just before imaging, cells were stained with the fluorescent
dyes by incubation in dye-containing growth medium at
37 ◦ C in the following conditions: LysoTracker® Green DND-
26 (75 nM, 15–30 min), used as a probe for check lysosomal
localization and photodamage of the Pc, and MitoTracker®
Green FM (100 nM, 45 min), used to study changes in
the mitochondria after PDT. Microscopic observations and
photographs were performed using a fluorescence micro-
scope (Axiophot, Carl Zeiss, Germany) equipped with a HBO
100 W mercury lamp, AxioCam HRc (Carl Zeiss, Germany)
camera and AxioVision Rel. 4.3 software. Images of green
LysoTracker and MitoTracker fluorescence were collected
using a DBP 450–490, FT 510, LP 515 filter (Carl Zeiss, Ger-
many).
2195
2.8. Detection of apoptotic cells
Cells grown on coverslips were fixed in PBS contain-
ing 3.7% formaldehyde. Cellular DNA was stained with
1–5 mg/ml Hoechst 33258 and examined by fluorescence
microscopy. Changes in nuclear morphology were exam-
ined after PDT, by labeling cells with HO258. Apoptotic
and necrotic cells were identified by characteristic fea-
tures of their nuclei. At least 200 cells were counted from
each sample, and the yield of apoptotic cells was expressed
as the percentage of the total population death. Since
detached cells, which are enriched in apoptotic cells, were
not included in this measurement, the estimated percent-
age of apoptosis determined here is a minimal estimation
of the true apoptosis levels. For the observations of the cel-
lular alterations occasioned after treatment, the MCF-7c3
tumor cells were stained with toluidine blue and visual-
ized under light microscope. Fluorescence of HO258 dye
was imaged using BP 365, FT 395 filters (Carl Zeiss, Ger-
many).
2.9. Animals and tumor model
Female Balb/c mice from 6 to 8 weeks old were obtained
from the Fundación Balseiro (Buenos Aires) and main-
tained in standard cages with free access to tap water and
normal dietary chow. For the generation of the experimen-
tal tumors, LM-2 cell line (obtained from Hospital Roffo,
Buenos Aires, Argentina) was implanted subcutaneously
by sterile injection of 106 cells in phosphate-buffered
saline (PBS, pH 7.0) into the right forward leg. When
the tumor size reached 0.7 cm on outer diameter, the
tumoral propagation was carried out extracting 2 mm of
tumoral tissue and subcutaneously reimplanted into the
right forward leg of the desired number of mice for each
experiment. The pharmacokinetic and/or phototherapeutic
experiments were performed on the 6th day after implan-
tation when tumors reached 0.6–0.7 cm on outer diameter.
Spontaneous necrosis was minimal or absent for these
tumor sizes.
When required, animals were anaesthetized with
Ketamine (150 mg/kg, by intraperitoneal injection). The
mice were treated according to the guidelines established
by the ANMAT Disposition N 6344/96, pp. 1–7 for human
care of experimental animals.
2.10. Dark toxicity and histopathology examination
S1 into DMPC liposome (0.1, 0.2 and 0.4 mg/kg bw)
was administered by intraperitoneal injection. The animals
were placed in metabolic cages in the dark and six mice
were sacrificed after 7 days S1 administration for histo-
logical examination. Internal organs such as liver, kidney
and spleen were excised and fixed in 4% formaldehyde, and
then embedded in paraffin. The blocks were cut 3 ␮m thick
and stained with hematoxylin-eosin (H&E) for microscopic
analysis.
The histopathological analysis was carried out in the
Animal Pathology Department of Agronomic and Veteri-
nary Faculty, UNRC, under PhD Silvia Romanini supervision.
2196 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205

2.11. Phototherapeutic studies 3. Results

After tumors reached ∼0.6–0.7 cm in size, the animals
were randomized into four groups to determine tumor
growth rate after the following treatments: (a) control,
not treatment, (b) 0.2 mg/kg bw S1-DMPC, (c) light reg-
imen alone: one diary dose of 108 J cm−2 , 600–800 nm,
60 mW cm−2 during three consecutive days (Visona et
al., 2000); previous experiments made in our laboratory)
and (d) PDT protocol: 0.2 mg/kg bw and light treatment
24 h dye administration. S1 was given by i.p. injection
and 24 h later a 1 cm diameter area encompassing the
tumor was irradiated with the light regime mentioned
above from slide projector. The light regimen administrated
was chosen based on previous S1 tumor accumulation
assays. We found that 24 h after dye administration
the tumor present the larger amount of S1 (data not
shown).
Growth curves representing tumor regrowth for the
control and treated groups were estimated by measur-
ing tumors in three dimensions using caliper. Tumor
volume (V) was determined by the following equation:
V = (L × W × H) × 0.5636, where L is length, W is the width
and H is the height of the tumor (Whitacre et al., 2000;
Jori, 1995). No spontaneous regression of the tumor was
observed during our investigations.
2.12. Evans blue assay for viability determination
With the aim to determinate phototherapeutic effect
of S1, tumor necrosis degree was registered following
Schastak et al. (2005). For this, vital stain was performed
by intraperitoneal injection of 0.4 ml 1% Evans Blue (EB)
solution. It was injected in the mice after 11 days that
received different treatments: (a) control, not treatment,
(b) 0.2 mg/kg bw S1, (c) light regimen alone, and (d) PDT
protocol as described above. Six hours after administra-
tion of the vital stain, animals were sacrificed, tumors were
excised, and 2–3 mm thick cross-section slices were cut. A
section from the central area of each tumor was examined
macroscopically and photographed using a magnification
of 4×, and analyzed with a PC ImageJ 1.36b program (Wayne
Rasband, National Institute of Health, USA). The unstained
area was attributed to necrotic tissue, whereas the stained
area showed tissue with preserved blood supply. Also, his-
tological cut of tumors were realized as was described
above.
3.1. Dissociation and uptake of S1 phthalocyanine in
MCF-7c3 cells
The chemical structure of S1 and the fluorescence spec-
tra in various media are shown in Figs. 1 and 2, respectively.
Fluorescence emission was recorded above 650 nm using a
low-cut filter. The excitation wavelength was set at 630 nm.
In solutions, the monomer and aggregated forms can be
clearly identified by their band positioned at 715 nm. The
spectra of S1 in DMF solution such as vehiculized in DMPC
and DPPEc liposomes are identical. They exhibit a peak at
715 nm, characteristic of the monomer form. However, the
fluorescence spectrum of S1 in culture medium indicates
a significant contribution by aggregated forms. From the
fluorescence spectra results, it seems that aggregation of
S1 can be prevented by an appropriate vehicle. Because
the aggregation phenomenon is counter productive for
effective photodynamic action, we decided to continue our
studies evaluating the liposomes as carriers which have
shown the drug in disaggregated form.
Uptake of S1 liposome formulations by MCF-7c3 tumor
cells was quantitatively analyzed by fluorescence, a method
for the rapid measurement of cell-associated dye. MCF-
7c3 cells were incubated with 1 ␮M of phthalocyanine at
37 ◦ C in the presence of 4 and 10% serum in incubation
media. After 3, 6, 18, and 24 h of incubation, phthalocya-
nine concentration was measured. The results reported in
Fig. 3A and B demonstrated that S1 formulations entered
the cells in a time-dependent manner. Moreover, the S1-
loaded DMPC liposomes were more rapidly internalized
to a greater extent than with the S1- DPPEc vehicle. They
exhibited different uptake kinetics: S1-DPPEc displayed a
maximum at 18 h (2.2 nmol/106 cells) whereas S1-DMPC
accumulated the most within MCF-7c3 cells, reaching the
highest point after 6 h incubation (35 nmol/106 cells). The
fluorescence intensities measured on MCF-7c3 cells treated

2.13. Statistics

All experiments were repeated at least three times inde-

pendently. Data is shown as the mean and the errors on
graphs represent one standard deviation for at least three
independent values.
Statistical analysis for in vivo experiments were per-
formed using two-way MANOVA’s test (factor 1: treatment,
animal control and treated with S1; factor 2: time). The
DUNCAN test was used to analyze the differences between
the experimental groups. It was considered significant at Fig. 1. Chemical structure of 2,3,9,10,16,17,23,24-octakis[(N,N-
P = 0.05. dimethylamino)ethylsulfanyl]phthlocyaninatozinc(II) (S1).
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205 2197

Fig. 2. Absorbance, fluorescence and emission measurements of S1 in solutions and in cell culture medium. (A) Absorption, excitation and emission spectra of
S1 in DMF solution. (B) Comparison of S1 (1 ␮M) emission spectra in DMF solution (solid squares) and; S1-DMPC liposome (open squares), S1-DPPEc liposome
(solid spheres), and S1 (open spheres) in RPMI medium supplemented with 4% FBS. The spectra were recorded on a SPEX FluoroMax spectroflorometer. The
excitation wavelength was set at 630 nm. DMPC: l-␣-dimyristoyl-phosphatidylcholine, DPPEc: d,l-␣-dipalmitoyl phosphatidylethanolamine containing
cholesterol. DMF: dimethylformamide.

with S1-loaded DMPC formulations were at least twenty- 3.2. Photocytotoxicity of S1

fold higher than those obtained with DPPEc by 24 h, at
equivalent dye concentrations. In all cases we observed
that incubation with 10% serum, instead of 4% serum, low-
ered S1 uptake. Similar was reported by Valduga et al.
(1996). Since factors that promote uptake are important
(lipophilicity, absence of aggregation and the presence of
4% serum), S1 incorporated into DMPC liposomes displayed
the most advantageous condition for loading MCF-7c3
cells and this liposome formulation was chosen for further
experiments.
Our results are in agreement with Ishikawa and Wohrl
who show that phthalocyanines easily aggregate in water,
while aggregated phthalocyanine dissociates in hydropho-
bic surroundings (Ishikawa, Ohno, Kaizu, & Kobayashi,
1992; Wohrle et al., 1994). Similar to this, observed cel-
lular uptake of porphyrins and phthalocyanines has been
increased with lipophilicity (Berg, Bommer, & Moan, 1989;
Moan, Berg, Steen, Warloe, & Madslien, 1992; Paquette, Ali,
Langlois, & van Lier, 1988).
Photodynamic activity of the S1 was determined on
MCF-7c3 cells after overnight incubation with 0.1, 0.5 and
1 ␮M followed by 27 J cm−2 dosage of light. Fig. 4A repre-
sents inhibition of the cell growth (%) plotted against the
drug concentration. Using the MTT assay to measure cell
growth at 24 h after photoirradiation, we show that treat-
ment resulted in 50% cell death (LD50 ) at ∼0.07 ␮M (value
obtained by extrapolation) and more than 90% cell death at
concentrations ranging from 0.5 to 1 ␮M (LD100 ). As shown
in Fig. 4B, S1 leads to photokilling of the cells with dif-
ferent efficacies indicating that cell photoinactivation was
dependent of both the Pc concentration and the light doses
applied. The different concentrations gave different levels
of killing or inhibition of function of MCF-7c3 cells as shown
by their half lethal dose (LD50 ) at 0.5 and 1 ␮M with light
doses of 17.8 and 8.8 J cm−2 , respectively. Cell survival was
not affected when cultures were maintained in the dark
with the S1 concentrations mentioned above.

Fig. 3. Cellular uptake of S1 in MCF-7c3 cells as a function of incubation time. Cells were incubated for the period of time indicated in the abscissa in
the presence of S1 (1 ␮M) under dark conditions. (A) Cells were incubated with S1 into DPPEc liposome and (B) DMPC. The amount of photosensitizer
incorporated by cells is determined by fluorescence measurements on the extracts as described in the text. Values represent mean ± S.D. of three separated
experiments. (Solid squares: culture medium containing 4% FBS; solid spheres: culture medium containing 10%.)
2198 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205
Fig. 4. Effect of S1 photoactivation on MFC-7c3 cell survival. (A) Cells were incubated for 18 h in the presence of different amounts of S1and finally irradiated
(27 J cm−2 ). (B) Cells were pre-treated with S1 (0.5 and 1 ␮M) for 18 h and then irradiated with various light dose. MTT cytotoxicity assay was carried out
24 h after PDT. Results are expressed as the mean ± S.D. of three replicates of each treatment.
3.3. Intracellular localization and sites of photodamage
The subcellular localization of S1 was investigated by
fluorescence microscopy, using MCF-7c3 cells. Fig. 5 shows
that cells incubated 3 h with 1 ␮M of S1 formulation exhib-
ited red fluorescence typical of ZnPc derivatives and mainly
localized in a cytoplasmic region. In order to determine
whether the staining pattern represented lysosomes, we
attempted to colocalize S1 with the organelle-specific flu-
orescent probe LysoTracker Green® (Fig. 5). LysoTracker
Green® also exhibited the expected punctuated cytoplas-
mic distribution in MCF-7c3 cultures. The merged stained
images revealed a partial overlap of S1 and LysoTracker
signals (yellow signal) suggesting that lysosomes are sites
of intracellular distribution of S1 dye, similar results were
published previously (Bonneau, Morliere, & Brault, 2004;
Reiners et al., 2002). The column graph also indicated
the percentage of overlap as spots color quantification
(red, green, and yellow) from the merged pictures which
one revealed more than 70% yellow spots scored corre-
sponding to signal of co-localization S1 with lysosome
probe.
When S1 was combined with 27 J cm−2 of visible light,
an immediate phototoxic effect was noted 1 h after PDT at
the level of the mentioned organelle, leading to the loss of
the punctuated LysoTracker Green® staining pattern, and
this effect was maintained for the first 3 h post-PDT (Fig. 6).
S1 in combination with visible light, but not S1 or light
alone, caused lysosome pattern dye loss, indicating perme-
abilization of lysosomal membranes.
We next investigated whether PDT induces mitochon-
drial morphology changes. MitoTracker Green® is taken up
by the polarized mitochondria. Once inside mitochondria,
MitoTracker Green® binds covalently to sulfhydryls and
is retained by mitochondria after depolarization. There-
fore, MitoTracker Green® is a suitable dye to monitor
mitochondrial volume changes under conditions that pro-
duce mitochondrial depolarization. As shown in Fig. 6,
before PDT MitoTracker Green® -labeled mitochondria were
bright fluorescent spheres and rods. By 3 h after PDT, mito-
chondrial morphology changed; mitochondria began to
round and increase in diameter. An examination of the
typical mitochondrial-labeling pattern indicates that S1
clearly does not exhibit mitochondrial localization but
photodynamic treatment using S1 induced mitochondrial
alternations.
3.4. Mode of cell death
We next monitored the MCF-7c3 cells to elucidate the
type of cell death induced by PDT treatment. Analyses
of morphology and nuclear condensation by HO258 indi-
cate that some of the cells in irradiated cultures preloaded
with S1-DMPC were undergoing apoptosis by within 8 h
of irradiation. The percentage of cells displaying nuclear
chromatin condensation suggestive of apoptosis is shown
in Fig. 7A. The cells with the mentioned features were
increased by more than 60% after photodynamic treatment
with 1 ␮M S1 and 8.8 J cm−2 , corresponding to LD50. Cells
treated with S1, but not exposed to light or without S1
but exposed to light, maintained an unaltered appearance.
Cells began to display nuclear condensation and fragmen-
tation as indicated by the fluorescent dye at 8 h post-PDT.
The same results were also observed with toluidine blue
staining including cell rounding, shrinkage and membrane
blebbing (Fig. 7).
3.5. S1 in animals: dark toxicity and histopathology
examination
With the purpose of determining the non-toxic PS dose
for further phototherapeutic treatment, the cytotoxic effect
caused by S1 on organs (liver, kidney and spleen) was stud-
ied by histological examinations. For this reason, animals
were treated with different concentrations of S1, and then
were sacrificed and the above-mentioned organs were ana-
lyzed.
Microscopic analyses of liver taken from mice adminis-
tered with 0.1 and 0.2 mg/kg S1 did not show any critical
effect on the normal morphology of the organ (Fig. 8A
and B). The biggest concentration evaluated (0.4 mg/kg
S1) leads to hepatocites hypertrophy, degenerative dis-
turbances (general hydropic degeneration) and loss of
lobulillar structure, as well as travecular structure. Indeed,
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205 2199

Fig. 5. Intracellular localization of 1 ␮M of S1 in MCF-7c3 cells after incubation for 3 h. The intracellular distribution and co-localization of S1 with lysosome
were assessed by fluorescence microscopy. MCF-7c3 cells were incubated with both S1 and Lyso Tracker Green DND-26 (75 nM, 15–30 min). Red fluorescence
corresponds to S1 while green fluorescence represents the signal from Lyso Tracker Green DND-26. Yellow fluorescence indicates regions of S1 and Lyso
Tracker Green DND-26 signal overlap. Arrows indicate co-localization spots. Images are representative of those obtained in 3 independent experiments.
Scale bar: 20 ␮m. The column graph represents the percentage of S1 + Lyso Tracker Green signal from merged picture. At least 200 cells were scored. (For
interpretation of the references to color in this figure legend, the reader is referred to the web version of the article.)

hypertrophy of Von Kupffer cells and minor indication of
necrosis was observed (Fig. 8C).
Sections of the kidney stained with H&E showed that
neither 0.1 nor 0.2 mg/kg of S1-DMPC caused any distur-
bance in the physiological morphology of this organ (Fig. 8D
and E). However, a dose of 0.4 mg/kg led to a low degree of
tumefaction from epithelial cells of the proximal and distal
contorneated tubes (Fig. 8F).
At the concentrations employed (0.1 and 0.2 mg/kg), S1-
DMPC caused no detectable alterations on the structural
properties of tissue from the spleen (Fig. 8G and H).
On the contrary, spleen from S1-treated animals
(0.4 mg/kg) revealed large degeneration and cell death
areas (Fig. 8I).
These results allowed us to selection the therapeutic
concentration of 0.2 mg/kg for further photodynamic treat-
ment.
3.6. Phototherapeutic studies
The tumor size was measured again 11 days after
the mice were treated. Tumors that received either no
treatment, S1 alone or light alone increased their size
during the period of observation and there were no sta-
tistically significant differences between these groups.
In contrast, treatment with S1-PDT resulted in partial
tumor regression in all tumors within 10 days after PDT.
Measurement of the tumor volume during the treat-
2200 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205

Fig. 6. S1-PDT causes loss of lysosomal integrity and mitochondrial swelling. Cells grown on coverslips were exposed to 75 mM of LysoTraker Green DND-26
during 30 min (upper panel) or 100 mM of Mito Traker Green FM during 45 min (lower panel). After that, the cells were treated with S1 1 ␮M alone or with
S1 1 ␮M and 27J cm−2 . Images were collected 3 h after PDT treatment. Scale bar: 10 ␮m.

ment revealed a growth delay of ∼5–6 days in tumor
growth in the S1-PDT treated mice. Estimates of contrast
between S1-PDT and each of the other three treatments
mentioned above were statistically different (p < 0.05)
(Fig. 9).
3.7. Tumor viability assay and histological analysis of
S1-PDT treated-tumors
In the tumors of the untreated, light and S1-
dark groups, a total staining with Evans Blue was

Fig. 7. PDT induces apoptosis in MCF-7c3 cells. (A) Percentage of cell death from PDT-treated cultures 8 h later (1 (M S1 and 8.8 J cm−2 , corresponding
to LD50). (B) Upper panel, fluorescence photomicroghaphs of MCF-7c3 cells stained with Hoechst 33258: (left) untreated; (right) MCF-7c3 cells 8 h after
treatment with LD50. Arrows indicate apoptotic nuclei. Lower panel, micrographs of MCF-7c3 cells stained with TB: (left) untreated; (right) cells after 8 h
PDT treatment. Arrows indicate plasma membrane blebbing. Scale bar: 10 ␮m. TB: toluidine blue dye.
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205 2201

Fig. 8. Histological examinations on different organs from mice treated with several concentrations of S1. Microscopic appearance of liver (A–C), kidney
(D–F), and spleen (G–I) obtained at 7 days from mice treated with S1: (A, D and G) 0.1 mg kg−1 ; (B, E, and H) 0.2 mg kg−1 ; (C, F, and I) 0.4 mg kg−1 (×400).
The specimen was stained with H&E. The discontinuous circles on the photograph are representatives of the histopathological alterations described in the
text.

observed (Fig. 10), which indicates that death in
those tumors does not occur. Mice which received
Evans Blue after PDT, showed an increased tumor
death.
In the tumors of the control group the histological cut
showed evidences of malignancy, such as imprecise mar-
gins with infiltrative growth in muscle, mitotic figures,
anisokaryosis, pleomorphism and vascularization. Those
characteristics have also been observed for light and S1-
dark groups (data not shown). In PDT-treated tumors, large
quantity of dead tumor cells can be observed. The sections
presented pycnotic nucleus, sign of injury with principles
of necrosis. The evaluated tumor sections did not present
capsule of connective tissue and we observed intratu-
moral hemorrhages, as a consequence of the S1-PDT effects
(Fig. 11).

4. Discussion

A novel zinc(II) phthalocyanine derived photosensi-

Fig. 9. Effect of S1-PDT on tumors. Tumors were either untreated or
treated with red light alone, S1alone or S1-PDT (0.2 mg kg−1 and red light).
The data represent the means ± S.E.M. of the volumes of four tumors in
each experimental group. Black arrow indicates the beginning of the treat-
ment and grey arrows indicates the light dose applied. Dosage of light:
108 J cm−2 . p < 0.05 S1-PDT vs untreated, S1 and light.
tizer was evaluated for the potential use in PDT. In
vitro screening of S1 administration systems efficacy
should be helpful in determining the most satisfactory
formulation, which would then merit further in vivo
evaluation. Comparison of different formulations in the
same system might lead to a better understanding of the
mechanisms involved in the cellular toxicity of S1 and
would be useful in defining which parameters should
2202 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205

Fig. 10. Evans blue assay on PDT-treated tumors for viability determination. The vital stain was performed by intraperitoneal injection of 0.4 ml 1% EB
solution after 11 days that received different treatments: (A) untreated, (B) light, (C) S1, and (D) S1-PDT. A section from the central area of each tumor was
examined macroscopically and photographed using a magnification of 4×. Unstained area—tumor necrosis, stained area—viable tissue.

be controlled in the preparation of an optimal formula-
tion.
The first part of our study was to determinate that the
DMPC-liposome fusion was the best way for delivering the
dye. Previous reports have shown that Pc aggregation is
easily detected spectroscopically due to the fact that aggre-
gated Pcs do not fluoresce, Pcs in their monomeric forms do,
and thus aggregated Pcs do not serve as PSs (Allen, Langlois,
Sharman, La Madeleine, & Van Lier, 2002; Oda, Ogura, &
Ocurra, 2000). Our data from fluorescent spectra clearly
demonstrates that S1 in culture media was as monomer
employing liposomal formulations, whereas was found in
aggregated form without carriers.
The results of the uptake assays, where this mechanism
has been influenced by the PS aggregation grade, support
the above mentioned results. PSs may be taken up by cells
either directly through the diffusion-mediated pathway or
by endocytosis (Berg & Moan, 1997). We could gain some
insight into the entry process by considering the differences
found in the amount of S1 after incorporation employ-
ing DMPC vesicles. The normal function of these processes
could be impeded in the case of S1 administered without
vesicles leading to a possible accumulation of larger aggre-
gates on the plasma membrane that could be inhibiting
the penetration and subsequent action of the dye. More-
over, in the case of S1 delivery using DPPEc liposome, we
found that the intracellular concentration level of the Pc
was lower than using DMPC (Fig. 3). The liposomal formu-
lations used differ in terms of phospholipid composition;
these differences could explain in part the variability of the
results which have been reported concerning the reduced
uptake of S1-DPPEc liposome by cells. The differences were
the length of the saturated carbon chain (14 carbon atoms
in DMPC, 16 in DPPE), the transition temperature (22 ◦ C for
DMPC and 41 ◦ C for DPPE), and the presence of cholesterol
in DPPE.
In addition, it is known that the effect of the serum
on cellular uptake is counter productive; we observed this
event when overnight incubation with S1-DMPC was car-
ried out with 10% serum, probably molecules are bound to
serum which prevents diffusion and limits cell penetration
mechanisms by endocytosis. In vitro, serum proteins have
been reported to compete with cells for Pc derivatives, thus
decreasing cell uptake of the dye (Konan, Chevallier, Gurny,
& Allemann, 2003; Rodal, Rodal, Moan, & Berg, 1998).
It is evident that S1-DMPC has photodynamic activity on
MCF-7c3 cancer cells. The irradiation regime of sensitized
cells showed a decrease in cell viability (MTT assay 24 h
post irradiation) in a concentration and light dose depen-
dent manner (Fig. 4A). Our results demonstrated that in
the cultures incubated with S1-DMPC followed by 27 J cm−2
irradiation level, there was a loss in viability, leading to a
∼70% inactivation at 0.5 ␮M whereas a substantially greater
phototoxic effect was achieved at 1 ␮M S1-DMPC, which
resulted in ∼100% cell death (Fig. 4B).
PS uptake by cancer cells is crucial for effective PDT and
therefore, to a certain degree, the type of photodamage that
occurs in cells loaded with a PS and illuminated depends
on the precise of subcellular localization of the PS within
the cell.
In the present study the visualization of subcellular
localization of S1 was achieved by the use of fluores-
cence microscopy techniques. The fluorescence localization
pattern observed suggest that this agent would be accu-

Fig. 11. Tumor specimen obtained from PDT-treated mice. H&E stained micrographs of PDT-treated tumor 11 days later. PDT dose: 0.2 mg kg−1 bw S1 by i.p.
injection and red light exposure, 108 J cm−2 three consecutive days. The micrograph (×400) shows a large area of cell death (discontinued circles).
 N.B.R. Vittar et al. / The International Journal of Biochemistry & Cell Biology 40 (2008) 2192–2205
mulated by lysosomes. Similar results have been reported
for PS with affinity for this organelle (Castino, Demoz, &
Isidoro, 2003; Kessel, Caruso et al., 2000; Kessel, Luo et al.,
2000; Reiners et al., 2002; Bonneau et al., 2004). Colocaliza-
tion of subcellular organelle specific probes with differing
fluorescence emission maxima than that of the PS can
be used to more closely identify the site of localization
(Wilson, Olivo, & Singh, 1997) and these probes can also be
used to identify sites of damage after illumination (Kessel
et al., 1997). Our findings from fluorescence localization
patterns of LysoTracker Green sensor correlated with the
photosensitizer thereby demonstrating an affinity for this
cellular compartment (Fig. 5). In addition, the fluorescence
of lysosomal probe became much more diffuse 1 h after PDT
dose application. This observation clearly places the lyso-
some as a primary target of S1 photoactivation due to the
intracellular localization of oxidative stress generated by
PDT coinciding with the intracellular localization of the PS
(Fig. 6).
Moreover, using MitoTracker Green sensor, a marked
effect was noted at the level of mitochondria, showing com-
plete alteration of the morphology within the first 3 h after
PDT, seen as a mitochondrial swelling (Fig. 6). Under these
PDT conditions, we have found morphologic features of
apoptosis such as cell shrinkage, chromatin condensation,
and nuclear fragmentation in MCF-7c3 tumor cells after S1
treatment (Fig. 7). Similar to our findings, photoactivation
of the phthalocyanine Pc4 was reported to induce mito-
chondrial swelling after inner mitochondrial membrane
permeabilization and depolarization, resulting in apoptotic
death (Lam, Oleinick, & Nieminen, 2001).
Apoptosis may be initiated by a wide array of stimuli
that trigger a variety of signaling pathways that, for the
most part, converge at the mitochondrion (Ferri & Kroemer,
2001). Thus, it has been shown that an apoptotic response
can be obtained when lysosomes (Berg & Moan, 1994;
Berg & Moan, 1997; Nagata, Obana, Gohto, & Nakajima,
2003; Woodburn et al., 1997) or mitochondria (Ali, Chee,
Yuen, & Olivo, 2002; Morgan & Oseroff, 2001; Theodossiou,
Noronha-Dutra, & Hothersall, 2006) are targeted for light
induced damage. Furthermore, several reports have related
a variety of cytotoxic agents that trigger lysosomal damage-
induced apoptosis (Neuzil, Svensson, Weber, Weber, &
Brunk, 1999; Roberg, Johansson, & Ollinger, 1999). Given
the susceptibility of lysosomes to oxidant-induced injury; it
is conceivable that lysosomal disruption may be a relatively
common initiator of the mitochondrial/intrinsic apoptotic
pathway.
We concluded that S1 had a primary site of accumula-
tion and photodamage in lysosomes, and cells underwent
apoptosis upon illumination doses leading to 60% cell
death, suggesting that apoptotic pathways caused by PDT
may be activated via lysosomes followed by mitochondrial
destabilization, which leads to amplification of the stim-
uli. The same phenomenon was observed in reports with
the chlorin-based PS, ATX-S10(Na) a lysosomally located
photosensitizer (Nagata et al., 2003).
Our in vivo studies clearly showed that the treatment
was well tolerated and without any significant side effects
as well as its potential tumor regrowth. This regression was
attributed to the combination of S1 and light. Moreover,
2203
microscopically tumor tissue after the therapy confirmed
cell death by mean of necrosis pathways. These data did not
reflect our previously in vitro results where the preferential
cell destruction was by apoptosis. The extrapolation of in
vitro observations to in vivo mechanisms is not always pos-
sible due to the tumor environment differs completely with
the in vitro conditions. In an interesting approach to exploit-
ing immune effects, Castano and colleagues (Castano, Mroz,
& Hamblin, 2006) reported that PDT kill malignant cells by
necrosis stimulating the host immune system.
The photosensitizing agent used in the present report
give an approach that the development of experimental
protocols in vivo can contribute to understanding of the
photokilling effect in basic oncological research and to
assess the potential for clinical applications in PDT of can-
cer.
Acknowledgments
Authors are grateful to Consejo Nacional de Investiga-
ciones Cientı́ficas y Técnicas (CONICET) Argentina, Agencia
Nacional de Promoción Cientı́fica y Tecnológica and SECYT,
Universidad Nacional de Rı́o Cuarto for financial support.
V.R. and J.A. holds a position of Researchers at CONICET.
R.V.N.B. thanks CONICET for a research fellowship. P.C.G.
thanks UNRC for a student fellowship.
The authors thank to Romanini S. for histological exam-
inations. R.V., R.V.N.B., and P.C.G. thank to Argenta Price for
her critical review of the manuscript.
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