Journal of Porphyrins and Phthalocyanines

J. Porphyrins Phthalocyanines 2001; 5: 161–169

Current status of phthalocyanines in the photodynamic

therapy of cancer

CYNTHIA M. ALLEN, WESLEY M. SHARMAN and JOHAN E. VAN LIER*

MRC Group in the Radiation Sciences, Faculty of Medicine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4

Received 7 January 2000; Revised 15 March 2000

Accepted 3 April 2000

ABSTRACT: Photodynamic therapy is a binary treatment now accepted in clinic for various malignancies in several
countries around the world. Phthalocyanine molecules are second-generation photosensitizers with enhanced
photophysical and photochemical properties over those of porphyrins. They have been shown to be phototoxic against
a number of cell types and tumor models. A great deal of research has been devoted to the elucidation of their
mechanism of action and mode of cell death. The present paper reviews phthalocyanine pre-clinical anti-cancer
research with emphasis on phthalocyanine induced apoptosis using a silicon phthalocyanine, Pc4. A brief summary of
the latest clinical results using phthalocyanines is presented. Copyright # 2001 John Wiley & Sons, Ltd.

KEYWORDS: apoptosis; photodynamic therapy; phthalocyanines; Pc4; Photofrin1; photosensitizer; clinical trials

INTRODUCTION agents produced during PDT. This induces a two-fold

In North America, cancer is one of the leading causes of
death and disease. In 1999, over 1.2 million new cases of
malignancies were diagnosed in the United States with
Canada having similar statistics per capita, 4 cases per 1000
individuals (Canadian and American Cancer Societies).
Traditional cancer therapies such as surgery, chemotherapy
and radiation therapy involve a delicate balance between
removing or destroying diseased tissue and sparing
surrounding healthy cells. These conventional treatments
procure serious side effects caused by the loss of normal cell
function due to relatively indiscriminate cytotoxic proper-
ties. The development of new cancer therapies with
improved selectivity is required.
PHOTODYNAMIC THERAPY
A novel cancer treatment that is receiving increased
attention is photodynamic therapy (PDT). PDT is a binary
therapy that involves the combination of visible light and a
photosensitizer. Each component is harmless by itself, but,
in combination with molecular oxygen, can lead to the
generation of reactive oxygen species (ROS), oxidative cell
damage and cell death. The duality of this treatment leads to
greater selectivity towards destroying diseased tissue since
only cells that are exposed simultaneously to the photo-
sensitizer, light and oxygen are subjected to the cytotoxic
selectivity, as there is a preferential uptake of the
photosensitizer by the diseased tissue as well as the capacity
to confine activation of the photosensitizer to the tumor by
restricting the illumination to that specific region. As such,
PDT allows for the exclusive eradication of tumor tissue
while sparing surrounding healthy cells from damage.
The phenomenon of photodynamic action may well date
as far back as primitive man with the use of plant extracts
and sunlight to cure various skin afflictions. In India, as
early as 1400 B.C., psoralens, obtained from the seeds of
Psoralea corylifolia, were used in the treatment of vitiligo
[1]. Photodynamic action, however, was not formally
reported until the turn of the twentieth century when a
medical student, Oscar Raab, working under the direction of
Dr H. von Tappeiner in Munich, noticed the lethal effects of
an acridine orange solution on Paramecia, which were
dependant on the light intensity in the laboratory [2]. This
phenomenon was more extensively studied by von Tappei-
ner and Jodlbauer [3] who coined the term “photodynamic
action”. It was noted that other such compounds such as
eosin were able to induce rapid cell kill in the presence of
light. It was established that dissolved molecular oxygen is
needed for the reaction to occur. Hausmann in 1908 reported
the photohemolysis of rabbit erythrocytes using hemato-
porphyrin (Hp) [4, 5]. Various scientists [6–8] went on to
demonstrate photosensitization of mice, guinea pigs and
man respectively, by inoculation with hematoporphyrin
(Hp). An excellent historical perspective has recently been
published [9].

———————
*Correspondence to: J. E. Van Lier, Department of Nuclear Medicine
and Radiobiology, Faculty of Medicine, Université de Sherbrooke,
Sherbrooke, Québec, Canada J1H 5N4.
E-mail: jvanlier@courrier.usherb.ca
Copyright # 2001 John Wiley & Sons, Ltd.
Mechanisms of Photosensitization
Upon illumination, the photosensitizer used in PDT is
excited to its first excited singlet state. This excited state is
far too short-lived to effectively interact with its surround-
ings and rapidly loses its energy via radiative and non-
162 C. M. ALLEN ET AL.
radiative decay. In terms of PDT, the most important of
these is intersystem crossing to populate the much longer
lived triplet state. This excited state usually has a lifetime in
the ms range [10], which is sufficient time for interaction
between the excited photosensitizer and surrounding mol-
ecules. As such, it is generally accepted that generation of
cytotoxic species during PDT involves interactions with the
excited triplet state of the sensitizer, although examples
exist where the singlet state is involved [11].
This excited triplet state can interact in one of two ways,
defined as Type I and Type II. A Type I mechanism involves
a hydrogen atom abstract or electron transfer reaction
leading to the generation of free radicals and radical ions.
Such species can readily react with molecular oxygen,
inducing irreparable oxidative cellular damage and the
production of ROS, which can lead to further biological
destruction. The role of reactive oxygen species in the
photodynamic process has recently been reviewed [12]. On
the other hand, an energy transfer reaction to molecular
oxygen is defined as a Type II mechanism. The resulting
reactive species, singlet oxygen, is a zwitterionic species
and reacts rapidly with numerous biologically important
substrates, again leading to important oxidative damage and
ultimately cell death. It is generally accepted that the Type
II mechanism predominates during PDT and that singlet
oxygen is the most important cytotoxic species produced
[12–15]. The range of singlet oxygen in cellular media is
limited to approximately 45 nm [16, 17]. With the diameter
of human cells ranging from 10 to 100 mm, the site of
primary generation of singlet oxygen consequently deter-
mines which subcellular target is attacked, either initiating
an apoptotic or necrotic response. Type I reactions become
more important at low oxygen concentrations or in polar
environments [18, 19] and produce reactive oxygen species
such as superoxide anion and hydroxyl radicals that have
Fig. 1. Photofrin1 structure consisting of a complex mixture of dimers and oligomers ranging from
two to nine porphyrin units linked via ether or ester bonds.
Copyright # 2001 John Wiley & Sons, Ltd.
similar short ranges in cellular systems. Furthermore, it has
been recently shown that Type I and Type II mechanisms
probably act in harmony in most cases [20–22]. Overall,
however, the initial reaction, whether via a Type I or a Type
II mechanism, is not of such importance as both lead to
similar oxidative damage and free radical chain reactions.
Ultimately, both mechanisms lead to important oxidative
damage within the target tissue, inducing cell death and
tumor destruction.
PHOTOFRIN1
The first photosensitizers accepted for clinical use by
governmental regulatory agencies are the first-generation
hematoporphyrin derivatives such as Photofrin1 (Fig. 1).
Photofrin1, a mixture enriched in the more active dimeric
and oligomeric components, has been accepted in several
countries for the treatment of early- and late-stage lung
cancer, superficial and advanced oesophageal cancer,
bladder cancer, superficial and early-stage gastric cancer,
early-stage cervical cancer and cervical dysplasia. In
addition, Photofrin1 is being investigated as a possible
therapy for a number of other malignant and non-malignant
conditions. Other hematoporphyrin derivatives are also
being used, with Photoheme, an enriched hematoporphyrin
derivative similar to Photofrin1 accepted for clinical use in
Russia for easily accessible cancers including skin, breast,
oropharingeal, lung, larynx and gastrointestinal cancers
along with psoriasis and prophylaxis for corneal transplant
opacity and recurrent blindness, where newly formed blood
vessels result in loss of corneal transparency [23–26].
Despite the clinical success achieved using Photofrin1,
this first-generation photosensitizer has several important
J. Porphyrins Phthalocyanines 2001; 5: 161–169

drawbacks. It is a complex chemical mixture produced by
the reaction of hematoporphyrin with 5% sulfuric acid in
acetic acid followed by treatment with aqueous base and
neutralization [27]. The resulting dimers and oligomers,
linked primarily with ester and ether bonds, may vary with
different preparations and storage times and make struc-
ture–activity relationships impossible to determine [28].
However, since market approval, Photofrin1 is manufac-
tured using stringent chemical composition and stability
guidelines, insuring product homogeneity. It absorbs very
weakly at the therapeutic wavelength of 630 nm, where
tissue penetration of light is not optimal, limiting treatment
to tumor depths of no more than 5 mm [29]. While suitable
for the more superficial lesions presently being treated,
absorption at longer wavelengths is necessary to treat more
deep-seated or larger tumors. In addition, hematoporphyrin
derivatives have proven to be ineffective for such cancers as
pigmented melanoma due to overlapping absorption spectra
of the photosensitizer and melanin in the malignant tissue
[30]. Most importantly, hematoporphyrin derivatives ex-
hibit an extended retention in cutaneous tissue for up to 10
weeks post-injection [19]. This results in a prolonged skin
photosensitivity, an obvious disadvantage especially for
patients with late-stage malignancies. Nonetheless, the
prolonged skin photosensitivity induced by PDT with
Photofrin1 is only a mild inconvenience as compared to
the severe adverse effects of standard chemotherapeutic
regimens. While these disadvantages have not prevented
Photofrin1 from becoming a useful drug against cancer and
other conditions, the search for new second-generation
photosensitizers with improved physical, chemical and
spectral properties remains an important goal [23, 31].
Ideal photosensitizers for PDT must meet certain criteria,
[9, 27, 29, 32, 33] namely:
. It should be chemically pure and maintain a constant
composition throughout treatment, undergoing minimal
photobleaching.
. It should have minimal dark toxicity.
. The photosensitizer should be preferentially retained by
the target tissue, whether malignant tumor cells or viral
components, so as to induce only marginal toxicity to
surrounding healthy biological matter. In addition, the
excess dye should be rapidly excreted from the body
exhibiting low systemic toxicity.
. The dye should have high photochemical reactivity with a
high quantum yield of long-lived triplet-states energetic
enough to produce singlet oxygen.
. The sensitizer should have a large absorption coefficient
at a long wavelength (600–800 nm) where there is optimal
tissue penetration by light with a low degree of
attenuation by hemoglobin. Furthermore, cheaper diode
lasers can be used in this range, thus increasing the
potential utility of PDT in a clinical setting.
While no photosensitizer can be expected to fulfill all of
these parameters, numerous second-generation photosensi-
tizers have been investigated that can overcome the
shortcomings of hematoporphyrin derivatives and take
advantage of their more highly suited properties in order
to treat a number of conditions. For instance, the use of 5-
aminolaevulinic acid (ALA)-induced endogenous photo-
sensitizers has recently gained clinical acceptance for the
treatment of actinic keratoses of the face and scalp. In the
meanwhile, verteporfin (benzoporphyrin derivative mono-
Copyright # 2001 John Wiley & Sons, Ltd.
CURRENT STATUS OF PHTHALOCYANINES 163
acid ring A) has received market approval in Switzerland
under the tradename Visudyne1 as a therapy for wet age-
related macular degeneration, a leading cause of blindness
in people over the age of 50. Visudyne1 has also recently
gained acceptance as a treatment for AMD in several
countries including the United States and Canada and is
undergoing further clinical trials for other diseases causing
vision loss due to choroidal neovascularization. Overall, the
photosensitizer used to treat a given condition will greatly
depend on the characteristics of the compound and the
condition to be treated, with a wide range of different
photosensitizers being used in clinic, each for its own
particular use.
PHTHALOCYANINES
Among the more promising second-generation photosensi-
tizers are phthalocyanines (Fig. 2). The term phthalocyanine
finds its origin in the Greek word ‘naptha’ which means rock
oil and “cyanine” which means dark blue. It was first used to
describe this class of macrocyclic compounds by Sir
Reginald Linstead in 1933 during his pioneering work on
the subject [34]. In hindsight, the first recorded observation
of a phthalocyanine occurred in 1907 [35]. During the
synthesis of o-cyanobenzamide from phthalamide and
acetic anhydride, Braun and Tcheriac observed the produc-
tion of a colored impurity of unknown structure and origin.
However, this colored by-product was not further studied. It
was not until 1927 that a second preparation of phthalo-
cyanine was reported [36]. Scottish Dyes, Ltd. went on to
further exploit the usefulness of these macrocycles and was
granted the first patent with respect to phthalocyanines [37].
In addition to the immediate applications as dyes and
colorants, it was realized that such compounds would be of
great academic interest. As such, starting in 1929, Linstead
and his group began their work on phthalocyanines which
lead to the determination of their structure in the early 1930s
[38–40]. The synthesis of Pcs [41–43] and their relationship
to porphyrins [44], along with further examination into the
intricate structure of Pcs [43], their planar nature, their
complexes with metal ions [40, 43, 45, 46] and their
stability [38, 43] were subsequently studied. The structure
of phthalocyanines was later confirmed by Robertson via
Fig. 2. Typical phthalocyanine structure where M is the central
metal atom (Al, Co, Ga, Si, Zn) and R represents a multitude of
possible ring substituents including SO3H, F, COOH, etc.
J. Porphyrins Phthalocyanines 2001; 5: 161–169
164 C. M. ALLEN ET AL.
X-ray crystallography in a series of classic papers [47–50].
In the seventy years to follow this initial work, a plethora of
information emerged concerning diverse synthetic routes,
Pc photochemical and photophysical properties and poten-
tial applications. They have long been used as dyes and
coloring agents [51] and have recently gained an industrial
application as photoconducting agents in photocopying
devices [52]. Other potential applications for phthalo-
cyanines include chemical sensors [53], optical storage
devices [54], molecular metals and conducting polymers
[55] and catalysts for numerous chemical reactions [51].
Of the utmost importance is their role in photodynamic
therapy. These azaporphyrin derivatives have stronger
absorbances at longer wavelengths than do porphyrins.
Pcs have favorable photophysical and chemical properties.
In addition, these properties can be altered through the
addition of substituents to the periphery of the macrocycle
or axial ligands to the chelated central metal ion. This makes
them interesting photosensitizing agents.
Phthalocyanine derivatives have attractive photophysical
and photochemical properties as compared to hematopor-
phyrin derivatives. Monomeric Pcs have a characteristic
absorption spectra with a strong Soret absorption peak at
approximately 350 nm, a weak maximum around 600 nm
and a narrow, very strong, absorption peak in the far-red
region of the visible spectra (Pc max  680 nm; HpD
max  630 nm) where tissue penetration by visible light is
superior. In addition to the red-shift of the phthalocyanine Q
band absoption maxima, Pcs have an improved capacity to
absorb light, by two orders of magnitude over that of the
highest Q band absorption of HpD (Pc molar extinction
coefficient, e  105 Mÿ1 cmÿ1; HpD e  103 Mÿ1 cmÿ1)
[56, 57]. These qualities therefore lead to enhanced photo-
physical and photochemical properties.
The nature of the central metal ion influences the Pc
photophysical properties (triplet quantum yield and life-
time). Complexation of Pcs with open shell or paramagnetic
metal ions such as Cu2‡, Co2‡, Fe2‡, Ni2‡, VO2‡, Cr3‡ and
Pd2‡ give dyes with shortened triplet lifetimes (nanosecond
range) due to increased intersystem crossing back to the
ground state, which renders the dye photoinactive [58]. Pcs
containing closed d shell or diamagnetic metal ions, such as
Zn2‡, Al3‡ and Ga3‡, are dyes producing high triplet yields
(fT > 0.4) with long lifetimes (tT > 200 ms) [59]. These
triplets vary in energy from 110–126 kJ/ molÿ1 which is
ample energy to generate singlet oxygen (94.5 kJ/ molÿ1)
with high quantum yields (f) of approximately 0.3–0.5 [60].
Singlet oxygen yield is affected by ring substituents and
axial ligands. In aqueous solutions, Pcs, either non-
substituted or substituted with hydrophilic groups, tend to
aggregate or dimerize which affects cell penetrating
properties and results in a loss of photochemical activity.
Upon excitation with light, energy is dissipated via internal
conversion to the ground state rather than triplet formation
with subsequent singlet oxygen production [59]. Decreasing
the number of sulfonate substituents increases the potential
to aggregate, however monomeric Pc molecules with
identical central metal ions, and varying degrees of
sulfonation, retain the same photochemical activity. The
extent of aggregation determines the MPcSn activity [60].
Aggregation is easily detected spectroscopically as absorp-
tion occurs at shorter wavelengths in the Soret region and
there is a 30–50 nm blue-shift of the Q band absorption peak
which appears more broad and less intense. Disaggregation
to form monomeric dyes can be accomplished by adding
Copyright # 2001 John Wiley & Sons, Ltd.
detergents, serum, plasma proteins or organic solvents
[57, 61].
Phthalocyanine Delivery
Increased photodynamic action has been attributed to
increased monomerization of the photosensitizer. Lipophilic
photosensitizers require biologically compatible delivery
systems. Nanoparticles have received attention as potential
delivery vehicles for drug targeting. A hexadecafluoro zinc
phthalocyanine was formulated in polyethylene–glycol-
coated poly(lactic acid) nanoparticles and was evaluated
for PDT efficiency. Using the EMT-6 tumor model,
improved tumor response was observed using the nanopar-
ticle formulation as compared to the same dye solubilized in
Cremophor EL [62]. Nanoparticle formulation allows for
increased serum levels of the dye as compared to
Cremophor EL formulation. Increased blood levels and
enhanced tumor uptake lead to higher tumor-to-organ
concentration ratios enhancing the overall dye efficacy
[63]. Likewise pH-responsive, polymeric micelles consist-
ing of random copolymers of N-isopropylacrylamide,
methacrylic acid and octadecyl acrylate have been loaded
with water-insoluble AlPcCl and shown to exhibit higher
photocytotoxicity against EMT-6 cells in vitro than the
control Cremophor EL preparation [64].
In addition to emulsifiers such as Cremophor EL, Solutol
HS or lipid-based vehicles such as liposomes, a substantial
amount of research has focused on proteinic carriers for
improved targeting of malignant cells. Serum lipoproteins
can incorporate hydrophobic sensitizers into their lipid
portion. Frequently, neoplastic cells have increased low-
density lipoprotein (LDL) receptors on the cell surface as
increased proliferation requires cholesterol for membrane
synthesis [65]. Subsequently, LDL are known to deliver
photosensitizers to neoplastic cells through a receptor
internalization mechanism [66–69]. Jori and associates
have done extensive studies using ZnPc incorporated into
liposomes of varying composition. In addition, they have
investigated the role of serum lipoproteins and found them
to be effective delivery vehicles for photosensitizers such as
phthalocyanines [70–73].
Recently, a series of publications has reported the use of a
modified aluminum tetrasulfophthalocyanine covalently
coupled to various proteins via one or two caproic acid
spacer chains. Brasseur et al. targeted the scavenger
receptor of macrophages using a maleylated bovine serum
albumin (BSA) covalently labeled with AlPcS4. Relative
photocytotoxicities were reported using a receptor positive
and a receptor negative cell line, where their lethal effects
correlated with receptor affinity for the BSA–Pc conjugate
[74].
Using the same phthlocyanine derivative, it was found
that covalently labeling a monoclonal antibody for the
CD3‡ antigen in a similar manner did not hinder receptor
recognition. There was increased uptake of the antibody–Pc
conjugate in a receptor positive cell line as compared to the
free phthalocyanine and antibody–Pc uptake was less in a
cell line void of receptors [75]. However, the Pc–antibody
conjugates were photoinactive, most likely due to inefficient
subcellular targeting. Employing similar compounds,
AlPcS4 was covalently coupled to monoclonal antibodies
directed against carcinoembryonic antigen (CEA) [76]. In
vivo studies using nude mice bearing human colon
J. Porphyrins Phthalocyanines 2001; 5: 161–169

carcinoma xenografts (T380) demonstrated increased up-
take of the antibody–Pc conjugate as compared to the free
Pc. In vitro results were also encouraging using this anti-
body complex.
Using the same principle, adenoviral proteins were
employed to target lung cancer cells rich in the appropriate
class of integrin receptors [77]. It was concluded that
adenoviral capsid proteins, principally the penton base,
when covalently bound to a modified AlPcS4 are able to
target receptor-bearing cells. However, in vitro photocyto-
toxicity was limited. Using the EMT-6 murine model, in
vivo results were more encouraging using the Pc–adenoviral
protein conjugates.
Urizzi and co-workers described parallel studies using
both covalently labeled and a non-covalently labeled LDL
to target human adenocarcinoma lung cancer cells (A549).
Covalently labeling the apoprotein portion did not hinder
receptor recognition yet cytotoxic effects were marginal,
presumably due to photosensitizer entrapment in the
endosome. Amphiphilic AlPcS4 molecules bearing long
aliphatic side chains on the Pc macrocycle were non-
covalently inserted in the LDL phospholipid layers. There
was a substantial increase in cell uptake of the phthalocya-
nine resulting in a 10-fold increase in cytotoxicity of the Pc–
LDL conjugate in vitro [66].
A novel approach to photosensitizer targeting has
recently been reported using epidermal growth factor
(EGF) [78]. Both AlPc and CoPc were covalently labeled
with EGF. Lutsenko and associates report a five-fold
increase in photocytotoxicity using the EGF–Pc complex
as compared to the uncoupled Pc. Cytotoxicity was
measured in both the human MCF-7 and the murine B16
cell lines. One month post-PDT, in vivo studies using the
melanoma B16 tumors implanted in C57B1/6 mice resulted
in tumors being half the volume using the CoPc–EGF
conjugate as compared to untreated tumors or tumors treated
with CoPc alone.
Pc4
A promising phthalocyanine receiving a great deal of
attention is the silicon Pc, denoted as Pc4, bearing a
long-chain amino axial ligand (HOSiPcOSi(CH3)2
(CH2)3N(CH3)2) (Fig. 3). This silicon phthalocyanine has
shown promising results both in vitro and in vivo and is
presently entering clinical trials for the treatment of
neoplasms and has been examined for the sterilization of
Fig. 3. Silicon phthalocyanine designated as Pc4.
Copyright # 2001 John Wiley & Sons, Ltd.
CURRENT STATUS OF PHTHALOCYANINES 165
blood components [23, 79]. Axial ligated Pcs have an
important advantage over Pcs substituted on the periphery.
Not only does the axial ligand impart greater solubility and
prevent aggregation, additionally these compounds do not
exist as isomers. As such, obtaining a pure sample is more
apparent.
Since the first reports in 1993, Pc4 has been studied
extensively using various cell lines and protocols. Initial
reports using V-79 Chinese hamster lung fibroblast indi-
cated a three-fold increase in Pc4 cytotoxicity as compared
to a reference AlPc [80]. No difference in intracellular
uptake was observed between these two photosensitizers,
seeming to indicate that this increased photocytotoxicity
was due to differences in the intracellular distribution and/or
the aggregation of the phthalocyanines in vitro [81]. Pc4
was also shown to be highly phototoxic on human erythro-
cyte ghosts, photoinducing lipid peroxidation. Using singlet
oxygen quenchers such as sodium azide and histidine, it was
determined that Pc4, at least in part, induces cytotoxicity via
a Type II mechanism [82].
Early in vivo reports were also promising using RIF-1
fibrosarcoma tumors in mice as a test model. At reasonable
Pc4 doses (1 mg kgÿ1) in 25% Cremophor EL, tumor
regression was evident with no re-growth using a light dose
of 135 J cmÿ2 [80, 83, 84]. Cutaneous photosensitivity was
transient using this photosensitizer, lasting only 24 hours
after onset occurring 10 days post-Pc4 administration.
Photosensitivity was no longer a factor. Photofrin1, under
similar conditions, invoked skin photosensitivity over a
period of 4 days. One month post-injection, mice remained
sensitive to solar light [85, 86]. Similarly, skin photosensi-
tivity using a silicon napthalocyanine having two long alkyl
chain axial ligands was investigated. At minimal photo-
therapeutic doses of 0.1 mmol kgÿ1, the dye elicited only a
mild and transient skin photosensitivity which subsided in a
few days. Photofrin1, on the other hand, induced a severe
reaction, requiring 3 to 8 weeks for complete recovery [87].
This would seem to indicate that such phthalocyanines do
not exhibit an appreciable and prolonged cutaneous tissue
uptake.
The photocytotoxicity of Pc4 was also determined against
L5178Y-R leukemic lymphoblasts [88]. Similar results
were obtained as for RIF-1 cells. In addition, this study
concluded that the number of axial ligands did not appear to
affect the photocytotoxicity of the compound. Contrarily,
the length of the axial ligand was shown to influence
phototoxicity as extended amino axial substituents were less
effective against these murine cell lines. It was theorized
that the shorter aminosiloxy ligands in Pc4 lead to binding at
more important intracellular targets.
Apoptosis or Necrosis
Preliminary results were promising using phthalocyanine
derivatives, thus prompting more in-depth studies regarding
mode of cell death. Apoptosis is a notable mode of cell
death in response to PDT using phthalocyanines [89]. Pc4
has been shown to initiate an apoptotic response in several
cell lines including L5178Y-R cells. DNA fragmentation, a
hallmark of apoptosis, was observed [88]. This same
research team demonstrated the importance of cell mem-
brane damage with respect to apoptosis induction. Photo-
damaging the cell membrane initiates a cascade of events
such as phospholipase C activation, release of second
J. Porphyrins Phthalocyanines 2001; 5: 161–169
166 C. M. ALLEN ET AL.
messengers and an increase in intracellular calcium con-
centration, all leading to cell suicide [90]. Furthermore, an
increase in intracellular calcium concentration also leads to
the formation of nitric oxide (NO), an important bioactive
signaling molecule. It has been demonstrated that NO is
involved in PDT-mediated apoptosis [91]. In addition to
NO, other well-documented apoptotic pathways are in-
itiated by Pc4-PDT. Cytochrome c is released into the
cytosol and subsequent activation of proteases [92], now
known as caspases, resulting in the cleavage of various
proteins including poly(ADP-ribose)polymerase have been
documented [93]. Cell cycle arrest in the G0/G1 phase of the
cell cycle as a result of p21 induction has been observed
following Pc4 therapy [94]. In addition, the participation of
retinoblastoma protein has been studied following photo-
therapy [95]. Several other apoptotic pathways and proteins
involved in PDT-mediated apoptosis are under investiga-
tion.
Furthermore, enhanced apoptotic response to photody-
namic therapy with AlPc was observed after Bcl-2
transfection [96]. Bcl-2 is a known inhibitor of apoptosis
via antagonizing the release of mitochrondrial cytochrome
c. In this case, using immortalized MCF-10A cells, over-
expression of Bcl-2 and bax was revealed and PDT caused
selective destruction of Bcl-2, leaving bax unaffected. As
such, the greater apoptotic response towards PDT in this
case is probably attributed to the higher bax:Bcl-2 ratio after
photodamage. On the other hand, CHO-K1 cells transfected
with Bcl-2 exhibited a partial resistance to PDT-induced
cell death, whether via apoptosis or non-apoptotic mechan-
isms [97].
Subsequently, it was demonstrated that mode of cell
death is not only dependant on cell type (i.e. fibroblasts do
not die via organized DNA fragmentation [88]) but also on
the photosensitizer, its intracellular localization and the
drug and energy dose administered. In addition, it has
recently be shown that the mode of cell death also varies
with cell density due to the bystander effect [98]. Using
AlPc and lower PDT doses resulted in the induction of
apoptosis as opposed to necrosis. Organelles such as the
mitochondria and lysosomes were mainly affected. At
higher PDT doses, cells underwent severe membrane
damage resulting in necrosis [99]. Interestingly, Dellinger
reported a mixed response when using Photofrin1 and CV-1
cells [100]. At higher irradiation doses, the cells lysed
instead of exhibiting fragmentation associated with apop-
tosis. However, morphological changes typical of apoptosis
were observed initially, such as plasma membrane blebbing,
condensation of the cytoplasm and nuclear fragmentation.
These cells appear to enter an apoptosis-like death, yet this
pathway is stopped by plasma membrane lysis and
ultimately, the cells undergo necrosis. In vivo studies using
the murine MS-2 fibrosarcoma tumor model demonstrated
random necrosis of tumor cells post-ZnPc-PDT. Interest-
ingly, apoptotic pathways were also detected, suggesting
different localization of photosensitizers within the cell
upon illumination [101].
Photodynamic therapy using phthalocyanines is known to
induce a number of responses that ultimately lead to
apoptosis. For instance, PDT using Pc4 induces an oxidative
stress associated with increased ceramide generation [102],
which has been associated with apoptosis in several malig-
nant and non-malignant cell lines. The increased production
of ceramide is due to the stress-induced activation of
sphingomyelinase (SMase). It has been shown that normal
Copyright # 2001 John Wiley & Sons, Ltd.
human lymphoblasts accumulated ceramide and underwent
apoptosis after Pc4-PDT. In contrast, Niemann–Pick disease
lymphoblasts, which are deficient in acid sphingomyelinase,
fail to respond to Pc4-PDT via ceramide accumulation and
apoptosis [103]. However, addition of exogenous bacterial
SMase during Pc4-PDT induced a significant apoptotic
response. This lead to the hypothesis that SMase may be an
important proapoptotic factor determining responsiveness
of cells to Pc4-PDT. Previous work showed that Pc4-PDT
was able to induce apoptosis in numerous cell lines. Three
cell lines were studied in vitro revealing the accumulation of
ceramide post-PDT and induction of apoptosis. Interest-
ingly, there were increased levels of ceramide in RIF-1 cells
post-PDT with no evidence of apoptosis. Ceramide clearly
is associated with PDT-mediated cell death either by
inducing apoptosis or necrosis [104].
Indirect or Direct Effect
Aluminum disulfonated phthalocyanine with sulfonate
groups on adjacent isoindoline units (AlPcS2adj) has been
used to photoinactivate human epidermal keratinocyte (UP)
and oral squamous cell carcinoma-derived cells (H376) in
vitro. This direct cell kill in vitro suggests that direct killing
of tumor cells may contribute to tumor regression in vivo
[105]. Using the murine EMT-6 model, disulfonated
aluminum phthalocyanine induced a significant direct tumor
cell phototoxic effect. Tumor vasculature damage, an
indirect effect, was assessed using 99mTc–methoxyisobutyl
isonitrile (99mTc–MIBI) retention in the tumor. The blood
flow declined to 50% of that of the control tumor and
remained constant. This is indicative of direct cell kill as
opposed to tumor vasculature shut down. Photofrin1-
treated tumors immediately exhibited decreased blood flow
to the treated tumor with almost no blood entering the tumor
stroma 24 hours post-PDT. This is indicative of an indirect
tumor cell death by starving the cells of vital nutrients
[106, 107]. A recent study confirmed these results using
positron emission tomography (PET). Mice-bearing EMT-6
tumors were subjected to either Pc-PDT or PII-PDT. Thirty
minutes post-Pc-PDT, [18F]fluoro-2-deoxy-D-glucose
(FDG) activity falls to about 50% of the control tumor
value while remaining well above the blood activity level.
In contrast, at 2 h post-irradiation, tumor activity drops to
the blood activity level. These results are consistent with an
action mechanism proceeding primarily by initial direct cell
kill, resulting in a gradual decrease in tumor cell survival
while sparing tumor microvasculature. PII-PDT affected
tumor metabolism immediately following PDT, as seen by
limited FDG uptake, thus suggesting reduced blood flow
and poor delivery of the tracer at the tumor site, indicative of
an indirect cell death mechanism [108].
Phthalocyanines In-Clinic
As was stated previously, Pc4 is about to enter clinical trials
for the treatment of neoplasms and has been investigated for
the sterilization of blood components [23, 78]. In addition, a
liposomal preparation of zinc phthalocyanine (CGP55847)
has been in early Phase I/II clinical trials in Switzerland for
the treatment of patients with squamous cell carcinomas of
the upper digestive tract [109]. Attempts were also under-
way to develop a topical formulation of this photosensitizer
J. Porphyrins Phthalocyanines 2001; 5: 161–169

for use in the treatment of skin disorders such as psoriasis
[110].
The onocological center of the Russian Academy of
Medical Sciences and the Moscow Medical Academy
treated 42 patients from 1992 to 1995 using a mixture of
AlPcSn, trade named Photosense. Cancers such as basal and
squamous cell skin cancers, breast cancer, cancer of the
tongue, oral mucous and lower lip, lung and larynx cancer
and various others have been treated using dye doses of
0.5–1 mg kgÿ1 body weight with success. Fifty percent
underwent complete tumor regression without re-growth
while 43% exhibited a partial response. Only 7% gave no
response at all [25]. The phthalocyanine mixture exhibited
no systemic toxicity. No immune system modifications were
observed [111]. More precisely, 93 tumor sites with
different head and neck tumors were treated with Photo-
sense. Over 50% of the tumors exhibited a complete
response where there was no clinical evidence of malignant
tissue. Various cancer types responded well to Photosense
therapy except for melanoma, where none of the treated
neoplasms gave a complete response [112]. The State
Research Center for Laser Medicine (Moscow, Russia)
reported excellent results for skin cancer when using the Pc-
PDT. Overall, 85% responded completely to the Pc-PDT
treatment of skin cancers [24]. A recent review of phthalo-
cyanines as photosensitizers has been published outlining
the success of the clinical studies carried out in Russia
[113].
CONCLUSION
In this new millennium, photodynamic therapy is becoming
a tangible treatment not only for malignancies but for
various afflictions too numerous to mention in this
publication [23, 114–122]. It is interesting that in this age
of technology the simplicity of this bimodal treatment
contributes to its versatility and usefulness. Phthalocyanines
are extremely powerful photosensitizers and pre-clinical
work clearly show their immense promise in photodynamic
therapy. Only the future will dictate whether phthalo-
cyanines will fulfill their potential as photosensitizing
agents for the treatment of cancer and other conditions.
Acknowledgements
The Canadian Institutes of Health Research is gratefully
acknowledged for their financial support. J.E.vL. is the
holder of the Jeanne and J.-Louis Lévesque Chair in
Radiobiology. C.M.A. and W.M.S. are recipients of
doctoral stipends from the Minister of Education of Québec
(Fonds pour la formation de Chercheurs et l’Aide á la
Recherche, FCAR).
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