Journal of Porphyrins and Phthalocyanines

J. Porphyrins Phthalocyanines 2001; 5: 105–129

REVIEW

Basic principles of photodynamic therapy

IAN J. MACDONALD* and THOMAS J. DOUGHERTY

Photodynamic Therapy Center, Roswell Park Cancer Institute, Buffalo, NY 14263, USA

Received 15 April 2000

Accepted 8 June 2000

Table of Contents: 1.6.1 Vascular Shutdown and Inflammation

1.6.2 Direct Cell Effects
1. INTRODUCTION 1.6.2.1 DNA Damage
1.1 History of Photodynamic Therapy 1.6.2.2 Lysosome Damage
1.2 Photochemistry of Photodynamic Therapy 1.6.2.3 Mitochondrial
1.3 Oxygen in PDT Damage
1.3.1 Singlet Oxygen 1.6.2.4 Membrane Damage
1.3.2 Tissue Oxygenation 1.7 Apoptosis
1.4 Photosensitizers 1.7.1 Apoptosis and Necrosis
1.4.1 Porphyrin Photosensitizers 1.7.2 Apoptosis in PDT
1.4.2 Photofrin 1.7.3 Mitochondrially Controlled Apoptosis
1.4.3 New Photosensitizers 1.7.3.1 Reactive Oxygen
1.4.4 Photosensitizer Pharmacology Species (ROS) and the
1.4.4.1 Structure–Activity and Mitochondrial Membrane
Quantitative Structure– Potential (D m) in Apoptosis
Activity Relationships 1.7.3.2 Permeability
1.4.4.2 Targeted Photosensitizers Transition (PT)
1.4.4.3 Amphiphilicity 1.7.3.3 Apoptosis Inducing Factor
1.4.4.4 How Amphiphilicity, and Cytochrome c in
Aggregation, and Apoptosis
Serum Protein Interactions 1.7.4 Summary, PDT and
Affect the Photodynamic Apoptosis
Activity of Photofrin1 1.8 Immune Effects in PDT
1.4.4.5 How Amphiphilicity, 1.8.1 Immune System Activation and
Aggregation, and Suppression
Serum Protein Interactions 1.8.2 Immune System Modulators
Affect the Photodynamic 1.9 Clinical Photodynamic Therapy
Activity of Other
Photosensitizers Acknowledgement
1.5 Light
1.5.1 PDT Dose REFERENCES AND NOTES
1.5.2 Light Penetration
1.5.3 Photobleaching
1.5.4 Light Sources and Delivery
1.6 Biological Mechanisms of Action
1. INTRODUCTION
1.1 History of Photodynamic Therapy

——————— The therapeutic use of light, as we know it, begins in 1900

*Correspondence to: I. MacDonald, Photodynamic Therapy Center, when Raab reported that the combination of acridine orange
Roswell Park Cancer Institute, Buffalo, NY 14263, USA. and light could destroy living organisms (paramecium) [1].
In the 1920s Policard [2] noted that tumor tissue was
Copyright # 2001 John Wiley & Sons, Ltd. inherently more fluorescent than healthy tissue. In the 1950s
106 I. J. MACDONALD AND T. J. DOUGHERTY
Ronchese [3] attempted to activate endogenous fluorescent
molecules in tumor tissue to delineate its boundaries more
accurately. Between 1940 and 1960 Figge et al. [4–6] and
Rasmussen-Taxdal et al. [7] administered natural porphy-
rins to patients and tumor-bearing animals in an attempt to
more accurately detect tumor tissue by fluorescence. During
the 1960s Winkelman [7–10] used synthetic porphyrins
(tetraphenylporphines) to detect tumor tissue. Throughout
the 20th century a few attempts were made to treat tumor
tissue with photosensitizing agents, mainly with non-
porphyrin photosensitizers. Tappeiner et al. and Jesionek
et al. [11, 12] used sunlight to activate eosin in tumor tissue
in vivo. Bellin et al. examined the photosensitizing activity
of many different dyes in vitro. However, until the 1960s the
field of photodynamic therapy lay fallow until a chance
connection between Richard Lipson and Samuel Schwartz
occurred. Schwartz had isolated a tumor localizing impurity
from hematoporphyrin preparations that was later named
hematoporphyrin derivative (HpD). Lipson was investigat-
ing how to detect tumor tissue by observing the intratumoral
fluorescence of hematoporphyrin. Unable to obtain repro-
ducible results with hematoporphyrin, Lipson began
experimenting with Schwartz’s HpD. He used it as a tumor
detection agent [13], and recognized that it could be used as
a photosensitizer to destroy tumor tissue [14].
In the 1970s Dougherty rediscovered that fluorescein
diacetate could photodynamically destroy TA-3 cells in
vitro [15]. Dougherty then began treating tumor-bearing
animals with fluorescein and found that it could work as a
photosensitizer [16]. Weishaupt et al. identified the cyto-
toxic product of the photochemical reaction to be singlet
oxygen [17]. However fluorescein has a low singlet oxygen
quantum yield and a long wavelength absorption in the
green portion of the electromagnetic spectrum that does not
penetrate deeply into tissue. Porphyrin photosensitizers
were then examined as photosensitizers because they are
efficient singlet oxygen generators and have absorption
maxima in the red portion of the electromagnetic spectrum.
Eventually Dougherty rediscovered Schwartz’s HpD, which
by then was known to have a high singlet oxygen quantum
yield, an absorption maximum in the red, and is selectively
retained in tumor tissue [15]. After several years spent
isolating and identifying the active fractions of HpD, a
purified version named Photofrin1, was produced. In the
following years Photofrin1 was approved for use in the
United States against early-and late-stage lung cancers and
esophageal cancers and dysplasias with other indications
pending [15]. At the time of this review it has been used on
nearly 10 000 patients in the United States, Canada,
Netherlands, Japan, France, and Italy (and is pending
approval for use in eight other countries). In addition to the
approved indications, Photodynamic therapy (PDT) has
been used against bladder cancers, brain cancers, breast
metastases, skin cancers, gynecological malignancies,
colorectal cancers, thoracic malignancies, and oral, head
and neck cancers [18]. Since Photofrin has gained legal
status many of the mechanisms by which PDT works have
been elucidated, many new photosensitizers have been
created [18] and research and development continues.
1.2 Photochemistry of Photodynamic Therapy
The photochemical reaction that generates singlet oxygen,
the putative cytotoxic agent in PDT [17], from ground-state
oxygen is represented by the Jablonski diagram (Fig. 1)
Copyright # 2001 John Wiley & Sons, Ltd.
Fig. 1. A Jablonski diagram showing the various modes of
excitation and relaxation in a chromophore: (A) excitation; (B)
fluorescence; (C) intersystem crossing; (D) phosphorescence;
(E) non-radiative transfer of energy to singlet oxygen; (F)
substrate oxidation by singlet oxygen; (G) internal conversion.
[19]. Illuminating the chromophore with light of the
appropriate wavelength excites it to excited singlet states
(A). The chromophore can relax back to the ground state by
emitting a fluorescent photon (B) or to excited triplet states
via intersystem crossing (ISC) (C). From triplet excited
states the chromophore can relax back to the ground state by
emitting a phosphorescent photon (D) or transferring energy
to another molecule via a radiationless transition (E). In the
first-order approximation, radiative, triplet to singlet transi-
tions are quantum mechanically ‘forbidden’ since a change
of electron spins is required, however these transitions occur
to a very small extent [20]. Additionally, the chromophore
can also lose energy through internal conversion or
radiation-less transitions during collisions with other mol-
ecules [19] (G). In oxygenated environments the chromo-
phore readily transfers its energy to ground-state molecular
oxygen (3O2) to produce singlet oxygen (1O2) which can
form adducts with organic substrates [S(O)](F).
The photosensitizer and oxygen interact through the triplet
states because oxygen has a unique, triplet-ground state and
low-lying excited states. The energy required for the triplet
to singlet transition in oxygen is 22 kcal molÿ1, which
corresponds to a wavelength of 1274 nm (infrared light)
[21]. Thus relatively low energy is needed to produce singlet
oxygen. Photochemical reactions of this type are known as
Type-II photoreactions and are characterized by a depen-
dence on the oxygen concentration (O2) (Fig. 2) [22].
Although Type-II photoreactions are commonly associated
with singlet oxygen production some other compounds have
triplet-ground states and can be involved in Type-II
photoreactions, among these are nitric oxide and vitamin A
[22]. In anoxic environments excited photosensitizers (3P*)
can react directly with organic substrates (S) by electron
exchange producing an oxidized substrate (S‡) and reduced
photosensitizer (Pÿ). In hypoxic environments the reduced
photosensitizer (Pÿ) can react with oxygen to produce
superoxide anions …Oÿ 2 † which. can then form the highly
reactive hydroxyl radical (OH ) [22]. The excited photo- .
sensitizer (3P*) can also react with superoxide radicals …O2 †
ÿ
to produce superoxide anions …O2 † which can then create the
.
highly reactive hydroxyl radical (OH ) [22]. Collectively
these reactions are classified as Type-I photoreactions and
are characterized by a dependence on the target–substrate
concentration (Fig. 2) [22]. Although Type-II reactions are
reported to dominate during PDT [23], Foote has suggested
J. Porphyrins Phthalocyanines 2001; 5: 105–129

Fig. 2. Type-I and Type-II photoreactions, where 1P is a
photosensitizer in a singlet ground state, 3P* is a photosensi-
tizer in a triplet excited state, S is a substrate molecule, Pÿ is
reduced photosensitizer molecule, S‡ is an oxidized substrate
molecule, O2 is molecular oxygen (triplet ground state), Oÿ 2 is
the superoxide anion, O2 is the superoxide radical, P‡ is the
oxidized photosensitizer, 3O2 is triplet ground-state oxygen,
1
O2 is oxygen in a singlet excited state, and S(O) is an oxygen
adduct of a substrate.
Type-I reactions may become more dominant under
conditions where the photosensitizers are highly concen-
trated, and especially under hypoxic conditions [22].
1.3 Oxygen in PDT
1.3.1 Singlet Oxygen. Singlet oxygen, the predominant
cytotoxic agent produced during PDT [17] is a highly
reactive form of oxygen that is produced by inverting the
spin of one of the outermost electrons. Normally, ground-
state oxygen has two unpaired electrons residing separately
in the outermost antibonding orbitals (Fig. 3). In the absence
of a magnetic field the electronic configuration is indis-
tinguishable, however in a magnetic field (B) the spins of
the electrons can be revealed to be in one of three possible
configurations, both spins aligned up, both spins aligned
down, or one up and one down [24]. When the spins are both
aligned up the oxygen molecule will deflect down in a
magnetic field, when both spins are aligned down they will
deflect the molecule upwards in a magnetic field, and when
the spins are anti-parallel the molecule will pass unper-
turbed through the magnetic field. Because of these three
possible states the ground state of oxygen is called a triplet
state. Any molecule with such a valence electron config-
uration is considered to be in a triplet state [19, 24].
The extreme reactivity of singlet oxygen arises from the
pairing of two electrons into one of the 2p antibonding
orbitals (Fig. 3). In the ground state the outermost electrons
are distributed, according to Hund’s rule, in the Px and Py
antibonding orbitals [24]. Since the orbitals are degenerate
and the electrons have aligned spins, the quantum numbers
of each electron are identical and thus are forced to occupy
separate orbitals to comply with the Pauli exclusion
principle [21]. During an interaction with the excited
photosensitizer the spin of one electron inverts, making its
quantum numbers unique, allowing them to pair together
into the antibonding orbital which destabilizes the molecule.
Although singlet oxygen is often depicted as diradical, it is
actually a highly polarized zwitterion (Fig. 3, bottom) [21].
Singlet oxygen is so reactive that it has a lifetime that
ranges from 10–100 ms in organic solvents. This restricts its
activity to a spherical volume, 10 nm in diameter, centered
at its point of production. In an aqueous environment singlet
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 107
Fig. 3. Molecular orbital diagrams showing the electron
distribution in triplet and singlet oxygen (top). Lewis structure
depicting the zwitterionic character of singlet oxygen (bottom).
oxygen’s lifetime is reduced to approximately 2 ms because
the energy of oxygen–hydrogen (O–H) stretching in water
molecules nearly equals the excited-state energies of singlet
oxygen. The energy is dissipated as heat by the stretching
and vibrational motions of water molecules. Because singlet
oxygen reacts so rapidly, PDT-induced oxidative damaged
is highly localized to regions no larger in diameter than the
thickness of a cell membrane. Photodynamic damage is
probably confined to targets near to or within hydrophobic
regions of the cell due to the hydrophobic character of most
photosensitizers.
1.3.2 Tissue Oxygenation. The importance of tissue
oxygenation in PDT was demonstrated by several research-
ers [25–27]. Hypoxic cells, those with less than 5%
oxygenation, were found to be resistant to PDT. Henderson
et al. have reported that oxygen consumption depends on the
fluence rate of the light [28], and that tissue destruction
might be enhanced by using lower fluence rates. Lower
fluence rates do not deplete the tissue’s oxygen supply as
rapidly and as a result the tissue is exposed to singlet oxygen
for a longer time during treatment [25, 26].
d‰1 O2 Š "C DPDT
ˆ ˆ  ˆ k‰3 O2 Š …1†
dt E tE
The rate of singlet oxygen production …d‰ O2 Š† is shown
1
dt
mathematically by equation (1) as a function of the light
fluence rate ( ), the total PDT dose (DPDT) and the
concentration of ground-state oxygen. The rate of singlet
oxygen production also varies as a function of the
concentration (C), the molar extinction coefficient (e), and
the singlet oxygen quantum yield of the compound (Φ).
Equation (1) shows the rate of oxygen consumption
increases linearly with the fluence rate. Clinically, high
fluence rates are usually chosen to decrease treatment times
[18]. However it was reported that high fluence rates may
not be appropriate for eliciting an optimal effect. Foster et
al. showed that a fluence rate of 50 mW cmÿ2 produced a
greater therapeutic response than 200 mW cmÿ2, when
tumor-bearing rats were treated with 5 mg/kgÿ1Photofrin1
and 360 J cmÿ2 of 630 nm light [29]. Similar results were
J. Porphyrins Phthalocyanines 2001; 5: 105–129
108 I. J. MACDONALD AND T. J. DOUGHERTY
also reported by Reed et al. [30], Tromberg et al. [31], and
Sitnik et al. [32]. Data from the reported experiments and
oxygen diffusion models suggest that lower fluence rates
expose greater portions of the tumor to singlet oxygen for a
longer period of time [29, 31]. Direct measurements of
tumor tissue oxygenation showed a rapid decrease in the
oxygen tension during the first seconds of treatment. The
extent of the decrease during treatment was fluence rate
dependent, as was the treatment response [33]. The degree
of damage to healthy tissue was also reduced, possibly
because healthy tissue can repair minor damage more
efficiently, avoiding a greater inflammatory response [32].
While an ample oxygen supply is necessary during PDT
treatment, eventual destruction of the tumor microvasculature
is also crucial to starve tumor cells of oxygen and nutrients
[30]. Sitnik et al. have suggested that fluence rates could be
judiciously chosen to maximize exposure to singlet oxygen
during treatment, while the total fluence could be adjusted to
ensure that the tumor microvasculature eventually collapses,
starving the tumor of oxygen and nutrients [33].
1.4 Photosensitizers
1.4.1 Photophysics of Photosensitizers. Porphyrins,
chlorins, and bacteriochlorins (Fig. 4) are among the most
useful photosensitizers for in vivo PDT, although other
classes of porphyrinoids such as phthalocyanines and
texaphyrins are also used. Porphyrins, chlorins, and
bacteriochlorins have absorption maxima in the red portion
of the electromagnetic spectrum and are efficient singlet
oxygen generators [34]. Red absorption maxima allow
activating light to penetrate deeper into tissue. They are
generally planar–aromatic molecules composed of four
symmetrically arranged pyrrol units linked by methine
bridges. Although texaphyrins have three pyrrolic units and
phthalocyanines are linked by azone bridges, both
molecules are planar and aromatic and have similar
photophysical properties as porphyrins, chlorins and
bacteriochlorins. Porphyrins, chlorins and bacteriochlorins
are all fully conjugated, but chlorins have one pyrrolic
double bond reduced and bacteriochlorins have two pyrrolic
double bonds reduced. It is because of their extensive
aromatic systems that these molecules absorb light in the
visible spectrum [34].
All porphyrin-like compounds have a strong absorption
band around 400 nm called the Soret band. Unfortunately
this band is not useful for PDT since blue light does not
penetrate very deeply into tissue; thus the weaker satellite
absorption bands (Q-bands) between 600 nm and 800 nm
are used for treatment. Porphyrins exhibit weak absorption
maxima around 630 nm while chlorins and bacteriochlorins
have strong absorption maxima around 650 nm and 710 nm
respectively. The different absorption spectra can be
Fig. 4. The basic chemical structures of porphyrins, chlorins
and bacteriochlorins.
Copyright # 2001 John Wiley & Sons, Ltd.
Fig. 5. Diagram depicting an electron (fuzzy ball) in a region of
length L bounded by infinitely high potential energy barriers.
explained by assuming that the delocalized p-electrons in
the aromatic ring behave as particles (fuzzy ball) in a one-
dimensional square-well potential, as described by Hoff-
man. The square-well potential consists of a region of length
L bounded on each side by an infinitely high potential
energy barrier (Fig. 5). Equation (2) shows the energy
levels, calculated from solutions of the Schrödinger equa-
tion. In equation (2), h is Plank’s constant, n is the energy
state of the electron (or valence electron in the case of an
ensemble of electrons), m is the mass of the electron and L is
the length of the confining region. The term E(n,N) is a
perturbation to the energy levels due to electron–electron
interactions when there is more than one electron in the box;
N is the number of additional particles in the box.
h2 …n ‡ 1†2
En ˆ ‡ E…n; N † n ˆ 0; 1; 2 . . .
8mL2
N ˆ 1; 2; 3 . . . …2†
The perturbation term E(n,N) is equal to zero when there is
only one electron in the potential well but raises the energy
of the levels as the number of electrons in the box increases.
Equation (2) is an great oversimplification but can be used
to illustrate the principle. Fig 6 shows how the energy states
accessible to a valence electron rise in the presence of other
electrons. In this example the energy required to excite an
electron from the ground state (n = 0) to an excited state
(n = 9) is greater when more than one electron (N > 1) is
confined in the potential well. Since the energy of light is
inversely proportional to the wavelength via the relation
DE = hn = hc/, where n is the frequency,  is the wave-
length, and c is the speed of light, the wavelength of light
needed to excite the valence electron is shorter, i.e. bluer
than the light needed to excite the single particle. Porphy-
rins, chlorins, and bacteriochlorins (Fig. 4) have 22-, 20-,
and 18-p electrons in their aromatic rings confined to
approximately the same region in space. Therefore the
absorption maxima of porphyrins are more blue-shifted than
chlorins, and chlorins more than bacteriochlorins because of
increasing numbers of p-electrons interacting in the ring.
All of these classes of compounds are highly efficient
singlet oxygen generators. The efficiency of production of
singlet oxygen is an empirically determined quantity called
the singlet-oxygen quantum yield (Φ). The singlet-oxygen
J. Porphyrins Phthalocyanines 2001; 5: 105–129

Fig. 6. A diagram depicting how increasing numbers of p-
electrons in a ring system cause a given transition to blue shift
to higher energies.
quantum yield describes the number of singlet oxygen
molecules that are formed per photon of energy absorbed.
1.4.2 Photofrin. The most commonly used and studied
photosensitizer to date is Photofrin1, the only commercially
available photosensitizer [18]. Photofrin is easily synthe-
sized by reacting hematoporphyrin in a mixture of sulfuric
and acetic acid to form hematoporphyrin-IX (di- and mono-)
acetates. Hydrolytic treatment with base yields a crude
mixture commonly referred to as hematoporphyrin deriva-
tive (HpD) [35]. Commercial Photofrin1 is made by
removing low-molecular weight components (Fig. 7). The
purified fraction was found to be optimally active against
tumor tissue, and is composed of monomers, dimers,
trimers, and larger oligomers [35, 36] up to eight or nine
porphyrin units [37]. Pandey et al. demonstrated that ether-
linked hematoporphyrin dimers and trimers, synthesized to
mimic Photofrin1 components, were more efficacious in
mice than carbon–carbon and ester-linked dimers [35,
38, 39]. The active HpD fraction that comprises Photofrin1
still contains many porphyrins oligomers of varying lengths
[18, 36, 40], linkages [38, 39, 41], and stereochemistries
[35, 39].
Photofrin’s longest wavelength absorption maximum is a
relatively weak (e630  3000 Mÿ1 cmÿ1) Q-band at 630 nm.
Light at 630 nm can adequately activate Photofrin1 to a
depth of about 5 mm in tissue.
Clinically Photofrin1 has exhibited no systemic toxicity
and does not appear to be carcinogenic or mutagenic at the
doses used [42]. Photofrin1 has been used successfully
against a wide variety of cancers, does not appear to
succumb to multidrug resistance, and Photofrin1-PDT has
no known cumulative dose ceiling as do radiation and
chemotherapy. Thus patients can be treated repeatedly as
needed. Photofrin1 does accumulate in the skin so that a
possible complication can be a severe sun burn or photo-
reaction. These are obviated by avoiding sunlight and high-
intensity light, and wearing protective clothes and sun-
glasses for approximately 6 weeks after treatment.
1.4.3 New Photosensitizers. Many new compounds have
been synthesized in an attempt to create a better photo-
sensitizer than Photofrin1. Often, researchers examine new
chromophores with absorption maxima at wavelengths
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 109
Fig. 7. The synthesis of Photofrin1.
longer than 630 nm, citing that Photofrin’s relatively weak
absorption band at 630 nm does not allow optimal light
penetration. After identifying a useful chromophore it is
tested in vitro and in vivo for PDT activity. The
pharmacological properties of the new compounds are also
evaluated. Many porphyrin-like compounds allow a large
number of alterations to be made at the peripheral positions
of the chromophore [34]. A quantitative structure–activity
relationship (QSAR) study can be performed to identify
optimally active compounds as was done by Henderson et
al. with the pyropheophorbide-a derivatives [43]. Ideally, a
photosensitizer should be non-toxic, selectively retained in
tumor tissue in high concentrations, water soluble, and
cleared in a reasonable time from the body and rapidly from
the skin to avoid photosensitivity reactions. Although the
literature is rife with reports of ‘novel’ photosensitizers for
PDT only Photofrin1 has been legalized by health agencies
for use around the world [18].
Several benzoporphyrin derivatives (BPDs) were synthe-
sized from protoporphyrin [44, 45]. The BPD chromophores
have absorption maxima around 690 nm. A comparative
study of several BPD analogues showed that the monoacid
derivative with the benzo ring attached to the A-ring (BPD-
MA) (Fig. 8A) was more photodynamically active than the
diacid or dimethyl ester versions [45]. The pharmacological
properties of BPD-MA are unfavorable for PDT treatment
of some cancers because it clears too rapidly from serum
and tissues including cancerous lesions [46]. Three hours
after injection BPD-MA reaches its maximum tumor con-
centration and quickly decreases thereafter [46]. Although
an adequate PDT response can be achieved by treating three
hours after injection, tumor selectivity is low because of the
large circulating concentration in all tissues. While BPD-
MA may not be entirely suitable for treating cancer it has
been proven useful for treating the wet form of age-related
macular degeneration (AMD) [47, 48]. Since most of the
BPD-MA remains in circulation at short times after injec-
tion, laser light can be focused to destroy the neovasculature
that grows near the macula and causes blindness in AMD
[47, 48]. The FDA has recently recommended that BPD-
MA, given the tradename Visudyne1 (verteporfin for
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110 I. J. MACDONALD AND T. J. DOUGHERTY
Fig. 8. Photosensitizer candidates for photodynamic therapy.
injection), be approved for use in Visudyne1 therapy, which
is essentially PDT to destroy the neovasculature on the
retina [49].
Tin etiopurpurin (SnEt2), tradenamed Purlytin1, is a
chlorin photosensitizer being investigated for use in PDT
(Fig. 8B) [44]. Purlytin1 has a tin atom chelated into its
center and has an absorption maximum near 650 nm. It has
been reported that Purlytin1 localizes into the skin and can
produce a photoreaction for 7 to 14 days after administration
[50, 51]. Purlytin1 has been evaluated for use against
recurrent metastatic breast cancer [50] and is currently
being evaluated for use in treating corneal neovasculariza-
tion [51].
Another chlorin, meta-tetrahydroxyphenyl chlorin (m-
THPC), tradenamed Foscan1 (Fig. 8C), was the most active
photosensitizer in a series of tetrahydroxyphenyl analogues
[44, 52, 53]. Although Foscan1 has a singlet-oxygen
quantum yield comparable to other chlorins, only low drug
(typically 0.1 mg kgÿ1) and light doses (<20 J cmÿ2) are
needed to achieve photodynamic responses equivalent to
Photofrin [18]. Foscan1 is currently under investigation for
use in treating esophageal, lung, laryngeal [54], thoracic
[55], and skin cancers [56]. Foscan1 can induce photo-
sensitivity for up 20 days after administration [56]. Foscan1
is so photodynamically active that efforts are underway to
treat with less penetrating green light and lower drug doses
to reduce this compounds phototoxicity [18].
Monoaspartyl chlorin e6 (Npe6) is another synthetic
chlorin developed to utilize the long wavelength absorption
maximum of chlorins (Fig. 8D). Npe6 has been investigated
for use against adenocarcinoma of the breast, basal cell
carcinoma, and squamous cell carcinoma [57]. Like BPD-
MA, Npe6 clears rapidly from tissues, therefore light
Copyright # 2001 John Wiley & Sons, Ltd.
treatment can be administered within four hours of injection
[44, 53]. At low doses (<1.65 mg kgÿ1 ‡ 100 J cmÿ2)
tumor regression was short lived while at high doses
(>2.5 mg kgÿ1 ‡ 100 J cmÿ2) regression was prolonged but
tissue selectivity was sacrificed [57]. Skin photosensitivity
was noted to be ‘temporary’ [57]. This compound is also
being tested to destroy neovasculature on the retina, and
initial results suggests that it can effectively destroy
choroidal vessels with minimal damage to the neurosensory
retina [58].
Lutetium texaphyrin, tradenamed Lutex (Fig. 8F), is a
non-porphyrin photosensitizer currently in Phase-II clinical
trials for the treatment of cancer [59]. Lutex has been used
to treat malignant melanoma [60] because it is highly
selective for tumor tissue and it can be activated with
732 nm light that penetrates more deeply into pigmented
lesions than the light needed to excite Photofrin1,
Verteporfin1, Foscan1, or Npe6. Lutex is also being used
to prevent re-stenosis of vessels after cardiac angioplasty
[61] by photo-inactivating foam cells that accumulate
within arteriolar plaques. For this indication Lutex has been
given the tradename Antrin1.
A prodrug being used clinically to treat superficial
cancers is D5-aminolevulinic acid (ALA, Fig 8E) ALA is
a precursor in the biosynthesis of heme [62]. Overloading
cells with ALA forces them to produce protoporphyrin-IX
(the photosensitizer) faster than iron ferrochelatase can
convert protoporphyrin-IX to heme, a non-photosensitizer.
Because the ALA used for treatment is endogenous the
feedback mechanisms are unable to prevent the over-
production of protoporphyrin-IX, which accumulates in
high concentrations in the cells making them photosensitive
[62]. ALA-PDT destroys superficial disease well but is not
J. Porphyrins Phthalocyanines 2001; 5: 105–129

well suited for destroying tumor tissue greater than one
millimeter in depth. Aminolevulinic acid has recently been
used successfully in the treatment of actinic keratosis and
may have a role in aiding detection of certain bladder
tumors [62]. Furthermore, esterified ALA derivatives may
have improved pharmacological properties improving
retention in tumor tissue, and thus use as a detection agent,
and possibly as a prodrug for PDT treatment [63].
1.4.4 Photosensitizer Pharmacology
1.4.4.1 Structure–Activity and Quantitative Structure–
Activity Relationships. Quantitative structure–activity re-
lationships (QSARs) and structure–activity relationships
(SARs) have been performed with several series of
porphyrin and porphyrinoid compounds to identify opti-
mally active derivatives within a series of structurally
related analogues [43, 45, 64–66]. Structure–activity rela-
tionship studies are performed to identify structural charac-
teristics that affect the biological activity (other endpoints
can also be studied) of a compound [67]. Quantitative
structure–activity relationship studies are performed to
quantify the contributions from physicochemical charac-
teristics that effect the compound’s activity; in this case a
photosensitizer’s photodynamic activity in biological sys-
tems [67]. In QSAR studies it is assumed that the biological
activity of a compound is determined by its ability to bind to
a target receptor. Theoretically, the affinity for the receptor
is related to the steric, hydrophobic, and charge interactions
between the receptor and the compound [67]. The general
regression equation describing a QSAR takes the form of
equation (3) where BA is the biological activity of the
compound which is related to the sum of the contributions
from steric, hydrophobic and charge interactions [67].
X X X
BA ˆ steric ‡ hydrophobic ‡ charge …3†
Most of the photosensitizers described in Section 1.4.3
have been optimized through SAR studies [45, 64, 66] while
few have been put through rigorous QSAR studies [43, 65].
Some compounds, such as sulfonated phthalocyanines [65]
and pyropheophorbide-a ether derivatives [43], exhibit
relationships between the hydrophobicities and photody-
namic activities of the compounds. The QSAR exhibited by
the sulfonated phthalocyanines [65] shows a dependence on
the log P values, possibly the result of optimal binding to the
outer membrane of the cell. The pyropheophorbide-a
derivatives are thought to exhibit a parabolic QSAR because
the optimal members bind most selectively to a protein
target within the cell [43].
A photosensitizer’s structure, charge, and hydrophobicity
determine how it interacts with itself and its surroundings,
these factors influence how it is accumulated by cells, to
what site, etc. In vivo there are many serum proteins and
blood cells with which the compounds can interact. Within
tumor tissue, pH gradients, irregular leaky vasculature, and
abnormal blood flow contribute to where the photosensitizer
will localize and to what degree. A photosensitizer that is
considered useful has to bypass these pharmacological
hurdles to force lethal concentrations into susceptible
regions of the tumor tissue. Theoretically photosensitizers
can be rationally designed, but more realistically they are
discovered during blind probing. Usually a structure with
reasonable photophysical characteristics is found; then the
pharmacology is tweaked by chemically modifying the
structure.
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 111
Some attempts have been made to create photosensitizers
that are specific to tumor cell targets. In vitro, cationic
photosensitizers localize selectively in high concentrations
within mitochondria which have been shown to be
susceptible PDT targets (see Section 1.6.2.3). Nile blue
derivatives (NB) [68], rhodamine-123 (R-123), chalcogen-
pyrylium dyes, and cationic porphyrins all localize to the
mitochondria, however some NB derivatives, R-123, and
some chalcogenpyrylium dyes have very low singlet-
oxygen quantum yields (<10%). The NB derivatives also
have low pKa values and are protonated only below pH 5. At
physiologic pH (7.4) NB derivatives are neutral and have
unfavorable absorption maxima around 500 nm [64]. The
largest drawback that most cationic photosensitizers have is
that they clear very rapidly from tissues [69, 70].
Highly anionic photosensitizers also appear to be less
effective photosensitizers. Generally photodynamic activity
decreases with increasing numbers of negative charges on
the chromophore. Mono-acid benzoporphyrin derivatives
were 10 times more photodynamically active then di-acid
benzoporphyrin derivatives in vitro [45]. However, in vivo
mono-acid BPD clears so rapidly that treatment must be
administered three hours after injection (see Section 1.4.3)
[46, 64]. Di and tri-sulfonated phthalimidomethyl deriva-
tives were 200 times more active then the tetra-sulfonated
derivative, despite the fact that cells accumulated higher
levels of the tetra-sulfonated compound [66]. Tetra-
sulfonated zinc phthalocyanine and tetra-sulfonated phtha-
limidomethyl aluminum phthalocyanine both required high
drug concentrations to achieve a photodynamic response in
vivo [65, 66]. Highly anionic compounds appear to be less
photodynamically active because they are cleared rapidly in
vivo [71]. The exception is Photofrin which is a di-acid that
is retained to a high degree in tumor tissue and is
photodynamically active. Unlike the other compounds
described Photofrin1 consists of high-molecular weight
components that are highly aggregated, possibly because of
amphiphilicity as well as the hydrophobicity of the
components.
1.4.4.2 Targeted Photosensitizers. Porphyrins have been
determined to be ligands for the mitochondrial peripheral
benzodiazepine receptor (PBR) [72] and a correlation
between the photodynamic activities of several porphyrin
photosensitizers and their binding affinities for the PBR was
shown by Verma et al. [73]. The benzodiazepine binding
site (Site-II) on human serum albumin (HSA) has also been
found to be a binding site for several porphyrins, and
chlorins. A correlation between the binding of certain
putative Photofrin1 components to Site-II and photo-
dynamic efficacy of the components was also demonstrated
by Tsuchida et al. [74]. Tsuchida et al. suggested that HSA
is not a photodynamic target but that Site-II binds photo-
sensitizers with a similar affinity as the PBR because Site-II
and the PBR both bind benzodiazepines [74]. Therefore
HSA binding could be used to screen for active photo-
sensitizers.
Some attempts have been made to direct photosensitizers
to known cellular targets by creating a photosensitizer
conjugate, where the other molecule is a ligand that is
specific for the target. To improve localization to cell
membranes cholesterol conjugates were synthesized [75].
Antibody-conjugates have also been synthesized to direct
photosensitizers to specific tumor antigens [76–78] and
insulin–porphyrin conjugates have been created to target the
J. Porphyrins Phthalocyanines 2001; 5: 105–129
112 I. J. MACDONALD AND T. J. DOUGHERTY
chromophore into cells in high concentrations through a
known uptake mechanism [79]. Chemotherapeutic agents
have been attached to porphyrin chromophores to increase
the lethality of the PDT treatments [80], and protein- and
microsphere-conjugates were made to improve the pharma-
cology of the compounds [81]. These strategies seldom
work well because the pharmacological properties of both
compounds are drastically altered.
1.4.4.3 Amphiphilicity. Amphiphilicity describes mol-
ecules that are partially hydrophobic and hydrophilic at
different positions so that each region interacts with the
solvent independently. No method has been established to
quantify amphiphilicity of small molecules, however
amphiphilicity is analogous to zwitterionicity. Margaron
et al. [65] and others [53] have reported that amphiphilic
photosensitizers are generally more photodynamically
active than symmetrically hydrophobic or hydrophilic
molecules. They clearly demonstrated the importance of
amphiphilicity by comparing the in vitro photodynamic
activity of sulfonated tetraphenylporphine derivatives
(TPPS), each derivative having a different amphiphilicity
depending on the number of sulfonic acid moieties [65]. The
amphiphilicity of TPPS derivatives (Fig. 9) is readily
apparent because the phenyl sulfonic acid moieties are fixed
in space (to the meso position of the porphine). It is harder to
determine the amphiphilicity of more complicated struc-
tures. Several researchers have suggested that amphiphilic
photosensitizers are more photodynamically active because
they can localize to the hydrophobic–hydrophilic interfaces
in membranes and at the surface of proteins [45, 53,
65, 66, 82–84]. Amphiphilicity is also important because it
can affect the degree to which a compound aggregates
which, in turn, can affect the photophysical properties of a
photosensitizer. In this thesis amphiphilicity has been
identified as a contributing factor in the photodynamic
activity of the pyropheophorbide-a derivatives. It appears
that amphiphilicity modulates aggregation which in turn
determines how the compounds are accumulated to various
Fig. 9. The structures of sulfonated tetraphenylporphines.
Copyright # 2001 John Wiley & Sons, Ltd.
targets within a tumor cell. This will be discussed in detail in
the following sections.
1.4.4.4 How Amphiphilicity, Aggregation, and Serum
Protein Interactions Affect the Photodynamic Activity of
Photofrin1. It has been shown that aggregation changes the
photophysical properties of porphyrin, chlorin, and phthalo-
cyanine photosensitizers [85–96]. Aggregation shortens the
triplet-state lifetime and decreases the singlet-oxygen
quantum yield by dissipating the energy through internal
conversion [92–97]. Therefore highly aggregated photo-
sensitizers in vivo are generally considered to be less
effective photosensitizers [87, 98]. Aggregation can some-
times be avoided by using emulsifying detergents such as
polysorbates, Cremophore EL1, polyethylene glycol, or
lipid emulsions. Solvents such as DMSO and ethanol can
also be used to dissolve the compounds. Emulsifying agents
increase the solubility of some compounds allowing them to
be administered easily [99, 100], however, not much is
known about the behavior of these agents in vivo [101–104].
Amphiphilic molecules are thought to bind most
efficiently to proteins at the interface between the hydro-
philic surface and hydrophobic interior, thus the interaction
between serum proteins and photosensitizers has been
examined in detail. Photofrin1 has the largest body of
pharmacology data related to photosensitizers. While it is
clear that serum proteins interact with Photofrin1 the exact
nature of the interactions is still not fully understood
[105, 106].
Since albumin is the most abundant serum protein and is
known to associate with many different compounds [107],
including porphyrins [98, 108, 109], the interaction between
Photofrin and albumin has been examined in detail
[98, 108–115]. In vitro, HpD, Photofrin, hematoporphyrin,
and deuteroporphyin all bound to albumin in solution
[98, 108, 109]. Hepatocytes incubated in vitro with HpD in
phosphate-buffered saline (PBS) containing one percent
bovine serum albumin accumulated lower levels of HpD
than hepatocytes incubated with HpD in PBS with no
albumin [110]. The experiments indicate directly and
indirectly that albumin binds these photosensitizers [110].
To determine if binding to albumin is important to achieve
an adequate PDT response Hamblin et al. conjugated
hematoporphyrin to albumin and tested the conjugate in
vivo [106]. Non-phagocytic cells (e.g. tumor cells) accu-
mulated small amounts of the hematoporphyrin–albumin
conjugate on their outer membranes but did not accumulate
any intracellularly [106]. However, the hematoporphyrin-
albumin conjugate was very efficiently accumulated by
macrophages [106].
Lipoproteins are the next most abundant serum proteins
after albumin. Kessel reported that in vitro HpD initially
binds to albumin but rapidly redistributes to lipoproteins
long before light treatment would occur in vivo [112]. Jori et
al. also showed that HpD was undetectable in the albumin
fraction of the serum 48 hours after administration in vivo
[111]. Other researchers have used electrophoresis, ultra-
centrifugation and spectroscopic analysis to confirm that
lipoproteins are the main reservoir for circulating Photo-
frin1 [98, 116].
The apparent reason that lipoproteins have a high affinity
for photosensitizers (especially amphiphilic photosensiti-
zers) is that they are composed of a core of 1500
cholesteryl esters encapsulated in a monolayer made of
800 phospholipid and 500 cholesterol molecules
J. Porphyrins Phthalocyanines 2001; 5: 105–129

[117, 118]. The apoprotein is approximately 50 kD and
wraps around the ‘equator’ of a hydrophobic core. With a
total molecular weight of 150 kD, lipoproteins are less
than 50% protein by weight and are more like hydrophobic
reservoirs.
The interaction between Photofrin1 and lipoproteins and
the effect this interaction has on the photodynamic proper-
ties of Photofrin1 was examined by Korbelik who exposed
cultured cells to Photofrin1 and high-density lipoprotein
(HDL), low-density lipoprotein (LDL), or very low-density
lipoprotein (VLDL) [113]. Summarizing Korbelik’s results,
the cells accumulated less Photofrin1 in the presence of the
lipoproteins than in the presence of albumin [115]. After
incubating the cells for 24 hours with Photofrin, and
lipoproteins or albumin, efflux of Photofrin1 was most
enhanced by medium-containing albumin [115]. Interest-
ingly, in both experiments the globulin fractions of the
serum bound the Photofrin1 more tightly than the
lipoproteins; however globulins comprise a much smaller
fraction of the serum.
Tumor cells display higher numbers of LDL receptors on
their surfaces, presumably because they require more
cholesterol than normal cells to build membranes [119].
Hamblin et al. created hematoporphyrin–lipoprotein con-
jugates to direct the photosensitizer into cells through the
LDL receptor-mediated pathway. The VLDL and HDL
conjugates were not accumulated by cells and the LDL
conjugate was accumulated to a lesser extent than hemato-
porphyrin alone [106]. Significantly, Hamblin et al. reported
that minor alterations to the LDL complex greatly increased
the probability that it would bind to scavenger receptors on
macrophages and become phagocytosed [105, 106]. Co-
valently binding hematoporphyrin to transferrin, a serum
protein with a known uptake pathway also did not increase
the cellular uptake of hematoporphyrin [106]. Korbelik’s
and Hamblin’s results demonstrate that lipoprotein binding
is important. However their results also show that Photo-
frin1 is not significantly accumulated by the LDL receptor-
mediated pathway.
Summarizing the reports cited, a description of the
pharmacokinetics and pharmacodynamics of Photofrin1
can be constructed. Photofrin1 consists of many compo-
nents, each having different hydrophobicities and amphi-
philicities. Therefore, it is reasonable to assume that each
component behaves independently from the others. Bellnier
et al. fitted a triphasic exponential curve to the pharmaco-
kinetic profile of Photofrin1, however it is likely that the
curve is actually an n-phasic curve where n is the number of
components [120]. Each component has two factors that
control its behavior in solution, the rates of aggregation and
disaggregation (related to the free energy [DG‡] barrier to
form the transition state) and the total free energy released
upon binding (DG). This is shown graphically in Fig. 9
where f is a reaction coordinate. In Fig. 9 four possible
situations are shown: (A) kinetically favorable, thermo-
dynamically favorable; (B) kinetically unfavorable, thermo-
dynamically favorable; (C) kinetically unfavorable,
thermodynamically unfavorable; and (D) kinetically favor-
able, thermodynamically unfavorable. The Photofrin1
components are probably described by these four examples
and many other possible combinations. Therefore two
components may have identical log P values [related to
DG by DG = ÿ RT ln (P)] but have different pharmaco-
logical properties because they have different amphiphili-
cities [related to DG‡ by].
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 113
Early in the distribution phase the most-soluble compo-
nents disaggregate and bind with albumin, possibly because
it is the most-available binding site. Simultaneously, the
concentration of soluble components decreases because of
clearance mechanisms. Since clearance is an irreversible
sink the system continuously shifts towards unbound
molecules to preserve equilibrium. As a result no Photo-
frin1 can be detected in the albumin fraction 10 hours after
administration in vivo [111]. The less-soluble oligomers
disaggregate more slowly and can bind to albumin
(especially if albumin is the only species present) [98,
114], however it is more likely that they will partition into
lipoproteins because the final free energy is lower than if
they were bound to albumin [112]. Although lipoproteins
make up a smaller portion of serum than albumin they are
considerably larger, more hydrophobic, and have a large
capacity for hydrophobic molecules [112, 117, 118]. At the
time of light treatment, 24–48 h after injection, mainly
lipoprotein-bound oligomers and some large aggregates
remain in circulation [112, 121, 122]. Interestingly, macro-
phages have a nearly unsaturable capacity for LDL (and the
photosensitizer bound to it) thus it has been speculated that
macrophages accumulate high concentrations of Photofrin
and then localize in the skin causing skin photoreactions
[105, 123].
The pathways by which Photofrin1 is accumulated by
cells is not well understood, however Maziere et al. and
Hamblin et al. demonstrated that a small amount of
material, bound to LDL, is internalized by the LDL-
receptor-mediated pathway [105, 123]. However the LDL-
receptor-mediated pathway does not appear to be the main
route of internalization. This was demonstrated by Moan et
al. who reported that cultured cells accumulate Photofrin1
components with a degree of selectivity that does not
correlate with their hydrophobicity or their affinity for LDL
[121, 124]. Therefore the main routes by which cells
accumulate Photofrin1 components may be by passive
diffusion through the outer membrane or some other
receptor-mediated transport process that is highly dependent
on the aggregation state of the molecules. This will be
discussed in more detail in the context of the current study.
1.4.4.5 How Amphiphilicity, Aggregation, and Serum
Protein Interactions Affect the Photodynamic Activity of
Other Photosensitizers. Allison et al. demonstrated that in
the presence of serum proteins BPD-MA disaggregates and
binds to lipoproteins [125]. They also showed that BPD-MA
was more photodynamically active if it is non-covalently
bound to LDL or HDL before use in vivo [126, 127]. When
BPD-MA was bound to LDL it was accumulated to higher
levels by cultured tumor cells and living tumor tissue than
when it was administered alone [127]. Allison et al. bound
BPD-MA to acetylated LDL, which cannot bind to the LDL
receptor, and demonstrated that the amount of drug
accumulated by the cells was markedly reduced compared
to the normal LDL-associated BPD-MA [127]. Unfortu-
nately, no direct comparison was made between the
intracellular levels of BPD-MA and the corresponding
photodynamic activity to assure that greater photodynamic
activity resulted from higher intracellular levels of BPD-
MA [127].
The interaction between sulfonated tetraphenylporphines
(TPPS) has also been examined in some detail. Although
these compounds cannot be used to treat human cancer
patients because they are neurotoxic, they are interesting
J. Porphyrins Phthalocyanines 2001; 5: 105–129
114 I. J. MACDONALD AND T. J. DOUGHERTY
because they have well-defined amphiphilicities and are a
useful model to study how amphiphilicity and hydrophobi-
city determine photodynamic activity (Fig. 10) [128]. The
TPPS1 derivative has one sulfonic acid group while TPPS2a
has two at opposing meso positions and TPPS2o has two at
adjacent meso positions. The TPPS3 and TPPS4 derivatives
have three and four sulfonic acid groups respectively. The
most interesting compounds in this series are the TPPS2
derivatives because they have identical log P values and
very different tumor localizing and photosensitizing proper-
ties in vitro [128]. The TPPS2a derivative is the most
photodynamically active member of this series followed by
the 2o-, 1-, 3- and 4- derivatives, respectively. The 2a-
derivative is accumulated the slowest, and in the highest
concentrations by cells in vitro. It also has the highest
affinity for lipoproteins and a low affinity for albumin. In
contrast the 4-derivative binds mainly to albumin and does
not bind to lipoproteins. It is accumulated to the highest
levels by tumor cells and is considered to be the most tumor
selective compound in the series; however it is also the least
photodynamically active [129].
Phthalocyanines and naphthalocyanines are classes of
photosensitizers that also have clearly defined amphiphili-
cities. They can be used as models, like the TPPS
derivatives, because they can have up to four sulfonic acid
groups, one at each pyrrol position. Di-sulfonated phthalo-
cyanines also have two configurations either on opposite
sides of the ring or at adjacent pyrrolic positions. Berg et al.,
Brasseur et al., and Margaron et al. have demonstrated that
the 2a-sulfonated phthalocyanine is the most efficient
photosensitizer in vitro followed by the 1-, 3-, 2o-, and
4-derivatives [65, 130, 131]. This is the same trend
exhibited by the TPPS series. Other phthalocyanine and
naphthalocyanine derivatives exhibit the same general
trend; the more amphiphilic they are the more photo-
dynamically active they are.
Other new photosensitizers, m-THPC and Npe6 [113,
115, 132] were also found to interact with serum proteins
Fig. 10. The diagrams depict four possible interactions
between photosensitizer molecules and serum proteins:
(A) thermodynamically favorable, kinetically favorable;
(B) thermodynamically favorable, kinetically unfavorable;
(C) thermodynamically unfavorable, kinetically unfavorable;
(D) thermodynamically unfavorable, kinetically favorable.
Copyright # 2001 John Wiley & Sons, Ltd.
especially lipoproteins. Glanzmann et al. and others have
reported that m-THPC exhibits an unusual pharmacokinetic
profile in vivo [132, 133]. Initially, half of the m-THPC
administered disappears from circulation gradually re-
appearing in the serum after about 10 h and decreases
thereafter [132, 133]. The shape and extinction coefficient
of the absorption maxima and the fluorescence signal of
m-THPC, by comparison with disaggregated m-THPC, did
not change during the time that the drug disappeared
indicating that the disappearance is not an artifact of
aggregation or binding to serum proteins [133]. Glanzmann
et al. and others have suggested that m-THPC is initially
sequestered in the liver and then released over time. Others
[134] have suggested that m-THPC metabolites are released
from the liver, although Glanzmann et al. have suggested
that this is not the case and have shown that the reappearing
fraction is as photodynamically active as m-THPC [133].
Bellnier et al. have shown that when the hexyl-ether
derivative of pyropheophorbide-a is administered in
ethanol, as opposed to 1.0% (v/v) Tween-80, the same
pharmacokinetic profile as that of m-THPC was obtained in
mice [134]. Bellnier et al. suggested that ethanol stimulates
the liver to rapidly accumulate the compound which is then
released back into circulation over time [134]. Since m-
THPC is formulated for clinical use in 20% (v/v) ethanol,
30% (v/v) polyethylene glycol, and 50% (v/v) water, it may
be possible that ethanol is responsible for the unusual
pharmacokinetic profile.
In the previous section the pharmacological properties of
Photofrin1 were described. They consisted of independent
actions of many different species, having various combina-
tions of hydrophobicity and amphiphilicity. The informa-
tion presented in this section shows that amphiphilic
photosensitizers, even among varied classes of compounds,
are more photodynamically active than symmetrically
hydrophobic or hydrophilic ones suggesting that the most
active fractions of Photofrin1 may be those that are most
amphiphilic.
1.5 Light
1.5.1 PDT Dose. Selective tumor destruction can be
achieved if the amount of photosensitizer accumulated in
tumor tissue is greater than that accumulated in the
surrounding healthy tissues. Selectivity can also be
enhanced by selectively delivering the light to the tumor
tissue and excluding it as fully as possible from the healthy
tissues nearby. Star et al. and Svaasand et al. have stated the
PDT dose mathematically (equation (4)) as the product of
the light fluence rate [ (,q,t)], the molar-extinction
coefficient of the photosensitizer ["()], and the photo-
sensitizer concentration in the region of treatment [C(q,t)]
[135, 136]. In equation (4), t is the illumination time, q is a
generalized spatial coordinate, and  is the wavelength of
the activating light.
Z Z
DPDT ˆ "…† C…q; t† …; q; t†dtdq …4†
0 q
The PDT dose is a measure of the energy absorbed by the
photosensitizer in a volume of tissue. Theoretically the PDT
dose could be calculated exactly if the fluence rate and drug
concentration and the extinction coefficient are known at
every point and at all times in the treatment volume
[135, 136]. Equation (4) is general so that the target
J. Porphyrins Phthalocyanines 2001; 5: 105–129

concentration C(q,t) could pertain to the amount of
photosensitizer in a volume of tissue, or to smaller regions
such as a cell, organelles within the cell, or even specific
proteins. Equation (4) shows that if a photosensitizer
localizes to a target with a concentration (Ct), localizes to
regions around the target with a concentration (C), the PDT
dose to the target will be scaled by a factor equal to the ratio
of the two concentrations (Ct/C). In reality photosensitizer
concentrations usually differ only slightly between tumor
and healthy tissue; therefore the light must be administered
as specifically as possible with fiber optics and specialized
lenses.
1.5.2 Light Penetration. Svaasand [137], Wilson et al.
[138], Bolin et al. [139] and others have demonstrated that
light with a wavelength near 800 nm passes most freely
through tissues. This is illustrated nicely by placing a
flashlight to one’s cheek and observing the red glow on the
inside surface (personal observation). We see the red
because red-light penetrates most deeply into tissue, and
not due to the color of blood as is sometimes thought. The
depth that light penetrates into tissue is dependent upon the
optical properties of the tissue and the wavelength of the
light [137–140]. When photons enter tissue a portion is
reflected by the surface and the rest scatter around in the
tissue until they either escape or are absorbed by chromo-
phores (either endogenous tissue chromophores or exogen-
ous molecules such as the photosensitizer) [135, 137, 138,
140, 141]. Theoretically a few photons may pass completely
through the tissue volume although this number is very
small [140]. Wavelengths less than 800 nm are scattered
with increasing efficiency by macromolecules because they
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 115
Equation (7) shows why there is a window around 800 nm
where light penetrates most deeply into tissue. At
wavelengths longer than 800 nm water molecules begin to
absorb the light and convert its energy to heat. Also highly
pigmented tissues can dramatically reduce the depth that
light can penetrate into tissue. This was demonstrated by
treating separate groups of black and white guinea pigs with
graded doses of dihematoporphyrin ether (DHE)-PDT until
they achieved equal responses. White guinea pigs required
26 J cmÿ2 of green light to produce eschars while black ones
needed 58 J cmÿ2 because of the high absorptivity of
melanin [143]. Dougherty et al. also reported that the
photosensitizer can decrease the attenuation depth if it has a
high extinction coefficient and is present in high concentra-
tions in tumor tissue [141]. In equation (7) the reduced
absorptivity has been expanded (equation (6)) to include the
absorptivity of endogenous chromophores and the concen-
tration of the photosensitizer (C), the distance from the light
source (r), and the molar extinction coefficient (e).
 ˆ f3…ma ‡ "Cr†‰m0s ‡ …ma ‡ "Cr†Šgÿ1=2 …7†
Increases in any one of these values without an equivalent
decrease in the others will result in a decrease in the
attenuation depth [141].
1.5.3 Photobleaching. Photobleaching is destruction of
the photosensitizer by light-mediated processes. It was
observed that light degrades photosensitizers in solution
according to equation (8), where b is the experimentally
determined decay constant that is specific for each
compound, and J is the total fluence (J = t) [144].
are equal to or less than the size of the particles [140].
The attenuation coefficient [a (cmÿ1)] describes the
exponential decrease of a specific wavelength of light as it
penetrates tissue and is different for each tissue [137–140].
The fluence rate of light at a depth (r) from a point light
source within a volume of scattering medium with an
attenuation coefficient a is given by equation (5) and was
derived from diffusion theory and describes experimental
data well [135, 136].
eÿr
ˆ 0 …5†
r condition [147]. In aqueous solutions Photofrin photo-
Fluence rates decrease exponentially with depth. Thus small
changes in the attenuation coefficient lead to large changes
in the fluence rate with depth. Therefore it is advantageous
to have photosensitizers that absorb near 800 nm to
maximize the treatment depth in many different tissues.
The inverse of the attenuation coefficient is the attenuation
depth [ (cm)] which describes the depth that light entering
tissue is reduced to 1/e (approximately 37%) of its initial
intensity (equation (6)). Typical values of the attenuation
depth range from 1 to 3 mm for non-pigmented tissues
[137–140, 142]. Dougherty has reported that the destructive
effects of PDT extend to two attenuation depths or about 5
to 6 mm, indicating that as little as 10 percent of the entering
light is required for a photodynamic effect [64]. Although
the attenuation depth is dominated by scattering at the
wavelength used for PDT, the attenuation depth actually
depends on the reduced scattering coefficient (m's) and the
reduced absorptivity coefficient (ms) (equation (6)) [64,
135, 138, 140–142].
 ˆ ÿ1 ˆ ‰3ma …m0s ‡ ma †Šÿ1=2 …6†
Copyright # 2001 John Wiley & Sons, Ltd.
C ˆ C0 eÿJ …8†
Generally photobleaching is measured as the decrease in the
fluorescence over time since fluorescence intensity and
concentration are proportional [144]. Measuring photo-
bleaching in vivo is more difficult and less reliable because
aggregation, clearance mechanisms, and changes in the
optical properties of the tissue can also decrease the
observed fluorescence [64, 145]. Photobleaching has been
shown to be mediated by singlet oxygen [146, 147],
independent of oxygen [64, 148], or indifferent to either
bleaches very slowly, presumably because it is so highly
aggregated. Since in the aggregated state Photofrin1 has a
low singlet-oxygen quantum yield it was assumed that
Photofrin1 was bleached by an oxygen-dependent mech-
anism [64]. However, Photofrin1 bleached rapidly in de-
oxygenated solutions suggesting that a Type-I photoreaction
may mediate photodestruction. In the first experiment,
aggregation probably inhibits photobleaching by inhibiting
Type-I photoreactions. Dougherty suggested that in bio-
logical systems proteins and other cellular components
disaggregate Photofrin1 so that photobleaching may be an
important factor in the PDT response [64]. Experimentally it
was determined that Photofrin was nearly 60% photo-
bleached after a typical PDT treatment in vivo.
While oxygen-mediated photodestruction of photo-
sensitizers has been theoretically and experimentally
demonstrated, most experiments suggest that Type-I photo-
degradation dominates [146].
Although photobleaching decay constants for several
porphyrins have been measured in solution, in vitro, and in
vivo, there is no agreement as to whether photobleaching has
J. Porphyrins Phthalocyanines 2001; 5: 105–129
116 I. J. MACDONALD AND T. J. DOUGHERTY
any discernible effect on PDT activity [144, 145]. It is
thought that photobleaching may rapidly destroy large
amounts of the drug that is localized in the healthy tissue
surrounding the tumor. If this happens fast enough the
normal tissue should be spared from some of the photo-
dynamic damage thus increasing tumor selectivity. There is
also some anecdotal evidence that suggests that low level
illumination or controlled illumination of patients could
effectively photobleach Photofrin out of the skin and that
this is the mechanism by which Photofrin is finally removed
from the skin [149, 150]. There is no clear proof that
photobleaching affects PDT treatments [136].
1.5.4 Light Sources and Delivery. The activating light is
most often generated by lasers, or in some cases by arc
lamps or fluorescent light sources. Lasers are most
frequently used because they produce highly coherent
monochromatic light that can be efficiently channeled into
quartz fibers used as light delivery devices. Since the lasers,
their power sources and cooling apparatus are often noisy,
bulky, and labor intensive they usually cannot be used in the
operating room or clinic. Therefore light is usually delivered
through fiber-optic cables to the treatment site, often
through an endoscope. The fibers can also be tipped with
a variety of lenses and diffusers that allow optimal
illumination of the tissues to be treated. For example
hollow, spherical organs (e.g. bladder) can be illuminated
from within with spherical diffuser bulbs, while tubular
regions (e.g. esophagus) can be illuminated with cylindrical
diffuser fibers of varying lengths [151, 152]. Graded index
(GRIN) lenses can be used to illuminate flat surfaces [28].
Conveniently the thin fiber-optic cables can also be fed
through the existing channels in bronchoscopes and
endoscopes eliminating the need for more complicated
surgical procedures. In the future light patches may be
available to illuminate surfaces even more effectively.
Inexpensive diode lasers are currently in use in Europe and
may soon be available in the United States, eliminating the
labor intensive argon-pumped dye lasers.
1.6 Biological Mechanisms of Action
1.6.1 Vascular Shutdown and Inflammation. Although it
was demonstrated that HpD-PDT killed tumor cells in vitro,
Henderson et al. reported vascular destruction occurred to a
greater extent in vivo [153]. Blood stasis and hemorrhaging
were found to starve tumor cells of oxygen and nutrients.
This was confirmed by Fingar et al. who treated tumor-
bearing animals within minutes of injecting Photofrin1, to
confine photodynamic damage to the vasculature, and
observed pockets of necrotic-tumor cells remote from the
damaged vessels [154]. Henderson et al. also demonstrated
that tumor bed effects (destruction of the vasculature) are
crucial to reducing the survivability of EMT-6 and RIF cells
in vivo [155]. Henderson et al. also showed directly, by
measuring oxygen concentrations in tumor tissue, that
oxygen deprivation kills tumor cells after PDT [156].
Korbelik et al. reported that vascular destruction,
observed after PDT is similar to the inflammatory response
observed after tissue injury or infection [157]. Thus
eicosinoids and other inflammatory agents have been
examined as mediators of the PDT response. Endothelial
cells normally produce a balance of vasoactive and
vasoconstrictive substances that maintain a healthy vascular
tone [158]. For example, prostacyclin and endothelium-
Copyright # 2001 John Wiley & Sons, Ltd.
derived growth factor are vasodilators and inhibit platelet
aggregation. They are produced simultaneously with
endothelin-1, an agent that sensitizes endothelial cells to
vasoconstricting stimuli [158]. When some event exposes
the basement membrane to the blood serum, in this case
PDT [159], cascades of eicosinoids tip the system towards
vasoconstriction. In the region of injury vessel constrictions
form. Platelets and neutrophils adhere to the vessel wall, roll
toward the constriction and aggregate, at which point they
migrate into the surrounding tissues following chemokine
gradients [160].
McMahon et al. showed that thromboxane and other
vasoactive leukotrienes were produced in large quantities in
vivo after Photofrin1-PDT. McMahon et al. showed that the
inflammatory response induced by Photofrin1-PDT is not
general since other photosensitizers do not necessarily
produce the same degree or type of vascular response [161].
Although NPe6-PDT produced blood stasis it was mainly
due to platelets aggregated on the artery walls. Throm-
boxane was not detected and arteriolar constriction was not
observed after NPe6-PDT. Although not confirmed directly,
McMahon et al. suggested that the vascular response
observed after Npe6-PDT resulted from the lack of
thromboxane production. They suggested that NPe6-PDT
stimulates the excretion of prostacyclin which inhibits
thromboxane production [161]. Although the mechanisms
are unknown, SnEt2 produces an inflammatory response but
does not cause vessel constriction or platelet aggregation
[161].
Many eicosinoids are derived from arachidonic acid,
which is produced by phospholipases that hydrolyze
phospholipids stored in cells [160, 162–164]. Arachidonic
acid is converted to prostaglandins by prostaglandin
endoperoxide synthase, which catalyzes the addition of
two molecules of oxygen to arachidonic acid [165].
Agarwal et al. demonstrated that phospholipases C and A2
were active in RIF-1 cells in vitro following Photofrin1-
PDT [166]. Inhibiting these phospholipases reduced the
tendency for these cells to undergo apoptosis (See Section
1.6). Fingar et al. also showed that Indomethacin, which
inhibits the cyclooxygenase activity of prostaglandin
endoperoxide synthase, decreases the amount of throm-
boxane produced after Photofrin1-PDT, in turn reducing
the PDT response by inhibiting vessel constriction [154].
Although vascular destruction is generally considered to
be one of the major contributing effects, direct cytotoxic
effects are not entirely unimportant and are described more
fully in the next section [155].
1.6.2 Direct Cell Effects. Originally PDT was thought to
destroy tissue by damaging cells which had accumulated
enough photosensitizer and received enough light to
produce lethal amounts of singlet oxygen. This had been
demonstrated effectively in vitro by Christensen et al. [167],
Berns et al. [168], Salvatore et al. [169] and others.
Henderson and others showed (see previous section) that a
greater percentage of tumor cells in vivo die after PDT
treatment because vascular shutdown starves them of
oxygen and nutrients [153–156]. However, Henderson
acknowledged that direct cell cytotoxicity is not entirely
unimportant [155, 156]. Fingar et al. confirmed this by
injecting DBA mice bearing SMT-F tumors with 5 mg kgÿ1
of Photofrin1 24 h before light treatment or 0.1 mg mlÿ1
Photofrin1 5 min before treatment. Both doses achieved
equivalent circulating concentrations of Photofrin1
J. Porphyrins Phthalocyanines 2001; 5: 105–129

(1.2 mg mlÿ1), except that the 24 h delay allowed time for
the photosensitizer to accumulate in tumor cells while the
five-minute delay confined most of the drug in the
vasculature. The five-minute treatment produced no tumor
responses while the 24-hour treatment elicited curative
responses, proving that some degree of cellular damage was
important to obtain complete tumor destruction [170, 171].
Tumor responses could be obtained with the short time
treatment regimen by using doses as high as 10 mg kgÿ1 of
Photofrin, however selectivity was poor and damage to the
surrounding tissues was extensive [170, 171].
Wilson et al. [172], Sharkey et al. [173], and Adams et al.
[174], have used Photofrin1-PDT-resistant RIF cells to
demonstrate the importance of direct cytotoxicity. Although
these cells were created in vitro, they were also found to be
resistant to PDT in vivo although the vascular damage
produced by the treatments was similar [174].
1.6.2.1 DNA Damage. Initially it was thought that the
DNA could be susceptible to photodynamic modification.
Gutter et al. and Fiel et al. showed DNA damage could be
observed after HpD- or cationic-porphyrin-mediated PDT in
vitro [175, 176]. Modification of the guanine moiety was
most commonly observed [175]. Indirect evidence of PDT-
induced DNA damage was demonstrated by Gomer et al.
who noted decreased cell survivability in cells with retarded
DNA-damage repair mechanisms in vitro [177]. However,
when Gomer compared the PDT-DNA damage to X-ray-
induced DNA damage, after equitoxic doses, there was less
DNA damage from PDT treatment than from X-ray
treatment. Moan et al. and Evensen et al. also showed that
X-ray treatment produced 80% more strand breaks, 5%
more sister-chromatid exchanges, and more chromatid
aberrations than PDT treatment. Furthermore PDT-induced
strand breaks were repaired more efficiently than those
caused by X-ray irradiation [178, 179] and the number of
chromatid aberrations did not correlate with the photo-
toxicity of HpD [179]. Evans et al. compared the
Table 1. Intracellular localization sites of various photosensitizers
Photosensitizer, dose, (incubation time) Cell line
Npe6, 15 mg mlÿ1 (24 h) Rat Heart, CHO
TPPS2o, S2a, S4, 30 (40 h), 3.0 (18 h), 75 mg mlÿ1 (18 h) NHIK3025
TPPS1, 5 mg mlÿ1 (18 h) NHIK3025
ÿ1
TPPS1, 1 mg ml (18 h) CT26 murine colon
carcinoma
AlPcS4, 75 mg mlÿ1 (18 h) NHIK3025
AlPcS4, 10 mM (4 h) RIF
ÿ1
AlPcS4, 50 mg ml (18 h) V79
AlPcS2, S1 40 mg mlÿ1 (18 h) NHIK3025
ALPcS2, 25 mg mlÿ1 (5 h) V79
SnEt2, 2 mM (15 min) P388
ÿ1
BPD-MA, 1 mg ml (4 h) NHIK3025
m-THPC, 0.5 mg mlÿ1 V79
ÿ1
Lu-Tex, 20 mg ml (4 h) EMT-6
1
Photofrin
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 117
carcinogenic potential of Photofrin1 and aluminum
phthalocyanine-mediated PDT to X-ray irradiation in
B-lymphoblast cell lines in vitro [42]. Both cell lines are
p53 deficient but one was transfected with a functional p53
gene. The cells with functional p53 genes did not accu-
mulate the numbers of mutations as did the p53 mutants. In
either case the mutation potential of PDT was determined to
be insignificant compared to that of X-ray irradiation [42].
Recently McNair et al. examined the ability of HpD,
m-THPC, and methylene blue to damage DNA in vitro. No
DNA damage was detected after m-THPC-PDT, but minor
DNA damage was observed after HpD-and methylene blue-
PDT. The damage from methylene blue and HpD was
transient and was resolved 4 h after treatment [180]. Thus
the published reports suggest that DNA damage is not
initially a major contributor to the PDT response (DNA
alterations will be a major factor during the apoptotic
response).
1.6.2.2 Lysosomal Damage. Many porphyrinoid photo-
sensitizers can be observed intracellularly with a fluores-
cence microscope. Most photosensitizers localize to
cytosolic targets such as the Golgi, endoplasmic reticulum,
mitochondria, lysosomes, and membranes [172, 181–185].
The localization sites, as reported in the literature, of the
most commonly studied photosensitizers are shown in
Table 1.
The data in Table 1 show that many photosensitizers
localize to the lysosomes, thus it was suggested by Berg et
al. that the lysosomes were highly susceptible PDT targets
[194]. Okada et al. demonstrated that lysosomal disruption
was not directly cytotoxic [195] but Geze et al. [183] and
others [196–198] suggested that hydrolases and acid that
leak out of the damaged lysosomes may degrade cellular
components. However Berg et al. showed that TPPS-
mediated PDT disrupted lysosomes but did not significantly
decrease cell viability [194]. The activities of the lysosomal
enzymes b-N-acetyl-D-glucosaminidase (b-AGA) and ca-
Localization target Ref.
Lysosomes [186]
Lysosomes [128]
Perinuclear [128]
Perinuclear [187]
Lysosomes [128]
Lysosomes [188]
Lysosomes [189]
Cytoplasm/lysosomes [128]
Lysosomes [189]
Lysosomes [190]
ER/Golgi [191]
Diffuse cytoplasm [52]
Lysosomes [192]
Outer membrane then mitochondria [193]
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118 I. J. MACDONALD AND T. J. DOUGHERTY
thepsins L and B were also measured in the cytosol of
cultured cells after photodynamic treatment with TPPS
derivatives. The TPPS4 derivative localizes mainly to the
lysosomes (Table 1), however after PDT treatment no extra-
lysosomal enzyme activity was detected. Some cytosolic
enzyme activity could be detected after treating the cells
with TPPS-1 or -2a, but only after non-lethal doses were
used. Apparently cytosolic hydrolase inhibitors and the pH
change (7.3 compared to 4.2 in the lysosomes) block the
enzyme activity. Lastly, Berg suggested that the enzymes
detected might have been actually released during the
extraction procedure rather than during PDT [194]. It was
concluded that TPPS-mediated PDT does not kill cells by
damaging lysosomes or facilitating the release of hydrolytic
enzymes or acid [194].
To reconcile the inconsistency between lysosomal
localization and cellular insensitivity to lysosomal damage
Moan et al. suggested that lysosomes are a primary
localization site but not the photodynamic target [189].
Moan explained that upon illumination the lysosomes may
rupture and the photosensitizer could relocalize to more
susceptible organelles such as the membranes of the
mitochondria or nuclei. Moan supported this theory by
demonstrating that di- and tetra-sulfonated aluminum
phthalocyanines (AlPcS2 and AlPcS4) exhibit large in-
creases in their fluorescence intensities within cells after the
cells were treated with low doses of light. Presumably the
compounds are disaggregated, thus more fluorescent, after
being released from the lysosomes. Relocalized into new
cellular targets the compounds are more photodynamically
active because they are disaggregated and have higher
singlet oxygen quantum yields. However, it has not been
determined whether the lysosomes rupture after photody-
namic damage and release the compounds or whether some
other cellular damage, caused by unobservable fractions of
the photosensitizer elsewhere in the cell, causes the
lysosomes to rupture and subsequently release the photo-
sensitizer.
1.6.2.3 Mitochondrial Damage. Since ATP, required to
power cellular functions, is produced by mitochondria Hilf
et al. considered this organelle to be susceptible to
photodynamic damage. In support of this, Hilf et al.
demonstrated that HpD-PDT inhibits several mitochondrial
proteins in vivo. Malate dehydrogenase, succinate dehy-
drogenase, and cytochrome c oxidase were inhibited by
15%, 20–25%, 50–60% respectively by HpD-PDT [82].
These three proteins are required to maintain the electro-
chemical gradient across the inner-mitochondrial mem-
brane. Murant et al. also examined the functioning of the
mitochondrial proteins adenylate kinase (AK) and mono-
amine oxidase (MAO) after in vivo HpD-PDT [199].
Adenylate kinase and MAO were inhibited by 30% and
80% respectively, but regained 100% activity within two
hours of treatment possibly because of the presence of
antioxidants in the mitochondria. Hilf et al. also examined
the functioning of glucose phosphate isomerase, pyruvate
kinase, and lactate dehydrogenase, all cytosolic proteins.
None of the these proteins were inhibited by HpD-mediated
PDT [82]. They suggested that cytosolic proteins may not be
as susceptible to photochemical oxidation as membrane-
bound proteins because the singlet-oxygen lifetime is
drastically reduced in aqueous environment (see Section
1.3.1). This was supported by the fact that pryruvate kinase
is anchored to the inside of the outer membrane yet was still
Copyright # 2001 John Wiley & Sons, Ltd.
unaffected by PDT, probably because the active portion of
the protein juts out into the cytosol. It was further
demonstrated that the susceptibility of the protein to PDT
correlates with the degree to which it is embedded into a
membrane. Cytochrome c oxidase resides within the inner
mitochondrial membrane, succinate dehydrogenase is
anchored in the inner mitochondrial membrane but
protrudes out into the mitochondrial matrix, and malate
dehydrogenase floats free in the mitochondrial matrix.
Cytochrome c oxidase was inhibited to a greatest extent
followed by succinate dehydrogenase which was inhibited
to a greater extent than malate dehydrogenase by HpD-PDT
[82]. Hilf suggested that cytochrome c oxidase was exposed
to the highest porphyrin concentrations because it is
embedded in a hydrophobic environment where active
HpD components tend to localize.
Gibson et al. compared the photosensitizing ability of
HpD, hematoporphyrin (Hp), hydroxyvinyldeuteroporphyin
(HVDP), and protoporphyrin-IX (PP-IX) to the degree to
which they inhibited cytochrome c oxidase activity in vitro
[200]. Reduced mitochondrial functioning and cytochrome
c oxidase activity were observed after HpD, HP and HVDP-
mediated PDT, but not after PP-IX-PDT. These results
further supported Hilf’s hypothesis that cytochrome c
oxidase is a susceptible target since the degree of inhibition
matched the in vivo efficacies of these compounds [200].
A protein complex, termed the permeability transition
pore (PT pore) [72, 201, 202], that consists of hexokinase,
the peripheral-benzodiazepine receptor (PBR), the voltage-
dependent anion channel (VDAC or porin), creatine kinase,
the adenine nucleotide translocator (ANT), and cyclophyllin
D [202] has been proposed to be a susceptible PDT target
[28, 203]. Miccoli et al. reported that photodynamically
damaging hexokinase may inhibit the enzyme’s ability to
phosphorylate glucose for entry into glycolysis, thereby
crippling the cell’s metabolic pathway [203]. Interestingly,
hexokinase was damaged by hypericin-PDT only after
hypericin was bound to hexokinase while it was bound to
the PT pore complex [203]. Dubbelman et al. considered the
ANT to be a susceptible-PDT target since its active site
contains several reactive thiol groups [28], and since Atlante
et al. showed that the functions of other anion translocators
decreased before ATP production declined [204]. The PBR
is also thought to be a susceptible target because it
transports cholesterol into the mitochondrial matrix for
steroidogenesis [205] and because hematoporphyrin and
protoporphyrin readily bind to it [72]. Verma et al. reported
a positive correlation between the doses needed to kill 63%
of the cells in culture and the PBR inhibition constants of a
series of porphyrin photosensitizers. Verma also demon-
strated that cells expressing higher levels of PBR were more
susceptible to Photofrin-PDT than cells expressing normal
levels of PBR [73].
Perlin et al. demonstrated that in vitro HpD-PDT reduced
the activity of the adenosine triphosphatase (ATPase),
which extracts energy from the proton gradient across the
mitochondrial membrane to form adenosine triphosphate
(ATP) [206]. Perlin demonstrated that PDT-induced cell
death occurs after the mitochondrial membrane potential
decreases. Leakage of markers from the mitochondria was
observed to occur only after oxidative phosphorylation
stopped due to photo-oxidation of mitochondrial proteins
such as cytochrome c oxidase [206]. Atlante et al. came to a
similar conclusion by showing that mitochondrial swelling
occurs only after disruption of the mitochondrial membrane
J. Porphyrins Phthalocyanines 2001; 5: 105–129

potential [204]. Atlante et al. treated mitochondria with
ammonium acetate to induce colloidosmotic swelling, and
compared the rates of swelling with Photofrin1-PDT-
treated mitochondria. They reported that Photofrin1-treated
mitochondria swelled faster than ammonium acetate-treated
mitochondria indicating that PDT was damaging the
mitochondria’s ability to regulate the membrane potential
rather than permeablizing the membrane, as does ammo-
nium acetate. Atlante et al. also spectrophotometrically
monitored the transport of substrates by mitochondria
during Photofrin-PDT. Before colloidosmotic swelling
occurred tricarboxylate (i.e. citrate, oxalosuccinate) and
oxodicarboxylate (i.e. malate and oxaolacetate) carriers
were observed to be inhibited by 50% and 65% respectively
[204]. Thus it appears photodynamic damage to the
mitochondrial proteins constitutes the primary event in a
chain leading to breakdown of the electron transport chain,
disruption of the mitochondrial-membrane potential and
mitochondrial swelling [204].
1.6.2.4 Membrane Damage. Photo-oxidizing cellular
membranes was initially thought to be an efficient way to
kill tumor cells. Furthermore Photofrin1 was found to
localize to the cellular membranes at early incubation times
[193]. Dubbelman et al. reported that PDT can cause
lysosomal rupture, potassium ions and small molecules leak
from cells, and increase the flip-flop rate of membranes lipid
but that these events usually occur after protein damage,
especially mitochondrial [28]. In general, cultured cells
seem to be unaffected by direct membrane oxidation, except
at very high levels. The increased flip-flop rates of inserted
lipid probes and the leakage of small molecules described
above appears to be the result of damage to the membrane
proteins band 3 and the 2-aminoisobutyric acid transport
protein (2-AATP) [28].
Although direct photo-oxidation of membrane lipids may
not be especially lethal to a cell, membranes may still have
an important role in PDT. Oxidation of lipids within the
mitochondrial membranes may not be directly cytotoxic but
these oxidized lipids may serve as potent signals that signal
a cell to die. Phospholipids are natural substrates for
phospholipases which, as reported in Section 1.6.1, can be
converted into arachidonic acid by phospholipases.
Membranes, especially mitochondrial membranes, may
also act as localization sites for photosensitizers. Photo-
dynamically active photosensitizers may have no inherent
affinity for specific protein targets but may partition into the
surrounding lipid layers. Therefore the photosensitizer’s
affinity for particular membranes determines the protein
target, i.e. those that reside in that membrane. Wilson et al.
have demonstrated that Photofrin1 competes with, 10-
nonyl acridine orange (which binds selectively to cardio-
lipin), for the cardiolipin-rich membranes of the inner
mitochondrial membrane [172].
1.7 Apoptosis
1.7.1 Apoptosis and Necrosis. Two ways in which
eukaryotic cells can die have been identified. Necrosis is
characterized by non-ATP-driven processes such as colloid-
osmotic swelling while apoptosis is mediated by ATP-
driven processes such as caspase activation [207]. Sub-
lethal genetic damage signals the cell to die apoptotically
while increasingly severe damage will destroy the cell’s
ability to produce ATP and it will die by necrosis.
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 119
Apoptosis is characterized by a series of cellular changes
that can be seen with a microscope; they include chromatin
condensation and the formation of apoptotic bodies.
Apoptosis can be also detected by examining the electro-
phoretic pattern of DNA extracted from cells. The DNA
from cells that die by apoptosis forms a ‘ladder’ pattern
because the DNA is cleaved into multiples of 180 kDa
fragments. This is the classical sign of apoptotic cell death,
although many new techniques have been devised, such as
the TUNNEL assay and flow cytometric techniques, to
detect apoptosis more easily and quntitatively.
1.7.2 Apoptosis in PDT. Argarwal et al. showed that
murine L5178Y lymphoma cells progressed to apoptosis in
as little as 30 min after PDT treatment in vitro [208].
Generally apoptotic responses require several hours to days
to complete. Argarwal suggested that PDT-induced cellular
damage bypasses the normal apoptotic mechanisms and
initiates late-stage processes directly [208]. Zaidi et al.
observed apoptosis in vivo (apoptotic bodies and DNA
laddering) in RIF-1 tumors treated with Photofrin-PDT
[209]. However Zaidi reported that the apoptotic bodies and
digested DNA may have come from host cells infiltrating
the tumor rather than the tumor cells [209]. He et al.
demonstrated that PDT does not induce apoptosis in all cell
lines in vitro [210]. Despite having similar sensitivities to
Photofrin1-PDT, an LD50 dose of Photofrin1-PDT induced
apoptosis in rat MTF7 cells while an LD85 dose was
required to induce apoptosis in PC3 human prostatic
adenocarcinoma cells, and apoptosis could not be induced
in H322a non-small cell carcinoma cells. He et al. suggested
that there are many different pathways through which PDT
can induce apoptosis [210].
Kessel et al. [190] and Luo et al. [211] showed that
different photosensitizers induce apoptosis to varying
degrees. They treated cultured cells with SnET2 or tin
octaethylpurpurin amidine (SnOPA, a cationic variant of
SnEt2) -PDT. Tin ethyl etiopurpurin induced apoptosis
within 60 min of treatment while SnOPA required 24 h or
longer [190, 211]. Tin octaethylpurpurin amidine localized
uniformly throughout the cell and its membranes while
SnET2 localized to the lysosomes and mitochondria, thus
Luo suggested that SnOPA damaged cellular membranes,
retarding the cell’s ability to regulate apoptosis [190].
Argarwal et al. demonstrated that phospholipases C and
A2, which are involved in apoptosis, are activated after
Photofrin1-PDT [166]. When the actions of phospholipases
C and A2 were blocked with inhibitors, the transient
increases in intracellular calcium levels and DNA fragmen-
tation commonly observed during apoptosis were elimi-
nated [166].
Kick et al. reported that the proto-oncogenes c-jun and c-
fos, which are commonly associated with apoptosis, were
activated after treating cultured cells with Photofrin1-PDT
[212]. C-jun and c-fos may be turned on to transcribe
apoptosis mediating factors. Fisher et al. showed that
cultured HL-60 cells expressing higher levels of p53 were
less sensitive to Photofrin1-PDT than cells expressing
normal levels or no p53 [213]. The tumor suppressor gene
p53 is known to arrest damaged cells in the S-phase of the
cell cycle and force them into apoptosis if they cannot repair
the damage [214].
In the last ten years there has been a shift away from
thinking that the nucleus is the only organelle to control
apoptosis. Guido Kroemer and other (next sections) have
J. Porphyrins Phthalocyanines 2001; 5: 105–129
120 I. J. MACDONALD AND T. J. DOUGHERTY
established that the mitochondria can induce and control
nuclear apoptosis. In light of this discovery and the
information presented in Section 1.6.2.3, Kessel et al.
hypothesized that PDT may kill cells by mitochondrially
controlled apoptosis [215, 216].
1.7.3 Mitochondrially Controlled Apoptosis
1.7.3.1 Reactive Oxygen Species (ROS) and the Mito-
chondrial Membrane Potential (D m) in Apoptosis.
Zamzami et al. showed that non-nuclear elements could
direct apoptosis by inducing apoptosis in enucleated cells
[217]. Zamzami also showed that splenocytes decreased
their uptake of dihexyloxacarbocyanine (DiOC6), a mito-
chondrially localizing dye, after forcing them to become
apoptotic with dexamethasone. The amount of DiOC6
accumulated by the mitochondria can be correlated with
the mitochondrial membrane potential (D m) which con-
trols its accumulation. In apoptotic splenocytes the DiOC6
signal decreased long before classical apoptotic signs could
be observed. While D m decreased, mitochondrial gluta-
thione levels decreased and reactive oxygen species (ROS)
production increased. All three signals (decreased D m,
decreased glutathione levels, and increased production of
ROS) were proposed to be very early, mitochondrially
controlled events that eventually lead to nuclear apoptosis
[217].
Zamzami et al. also reported a D m threshold value
below which the cell was irreversibly committed to die by
apoptosis [218]. This was demonstrated by treating
lymphocytes with various apoptosis-inducing agents (cyclo-
sporin A, rotenone, m-ClCCP, A23187, N-t-butyl-a-phenyl-
nitrone, trolox, and L-ascorbate), each which induces
apoptosis by different mechanisms. The lymphocytes were
then stained with DiOC6 as described above and separated
into two populations, those with high D m values and those
with low D m values. Neither population initially exhibited
classical apoptotic signs (DNA laddering, apoptotic bodies)
but within 18 h the low-D m lymphocytes progressed to
apoptosis while the high-D m lymphocytes did not, clearly
indicating that the D m collapse is an early apoptotic signal
[218].
1.7.3.2 Permeability Transition (PT). Zamzami reported
that mega-channels in the inner mitochondrial membrane
can open in response to certain compounds. When these
channels open (termed permeability transition [PT]) small
molecules (1.5 kDa) can flow in and out of the
mitochondrial matrix. When PT occurs the mitochondria
cannot maintain the proton gradient, D m collapses then the
electron transport chain uncouples, ROS production in-
creases (produced by an inefficiently functioning electron
transport chain) and consumes antioxidants. Eventually
antioxidants are depleted and ROS damage accumulates and
more PT-pores open, the mitochondria swell and burst,
spewing out detectable molecules such as glutathione
(GSH) and calcium ions, and the cell proceeds into
apoptosis [219].
The PT-pore complex consists of a conglomerate
hexokinase, VDAC, PBR, ANT, creatine kinase, and
cyclophyllin D and probably others that have not been
identified [202]. Zamzami et al. used attractyloside (ATT)
to force open PT-pores and bongkrekic acid (BA) to force
them closed on isolated mitochondria. If the isolated
mitochondria were exposed to ATT to induce PT and then
the mitochondria were mixed with isolated nuclei, the nuclei
Copyright # 2001 John Wiley & Sons, Ltd.
would undergo apoptosis. If the mitochondria were treated
with ATT, then with BA, and then mixed with isolated
nuclei the nuclei did not undergo apoptosis. Thus it was
clearly demonstrated that PT can be a early apoptotic event
that precedes the loss of D m and ROS generation. It was
thought that many apoptosis inducers may function by
triggering PT [219].
1.7.3.3 Apoptosis Inducing Factor and Cytochrome c in
Apoptosis. Zamzami et al. and Petit et al. reported that
cytochrome c and a 50 kDa protein, named apoptosis
inducing factor (AIF), were released after mitochondrial
swelling [219, 220]. Cytochrome c and AIF both induce
apoptosis but by different mechanisms. Apoptosis inducing
factor directly initiates apoptosis in the nucleus while
cytochrome c activates caspases. In the presence of cyto-
chrome c and dATP the Apaf-1/procaspase-9 complex
(Apaf-1 is the human homologue of the Caenorhabditis
elegans cell-death gene that forms a complex with
procaspase-9) can activate caspase-9 which in turn activates
other death signals such as caspase-3, 6, and 7 [221, 222].
The Apaf-1/procaspase-9 complex has also been shown to
be an essential component of p53 mediated apoptosis [222].
1.7.4 Summary, PDT and Apoptosis. There are many
similarities between the mechanisms by which mitochon-
dria regulate apoptosis and mechanisms by which PDT kills
cells. Kessel et al. have proposed that mitochondrially
controlled apoptosis, similar to the mechanisms described
above, is occurring after PDT treatment [215, 216]. Active
photosensitizers may localize to mitochondrial targets such
as the membranes surrounding the PT pore or specific
proteins in the PT pore. Hilf et al. and others have shown
other important mitochondrial proteins may be involved as
well. During PDT singlet oxygen attacks mitochondrial
proteins and possibly alters their amino acid structures,
thereby disrupting the protein’s folding conformation. This
may force them into other conformations that disrupt their
normal functioning. Ruck et al. recently demonstrated that
ANT, reconstituted into liposomes, could act normally as an
ADP–ATP exchange carrier or it could form nonspecific
channels with conductance properties similar to the PT-pore
[223]. The PT pores allow water molecules to flow into ions
and antioxidants flow out as the system settles towards
equilibrium. The membrane potential decreases and the
electron transport chain uncouples spewing out detrimental
ROS. The remaining antioxidants may scavenge some ROS
but if PT is not reversed damage will accumulate and the
electron transport chain will collapse. Petit et al. reported
that bcl-2 decreases the likelihood of apoptosis by inhibiting
PT-pore formation [224] and He et al. have shown that cells
overexpressing bcl-2 were more resistant to in vitro
Photofrin1-PDT than parental cells [225]. Kim et al. also
demonstrated that bcl-2 mediates PDT-induced cell death,
and that aluminum phthalocyanine-PDT selectively de-
stroys bcl-2, enhancing the apoptotic response after PDT
[226]. Both results imply that PT-pore formation is a
component of PDT-mediated cell death [225, 226]. Below a
certain D m threshold level the mitochondria will cease to
function and swell, bursting off the outer membrane because
it has less surface area than the inner membrane. This
releases cytochrome c and AIF, which reside between the
inner and outer membranes, and at this point apoptosis is
irreversible. Cytochrome c activates caspases and AIF
initiates nuclear apoptosis. Varnes et al., Kessel et al. and
J. Porphyrins Phthalocyanines 2001; 5: 105–129

Kim et al. have demonstrated that cytochrome c is released
during PDT and that it enhances the apoptotic response to
PDT [215, 216, 226, 227]. A diagram showing the pathways
described and the putative PDT targets is shown in Fig. 11.
The star bursts represent the putative PDT targets which
when damaged initiate this apoptotic pathway.
1.8 Immune Effects in PDT
1.8.1 Immune System Activation and Suppression.
Although it is not completely understood, researchers have
reported that PDT activates and suppresses the immune
system, depending upon certain variables. Using PDT to
generate tumor antigens to induce a specific immune
response against tumor tissue could be useful to treat a
primary tumor and its metastases. However, it has been
reported that PDT also suppresses immune system function,
limiting the effect of specific immune responses. Photo-
dynamic treatment seems to alter the balance of agents that
regulate the immune system and appears to be able to tip it
towards activation or suppression by the release of specific
cytokines. This is probably controlled by many complicated
factors including, but not limited to, the particular
photosensitizer, light dose, and dose rate.
The mechanism by which PDT induces specific immune
responses has been suggested by Korbelik [157]. Summariz-
ing, PDT-treated tissue releases large quantities of cell
debris, and inflammatory signals, cytokines and chemotactic
agents. Within minutes of light treatment large numbers of
neutrophils can be observed invading the treated tissue.
They secrete lysosomal enzymes and ROS that destroy
endothelial and tumor cells and further amplify the
inflammatory response. When the neutrophils die they
release their cellular contents which then act as chemotactic
signals for future waves of inflammatory cells. As the
neutrophil invasion wanes mast cells flood into the damaged
tissue and release granules containing vasoactive agents and
cytokines. Monoctyes and macrophages invade, proliferate,
collect cell debris, and destroy pockets of surviving tumor
cells. At the end of the inflammatory response the macro-
phages and monocytes secrete immunosuppressive factors
which down-regulate the response and may hinder any
future, specific immune response [157].
Musser et al. showed that PDT-induced immune sup-
pression was a function of the photosensitizer used [228].
Musser demonstrated that TPPS4 and HpD-PDT suppress
Fig. 11. A summary of the pathways and feedback loops involved in mitochondrially controlled
apoptosis. Star bursts represent possible points at which PDT initiates mitochondrially controlled
apoptosis.
Copyright # 2001 John Wiley & Sons, Ltd.
BASIC PRINCIPLES OF PHOTODYNAMIC THERAPY 121
the immune system immediately after treatment while it
required 72 h for immune suppression to develop in animals
treated with Photofrin and m-THPC-PDT. Musser et al.
suggested that different photosensitizers behave differently
because they have different hydrophobicities and localize to
different subcellular targets which in turn affects the
immune response [228]. Alternatively, Lynch et al.
suggested macrophages modulate immune suppression
because they accumulate large amounts of highly aggre-
gated compounds (i.e. Photofrin1) and then secrete
suppressive agents during PDT [229]. Lynch et al.
supported this theory by adoptively transferring macro-
phages from PDT-treated animals to di-nitrofluorobenezene
(DNFB) contact-sensitized animals. They reported a 60%
reduction in the immune response after a second DNFB
challenge [229]. Furthermore Lynch et al. showed that
PDT-treated macrophages produce and secrete cytokines,
such as interleukin-1 (IL-1). This seemed logical since IL-1
can facilitate production of acute phase proteins (i.e. serum
amyloid A, serum amyloid P, and C-reactive protein) which
modulate immune suppression. They reported that serum
amyloid A, serum amyloid P, and C-reactive protein were
detectable after PDT [229].
Korbelik et al. have demonstrated that PDT can induce
long-term specific immunity [230]. Korbelik used vitamin
D3 binding protein-derived macrophage activating factor
(DBPMAF), which hyper-stimulates macrophage prolifera-
tion, and may be important for retrieving antigens, to
overcome immune suppression. They reported increased
selective tumor destruction after DBPMAF and PDT [231].
1.8.2 Immune System Modulators. The immune system
produces cytokines, many of which appear to have
numerous and subtle roles depending on the situation, to
regulate itself. Cytokine signals have been studied during
and following PDT to determine how the immune system
functions at a particular time.
Nseyo et al. detected interleukin-1b (IL-1b), IL-2, and
tumor necrosis factor-a (TNF-a) in the urine of patients who
received whole bladder PDT, demonstrating that the
immune system was active in the tissues of treated patients
[232]. It was not reported whether these cytokines were
produced during an inflammatory or a specific immune
response.
Herman et al. showed that protoporphyrin-IX (PP-IX)
and Photofrin-PDT induced secretion of IL-2, IL-3, TNF-a,
J. Porphyrins Phthalocyanines 2001; 5: 105–129
122 I. J. MACDONALD AND T. J. DOUGHERTY
and interferon-g (IFN-g) from human and mouse monocytes
[233]. They also reported that PP-IX sensitized the immune
system without light and suggested that the PDT-induced
immune response was enhanced because of pre-sensitiza-
tion before light treatment [233]. Caution must be used
while interpreting these results since the doses of PP-IX in
the experiments used were excessively high so that even
small amounts of light may have induced PDT.
Bellnier demonstrated that a combination of low-dose
recombinant human TNF-a, administered adjuvant to
Photofrin-PDT doubled the phototoxicity of Photofrin and
improved the selectivity of the treatment. Since TNF
stimulates macrophage growth and differentiation, Bellnier
suggested that exogenous TNF may enhance the inflamma-
tory response at the treatment site [234]. Evans et al. and
Schumaker et al. also detected TNF-a in the supernatants
from mouse macrophages, and human cells treated in vitro
with PDT [235, 236].
Kick et al. detected IL-6 mRNA and IL-6 protein in
epithelial-HeLa cells treated with Photofrin-PDT [237].
Since IL-6 production from PDT peaked four hours earlier,
and in higher quantities than from cells treated with UV
irradiation Kick et al. suggested that that PDT killed cells by
a different pathway than radiation. They reported that PDT-
stimulated IL-6 production was more similar to that from
cells treated with 12-O-tetradecanoylphorbol-13-acetate
(TPA), an apoptosis inducer. However, IL-6 production
peaked nearly two hours earlier in the PDT-treated cells
than in the TPA-treated cells, suggesting that PDT induces
immune responses by unique pathways [237].
Henderson et al. examined the changes in levels of IL-1,
IL-3, IL-6, IL-10, IFN-g, granulocyte/macrophage-colony-
stimulating factor (GMCSF), TNF-a, and transforming
growth factor b (TGFb) after in vivo and in vitro PDT
[238]. After in vivo Photofrin1-PDT Henderson reported
that only IL-6 and IL-10 levels changed significantly.
Interleukin-6 mRNA and protein levels in the skin, spleen,
and tumor cells peaked three hours post-treatment and
decreased thereafter. At high light doses (>100 J cmÿ2),
IL-10 mRNA levels rapidly increased then decreased to
undetectable levels within six hours. However, in the skin
IL-10 levels increased up to 120 h after treatment (at that
point observation was stopped). Flow cytometry experi-
ments revealed that granulocyte and macrophage popula-
tions increased 89% and 40% respectively and tumor cells
decreased by 60% after treatment. These data demonstrate
that macrophage proliferation and activity was destroying
tumor cells long after irradiation had stopped [238].
1.9 Clinical Photodynamic Therapy
While the clinical aspects of PDT are not the main focus of
this review it is noteworthy to point out that Photofrin-PDT
has been approved for use in the United States against
advanced stage esophageal, advanced non-small-cell lung
cancer and early-stage lung cancer. Photofrin-PDT has also
been approved for use in five European countries, Canada
(including bladder cancer), and Japan. Other trials that are
pending approval use Photofrin-PDT to treat early-stage
esophageal cancer. Photofrin1-PDT is also being investi-
gated as an adjuvant therapy for treatment of the surgical
bed after resection of malignant glioma, malignant astro-
cytoma, malignant mesothelioma, head and neck cancers,
and some intraperitoneal cancers [18]. In addition numerous
new photosensitizers for both oncologic and non-oncologic
Copyright # 2001 John Wiley & Sons, Ltd.
uses are in various stages of clinical trials demonstrating the
broad applicability of PDT to human disease.
Acknowledgements
The authors thank the National Institutes of Health (CA
55791) and the Oncologic Foundation of Buffalo for
financial support.
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