Photodynamic therapy for pathogenic fungi

Abstract

Photodynamic therapy (PDT) is a minimally invasive approach, in which a photosensitiser compound is activated by exposure to visible light. The activation of the sensitiser drug results in several chemical reactions, such as the production of oxygen reactive species and other reactive molecules, whose presence in the biological site leads to the damage of target cells. Although PDT has been primarily developed to combat cancerous lesions, this therapy can be employed for the treatment of several conditions, including infectious diseases. A wide range of microorganisms, including Gram positive and Gram negative bacteria, viruses, protozoa and fungi have demonstrated susceptibility to antimicrobial photodynamic therapy. This treatment might consist of an alternative to the management of fungal infections. Antifungal photodynamic therapy has been successfully employed against Candida albicans and other Candida species and also against dermatophytes. The strain-dependent antifungal effect and the influence of the biological medium are important issues to be considered. Besides, the choice of photosensitiser to be employed in PDT should consider the characteristics of the fungi and the medium to be treated, as well as the depth of penetration of light into the skin. In the present review, the state-of-the-art of antifungal PDT is discussed and the photosensitiser characteristics are analysed.

Full text

Photodynamic therapy for pathogenic fungi
Juliana Pereira Lyon,
1,2
Leonardo Marmo Moreira,
3
Pedro Claudio Guaranho de Moraes,
3
Fa
´bio Vieira dos Santos
4
and Maria Aparecida de Resende
1
1
Laborato
´rio de Micologia, Departamento de Microbiologia, Instituto de Cie
ˆncias Biolo
´gicas (ICB), Universidade Federal de Minas Gerais (UFMG), Belo
Horizonte, Minas Gerais, Brazil,
2
Departamento de Cie
ˆncias Naturais (DCNAT), Universidade Federal de Sa
˜oJoa
˜o Del Rei (UFSJ), Sa
˜o Joa
˜o Del Rei, Minas Gerais,
Brazil,
3
Departamento de Engenharia de Biossistemas (DEPEB), Universidade Federal de Sa
˜o Joa
˜o Del Rei (UFSJ), Sa
˜o Joa
˜o Del Rei, Minas Gerais, Brazil and
4
Universidade Federal de Sa
˜o Joa
˜o Del Rei (UFSJ), Divino
´polis, Minas Gerais, Brazil
Summary Photodynamic therapy (PDT) is a minimally invasive approach, in which a
photosensitiser compound is activated by exposure to visible light. The activation of
the sensitiser drug results in several chemical reactions, such as the production of
oxygen reactive species and other reactive molecules, whose presence in the biological
site leads to the damage of target cells. Although PDT has been primarily developed to
combat cancerous lesions, this therapy can be employed for the treatment of several
conditions, including infectious diseases. A wide range of microorganisms, including
Gram positive and Gram negative bacteria, viruses, protozoa and fungi have
demonstrated susceptibility to antimicrobial photodynamic therapy. This treatment
might consist of an alternative to the management of fungal infections. Antifungal
photodynamic therapy has been successfully employed against Candida albicans and
other Candida species and also against dermatophytes. The strain-dependent antifungal
effect and the influence of the biological medium are important issues to be considered.
Besides, the choice of photosensitiser to be employed in PDT should consider the
characteristics of the fungi and the medium to be treated, as well as the depth of
penetration of light into the skin. In the present review, the state-of-the-art of
antifungal PDT is discussed and the photosensitiser characteristics are analysed.
Key words: PDT, Fungi, Candida, photosensitisers, antimicrobial photodynamic chemotherapy.
Photodynamic therapy
Photodynamic therapy (PDT) is a treatment that
employs a photosensitiser compound, which is activated
by exposure to visible light in a wavelength that is
excitatory to this compound. The activation of the
sensitiser drug results in several chemical reactions,
such as the production of oxygen reactive species and
other reactive molecules. The presence of these mole-
cules in the site to be treated leads to the damage of
target cells.
1
Photodynamic therapy is a selective, non-invasive, or,
at least minimally invasive modality of treatment for
several types of diseases. In fact, PDT was first developed
for the treatment of malignant diseases, and it has
been successfully employed for the treatment of skin
tumours,
1,2
cutaneous T-cell lymphoma
3
and for
tumours localised in the oral cavity, blade and others.
4
Besides precancerous lesions, such as BowenÕs disease,
early stages of cervix cancer and BarrettÕs oesophagus
can be treated with PDT.
5
However, in recent years, the
range of indications for PDT has been expanding. This
kind of treatment is also used for acne vulgaris and
leishmaniasis, and for treating premature skin ageing
due to sun exposure.
6
There are also lines of evidence
that PDT can be applied against bacteria, fungi and
viruses,
7
which will be discussed later.
Photodynamic therapy is performed in two stages. In
the first step, the photosensitiser is administered to the
Correspondence: Juliana Pereira Lyon, Rua Padre Machado, 152, Bela Vista,
Sa
˜o Joa
˜o Del Rei, Minas Gerais, Brazil.
Tel.: +55 32 3371 4013. Fax: +55 32 3379 2408.
E-mail: julianalyon@yahoo.com.br
Accepted for publication 18 August 2010
Review article
Ó2011 Blackwell Verlag GmbH doi:10.1111/j.1439-0507.2010.01966.x
mycoses
Diagnosis, Therapy and Prophylaxis of Fungal Diseases

patient as a cream, if the lesion is localised in the skin,
or by injection into a vein, for inner lesions, although
some drugs can be taken via oral, nasal or by
pulmonary administration.
8
The drug must act for a
time period to be concentrated in the target cells. Then,
in the second stage, the light of the appropriate
wavelength is applied though a light device, which is
directly driven to the target in the case of skin lesions, or
can be directed by an endoscope or a catheter to reach
inner sites.
Regarding the light sources, lasers and non-coherent
light sources are employed for PDT. An advantage of
using lasers is that the light can be focused into fibre
systems and led to otherwise inaccessible locations, such
as urinary bladder, digestive tract or brain. For derma-
tology, however, non-laser sources are superior to laser
systems because of their large illumination field, lower
cost, smaller size, reliability and easy setup.
9
Mechanism of action
The mechanism of action of PDT results from the
interaction between visible light photons of appropriate
wavelength with intracellular molecules of the photo-
sensitiser. Reactive species are generated by the inter-
action between the light and the biological tissue
causing an oxidative stress.
Oxidative stress has been defined as a disturbance in
the pro-oxidant–antioxidant balance, in favour of the
former, leading to potential damage. This imbalance
may be due to an increased production of various
reactive species and a decreased ability of the natural
protective mechanisms of the organism to inhibit the
action of these reactive compounds. Injury to cells
occurs only when the reactive oxygen species over-
whelm the biochemical defences of the cell.
1
Photosensitiser compounds possess a stable electronic
configuration, which consists of a singlet state in its
ground energy level, i.e. there are no unimpaired
electrons (diamagnetic electronic configuration). When
the photosensitiser absorbs one photon of a specific
wavelength, the electronic quantum jump occurs and
the molecule is promoted to an excited state, which is
also a singlet state, with short half life (10
)6
to 10
)9
s).
The photosensitiser can return to the ground energy
level through the emission of a photon, which consists
of the fluorescence phenomenon or by internal conver-
sion with loss of energy as heat by the interaction with
neighbourhood molecules. Alternatively, the molecule
can be converted to the triplet state (Fig. 1). This
conversion occurs through an intersystem crossing,
which involves a change in the electronic spin state.
10
The photosensitiser triplet state possesses a lower energy
level than the singlet, consisting of a meta-stable state,
and as consequence, showing a longer half-life.
10–12
The excited singlet state may interact with the
surrounding molecules via type I reactions, whereas
the triplet state interacts through type II reactions.
13
Type I reactions lead to the formation of free radicals
by hydrogen or electrons transference. These reactive
species, after the interaction with oxygen, might
produce oxygen reactive species, such as peroxide or
superoxide anions, which attack cellular targets.
14
However, type I reactions do not necessarily require
oxygen and could cause direct cellular damage by the
action of free radicals. On the other hand, type II
reactions need a mechanism to transfer energy from the
triplet state of the sensitiser to the molecular oxygen,
which usually occupies the triplet state
3
O
2
in the
characteristic electronic configuration of its ground
state.
15
In any case, the life time of the reactive species is
relatively low, implying that the representative damage
Light
Ground state
singlet PS
1
PS
1
PS*
1
PS*
1
PS*
Eletronic
transition
Fluorescence
Internal
conversion
HeatLight
3
PS*
Phosphorescence
Light
Triplet PS
Radicals, superoxide
Cytotoxic
species
Excited state
singlet oxygen
1O2*
3O2* Ground state
triplet oxygen
1
PS Spin permitted
energy transfe
r
Figure 1 Schematic representation of the photochemical process of excitation of the photosensitiser (PS), including the possibilities of
luminescence (fluorescence and phosphorescence), the singlet and triplet excited states and the reactive oxygen species generated by the
energy transfer from the photosensitiser (PS).
J. P. Lyon et al.
2Ó2011 Blackwell Verlag GmbH

action is focused on the target tissue, without affecting
the neighbourhood tissues in a significant way.
Antimicrobial photodynamic therapy
The employment of PDT in the treatment of cancer and
its effect in mammalsÕcells have been intensively
studied.
16
Although the selective destruction of micro-
organisms by the action of light is known for more than
a 100 years,
17
only recently, the susceptibility of
microorganisms to this treatment has received a greater
attention.
18
The technique can be employed against
bacteria, viruses, fungi and parasites.
7,19–29
Bacterial resistance to conventional antimicrobial
chemotherapy is an issue of major concern, leading to
the search for new therapeutic approaches. At this point
of view, PDT arises as a promising strategy, as the
mechanism of action involves multiple targets and
mutations leading to resistance are unlikely to happen.
It is important to notice that PDT has been tested
against skin and mucosal infections, where the drug
delivery and the light application are easier to achieve.
Two points must be considered when the efficacy of
photodynamic procedure is evaluated: (i) the concen-
tration of the photosensitiser in the target tissue; (ii) the
intensity of photons incident on the target tissue.
Antifungal photodynamic therapy
The great interest in alternative therapies for the
treatment of fungal infections comes from the fact that
the number of antifungal agents available for chemo-
therapy is very restricted when compared with the
number of antibacterial drugs. Furthermore, the cases of
recurrent infections are a major issue for certain kinds
of disease, such as candidiasis, dermatophytosis and
chromoblastomycosis. Antifungal photodynamic ther-
apy is a developing area of research,
10
and a majority of
the literature in this area is concerned with in vitro
experiments. Considering the potential of the technique
in the treatment of fungal infections and the importance
of developing new antifungal strategies, this is an area
of great interest for future research studies.
PDT against Candida species
The photosensitisation of Candida yeasts inducing cellu-
lar damage through the utilisation of several sensitiser
compounds has received special attention in several
works.
19,30,31
Candida yeasts may cause skin and
mucosal infection in patients with local predisposing
conditions and are also a major cause of systemic
infections, especially in immunocompromised
patients.
32
The resistance of this yeast to azole antifun-
gal agents has been increasingly reported.
33
The effect
of PDT has been already demonstrated in the inhibition
of germ tube formation,
30,34
biofilm formation
34,35
and
reduction in adhesion to epithelial buccal cells.
36
Although Candida albicans is the most prevalent
species involved in human infections, other species are
also important. It is worth mentioning that Candida
krusei is intrinsically resistant to fluconazole.
37,38
Considering this fact, the action of PDT against
different Candida species is very relevant. Dovigo et al.
[35] evaluated the efficacy of PDT against C. albicans
and Candida glabrata resistant to fluconazole, and
against cells in suspension or in biofilms. These
authors concluded that the fungicidal effect of PDT
was strain-dependent and that although PDT was
effective against Candida species, fluconazole-resistant
strains showed a reduced sensitivity to PDT. In another
study, Dovigo et al. [39] tested the efficacy of PDT with
Photogen, a porphyrin photosensitiser, against four
species of Candida. Interestingly, C. krusei was not
inactivated by any of the associations between light
and photosensitiser tested, while C. albicans,Candida
tropicalis and Candida dubliniensis were completely
eliminated.
Results achieved in vitro may not reflect the in vivo
situation. It is known that biofilms are less susceptible
to antimicrobial treatment than planktonic cultures.
The same situation seems to occur in PDT. According
to Dovigo et al. [35] biofilms were less susceptible to
PDT than their planktonic counterparts. Another
important point to be considered is the influence of
the biological medium. Bliss et al. [18] observed that
the uptake of Photophrin
Ò
(Axcan Pharma, Mont-
Saint-Hilaire, QC, Canada) by Candida yeasts was poor
when blastoconidia growth in nutrient broth, but was
increased when cultures were in chemically defined
medium. In addition, as expected, the uptake of
photosensitiser influenced the susceptibility to PDT.
Although the efficacy of PDT against Candida yeasts has
been already demonstrated by in vitro studies, this point
might impair its utilisation in vivo, and further studies
are necessary.
It is worth mentioning that other photosensitisers
may not have the same behaviour as that of Photoph-
rin
Ò
. Indeed, Teichert et al. [40] achieved a drug-
dependent photokilling of C. albicans in a murine model
of oral candidosis employing methylene blue. On the
other hand, Giroldo et al. [31] demonstrated that PDT
with methylene blue increases membrane permeability
in C. albicans, which could decrease the resistance of this
Antifungal PDT
Ó2011 Blackwell Verlag GmbH 3

microorganism to other drugs. In this way, PDT could
also be employed as a coadjutant to conventional
antifungal chemotherapy.
PDT against other fungal species
Fungi can cause infections varying from superficial
mycoses and cutaneous infections, to severe systemic
diseases. Due to the easier application of light and drug
delivery, the infections of the skin are more suitable to
be treated with PDT.
The genus Malassezia can be responsible for a number
of conditions. The most common infection is Pityriasis
versicolor, but other diseases can also be mentioned,
such as seborrheic dermatitis, folliculitis, neonatal
pustulosis and blepharitis. Lee et al. [41] employed
MAL-PDT (methyl 5-amino-levulinic acid) to treat
patients with recalcitrant Malassezia foliculitis. Three
from six patients achieved a strong improvement of the
lesions after three sessions of PDT and one patient
presented a moderate improvement.
A very interesting review was carried out by Calzav-
ara-Pinton et al. [42] regarding the employment of PDT
for the treatment of cutaneous fungal infections.
According to this work, the preliminary results obtained
in vitro are very promising and demonstrated that yeasts
and dermatophytes can be sensitised by the administra-
tion of photosensitiser drugs, such as phenothiazines,
phthalocyanines, porphyrins and the porphyrinic pre-
cursor aminolevulinic acid (ALA). In addition, the use of
these sensitisers did not lead to the selection of resistant
samples.
Dermatophytosis is very prevalent and the treatment
frequently leads to recidives. These infections may cause
great inconvenient to the patient and PDT could be a
helpful alternative. In fact, dermatophytes have been
evaluated in important research studies employing PDT.
The effect of PDT has been observed against the
dermatophyte Trichophyton rubrum by Smijs et al. [43].
These authors
44
also investigated the factors
involved on the susceptibility of Trichophyton rubrum
to PDT, employing two photosensitisers: 5,10,15-tris
(4-methylpyridinium)-20-phenyl-[21H,23H]-porphine
trichloride (Sylsens B) and deuteroporphyrin monome-
thylester (DP mme). It was observed that in acid
medium with low levels of calcium (obtained by the
addition of a chelant agent), the selective binding of
Sylens B is enhanced. The same does not occur with DP
mme. Calzavara-Pinton et al. [42] achieved promising
results regarding the treatment of mycological lesions of
the fingers in nine patients applying ALA as photosen-
sitiser. This preliminary study might encourage future
investigations on the use of PDT for the treatment of
fungal infections of the skin.
Onychomycosis, the fugal infection of the nail, is
one of the most difficult fungal infections to treat.
Watanabe et al. [45] describe two cases of onycho-
mycosis successfully treated with PDT with topical
application of an ointment containing ALA 20%.
These results are very important as not all cases of
onychomycosis are healed by conventional antifungal
chemotherapy.
45
Piracinini et al. [46] presented a
patient with onychomycosis caused by Trichophyton
rubrum to whom systemic antifungal agents were
contraindicated, and the therapy with topical antifun-
gal agents for 18 months had failed. Three sessions of
PDT with ALA with intervals of 15 days led to the
remission of the infection within a follow-up period of
24 months.
Some common environmental fungi may cause
opportunistic infections in patients with predisposing
conditions, which is the case of aspergillosis. Friedberg
et al. [47] demonstrated an in vitro fungicidal effect for
the photosensitiser Green 2W employed against Asper-
gillus fumigatus. These authors suggested that PDT could
be an efficient option for the treatment of the cavitary
lesions caused by this microorganism.
In this way, the treatment of fungal infections with
PDT could be an interesting area of study, especially
considering recurrent superficial and cutaneous myco-
logical lesions. The treatment might be an alternative to
conventional antifungal agents or a coadjutant to the
traditional drug therapy.
Photosensitisers employed in PDT
Photosensitisers are necessary for PDT as well as a
light source and the presence of significant concentra-
tions of molecular oxygen in the target tissue. For this
reason, more vascularised tissues generally achieve
better results when submitted to PDT.
48
Some features
are desirable on an ideal photosensitiser: absence of
toxicity and toxic by-products; lack of mutagenic effect;
selective accumulation on the target tissue, suitability
for topical, oral and intravenous administration, and
low cost.
48
The major groups of photosensitisers employed in
PDT are porphyrins, chlorines, phthalocyanines and
phenothiazines. Mainly methylene blue and ortho-
toluidine blue are the phenothiazines employed in
PDT. Phenothiazines are cationic compounds, which
have simple tricycle planar structures. The maximum
absorption wavelength is 656 nm for methylene blue
and 625 nm for ortho-toluidine blue.
10
J. P. Lyon et al.
4Ó2011 Blackwell Verlag GmbH

Porphyrins are tetraazamacrocycle compounds
widely encountered in nature.
49,50
The optimal wave-
length to photokilling is about 410 nm.
1
In turn,
phthalocyanines and chlorines are porphyrin-like
compounds that demonstrate longer wavelengths of
absorption, near infrared (650–700 nm).
1
It is also
relevant to mention that the ALA, which is not a
photosensitiser itself, but a porphyrin precursor, is
metabolised to protoporphyrin IX. ALA induced more
pronounced protoporphyrin IX synthesis and accumu-
lation in malignant and premalignant cells than in
normal mammalian cells.
1
Although there is a significant number of com-
pounds that may act as photosensitisers, only a few are
commercially available and approved for use in
humans. Among these, we can cite the porphyrins
Levulan
Ò
(Dusa Pharmaceuticals, Wilmington, DA,
USA), Photophrin
Ò
and Vysudine
Ò
(QLT, Vancouver,
BC, Canada), the porphyrin precussor ALA represented
by Levulan
Ò
and Metvix
Ò
(Photocure ASA, Oslo,
Norway), the chlorines Foscan
Ò
(BioLitec Pharma,
Jena, Germany) Photochlor
Ò
(RPCI, Buffalo, NY, USA)
and LS11
Ò
(Light Sciences, Snoqualmie, WA, USA),
besides the Phthalocyanine Photosens
Ò
(General
Physics Institute, Moscow, Russia).
1,48
These photo-
sensitisers were especially developed for the treatment
of malignant conditions. Currently, no clinical treat-
ment based on antimicrobial PDT is licensed.
8
When a photosensitiser is chosen for antifungal
PDT, the light penetration is an important concern.
Even if we consider infections of the skin, nails, hair,
oral cavity, oesophagus or lower feminine reproductive
tract, some degree of light penetration is required to
kill fungi localised below the skin surface.
10
Light in
the red region of the spectrum penetrates 3.0 mm
down the tissue, whereas light in the blue region
penetrates 1.5 mm. In this way, phthalocyanines and
methylene blue are employed in a larger number of
works, as they absorb near this desired wavelength.
10
Moreover, some fungi possess pigments that could
interfere with light absorption, such as melanin, which
is present in the dematiaceous fungi that cause
chromoblastomycoses. In these cases, to obtain a
photokilling effect, the photosensitiser employed must
absorb light in a different wavelength from that
corresponding to the absorption of the pigment present
in the fungi. It is important to register that photosen-
sitisers such as phthalocyanines and methylene blue
have a maximum absorption wavelength above
600 nm. Considering this, the employment of these
photosensitisers minimises the competition with the
melanin maximum absorption wavelength. It is also
known of the interference of compounds present in the
biological medium, such as haemoglobin, and the
choice of the photosensitiser is an important issue to
consider for clinical application. These considerations
allow observing that although the photodynamic
antifungal therapy consists of a promising therapeutic
alternative, there is a large field available for future
research studies.
Conclusion
Photodynamic therapy is a minimally invasive ap-
proach, primarily developed for the treatment of malig-
nant conditions. However, this therapy can be employed
for the treatment of several diseases, including infectious
diseases. The antifungal photodynamic therapy is a
promising area of research and its development could
benefit many patients, especially those with resistant or
recurrent mycological infections of skin and mucosa.
The antifungal action of PDT seems to be strain-
dependent, and the influence of the biological medium
must be taken into consideration because it can
diminish the efficacy of the therapy in vivo. In addition,
the photosensitisers to be employed in antifungal PDT
must overcome fungal pigments and other substances
that might be present in the medium to be treated, as
well as the depth of penetration of light into the skin.
Although positive results have been demonstrated
in vitro, there are considerably fewer in vivo investiga-
tions. There are many fungal species to be evaluated. In
addition, several issues must be improved in future
research studies, such as the investigation of appropri-
ate photosensitisers and drug delivery systems.
Currently, no clinical treatment based on antimicrobial
PDT is licensed. Considering this, antifungal PDT is an
area of great interest for future studies, and advance-
ments in this research should be strongly supported.
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