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The growing resistance against antifungal drugs has renewed the search for alternative treatment modalities, and antimicrobial photodynamic therapy (PDT) seems to be a potential candidate. Preliminary findings have demonstrated that dermatophytes and yeasts can be effectively sensitized in vitro and in vivo by administering photosensitizers (PSs) belonging to four chemical groups: phenothiazine dyes, porphyrins and phthalocyanines, as well as aminolevulinic acid, which, while not a PS in itself, is effectively metabolized into protoporphyrin IX. Besides efficacy, PDT has shown other benefits. First, the sensitizers used are highly selective, i.e., fungi can be killed at combinations of drug and light doses much lower than that needed for a similar effect on keratinocytes. Second, all investigated PSs lack genotoxic and mutagenic activity. Finally, the hazard of selection of drug resistant fungal strains has been rarely reported. We review the studies published to date on antifungal applications of PDT, with special focus on yeast, and aim to raise awareness of this area of research, which has the potential to make a significant impact in future treatment of fungal infections.
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Mycotic infections of skin and mucosae of humans and animals are common worldwide. The growing resistance against commercially available antifungal drugs has renewed the search for alternative treatment modalities, and in the 3 past decades, phenothiazine dyes, 5-aminolevulinic acid (ALA), cationic porphyrins and phthalocyanines have gained popularity as both antimycotic and antimicrobial PSs due to their abilities to inactivate yeasts, fungi, gram-positive and gram-negative bacteria. This has fueled the thought that these PSs could be successfully applied as broad spectrum antimicrobial agents. The treatment might be an alternative to conventional antifungal agents or a coadjuvant to the traditional drug therapy. A recent review by Smijs and Pavel (1) summarized the anti-microbial photodynamic therapy (PDT) applications to treat dermatophytes. The present review aims to provide an overview of investigation in vitro and in vivo studies of anti-fungal PDT with special focus on yeasts.
General Principles of Photosensitization of Fungi
Several PSs, mainly belonging to four chemical groups (phenothiazine dyes, porphyrins 5-ALA, and phthalocyanines), have been investigated. As a general rule, sensitizers that are medically interesting are those without dark toxicity, i.e., are devoid of toxicity in the absence of light activation, and, upon irradiation, lack genotoxicity and mutagenicity (2). The hazard of DNA damage in eukaryotic fungi is further reduced by the presence of a membrane that envelops the nucleus and may act as a barrier to the penetration of dyes or their high-energy photoproducts (3). The anti-microbial photodynamic effect has been found strictly dependent on physical and chemical parameters, e.g., absorption peak (kmax), intensity of absorption (emax), and quantum yield for singlet oxygen. In addition, the mechanisms and efficiency of cell inactivation are critically dependent on other chemical properties, e.g., the lipophilicity/hydrophilicity balance (log P), the degree of ionization (pKa), and the presence of electric charged groups that qualify the type and efficacy of cellular mechanisms of uptake of the dye and the pattern of its distribution among the various subcellular compartments (2). Unlike mammalian cells, fungi are surrounded by a rigid cell wall that is composed principally of soluble and insoluble polysaccharide polymers, like chitin, beta-glucans and glycoproteins, and, as a general rule, cellular uptake is negatively influenced by the lipophilicity of the molecules and positively by hydrophilicity and presence of electric charged groups (4). Following uptake, sensitizers are distributed to subcellular targets and the pattern of localization is very important because targets adjacent to the sensitizer have the greatest probability of being involved in the photoprocess due to the high reactivity and short intrinsic lifetime of the intermediate reactive oxygen species (ROS) (5). The photodynamic mechanism of fungal cell destruction is by perforation of the cell wall and membrane with PDT-induced singlet oxygen and oxygen radicals thereby allowing the dye to be further translocated into the cell. Subsequently the photodynamic dye damages inner organelles such as lysosomes, mitochondria and nucleus, inducing cell death (6). The multiplicity of cellular targets in fungi should reduce the risk of selection of photomutant resistant strains and this risk should be further minimized by the lack of mutagenic effects of PDT.
The spectrum of the light used for photoactivation is another critical issue. Light with wavelengths in the far red or near infrared regions can penetrate deeply into mammalian skin. Sensitizers with an absorption peak in these wavebands are needed for the treatment of dermatophytes that colonize both the stratum corneum and hair follicles. However, PSs absorbing in the blue region may be suitable for the treatment of Candida species that invade only the stratum corneum.
However, many yeasts, including Candida species, use specific mechanism to counteract antimicrobial therapy, such as the reduced uptake or the enzymatic alteration of the PS, the induction of drug efflux pumps to expel PS, the induction of enzymes (catalase and superoxide dismutase) capable of neutralizing oxidative species (7,8). In addition, the C. albicans ability to form biofilms is another mechanism to increase the resistance to treatment.
Biofilm is a complex aggregation of microorganism characterized by excretion of a protective and adhesive matrix, composed in yeasts by heterogeneous mixture of blastoconidia, pseudohyphae and hyphae embedded in extracellular polymeric substances that form channels and pores and exhibit different phenotypic characteristics than planktonic yeasts. The extracellular matrix is composed of polysaccharides, proteins, hexosamine and DNA to promote biofilm adhesion and formation, protect the cells from phagocytosis, maintain the integrity of the biofilm and limit the diffusion of substances, influencing the penetration of PS and light during PDT (9). The multi-species biofilms (bacteria and yeasts) are more resistant to PDT, suggesting that biofilm complexity increases resistance to PDT (9). The biofilm formation is in vivo a great problem involved in a wide variety of human fungal infections, such as oral candidiasis, and catheter infections (9).
By definition, PDT requires a source of light to supply the requisite energy for singlet oxygen production in situ. The energy required is determined by the molecular structure of the photosensitizer and, thus, a different light excitation range is required for the phenothiaziniums (ca 600–660 nm) than for the phthalocyanines (ca 630–690 nm). The porphyrins can be excited by light in the red region of the spectrum, but they absorb blue light more efficiently.
Ideally light sources should provide a strong output at the requisite wavelength for photoexcitation (10). Lasers, and the less expensive and easier to use, LED filtered and incoherent fluorescent lamps are the most commonly employed sources in PDT today. Typical power outputs for light sources used in antifungal PDT are in the range 10–100 mW cm−2 (11). Light fluence through tissue decreases exponentially with thickness and the decrease is determined by absorption, particularly by haemoglobin, and scattering, parameters that vary between tissue types (10). However, a high degree of tissue penetration is required only to kill fungi residing below the surface of the skin, in the matrix of the nail, or in the deep part of the hair follicle.
Oxidized phenothiazines have a simple planar, tricyclic skeleton and are normally encountered as cations. The most widely used compounds are methylene blue (MB) and toluidine blue (TBO). Both are efficient producers of singlet oxygen (12) and the wavelength of maximum absorption (kmax) in water is, respectively, 656 nm for MB and 625 nm for TBO (13). The phenothiaziniums are known to localize in the plasma membrane of yeasts. Consequently, this is the cellular structure damaged upon illumination and it has been proposed that the increased permeability resulting from such damage is the reason for cell death (14). The fungicide effect of low concentrations of MB (0.1 mg mL−1) followed by irradiation with 28 J cm−2 (685 nm) from a diode laser was demonstrated on various species of Candida genus: C. albicans, C. dubliniensis, C. krusei and C. tropicalis (15).
Recently a new MB (NMB) characterized by more lipophilic nature than traditional MB showed a better photodynamic inactivation of C. albicans in vitro and in vivo mouse model with infected abrasion wounds, irradiated with red light (16).
Soares et al. (17) assessed the effects of TBO and LED based PDT on C. albicans strains that were both sensitive and resistant to fluconazole and observed an average reduction of 3.41 log10 and a 55% reduction in adhesion to buccal epithelial cells. Soares et al. (18) more recently demonstrated that TBO (6.76 μg L−1) and LED light (630 nm; 54 J cm−2) were also effective in vitro against Cryptococcus gattii strains fluconazole-resistant, reducing significantly the cell viability after PDT treatment.
In vitro studies show that Candida species are effectively killed by PDT, whether grown planktonically or in biofilm. However, they are considerably less susceptible to photodynamic killing than a number of prokaryotic bacteria, including Staphylococcus aureus, Streptococcus mutans (19). In fact, doses of TBO as high as 2.0 mg mL−1 have been required to induce high levels (>99%) of kill in planktonic C. albicans upon illumination. Similarly, C. albicans biofilms required TBO doses of 5.0 mg mL−1 to achieve high levels of killing. Doses of red light (660 nm) required in the photodynamic killing of planktonic C. albicans or biofilm are also high (200 J cm−1) (11).
A possible explanation is that, according to the target theory, the killing effect of photoactivated MB and TBO in prokaryotic cells appears to be a single hit process where all the putative targets in the cell are equally susceptible and if damaged can lead to cell death, whereas in the eukaryotes a multihit process is needed where saturation of more than one molecular target is required before cell death occurs (11). These differences in susceptibility may be amplified by differences in the ratio of cell size to volume. Candida species are ca 25–50 times larger than bacteria and therefore contain a greater number of targets per cell. In the same experimental conditions, the killing rates for fungi were 18–200-fold higher than those determined for keratinocytes suggesting that MB-PDT of fungal infections of the skin may be not only effective but also highly selective (20). The same drug and light doses did not cause detectable genotoxic and mutagenic effects on both fungi and keratinocytes (20).
The interest raised by in vitro findings has prompted in vivo investigations. Oral azole-resistant candidiasis of severe combined immunodeficiency (SCID) beige nude mice, an immunodeficient murine model, was treated with the application of an aqueous solution of MB followed by irradiation with 275 J cm−1 of diode laser light with emission peak at 664 nm delivered with a cylindrical diffuser. Candida infection was sensitive to PDT and effects were dependent on MB concentration (250–500 μg mL−1). The most effective total eradication of C. albicans occurred with MB concentrations of 400 and 500 μg mL−1 (21). Other in vivo studies evaluated the effects of PDT on buccal candidiasis in rats.
Junqueira et al. (22) verified that the rats treated with laser light and MB developed less candidiasis lesions compared with the control group. Martins et al. (23) demonstrated that MB and laser light can also reduce microscopic lesions of candidiasis and the proteinase activity of C. albicans in a murine model of oral candidiasis without damaging effects to normal tissues. These findings suggested that MB-PDT could have the potential to become an effective, non-toxic, simple, inexpensive and repeatable therapy of oral candidiasis in immunodeficient HIV+ patients. However, it is necessary to develop a technique for the treatment of the concomitant esophageal infection which is noticed very often concomitantly in such patients (24).
Another difficulty is that the administration of these highly coloured photosensitizers to humans as a liquid mouthwash causes an undesirable staining of teeth, lips and buccal mucosa. Therefore targeted delivery of the photosensitizer directly to the site of infection should be the aim.
Donnelly et al. have formulated patches containing TBO, that were capable of resisting dissolution when immersed in artificial saliva (11). When released directly into an aqueous sink, patches containing TBO at a concentration between 2.0–5.0 mg mL−1 were capable of killing both planktonic and biofilm-grown C. albicans upon illumination, although the sterilization of biofilm structures needed much longer times of application of the patches. Therefore, the authors suggested that short application times of TBO-containing mucoadhesive patches should allow treatment of recently acquired oropharyngeal candidiasis, whereas longer patch application times are required for persistent disease where biofilms are implicated (11).
Although yeasts of the genera Candida and Cryptococcus continue to be the main opportunistic fungi responsible for invasive mycoses in humans, serious infections caused by Aspergillus spp. and by other genera of filamentous fungi have emerged all over the world. The primary reason is the increased numbers of immunocompromised individuals. Additionally, the continuous and indiscriminate use of fungicides has favored the selection of fungal strains resistant to currently used fungicide. Metarhizium anisopliae is a worldwide entomopathogenic deuteromycete that has been used for decades in programs of agricultural pest and disease-vector control in various countries. Although M. anisopliae is not considered to be pathogenic for humans or domestic animals, starting in the late 1990s some rare cases of mycoses caused by the fungus in both immunocompromised and immunocompetent individuals have been reported (25). Gonzales and colleagues (26) compared the conidial photosensitization of two fungal species (M. anisopliae and Aspergillus nidulans) with MB and TBO under different incubation and light conditions. Conidial inactivation reached up to 99.7%, but none of the treatments achieved a 100% fungicidal effect although PDT delayed the germination of the surviving conidia.
Photodynamic inactivation of dermatophytes has been studied as well. Eight strains of dermatophytes (Trichophyton mentagrophytes, Trichophyton rubrum, Trichophyton tonsurans, Microsporum cookei, Microsporum canis, Microsporum gypseum, Epidermophyton floccosum and Nannizia cajetani) were exposed to UVA irradiation after sensitization with two thiophenes (2,20:50 200-terthienyl and 5-(4-OH-1-butinyl) 2,20-bithienyl). A strong and dose-dependent inhibition of the growth of all tested strains was found although a complete inactivation was never obtained (27).
Recently, ten patients with chromoblastomycosis, an skin infection caused by traumatic inoculation of dematiaceous fungi species (Fonsecaea pedrosoi and Claphialophora carrionii), was positively treated with red light emitting diode (LED) light (660 nm, 28 J cm−2) after sensitization with MB for 4 h (28). Complete healing was not achieved, but all of the patients presented after 6 MB-PDT treatments a reduction of the lesions (80–90% of improvement in lesion volume and tissue cicatrization).
Costa et al. (29) investigated the antifungal activity of two new dye (rose bengal and erythrosine) against C. albicans. Rose Bengal (RB) and erythrosine are xanthenes dyes that are characterized by light absorption at wavelengths of 450–600 nm and 500–550 nm respectively. RB is usually used for the diagnosis of eye diseases and erythrosine is used in dentistry to reveal dental biofilms. The results revealed a significant reduction of planktonic cultures (3.45 log10 and 1.97 log10) and of biofilms (<1 log10) after blue LED irradiation (95 J cm−2) using erythrosine and RB respectively (29). Erythrosine was more effective than RB against C. albicans in planktonic cultures. The chemical structures of these dyes may contribute to a greater affinity with the external structure of the yeast cells than other phenothiazine dyes (29). The same authors (30) obtained interesting results also against C. dubliniensis using erythrosine and green LED light, although Candida biofilms were more resistant than their planktonic cultures.
The antimicrobial photodynamic activity of RB (200 μmol L−1) on C. albicans was also evaluated by Demidova and Hamblin (31) that observed log10 reduction of 4 and 6 for cellular densities of 107 and 106 cells mL−1 respectively. However the authors also showed that 5 μM of poly-L-lysine chlorine(e6) conjugate was more effective in killing C. albicans than 50 μM of TBO and 200 μM of RB after illumination at fluence ranging from 0 to 200 J cm−2.
Another new cationic dye is malachite green of the triarylmethane family. This dye showed strong absorption in the red region of the visible spectrum. Junqueira et al. (32) demonstrated the PDF with malachite green was effective in Candida strains reduction (<1 log10) when irradiated with low doses (26 J cm−2) of gallium-aluminum-arsenide (GaAlAs) laser. The photoinactivation efficacy of malachite green was also investigated by Souza et al. (33) at a concentration of 0.1 mg mL−1, followed by low-power GaAlAs laser irradiation, reducing the number of log CFU mL−1 of C. albicans. However in this study the malachite green was less effective than TBO and MB in reducing the viability of C. albicans.
The biological role of photodynamic reactions mediated by naturally occurring endogenous porphyrins is presently being debated. Irradiation with 20–50 J cm−2 of broadband visible light without the addition of exogenous sensitizers was found to produce oxygen dependent lethal effects of plasma membranes and mitochondria of C. guilliermondii (34). However in another study (35), the viability of suspensions of C. albicans was not affected by irradiation with 66 J cm−2 of red laser light (632.8 nm).
Hematoporphyrin derivative (HpD) is a complex mixture of mono- and oligo-meric porphyrins (Pp) derived from blood. It has various absorption peaks in the 400–650 nm region and its anti-fungal properties have been investigated since the early 1980s (36). Within a broad range of concentrations, HpD is not toxic for cells in dark conditions (2,37) but, after irradiation it can effectively kill fungal cells although mechanisms of photosensitization are quite different from those described for MB and other phenothiazine dyes. HpD is not uptaken by Saccharomyces cerevisiae and C. albicans (38) and the photocytotoxic activity is mainly promoted by unbound dye molecules in the bulk aqueous medium. These, after irradiation, cause an initial limited alteration of the cytoplasmic membrane that allows for the penetration of the dye into the cell, the translocation to the inner membranes and the consequent photodamage of intracellular targets. Freeze-fracture electron microscopy studies on C. albicans cells, which had been photosensitized by a meso-substituted cationic porphyrin, clearly indicate that the photodamage progresses from the outer leaflet of the plasma membrane to the inner leaflet (39). At a biochemical level, the photoprocess involves mainly the peroxidation of the lipids and, only to a minor extent, the inactivation of proteins of the cell wall (39) and is inhibited by albumin (40).
In order to determine the importance of sterol oxidation, sterol auxotrophic strains of S. cerevisiae were grown on media containing sterols with different levels of saturation before photosensitization (41). Cells grown on a completely saturated sterol, cholestanol, were markedly more resistant to the photosensitizing process whereas photodegradation of the native unsaturated yeast sterol, ergosterol, caused the substantial loss of cell viability.
The purified compounds derived from hematoporphyrin (HpD), Photofrin® (QLT Vancouver, BC, Canada), and Photosan III® (Seehof Laboratorium, GmbH, Munich, Germany) are also effective for the photoinactivation of bacteria and fungi upon red light exposures.
Using Photofrin® and laser light Mang et al. (42) demonstrated that strains of C. albicans, C. glabrata and C. krusei isolated from AIDS patients that had fluconazole and amphotericin B resistance were equally susceptible to photodynamic killing. Also Photogem® (Timtec Co. Newark, DE), another purified HpD derivative, was found effective in the inactivation of C. albicans, C. dubliniensis, C. tropicalis and C. glabrata, following blue light irradiation from a LED source (455 nm) (43). However, the experiments of this study were conducted with planktonic cultures of the microorganisms, which is not a good experimental model for investigating “in vivo” conditions.
Mima et al. (44) evaluated the susceptibility of C. albicans to Photogem®-PDT in a murine model of oral candidiasis. They obtained a significant reduction in the viability of C. albicans from tongues after 400, 500, and 1000 mg L−1 of Photogem® associated with LED light (305 J cm−2) (455 and 630 nm). There were no significant differences in effectiveness among the different concentrations and wavelengths tested. However no complete inactivation was observed and the CFU values obtained by this murine model of oral candidiasis are higher than those described by Teichert et al. (21). This difference may be attributed to the different PS and light source used. The molecule size of MB is smaller than porphyrins and a smaller molecule can more easily penetrate the inner layers of the biofilm and promote a better sensitization of the cells.
Another in vivo study was performed by Mitra et al. (45) that investigated the effectiveness of PS meso-tetra (N-methyl-4-pyridyl) porphine tetra tosylate (TMP-1363) in the PDT of C. albicans infection in a mouse model. Candida albicans was inoculated in the intradermal space of the ear pinna and irradiated after 30 min at 514 nm with a fluence of 90 J cm−2. Infected ears displayed complete healing over time without damage to the pinna.
Recently, several new synthetic Pp derived compounds have been evaluated for their possible anti-fungal activity. These drugs have been synthesized in order to obtain chemically pure dyes with an intense absorption peak in the 650–700 nm waveband, and high quantum yields for singlet oxygen. Polarity of the molecule is a critical feature. The hydrophilicity of porphyrins influences strongly their ability to photosensitize efficiently yeast cells, with highly water soluble porphyrins much more effective than their more hydrophobic counterparts (46,47).
Neutral amphiphilic meso-arylglycosylporphyrins (46) and water-soluble diamino-acid derivatives of Pp (47) are characterized by a greater uptake and, consequently, a greater activity against the yeast S. cerevisiae in comparison to hydrophobic molecules such as HpD, chlorines and benzoporphyrin derivatives.
Photo-activated hydrophilic porphyrinic compounds, e.g. deuteroporphyrin, deuteroporphyrin monomethylesther (DPmme) and 5,10,15-tris(4-methylpyridinium)-20-phenyl- (21H, 23H)-porphine trichloride (Sylsens B) showed a high activity against T. rubrum as well. Again, their activity was found greater than that of lipophilic phthalocyanines (ZnPc, PcS4 and AlPcS4) and Photofrin® that displayed nothing more than a fungistatic effect for about 1 week (48). It was also observed that in acid medium with low levels of calcium (obtained by the addition of a chelating agent) the selective binding of Sylsens B is enhanced, but the same does not occur with DPmme (49).
In an ex vivo model using human stratum corneum the application of a formulation containing 10 μM Sylsens B followed by the irradiation of 18 J cm−2 UVA-1 radiation (340–400 nm) proved to be effective against T. rubrum (50).
Ragàs et al. (51) described the susceptibility of C. albicans to a new cationic porphycene (a structural isomer of porphyrin), 2, 7, 12-tris (α-pyridinio-p-tolyl)-17-(p (methoxymethyl) phenyl) porphycene (3, Py3 MeO-TBPo). They showed a complete inactivation of both C. albicans and C. krusei using low light doses (30 and 15 J cm−2, 652 ± 5 nm and 635 ± 10 nm, respectively) and low porphycene concentrations (50 and 10 μM, respectively).
Recently a new Pp, the tetracationic porphyrin 5,10,15,20-tetra(4-N-methylpyridyl) porphyrin (TMPγP) demonstrated a high in vitro effectiveness for photodynamic inactivation of C. albicans colonies in both liquid suspensions and localized on a surface (52).
Cormick et al. (53) investigated the susceptibility of C. albicans to two tetracationic porphyrins (TMPγP4+) and 5,10,15,20-tetrakis(4-N,N,N-trimethylammoniumphenyl)porphyrin (TMAP4+) and a tricationic porphyrin 5-(4-trifluorophenyl)-10,15,20-tris(4-N,N,N-trimethylammoniumphenyl) porphyrin (TFAP3+). All the PSs had amphiphilic character of the structure and high affinity for C. albicans. They produce a ca 5 log decrease of cell survival when the cultures are incubated with 5 μM of PS and irradiated for 30 min with visible light.
5-ALA is not a PS by itself but all eukaryotic cells metabolize it into a very active endogenous sensitizer, protoporphyrin IX (PpIX), in the enzymatic pathway to heme (54). In general, the mechanisms used by fungi to internalize 5-ALA are poorly understood. Some studies showed that this metabolic pathway displays some differences to that of other eukaryotic cells in relation to its enzymatic machinery. In particular coproporphyrinogen oxidase is found in the cytoplasm and both ALA-synthase and ALA-dehydratase constitute the rate limiting steps of heme biosynthesis and their activity is controlled by the intracellular free heme pool (55). Therefore the content of PPIX may be increased by the addition of iron chelators, e.g., EDTA, hydroxypyridinone and 2,20-dipyridil, that inhibit the conversion of PpIX itself into heme which has been demonstrated in the cases of C. guilliermondii and S. cerevisiae (56). More recently it was shown that the administration of an iron chelator could induce the accumulation of PPIX in S. cerevisiae in the absence of ALA, because this yeast inherently possesses high levels of the PS (57).
PpIX may be activated by wavelengths ranging from UVA to the visible wavebands with a maximum peak in the Soret band at 375–405 nm and a lower peak at 630–633 nm. Upon exposure to light, PpIX induces cytotoxic effects through oxygen-dependent photochemical reactions that damage mainly the mitochondria, where PpIX is synthesized, and plasma membranes (58). Prolonged irradiation causes the additional alteration of other cytoplasmic structures and the inhibition of the synthesis of DNA and RNA (59), but genotoxic and mutagenic effects were never detected in yeasts (55).
ALA seems suitable for the treatment of human mycosis because in vitro studies have demonstrated that fungi can be effectively photosensitized by 5-ALA and killed at dose rates much lower than those that kill keratinocytes (60). Its clinical efficacy has been investigated with an open pilot study enrolling nine patients with interdigital mycosis of the feet (61) caused by C. albicans, T. mentagrophytes and T. rubrum. All colonies showed a strong red fluorescence after incubation with 20% ALA water solution and irradiation with Wood’s lamp (Fluolight, Saalman GmbH, Germany) (Fig. 1). The treatment protocol provided for the application of a 20% ALA preparation in eucerin cream under an occlusive dressing, followed, 4 h later, by the irradiation of 75 J cm−2 of broadband red light. Clinical and microbiological recovery was seen in six out of nine patients after one (four cases) or four (two cases) treatments. However, two patients (one with C. albicans and one with T. mentagrophytes) had a persistent remission at a 4 weeks’ follow up visit. Overall tolerability was good in all patients.
Quick relapse after ALA-PDT was also observed in patients with tinea cruris (62) and tinea pedis (63) caused by Trichophyton spp. Eight of 10 tinea cruris patients and six of 10 tinea pedis patients obtained a complete remission after 1–3 ALA-PDT treatment, but four tinea cruris patients and three tinea pedis patients had a persistent healing at the 8-week follow-up after the last treatment.
It was unclear why the apparently good therapeutic effect was followed by quick recurrence in almost all patients. A first possible explanation is that ‘‘in vivo’’ environmental conditions, i.e., temperature, humidity and pH of the interdigital skin, could induce a poor cell uptake of ALA and a deficient biosynthesis of PpIX. In addition the non-uniform delivery of light and/or ALA cream due to the irregular tridimensional shape of this peculiar anatomical area must be taken into account.
Another recent clinical open study conducted by Lee and co-workers (64) investigated the efficacy of methyl 5-amino-levulinic acid (MAL)-PDT in six patients with recalcitrant Malassezia folliculitis caused by Malassezia furfur. MAL cream was applied on the lesions on the trunk under occlusion for 3 h, followed by irradiation with 37 J cm−2 of red light (630 nm). After three sessions of MAL-PDT at 2-week intervals, inflammatory lesions decreased in four patients, slightly improved in one patient; the treatment was not effective in one patient.
Pityriasis versicolor is another skin disease caused by Malassezia yeasts (M. globosa). Kim and Kim (65) treated a patient with the disease affecting the axillae. Hyphal forms of Malassezia are also found in lesions. After twice (2 week apart) ALA-PDT with red light (70–100 J cm−2), a complete clearance was obtained and no fungal hyphae or spores were present 10 weeks after the last treatment.
PDT is also an attractive treatment option for fungal nail infections, given the extremely long duration of conventional treatment with oral and/or topical antifungal drugs, although antifungal PDT for onychomycosis requires repeated PDT sessions. Donnelly et al. developed a bioadhesive patch containing 50 mg cm−2 ALA that allowed achievement of a high ALA concentration on the ventral side of excised human nail (66). ALA penetration across the nail may be improved using penetration enhancers, or by filing the impenetrable dorsal surface of the nail and ALA efficacy may be enhanced by delivering also iron chelators with the patch (67).
Watanabe et al. (68) treated two patients with onychomycosis by applying a commercial 16% ALA methyl esther in cream (Metvix®, Galderma, F) after that the nail underwent a keratolytic treatment with a 20% urea ointment. The nail was then irradiated with 100 J cm−2 of 630 nm pulsed light from an excimer-dye laser. The treatment was repeated 6–7 times at weekly intervals leading mycological cure in treated nail (but not control nails) and no recurrence was observed clinically at a 6-month follow-up visit.
Another successful strategy was proposed by Piraccini et al. that described one patient with toenail onychomycosis caused by T. rubrum was treated with topical ALA-PDT acid after surgical removal of the nail plate and nail bed hyperkeratosis. Three PDT sessions were delivered during a period of 45 days. They were well tolerated and at the 12 and 24 months follow up visits, potassium hydroxide (KOH) examinations and cultures were still negative and the toenails were considered recovered with residual mild traumatic onycholysis (69).
An open trial conducted by Sotirou and colleagues reported quite different results. Thirty patients with distal and lateral subungual toenail onychomycosis were treated with the application of a 20% ALA cream after removal of the nail plate and subungual hyperkeratosis. The treatment was repeated 3 times at 2 weekly intervals. Cure rate of 43.3% and 36.6% was seen 12 and 18 months after treatment, respectively, while clinical and mycological recurrence was seen in other patients (70).
There were several possible explanations for the unsatisfactory outcome in these patients. In vitro studies demonstrated that formation of PpIX was restricted to selected parts of the fungal mycelium, leading to a significant but not higher than 50% growth inhibition of T. rubrum. Thus, according to the investigators, the growth of T. rubrum seems to be only time-delayed by PDT (71). Smijs et al. hypothesize that, although PDT inactivates both hyphae and spores, better fungicidal effect could be achieved by the application of a second treatment within 24–48 h, a time interval in which hyphal tip morphology could not change into more resistant globular structures (49).
Another possible limiting factor could be the adequate ALA uptake under in vivo conditions. Conditions such as temperature, humidity or pH of the biological environment could induce a poor uptake of ALA or a deficient biosynthesis of PpIX (61).
A high number of synthetic phthalocyanines (Pc) are currently available. All these compounds have in common a Pp like chemical structure characterized by the condensation of benzene rings with pyrrole moieties and are characterized by high singlet oxygen quantum yields and high extinction coefficient in the far red (680–720 nm) spectral region (2). However, they differ from each other in chemical properties that may be tuned by modifying the central metal/semi-metal atom and the number and type of side chains. The central metal ligand plays a crucial role in the photobiological activity influencing the triplet yield and the triplet lifetime of the compound and the introduction of polar substituents on the side chains which modify the overall balance of polarity. The hydrophilicity/hydrophobicity balance is a very important chemical property when considering Pc uptake and its localization into micro-organisms. The Pc macrocycle is essentially hydrophobic but hydrophobic compounds were found poorly effective (72). However, also hydrophilic mono- and tetra-sulfonated AlPc were not taken up efficiently and did not inactivate the yeast Kluyveromyces marxianus (72) whereas an appreciable amount of another water-soluble compound, the mono-sulfonated ZnPcS, was tightly bound to intracellular loci and showed a high photosensitizing activity (2) in S. cerevisiae. This discrepancy may be explained by recent findings showing that the presence of cationic charges is necessary for inactivation of C. albicans, but an excess of charges, especially if homogeneously distributed, causes a decrease in activity (73).
Similar results was obtained by Mantareva et al. (74) that demonstrated a higher susceptibility of C. albicans to cationic tetrakis-(3-methylpyridyl-oxy)-phthalocyanine zinc (II) (ZnPcMe) than anionic tetrakis-(4-sulfophenoxy)-phthalocyanines zinc (II) (ZnPcS), although both are water-soluble Pc.
On the basis of these findings, a tetracationic ZnPc bearing four aminoalkylated peripheral substituents proved highly capable of inactivating even strains of C. albicans which are multi-drug resistant (2).
A recent study of Lam and co-workers (75) characterized the antifungal activity of Pc-PDT against C. albicans; Pc-PDT impaired fungal metabolic activity and led to apoptosis substantiated by increased externalization of phosphatidylserine and DNA fragmentation.
Additional advantages of Pc-PDT consist of the lack of selection of PDT resistant strains even after multiple treatments and the much greater photosensitizing activity in C. albicans than in mouse and human keratinocytes and fibroblasts (76). Some authors have investigated the incorporation of Pc in liposomes as an alternative strategy for enhancing cell uptake of yeasts (2). However, results were disappointing because the size of liposomes ranges from 20 to 100 nm (77) and only globular structures with diameter of less than 5.8 nm can pass through the cell membrane of yeasts (78). Mutagenic effects of photodynamic treatment with ClAl-Pc and RPL068 were not found in K. marxianus (79) and C. albicans (76).
Mantareva et al. (80) investigated the photodynamic inactivation of planktonic and biofilm cultures of C. albicans by two new cationic water soluble tetra and octa-methylpyridyloxyl-substituted phthalocyanine Ga (III) complexes (GaPc1 and GaPc2). GaPc1 showed lower cellular uptake compared to the GaPc2. However photodynamic treatment with 3.0 μM GPc1 at mild light conditions (50 J cm−2, 60 mW cm−2) resulted in a high photoinactivation of planktonic phase; GaPc2 showed a complete inactivation of fungal biofilms at a higher concentration (6.0 μM).
The same authors (81) also demonstrated that planktonic cultures of C. albicans were completely inactivated by 1.8 μM of tetra-methylpyridyloxy-substituted Si (IV)-phthalocyanines (SiPc1) and red LED irradiation (50 J cm−2, 635 nm), but the inactivation of yeast biofilm needed higher concentration and fractionated light irradiation.
Mantareva’s group more recently investigated the antifungal activity of several new Pc complexes and they concluded that the ZnPcMe is more potent as antifungal PS for biofilm. This statement suggests that differing in molecular electronic structure of Pc due to replacement of Zn (II) with other elements tend to change sufficiently some photophysical and uptake properties (80).
Other new photosensitizers
Dovigo et al. (82) performed in vitro studies about susceptibility of Candida species to photodynamic effects of curcumin (CUR), a natural yellow pigment isolated from rhizomas of Curcuma longa, used as a cooking spice and colorant. CUR displays a high light absorption in the visible spectral region, around 400–500 nm. The authors found that low CUR concentration (40 μM) was an effective PS for inactivation of C. albicans, C. tropicalis and C. glabrata, both in planktonic and biofilm forms, exposed to blue LED (440–460 nm). The authors recommended longer application times of PS to obtain PDT response when biofilms are involved. They also showed that the therapy was more effective in inactivating yeast cells than a macrophage cell line, suggesting certain specificity of CUR-PDT.
The CUR-phototoxicity to microbial system seem to be mediated through the excited states of CUR, their subsequent reactions with oxygen and formation of reactive species (83).
Gasparetto et al. (84) investigated the photodynamic effect of hexane and ethanol extracts of Alternanthera maritima against C. dubliniensis. A. maritima is an herbaceous plant, commonly found on the Brazilian east coast. The photodynamic antifungal activity of A. maritima is not clear but the presence of flavonoids, saponins, steroids and triterpenes was identified in the analyzed extracts and they may be acting in a synergistic way. The authors demonstrated the inhibition of viability of C. dubliniensis incubated with A. maritima and irradiated with a 685 nm diode laser (28 J cm−2).
Strategies against Fungal Resistance and Biofilm
By modifying the treatment conditions such as the incubation time, surrounding solvents, drug dose, fluence rate and light energy, the photoinactivation of the resistant species can be enhanced.
Bliss et al. (85) observed that the uptake of Photofrin® by Candida was poor when blastoconidia growth in nutrient broth but was increased when cultures were grown in a chemically defined medium that stimulated germ tube production, demonstrating the influence of the biological medium and the morphological form of C. albicans.
Prates et al. (86) demonstrated that both the influx and efflux of PS (MB) may be regulated by multidrug efflux system present in C. albicans and may decrease the effectiveness of PDT. The authors reported that antifungal resistance can be reversed by blocking this system before PS incubation using verapamil, an angiotensin-converting-enzyme-inhibitor drug, that works as inhibitor of multidrug efflux system.
Giroldo et al. (14) demonstrated that PDT with MB increases membrane permeability in C. albicans which could decrease the resistance of this yeast to further treatments with other drugs.
Oriel and Nitzan (87) suggested that the use of more hydrophobic ALA derivatives could promote greater uptake through the membranes of C. albicans.
Garcez et al. (88) investigated the combination of MB and hydrogen peroxide (H2O2) to improve PDT efficiency. The exposure of C. albicans to H2O2 before or simultaneously with PDT leads to a higher uptake of MB inside the yeast resulting in an improved ROS production close to vital structures, leading to a consistent reduction in C. albicans viability.
Fuchs et al. (89) demonstrated the susceptibility of Cryptococcus neoformans to PDT with the photosensitizer chlorine (e6) conjugate of polyethyleneimine (PEI-ce6) and irradiation with red light. The authors showed that a C. neoformans rom2 mutant was more sensitive to the treatment than wild-type C. neoformans, because of cell wall defects found in the mutant, associated with increased accumulation and cell permeation by PS, leading to increased photoinactivation. An increase in photoinactivation was also achieved by exposing wild-type C. neoformans to caspofungin as a means to weaken the cell wall and promote increase penetration of PS.
Chabrier-Rosellòet al. (90) demonstrated that selective inhibition of electron transport chain function increases intracellular levels of ROS in yeast leading to a significantly increases the sensitivity of C. albicans, C. glabrata and S. cerevisiae to PDT performed with the cationic porphyrin meso-tetra (N-methyl-4-pyridyl)porphine tetra tosylate (TMP-1363).
Several authors reported techniques to improve the drug penetration into biofilms. Coleman et al. (91) characterized the antifungal activity of members of the saponins family, which are natural products composed of sugar moieties connected to a hydrophobic aglycone backbone and are an integral part of the plant’s defense antimicrobial mechanism. Saponins are able to form pores in lipid bilayers and are known to increase cellular permeability allowing uptake of molecules that would otherwise be excluded. The authors demonstrated that they can impede C. albicans biofilm formation and dramatically potentiate in vitro photodynamic inactivation when the saponins are coupled with PSs (RB and chlorine [e6]) incubated for 30 min with the yeasts and then irradiated with visible light.
Other studies reported that biofilm may be inactivated by using a variety of PS and light sources, but the best results against C. albicans biofilms was obtained by Zn (II) and Ga (III) phtalocyanines, such as ZnPcMe and GaPc2 (80).
[ Piergiacomo Calzavara-Pinton ]
Piergiacomo Calzavara-Pinton is Professor of Dermatology and Chairman of the Department of Dermatology and the Postgraduate School of Dermatology at the University and Spedali Civili of Brescia, Italy, where he has lectured in the Postgraduate Schools of Paediatrics, Microbiology, Clinical Immunology, Urology and Dentistry and the Faculty of Medicine. He has also taught at the School of Laser Therapy in Milan. Professor Calzavara-Pinton is a member of the Scientific Committee of the Italian Society of Dermatology (SIDEMAST) and Italian Society for Photobiology (SIFB), as well as many other scientific committees and societies. Professor Calzavara-Pinton’s research interests centre on phototherapy, photochemotherapy, photodermatology, connective tissue diseases and non-invasive diagnostic techniques. He has published nearly 170 peer-reviewed papers and numerous book chapters and meeting abstracts.
[ MariaTeresa Rossi ]
MariaTeresa Rossi studied medicine in the University of Brescia, where she graduated in 2006. Then she was appointed to the Phototherapy and Photobiology Unit in Spedali Civili Hospital, Brescia, where she focused her studies on photodynamic therapy, in particular on registered and off-label indications. She is also interested in idiopathic photodermatoses and genophotodermatoses. She has worked in the production of the Italian register for Psoriasis (PSOCARE). In 2012 she received a research grant in Dermatology, in the field of research of anti-TNF alpha therapies, and she’s now working on it.
[ Raffaella Sala ]
Raffaella Sala studied medicine at the University of Brescia, Italy, from which she graduated in 2002. She was appointed at the Department of Dermatology of Spedali Civili Hospital. Working in the Phototherapy and Photobiology Unit, she focused her studies on photodynamic therapy, photodermatoses and genophotodermatoses and on non-ivasive diagnostic technique, especially reflectance confocal microscopy. In 2007 she completed a Residency in Dermatology in the Department of Dermatology at the University of Brescia. She has published more than 40 full papers and book chapters concerning phototherapy.
[ Marina Venturini ]
Marina Venturini graduated in Medicine from the University of Brescia in 2001 and completed a Residency in Dermatology in 2006 at the Department of Dermatology, Spedali Civili Hospital of Brescia. Since 2002 she has worked in the Phototherapy and Photobiology Unit in Spedali Civili Hospital of Brescia and her research is mainly focused in the field of photodermatology, photodiagnosis and phototerapy, with special focus on photodynamic therapy. She also interested in heritable connective tissue disorders and non-invasive diagnostic techniques, especially reflectance confocal microscopy. She is author of numerous scientific articles and book chapters concerning phototherapy. Presently she is Assistant Professor at the Department of Dermatology at the University and Spedali Civili of Brescia.