Silicon Phthalocyanine 4 Phototoxicity in Trichophyton rubrum

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Minh Lam, Matthew L. Dimaano, Patricia Oyetakin-White, Mauricio A. Retuerto, Jyotsna Chandra, Pranab K. Mukherjee, Mahmoud A. Ghannoum, Kevin D. Cooper and Elma D. Baron Antimicrob. Agents Chemother. 2014, 58(6):3029. DOI: 10.1128/AAC.01448-13. Published Ahead of Print 10 March 2014.

Silicon Phthalocyanine 4 Phototoxicity in Trichophyton rubrum Minh Lam,a,b Matthew L. Dimaano,a Patricia Oyetakin-White,a Mauricio A. Retuerto,c Jyotsna Chandra,c Pranab K. Mukherjee,c Mahmoud A. Ghannoum,c Kevin D. Cooper,a,b,d Elma D. Barona,b,d Department of Dermatology,a Case Skin Diseases Research Center,b and Center for Medical Mycology,c Case Western Reserve University/University Hospitals Case Medical Center, Cleveland, Ohio, USA; Louis-Stokes VA Medical Center, Cleveland, Ohio, USAd

U

tilizing mainly keratin for their source of energy, dermatophytes can cause dermatophytosis or tinea of the skin, hair, and nails that impact the quality of life of infected patients, especially in immunocompromised individuals. Trichophyton rubrum is the main culprit of superficial mycosis of the nail (onychomycosis), which is often long lasting and has a high incidence of recurrence. Onychomycosis affects 10% of the general population, 20% of the population older than 60 years, 50% of people older than 70 years, and 30% of diabetic patients, and it can often result in pain, disability, and psychosocial stress, therefore significantly reducing quality of life (1–3). The conventional treatments involve excruciating surgical nail avulsion and toxic systemic antifungal drugs. Topical therapy is a less invasive and hence more attractive treatment for the eradication of fungal infection. However, dismal patient compliance often contributes to the high rate of unsuccessful treatment (4). It is for these reasons that alternative therapeutic agents need to be developed that can effectively target T. rubrum without significantly harming the host. In vitro studies have demonstrated that yeast-like fungi, such as Candida albicans and T. rubrum, are highly susceptible to photodynamic therapy (PDT) using Photofrin (porfimer sodium) (4), the protoporphyrin IX (PpIX) precursor 5-aminolevulinic acid (ALA), or other photosensitizers that are not yet approved by the FDA (5–9). However, before PDT can become a mainstream therapeutic strategy or be used in conjunction with an FDA-approved antifungal agent to combat dermatophytes, a comprehensive understanding of the cellular and molecular PDT pathways is necessary. Allylamines, such as terbinafine and naftifine, are a relatively new class of ergosterol biosynthetic inhibitors. As an essential component of fungal membranes, ergosterol is also known to be involved in the modulation of membrane fluidity and permeabil-

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ity (10). Specifically, terbinafine acts by inhibiting the early steps of ergosterol biosynthesis, which leads to the accumulation of squalene, the sterol precursor, and the absence of other sterol intermediates (11). In other words, terbinafine inhibition of sterol synthesis possibly occurs during the epoxidation of squalene, which is catalyzed by the enzyme squalene epoxidase (12). This enzyme is reportedly encoded by ERG1 (13), and mutations in this particular gene have been demonstrated to confer resistance to terbinafine in fungi (12, 14–16). Conversely, PDT with the combination of a photosensitizer, light, and molecular oxygen in producing cytotoxic reactive oxygen species (ROS) is generally known to target intracellular organelles, such as mitochondria, lysosomes, and endoplasmic reticulum (ER) (17, 18). In essence, upon absorption of a photon generated by light of an appropriate wavelength, the photosensitizer would undergo at least one energy transition to go into an excited triplet state. It then can either (i) react with cellular molecules to produce free radical intermediates which subsequently form ROS within the presence of molecular oxygen (photochemical type I) or (ii) directly transfer energy to ground state molecular oxygen (photochemical type II) to generate singlet oxygen, which can cause irrevocable damage to lipids, proteins, and other biological molecules that are vital for the

Received 3 August 2013 Returned for modification 8 December 2013 Accepted 5 March 2014 Published ahead of print 10 March 2014 Address correspondence to Elma D. Baron, [email protected]. Copyright © 2014, American Society for Microbiology. All Rights Reserved. doi:10.1128/AAC.01448-13

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Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of terbinafine for the skin and ciclopirox for the nails, whereas systemic agents, such as oral terbinafine and itraconazole, are also prescribed. These systemic drugs have severe side effects, including liver toxicity. Topical therapies, however, are sometimes ineffective. This led us to investigate alternative treatment options, such as photodynamic therapy (PDT). Although PDT is traditionally recognized as a therapeutic option for treating a wide range of medical conditions, including agerelated macular degeneration and malignant cancers, its antimicrobial properties have also received considerable attention. However, the mechanism(s) underlying the susceptibility of dermatophytic fungi to PDT is relatively unknown. As a noninvasive treatment, PDT uses a photosensitizing drug and light, which, in the presence of oxygen, results in cellular destruction. In this study, we investigated the mechanism of cytotoxicity of PDT in vitro using the silicon phthalocyanine (Pc) 4 [SiPc(OSi(CH3)2(CH2)3N(CH3)2)(OH)] in T. rubrum. Confocal microscopy revealed that Pc 4 binds to cytoplasmic organelles, and upon irradiation, reactive oxygen species (ROS) are generated. The impairment of fungal metabolic activities as measured by an XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay indicated that 1.0 ␮M Pc 4 followed by 670 to 675 nm light at 2.0 J/cm2 reduced the overall cell survival rate, which was substantiated by a dry weight assay. In addition, we found that this therapeutic approach is effective against terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) strains. These data suggest that Pc 4-PDT may have utility as a treatment for dermatophytosis.

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MATERIALS AND METHODS

cells (19, 20). Most studied photosensitizers for PDT are known to produce type I and II photochemistry (21), and for most microbial systems, both type I and II photochemical reactions are typically produced by photosensitizers following irradiation and can result in cell death due to the oxidative damage caused by the formation of ROS (22). PDT sensitized by silicon phthalocyanine (Pc) 4, a secondgeneration photosensitizer (Fig. 1) developed at Case Western Reserve University, induces biological responses (17). Pc 4-PDT may be effective in targeting T cell-mediated malignant and nonmalignant dermatological diseases, such as cutaneous T cell lymphoma and psoriasis, respectively, as multiple clinical trials have been undertaken here at the University Hospitals Case Medical Center. Unlike current FDA-approved PDT agents (e.g., 5-aminolevulinic acid or ALA, which is principally a metabolic precursor of the protoporphyrin IX [PpIX] photosensitizer), Pc 4 is a chemically pure photosensitizer which requires no bioconversion. Pharmacokinetic data in swine (M. Lam, K. D. Cooper, and E. D. Baron, unpublished data) and mice indicate that Pc 4 clears much more rapidly than other photosensitizers, such as Photofrin, when delivered systemically, thereby minimizing the possibility of prolonged generalized photosensitivity. In addition, with the absorbance maximum occurring at a wavelength of 672 nm (Fig. 1), Pc 4 is ideal for tissue penetration with minimal cutaneous photosensitivity (23, 24). Pc 4-PDT also demonstrated an excellent safety profile in a phase 1 trial on cutaneous neoplasms (25). Although the susceptibility of T. rubrum to PDT using other photosensitizers has been extensively investigated in vitro, the mechanism of phototoxicity has not been clearly defined (2, 9, 26–28). In this study, we showed that a common dose of Pc 4-PDT can induce cell death in microconidia (terbinafine-sensitive and -resistant strains of T. rubrum). Our data also showed specific intracellular localizations of the photosensitizer and diminished metabolic activity immediately following Pc 4 irradiation and subsequently cell death, suggesting that the mechanism of cell death in the two strains involves the mitochondrial pathway.

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FIG 1 Structure and absorbance spectrum of Pc 4. A silicon phthalocyanine with a dimethylamino-propylsiloxy ligand that links axially to the silicon in the center, Pc 4 is an effective second-generation chemically pure photosensitizer. In culture medium, the absorbance maximum occurred at 672 nm, a wavelength that may be ideal for tissue penetration and minimal cutaneous photosensitivity.

Fungal isolates and culture conditions. Clinical isolates of T. rubrum obtained from patients with onychomycosis were provided by the Center for Medical Mycology, University Hospitals Case Medical Center (Cleveland, OH). The MIC testing protocol was performed according to the CLSI M38-A2 standard methodology for dermatophyte susceptibility (29). In the current study, T. rubrum 24602, a terbinafine-sensitive strain (MIC, 0.016 ␮g/ml), and T. rubrum MRL666, a terbinafine-resistant strain (MIC, 4.0 ␮g/ml), were used for the in vitro analyses. Each T. rubrum strain was grown separately on potato dextrose agar (PDA) plates with 0.025% dextrose Sabouraud agar (BD Biosciences, Franklin Lakes, NJ) and 1.0% penicillin-streptomycin in a 30°C incubator. Grown colonies seen as visible white cotton-like growth (hyphae) were detected after approximately 1 week to 10 days. Microconidia were harvested by swabbing the colony surface with a sterile cotton tip applicator and filtering through cotton gauze using a 2-ml syringe with 1⫻ phosphate-buffered saline (PBS). The resultant microconidia were serially diluted and counted using a hemocytometer. The stock of T. rubrum microconidia was incubated at 30°C with a complete growth medium consisting of 1⫻ RPMI with L-cysteine and L-glutamine (Hardy Diagnostics, Santa Maria, CA), 3.5% MOPS (morpholinepropanesulfonic acid; Sigma, St. Louis, MO), and 10% fetal bovine serum (FBS) at 40,000 microconidia per ml for all experiments. Pc 4-photodynamic treatment conditions. Pc 4 was kindly provided by Malcolm E. Kenney (Department of Chemistry, Case Western Reserve University, Cleveland, OH). Stock solutions (0.5 mM) of Pc 4 were prepared in a vehicle of N=,N=-dimethylformamide (DMF; Thermo Fisher, Waltham, MA) and stored at 4°C. Unless otherwise indicated, isolated microconidia were incubated with 1.0 ␮M Pc 4 in complete growth medium containing 10% FBS for 2 to 16 h at 30°C in the dark and subsequently irradiated with red light using a light-emitting diode array (EFOS, Mississauga, Ontario, Canada) at a dose of 2.0 J/cm2 (with a fluence rate of 1.0 mW/cm2, ␭max(Ex) of ⬃ 670 to 675 nm) at room temperature. As a negative control, an equal volume of DMF (3.2 mM without Pc 4) was used and followed by irradiation with the same dose. Furthermore, Pc 4 application to microconidia was typically applied overnight prior to irradiation in the cytotoxicity assays described below. It was not uncommon to find hyphae or mycelia in all tested samples due to the complete medium, which might have facilitated the differentiation of these microconidia. Dry weight assay. We examined the effect of Pc 4-PDT by dry weight measurements (30), which represented the total amount of microconidia and hyphal masses of the two T. rubrum strains. Briefly, control and Pc 4-PDT microconidia/hyphae suspensions (see “Pc 4-photodynamic treatment conditions”) were filtered through a preweighed filter (0.45-␮m pore size), washed with 1⫻ PBS, air dried at 35°C for 24 h, and weighed on an analytical balance (model no. A-160; Denver Instrument Company, Bohemia, NY). Metabolic XTT assay. Within 5 h following irradiation of Pc 4-treated microconidia and hyphae, their metabolic activities were assayed using the colorless sodium salt of XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide inner salt) (Sigma-Aldrich, St. Louis, MO), which is converted by mitochondrial dehydrogenases of viable microconidia/hyphae into a water-soluble orange formazan derivative through the reduction of the tetrazolium ring of XTT. The absorbance of the resulting orange solution was measured using a spectrophotometer (Spectronic Genesys 5; Analytical Instruments, Golden Valley, MN) at a wavelength of 492 nm as previously described (30, 31). Note again that some microconidia may have become hyphal during the overnight Pc 4 incubation in complete medium. The XTT assay detected metabolic activities with and without Pc 4-PDT. Confocal microscopy. To investigate the uptake and localization of Pc 4, microconidia in a complete growth medium plus 10% FBS were loaded with 0.5 ␮M Pc 4 and incubated for 16 to 18 h. Thirty minutes prior to imaging Pc 4, 50 nM MitoTracker Red CMXRos (Invitrogen, Carlsbad,

Pc 4-PDT Targets Mitochondria in T. rubrum

to bind to the cell wall and the cytoplasmic compartments of the microconidia. Pc 4 displayed a punctate fluorescence, some of which was reminiscent of a mitochondrial pattern (a and b). To investigate the cellular localization of Pc 4, microconidia and hyphae were also labeled with 50 nM MitoTracker Red CMXRos and visualized by confocal microscopy. Pc 4 displayed a punctate pattern of fluorescence (pseudored) (c) that closely resembled that of MitoTracker (pseudoblue) (d), as indicated by the purple fluorescence in the merged image (e). The white arrows identify regions of clear overlap of Pc 4 and MitoTracker fluorescence. Scale bars, 10 ␮m.

CA) was loaded onto microconidia and hyphae. Five to 10 ␮l of stained microconidia/hyphae were placed on a slide with a glass coverslip, and all images were acquired using an UltraView VoX spinning-disk confocal system (PerkinElmer, Waltham, MA) mounted on a Leica DMI6000B microscope (Leica Microsystems, Inc., Bannockburn, IL) equipped with an HCX PL APO 100⫻/1.4 oil immersion objective. Confocal images of Pc 4 fluorescence and MitoTracker Red were collected using solid-state diode 640-nm and 561-nm lasers, respectively, and with the appropriate emission filters. Appropriate imaging controls were used to ensure that MitoTracker Red and Pc 4 fluorescences were spectrally distinct. The formation of ROS was monitored by the conversion of nonfluorescent 2=,7=-dichlorohydrofluorescein diacetate (H2DCFDA; Invitrogen, Carlsbad, CA) to fluorescent 2=,7=-dichlorofluorescein (DCF) as described previously (32–34). Briefly, Pc 4-treated microconidia and hyphae were loaded with 5 ␮M 2=,7=-dichlorohydrofluorescein diacetate in complete growth medium for 30 min at 30°C. After loading, microconidia and hyphae were washed twice with 1⫻ PBS. Images of DCF fluorescence were collected using a solid-state diode 488-nm laser and 500- to 550-nm bandpass barrier filters. Flow cytometry. To quantitatively measure the formation of ROS, terbinafine-sensitive (strain 24602) and terbinafine-resistant (strain MRL666) microconidia were suspended in complete growth medium and treated with 1 ␮M Pc 4. Following an overnight Pc 4 incubation, microconidia and hyphae were then resuspended in flow buffer, which contained 5% FBS and 2 mM EDTA in 1⫻ PBS, in flow tubes and then stained with 50 ␮M H2DCFDA and incubated on ice for 30 min. From each sample, 100,000 events were collected using an Accuri C6 flow cytometer (BD, Franklin Lakes, NJ) at 0, 5, 15, 25, and 33 min during the light treatment, which yielded 0, 0.3, 0.9, 1.5, and 2.0 J/cm2 doses, respectively.

RESULTS

Pc 4 is localized to mitochondria in T. rubrum. To have a PDT effect, the photosensitizer needs to be sufficiently taken up by the microconidia or hyphae. Utilizing confocal microscopy, the up-

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take and localization of Pc 4 were examined. As shown in Fig. 2b and c, the majority of Pc 4 red fluorescence appeared on the cell wall and in the cytoplasm of the microconidia and hyphae after overnight incubation. Pc 4 displayed a punctate pattern of fluorescence primarily localized in cytoplasmic organelles, resembling mitochondria. To test whether mitochondria are the primary sites where Pc 4 is localized, microconidia and hyphae were costained with MitoTracker Red, a mitochondrion-specific dye. The bright punctate fluorescence shown in the Pc 4 (pseudo-red) image (Fig. 2c) overlaps with the MitoTracker Red (pseudo-blue) fluorescence (Fig. 2d), as indicated in the merged image (Fig. 2e) of this particular hypha. Furthermore, Pc 4 binds to other intracellular organelles, presumably ER membranes, and other vesicular membranes. Similar Pc 4 uptake and localization were also found in a terbinafine-resistant strain (MRL666) of T. rubrum by confocal microscopy (data not shown). Photodynamic therapy inhibits metabolic activity in T. rubrum 24602 and MRL666 strain isolates. Having established that mitochondria are some of the Pc 4 binding sites, we then assessed the ability of Pc 4-PDT to affect the metabolic activities of these two strains. Following Pc 4-PDT, a decrease in absorbance, as demonstrated by a loss of water-soluble orange formazan derivative in the XTT assays, was an indication of metabolic impairment. As shown in Fig. 3a, metabolic activity was reduced by ⬎50% within 4 h compared to controls following irradiation of Pc 4-loaded terbinafine-sensitive (24602) and terbinafine-resistant (MRL666) microconidia or hyphae. Furthermore, in an effort to determine if the impaired metabolic effect can subsequently affect the rate of microconidial growth, irradiated Pc 4 microconidia and hyphae were weighed and compared to their respective light-only controls. Consistently, there was a marked attenuation of growth in the two T. rubrum isolates, based upon the dry weight measure-

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FIG 2 Pc 4 is localized to mitochondria in T. rubrum. Microconidial and hyphal forms of T. rubrum were loaded with 0.5 ␮M Pc 4 overnight. Pc 4 (pseudored) appeared

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isolates of strains 24602 and MRL666. Equal quantities of the two microconidial culture strains were treated overnight with 1.0 ␮M Pc 4 and then were irradiated with 2.0 J/cm2 of 670- to 675-nm light. (a) One hour following irradiation of Pc 4-loaded microconidia and hyphae, cultures were assayed for XTT reduction. (b) Pc 4 treatment was done as described for panel a; however, 3 h following irradiation of Pc 4-loaded microconidia, suspensions, including the control, were filtered through a preweighed filter (0.45-pm pore size), washed with 1⫻ PBS, air dried at 35°C for 24 h, and weighed on an analytical balance. Values represent means ⫾ standard errors of the means (SEM) from at least three independent experiments.

ments in 24 h following irradiation with 670- to 675-nm red light at a dose of 2.0 J/cm2 (Fig. 3b). Pc 4-PDT induces intracellular ROS generation. In this study, we demonstrated the formation of ROS with 2 T. rubrum isolates following Pc 4-PDT. With the dose of Pc 4-PDT used in previous experiments, we monitored intracellular ROS formation, as measured by the conversion of nonfluorescent H2DCFDA to fluorescent DCF. Confocal images showed that DCF fluorescence increased within 15 min after Pc 4-PDT (Fig. 4a, inset). To quantitatively measure the amount of intracellular ROS generation, we performed a dose-response study (Pc 4 at a concentration of 1.0 ␮M and light doses ranging from 0.3, 0.9, and 1.5 to 2.0 J/cm2) by flow cytometry. Our data consistently showed that a higher Pc 4-PDT dose correlated with a greater DCF fluorescence or ROS level in the 24602 and MRL666 strains (Fig. 4a and b). As a positive control, we used H2O2 to demonstrate the enhanced level of ROS in these microconidia and hyphae. Red light alone, without Pc 4, had no effect on DCF fluorescence (data not shown). DISCUSSION

Minimally invasive with limited side effects, PDT is commonly used in treating various medical conditions ranging from acne to

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FIG 4 Pc 4-PDT induces intracellular ROS in isolates of strains 24602 and MRL666. To monitor ROS generation by confocal microscopy and flow cytometry, Pc 4-treated microconidia were loaded with 2=,7=-dichlorofluorescin as described in Materials and Methods. The generation of ROS based upon DCF fluorescence can be quantified by flow cytometry. From each sample, 100,000 events were collected using an Accuri C6 flow cytometer at 0, 5, 15, 25, and 33 min during the light treatment, which yielded doses of 0, 0.3, 0.9, 1.5, and 2.0 J/cm2, respectively. (a) For a positive control, H2O2 was used. In addition, shown are confocal microscopy images collected before (inset, top) and 15 min after (inset, bottom) Pc 4-PDT. Scale bar, 5 ␮m. Values represent the percentages of DCF fluorescence at doses of 0, 0.3, 0.9, 1.5, and 2.0 J/cm2. (b) Results are expressed as means ⫾ SEM from three independent experiments. hv, hv (mJ/cm2).

age-related macular degeneration to malignant cancers (21). PDT has recently attracted considerable attention for its antimicrobial properties (35). In vitro, it has been reported that PDT using various photosensitizers can lead to cytotoxicity in T. rubrum (9, 26–28, 36, 37), a primary pathogen of superficial mycosis of the nail (known as onychomycosis). However, the antimicrobial effects of PDT with the tested photosensitizers and their precise mechanisms of cell death have not been well characterized. The silicon phthalocyanine Pc 4, which has undergone clinical trials for cutaneous T cell lymphoma and psoriasis, has also been shown to have antimicrobial effects in T. rubrum, as demonstrated in this in vitro study. Although the currently FDA-approved PDT agent ALA has demonstrated an antifungal effect in T. rubrum in vitro, its limitation is the need for bioconversion to PpIX (38) prior to irradiation. Unlike ALA, Pc 4 does not require the bioconversion step. In other words, upon treatment, its photocytotoxicity effect occurs as soon as Pc 4 is exposed to 670- to 675-nm light. This wavelength is also more advantageous for cutaneous penetration compared to 410-nm blue light used to activate PpIX (Fig. 1).

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FIG 3 Cytotoxic effects, as measured by XTT and dry weight, of Pc 4-PDT in

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the cell wall. Given the binding of Pc 4 to intracellular organelles, such as the mitochondria, it is not surprising that the T. rubrum isolate that is known to be terbinafine resistant demonstrated susceptibility to Pc 4-PDT (Fig. 3 and 4b). Furthermore, such cytotoxicity was confirmed by dry weight assays (Fig. 3b) and clonogenic assays (data not shown) for the terbinafine-sensitive and -resistant strains in vitro. In summary, data from this study indicate that the mechanism of action of Pc 4-PDT in T. rubrum involves the mitochondrial pathway, disrupting the overall metabolic activity and resulting in cytotoxicity. Based upon these findings, the development of Pc 4-PDT as a potential clinical antifungal therapy, or as an adjunctive therapy with a currently approved antifungal agent, warrants further exploration. ACKNOWLEDGMENTS This study has been supported in part by the National Institutes of Health (grant 5P30AR039750) via the Skin Diseases Research Center (SDRC) and the Ohio Department of Development, Center for Innovative Immunosuppressive Therapeutics (grant TECH 09-023). Funding support was also provided by the NIDCR to M.A.G. (R01DE17846), the Oral HIV AIDS Research Alliance (OHARA; grant no. BRS-ACURE-S-11-000049110229), NIAID/NEI to P.K.M. (NEI/R21EY021303 and NIAID/ R21AI074077), and the Infectious Diseases Drug Development Center (IDDDC, Case) to P.K.M. We thank Yuntao Li for his technical assistance and Scott J. Howell for critically reviewing the manuscript.

REFERENCES 1. Elewski BE. 1997. The effect of toenail onychomycosis on patient quality of life. Int. J. Dermatol. 36:754 –756. http://dx.doi.org/10.1046 /j.1365-4362.1997.00163.x. 2. Gulcan A, Gulcan E, Oksuz S, Sahin I, Kaya D. 2011. Prevalence of toenail onychomycosis in patients with type 2 diabetes mellitus and evaluation of risk factors. J. Am. Podiatr. Med. Assoc. 101:49 –54. 3. Thomas J, Jacobson GA, Narkowicz CK, Peterson GM, Burnet H, Sharpe C. 2010. Toenail onychomycosis: an important global disease burden. J. Clin. Pharm. Ther. 35:497–519. http://dx.doi.org/10.1111/j.1365-2710.2009.01107.x. 4. Kumar S, Kimball AB. 2009. New antifungal therapies for the treatment of onychomycosis. Expert Opin. Invest. Drugs 18:727–734. http://dx.doi .org/10.1517/13543780902810352. 5. Bliss JM, Bigelow CE, Foster TH, Haidaris CG. 2004. Susceptibility of Candida species to photodynamic effects of photofrin. Antimicrob. Agents Chemother. 48:2000 –2006. http://dx.doi.org/10.1128/AAC.48.6.2000-2006 .2004. 6. Chabrier-Rosello Y, Giesselman BR, De Jesus-Andino FJ, Foster TH, Mitra S, Haidaris CG. 2010. Inhibition of electron transport chain assembly and function promotes photodynamic killing of Candida. J. Photochem. Photobiol. B 99:117–125. http://dx.doi.org/10.1016/j.photobiol .2010.03.005. 7. Maisch T. 2009. A new strategy to destroy antibiotic resistant microorganisms: antimicrobial photodynamic treatment. Mini Rev. Med. Chem. 9:974 –983. http://dx.doi.org/10.2174/138955709788681582. 8. Monfrecola G, Procaccini EM, Bevilacqua M, Manco A, Calabro G, Santoianni P. 2004. In vitro effect of 5-aminolaevulinic acid plus visible light on Candida albicans. Photochem. Photobiol. Sci. 3:419 – 422. http: //dx.doi.org/10.1039/b315629j. 9. Smijs TG, Schuitmaker HJ. 2003. Photodynamic inactivation of the dermatophyte Trichophyton rubrum. Photochem. Photobiol. 77:556 –560. http://dx .doi.org/10.1562/0031-8655(2003)077⬍0556:PIOTDT⬎2.0.CO;2. 10. Lupetti A, Danesi R, Campa M, Del Tacca M, Kelly S. 2002. Molecular basis of resistance to azole antifungals. Trends Mol. Med. 8:76 – 81. http: //dx.doi.org/10.1016/S1471-4914(02)02280-3. 11. Roberts CW, McLeod R, Rice DW, Ginger M, Chance ML, Goad LJ. 2003. Fatty acid and sterol metabolism: potential antimicrobial targets in apicomplexan and trypanosomatid parasitic protozoa. Mol. Biochem. Parasitol. 126:129–142. http://dx.doi.org/10.1016/S0166-6851(02)00280-3.

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Other photosensitizing drugs, including Photofrin, TMP-1363, cationic porphyrin 5-phenyl-10,15,20-Tris(N-methyl-4-pyridyl)porphyrin chloride, and chlorin e6, have been shown to target different cellular domains, including ER, lysosomes, plasma membrane, or mitochondria; irradiation of these photosensitizers has been shown to generate oxidative damage that leads to cytotoxicity (18–20). Our in vitro findings demonstrated that PDT sensitized by Pc 4 can induce cell death via a pathway which involves the mitochondria in T. rubrum. Our confocal images showed that Pc 4 is taken up by microconidia as early as 2 h after treatment (data not shown). Figure 2 illustrates the uptake of Pc 4 following an overnight incubation. Due to the length of time that the microconidia had been resuspended in a complete medium during the Pc 4 treatment, hyphae began to form. Figure 1b depicts that Pc 4 uptake was detected in microconidia and hyphae. In addition, the Pc 4 fluorescence appears to be site specific, which is consistent with previous studies which showed that Pc 4 preferentially binds to intracellular organelles once the cellular uptake has occurred (31, 39). To examine whether the mitochondrion is one of the organelles to which Pc 4 binds, we coloaded the Pc 4-treated microconidia and hyphae with a commercially available mitochondrion-specific probe. The punctate fluorescence shown in the Pc 4 image (Fig. 2c) corresponded to the mitochondrial probe (pseudoblue), indicating the mitochondrial localization of Pc 4 (Fig. 2e). However, Pc 4 fluorescence was also presumably found on other membrane-bound organelles, such as Golgi complexes, ER, and vacuoles, due to the fact that its fluorescence did not exclusively overlap the mitochondrial dye (Fig. 2d). Interestingly, Pc 4 fluorescence was also detected on the plasma membrane/cell wall, especially in the microconidia. Consistently, XTT assays showed significant impairment of fungal metabolic activities in both strains of T. rubrum with 1.0 ␮M Pc 4 followed by 670- to 675-nm light at 2.0 J/cm2. The metabolic activity was reduced by almost 50% within an hour of PDT in the terbinafine-sensitive and -resistant strains (Fig. 3a). Furthermore, with the same dose, a dry weight assay indicated that it reduced cell survival by almost 40% (Fig. 3b), suggesting that the Pc 4-PDT effect on the metabolic activity plays a role in the reduced growth rate. Using confocal microscopy, we showed a rise of intracellular ROS (Fig. 4a, inset) immediately following the irradiation of Pc 4. These early PDT events are not uncommon in vitro using photosensitizers reported previously for most biological systems (20, 21, 40–42). Light activation of most photosensitizers, such as Pc 4, is known to produce singlet oxygen and other ROS (43, 44). Due to its highly reactive nature and extremely short lifetime (⬍50 ␮s), singlet oxygen is capable of damaging proteins and lipids in close proximity to the binding site(s) of the photosensitizer (45). In this study, we quantitatively demonstrated that Pc 4-PDT generated an intracellular rise of ROS in a dose-response fashion from the conversion of nonfluorescent H2DCFDA to the highly fluorescent DCF by flow cytometry (Fig. 4b). One can infer that some of the ROS generation occurred within the mitochondria, given the affinity of Pc 4 to target mitochondria (Fig. 2e). DCF reacts with hydroperoxides (46), not singlet oxygen, indicating that the formation of ROS other than singlet oxygen takes places during Pc 4-PDT. Nevertheless, sodium azide, a specific singlet oxygen scavenger, has been shown to block the DCF fluorescence, suggesting that singlet oxygen was the initial ROS generated after Pc 4-PDT (33). Most antifungal agents, like terbinafine, are designed to target

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29. Ghannoum MA, Chaturvedi V, Espinel-Ingroff A, Pfaller MA, Rinaldi MG, Lee-Yang W, Warnock DW. 2004. Intra- and interlaboratory study of a method for testing the antifungal susceptibilities of dermatophytes. J. Clin. Microbiol. 42:2977–2979. http://dx.doi.org/10.1128/JCM.42.7.2977 -2979.2004. 30. Chandra J, Mukherjee PK, Ghannoum MA. 2008. In vitro growth and analysis of Candida biofilms. Nat. Protoc. 3:1909 –1924. http://dx.doi.org /10.1038/nprot.2008.192. 31. Lam M, Jou PC, Lattif AA, Lee Y, Malbasa CL, Mukherjee PK, Oleinick NL, Ghannoum MA, Cooper KD, Baron ED. 2011. Photodynamic therapy with Pc 4 induces apoptosis of Candida albicans. Photochem. Photobiol. 87:904 – 909. http://dx.doi.org/10.1111/j.1751-1097.2011.00938.x. 32. Dawson TL, Gores GJ, Nieminen AL, Herman B, Lemasters JJ. 1993. Mitochondria as a source of reactive oxygen species during reductive stress in rat hepatocytes. Am. J. Physiol. 264:C961–C967. 33. Lam M, Oleinick NL, Nieminen AL. 2001. Photodynamic therapyinduced apoptosis in epidermoid carcinoma cells. Reactive oxygen species and mitochondrial inner membrane permeabilization. J. Biol. Chem. 276: 47379 – 47386. http://dx.doi.org/10.1074/jbc.M107678200. 34. Nieminen AL, Byrne AM, Herman B, Lemasters JJ. 1997. Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species. Am. J. Physiol. 272:C1286 –C1294. 35. Hamblin MR, Hasan T. 2004. Photodynamic therapy: a new antimicrobial approach to infectious disease? Photochem. Photobiol. Sci. 3:436 – 450. http://dx.doi.org/10.1039/b311900a. 36. Amorim JC, Soares BM, Alves OA, Ferreira MV, Sousa GR, Silveira Lde B, Piancastelli AC, Pinotti M. 2012. Phototoxic action of light emitting diode in the in vitro viability of Trichophyton rubrum. An. Bras. Dermatol. 87:250 –255. http://dx.doi.org/10.1590/S0365-05962012000200009. 37. Piraccini BM, Rech G, Tosti A. 2008. Photodynamic therapy of onychomycosis caused by Trichophyton rubrum. J. Am. Acad. Dermatol 59:S75– S76. http://dx.doi.org/10.1016/j.jaad.2008.06.015. 38. Kamp H, Tietz HJ, Lutz M, Piazena H, Sowyrda P, Lademann J, Blume-Peytavi U. 2005. Antifungal effect of 5-aminolevulinic acid PDT in Trichophyton rubrum. Mycoses 48:101–107. http://dx.doi.org/10.1111 /j.1439-0507.2004.01070.x. 39. Trivedi NS, Wang HW, Nieminen AL, Oleinick NL, Izatt JA. 2000. Quantitative analysis of Pc 4 localization in mouse lymphoma (LY-R) cells via double-label confocalfluorescencemicroscopy.Photochem.Photobiol.71:634–639.http://dx .doi.org/10.1562/0031-8655(2000)071⬍0634:QAOPLI⬎2.0.CO;2. 40. Dougherty TJ. 2002. An update on photodynamic therapy applications. J. Clin. Laser Med. Surg. 20:3–7. http://dx.doi.org/10.1089/104454702753474931. 41. Gomer CJ, Ferrario A, Hayashi N, Rucker N, Szirth BC, Murphree AL. 1988. Molecular, cellular, and tissue responses following photodynamic therapy. Lasers Surg. Med. 8:450–463. http://dx.doi.org/10.1002/lsm.1900080503. 42. Varnes ME, Chiu SM, Xue LY, Oleinick NL. 1999. Photodynamic therapy-induced apoptosis in lymphoma cells: translocation of cytochrome c causes inhibition of respiration as well as caspase activation. Biochem. Biophys. Res. Commun. 255:673– 679. http://dx.doi.org/10 .1006/bbrc.1999.0261. 43. He J, Larkin HE, Li YS, Rihter D, Zaidi SI, Rodgers MA, Mukhtar H, Kenney ME, Oleinick NL. 1997. The synthesis, photophysical and photobiological properties and in vitro structure-activity relationships of a set of silicon phthalocyanine PDT photosensitizers. Photochem. Photobiol. 65:581–586. http://dx.doi.org/10.1111/j.1751-1097.1997.tb08609.x. 44. Moan J, Berg K. 1992. Photochemotherapy of cancer: experimental research. Photochem. Photobiol. 55:931–948. http://dx.doi.org/10.1111/j .1751-1097.1992.tb08541.x. 45. Moan J, Berg K. 1991. The photodegradation of porphyrins in cells can be used to estimate the lifetime of singlet oxygen. Photochem. Photobiol. 53:549 –553. http://dx.doi.org/10.1111/j.1751-1097.1991.tb03669.x. 46. Taguchi H, Ogura Y, Takanashi T, Hashizoe M, Honda Y. 1996. In vivo quantitation of peroxides in the vitreous humor by fluorophotometry. Invest. Ophthalmol. Vis. Sci. 37:1444 –1450.

Antimicrobial Agents and Chemotherapy

Downloaded from http://aac.asm.org/ on June 3, 2014 by KAROLINSKA INST BIBL

12. Ryder NS. 1992. Terbinafine: mode of action and properties of the squalene epoxidase inhibition. Br. J. Dermatol 126(Suppl):2–7. 13. Leber R, Fuchsbichler S, Klobucnikova V, Schweighofer N, Pitters E, Wohlfarter K, Lederer M, Landl K, Ruckenstuhl C, Hapala I, Turnowsky F. 2003. Molecular mechanism of terbinafine resistance in Saccharomyces cerevisiae. Antimicrob. Agents Chemother. 47:3890 –3900. http: //dx.doi.org/10.1128/AAC.47.12.3890-3900.2003. 14. Leyden J. 1998. Pharmacokinetics and pharmacology of terbinafine and itraconazole. J. Am. Acad. Dermatol. 38:S42–S47. http://dx.doi.org/10 .1016/S0190-9622(98)70483-9. 15. Mukherjee PK, Leidich SD, Isham N, Leitner I, Ryder NS, Ghannoum MA. 2003. Clinical Trichophyton rubrum strain exhibiting primary resistance to terbinafine. Antimicrob. Agents Chemother. 47:82– 86. http://dx .doi.org/10.1128/AAC.47.1.82-86.2003. 16. Osborne CS, Leitner I, Hofbauer B, Fielding CA, Favre B, Ryder NS. 2006. Biological, biochemical, and molecular characterization of a new clinical Trichophyton rubrum isolate resistant to terbinafine. Antimicrob. Agents Chemother. 50:2234 –2236. http://dx.doi.org/10.1128/AAC.01600-05. 17. Oleinick NL, Evans HH. 1998. The photobiology of photodynamic therapy: cellular targets and mechanisms. Radiat. Res. 150:S146 –S156. http: //dx.doi.org/10.2307/3579816. 18. Plaetzer K, Kiesslich T, Oberdanner CB, Krammer B. 2005. Apoptosis following photodynamic tumor therapy: induction, mechanisms and detection. Curr. Pharm. Des. 11:1151–1165. http://dx.doi.org/10.2174/1381612053507648. 19. Plaetzer K, Krammer B, Berlanda J, Berr F, Kiesslich T. 2009. Photophysics and photochemistry of photodynamic therapy: fundamental aspects. Lasers Med. Sci. 24:259 –268. http://dx.doi.org/10.1007/s10103-008 -0539-1. 20. Robertson CA, Evans DH, Abrahamse H. 2009. Photodynamic therapy (PDT): a short review on cellular mechanisms and cancer research applications for PDT. J. Photochem. Photobiol. B 96:1– 8. http://dx.doi.org/10 .1016/j.jphotobiol.2009.04.001. 21. De Rosa FS, Bentley MV. 2000. Photodynamic therapy of skin cancers: sensitizers, clinical studies and future directives. Pharm. Res. 17:1447– 1455. http://dx.doi.org/10.1023/A:1007612905378. 22. Smijs TG, Pavel S. 2011. The susceptibility of dermatophytes to photodynamic treatment with special focus on Trichophyton rubrum. Photochem. Photobiol. 87:2–13. http://dx.doi.org/10.1111/j.1751-1097.2010 .00848.x. 23. Egorin MJ, Zuhowski EG, Sentz DL, Dobson JM, Callery PS, Eiseman JL. 1999. Plasma pharmacokinetics and tissue distribution in CD2F1 mice of Pc4 (NSC 676418), a silicone phthalocyanine photodynamic sensitizing agent. Cancer Chemother. Pharmacol. 44:283–294. http://dx.doi.org/10 .1007/s002800050979. 24. Oleinick NL, Antunez AR, Clay ME, Rihter BD, Kenney ME. 1993. New phthalocyanine photosensitizers for photodynamic therapy. Photochem. Photobiol. 57:242–247. http://dx.doi.org/10.1111/j.1751-1097.1993.tb02282.x. 25. Baron ED, Malbasa CL, Santo-Domingo D, Fu P, Miller JD, Hanneman KK, Hsia AH, Oleinick NL, Colussi VC, Cooper KD. 2010. Silicon phthalocyanine (Pc 4) photodynamic therapy is a safe modality for cutaneous neoplasms: results of a phase 1 clinical trial. Lasers Surg. Med. 42:728 –735. http://dx.doi.org/10.1002/lsm.20984. 26. Baltazar Lde M, Soares BM, Carneiro HC, Avila TV, Gouveia LF, Souza DG, Ferreira MV, Pinotti M, Santos Dde A, Cisalpino PS. 2013. Photodynamic inhibition of Trichophyton rubrum: in vitro activity and the role of oxidative and nitrosative bursts in fungal death. J. Antimicrob. Chemother. 68:354 –361. http://dx.doi.org/10.1093/jac/dks414. 27. Smijs TG, Bouwstra JA, Talebi M, Pavel S. 2007. Investigation of conditions involved in the susceptibility of the dermatophyte Trichophyton rubrum to photodynamic treatment. J. Antimicrob. Chemother. 60:750 – 759. http://dx.doi.org/10.1093/jac/dkm304. 28. Smijs TG, van der Haas RN, Lugtenburg J, Liu Y, de Jong RL, Schuitmaker HJ. 2004. Photodynamic treatment of the dermatophyte Trichophyton rubrum and its microconidia with porphyrin photosensitizers. Photochem. Photobiol. 80:197–202. http://dx.doi.org/10.1562/2004-04 -22-RA-146.1.

Silicon phthalocyanine 4 phototoxicity in Trichophyton rubrum.

Trichophyton rubrum is the leading pathogen that causes long-lasting skin and nail dermatophyte infections. Currently, topical treatment consists of t...
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