In vitro effects of photodynamic therapy on Aspergillus fumigatus

Joseph S. Friedberga,*, Cynthia Skemaa, Eric D. Bauma, Jeffrey Burdicka, Sergei A. Vinogradovb, David F. Wilsonb, Annamarie D. Horanc and Irving Nachamkind

a Departments of Surgery, b Biochemistry and Biophysics, c Radiation Oncology and d Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Photodynamic therapy is a technique for killing cells with visible light after pretreatment with a photosensitizing agent. We demonstrated significant in vitro fungicidal activity against Aspergillus fumigatus of the photosensitizer Green 2W, activated with 630 nm light. This effect was both inoculum- and light dose-dependent. At a Green 2W concentration of 31.5 mg/L, there was complete killing of 2.7 x 101 cfu/mL with a light dose of 110 J/cm2 and up to 2.7 x 106 cfu/mL with a light dose of 385 J/cm2.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Photodynamic therapy (PDT) is a technique for killing cells with visible light after pretreatment with photoactive compounds known as photosensitizers. Photosensitizers are generally macrocyclic structures, like porphyrins, which exhibit little or no toxicity on their own. Excitation of the photosensitizer by light of the appropriate wavelength results in the production of chemically active cytotoxic species of oxygen.1,2

The majority of work with PDT has been directed at neoplasms, with much less known about the effect of PDT on fungi. The majority of work that has been done on fungi has been performed on non-virulent organisms.2,3 Localized Aspergillus fumigatus infections in the lung, sinuses and brain are difficult to treat, frequently fatal and increasing in frequency.4 Demand for new treatments for this pathogenic organism is underscored by the emergence of drug-resistant strains.4 This report details the effects of PDT on A. fumigatus in an in vitro system.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Photosensitizer

Green 2W [Pd Ph4TBP(SO3Na)n, n = 4–8] (see FigureGo) was synthesized as described previously.5 A variant of Green 2W that lacked the palladium moiety was also tested.



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Figure. The chemical structure and absorption spectrum of the photosensitizing drug Green 2W [Pd Ph4TBP(SO3Na)n, n = 4–8] palladium tetraphenyltetrabenzoporphyrin sulphate, a sodium salt.

 
PDT

Light treatments and dosimetry measurements were performed in a custom-designed apparatus that allowed simultaneous, and equal, illumination of thirty 12 x 75 mm polystyrene tubes (Falcon, Franklin Lakes, NJ, USA). Five Watts of 630 nm light (Laserscope, San Jose, CA, USA) was delivered through a 3 cm cylindrical diffusing fibre (600 micron; Cardiofocus, West Yarmouth, MA, USA). Light dosimetry measurements were performed with a calibrated isotropic detector (Cardiofocus) placed inside a tube filled with fungal growth medium (RPMI 1640). Light dosage ranged from 0 to 440 J/cm2, delivered in 55 J/cm2 fractions (630 nm light at a fluence of 60 mW/cm2). Zero (no dose) to eight (maximum dose) fractions were delivered at 12 h intervals.

Organism

An isolate of A. fumigatus was obtained from a respiratory sample of a patient seen at the Hospital of the University of Pennsylvania. The fungus was identified according to conventional methods and was maintained in RPMI 1640 medium (pH 7.0).6 Culture tubes were incubated in the dark at 35°C in a 5% CO2 atmosphere, loose-capped and taped for gas exchange. Tubes were removed from these culture conditions during light treatments, which were performed at room temperature, and were then returned during the 12 h intervals between light dose fractions.

Experimental conditions

Four groups were tested in each experiment: (i) no light/ no drug; (ii) +light/no drug; (iii) no light/+drug; and (iv) +light/+drug. The concentration of Green 2W in the +drug experiments was 31.5 mg/L. Green 2W without palladium was also tested at the same concentration.

The density of fungal cultures was measured spectrophotometrically and adjusted to 80% transmittance at 530 nm.7 A stock culture of 2.7 x 107 cfu/mL was prepared for each experimental condition. Serial dilutions were performed to give cultures with initial inoculum concentrations ranging from 2.7 x 101 to 2.7 x 107 cfu/mL. Control samples from each dilution were grown to assure inoculation and organism viability. Experimental tubes were incubated under growth conditions for 3 h before the initial light treatment.

Upon completion of the final light treatments, all tubes were returned to the culture conditions for 72 h, after which time they were scored for ‘growth’ versus ‘no growth’ by visual inspection. By 72 h all tubes demonstrated either dense confluent growth or remained completely clear. Tubes that showed no-growth after 72 h were further tested as described below.

Fungicidal versus fungistatic activity of Green 2W

To determine whether the treatments were fungicidal or fungistatic, treated tubes were used to inoculate fresh cultures. Aliquots of 100 µL from the no growth tubes that received the lowest light doses were used to seed three separate cultures containing 10 mL of fresh, drug-free medium. These were then incubated for 4 days at which time no visible growth in any of the new cultures was considered evidence of fungicidal effect in the original no-growth tube.


    Results and discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
The results of the in vitro PDT treatment of A. fumigatus using Green 2W as the photosensitizer are shown in the TableGo. All controls showed confluent growth at the 72 h time point. All experimental tubes (+light/+drug) that contained Green 2W with palladium showed both a light dose- and inoculum-dependent response. There was complete killing of 2.7 x 101 cfu/mL at a light dose of 110 J/cm2 up to complete killing of 2.7 x 106 cfu/mL at a light dose of 385 J/cm2. Subsequent culture of the no-growth tubes established that the successful treatments were fungicidal rather than fungistatic. All cultures treated with Green 2W without palladium grew to confluence, indicating that palladium is necessary for the fungicidal effect of Green 2W in this in vitro system.


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Table. Effect of in vitro photodynamic therapy with the photosensitizer Green 2W at 31.5 mg/L on A. fumigatus
 
The findings from this study demonstrate that PDT can have a significant effect against A. fumigatus in vitro. PDT may have potential as a treatment option for select localized fungal infections that occupy cavities and lend themselves to minimally invasive access, which could be used for instillation of a photosensitizer and introduction of a light delivery fibre.8 Further studies to identify or develop the optimal photosensitizer for this application would be required, but the potential use of a non-toxic photosensitizer that has in vitro activity against A. fumigatus is illustrated by Green 2W. This sensitizer is well tolerated when delivered intravenously in mice at concentrations of 50–100 times that employed in this in vitro study.9 Furthermore, the light doses employed for this in vitro study are similar to those used clinically for PDT treatment of endobronchial lung cancers.2,10 The demonstration of the fungicidal effect of PDT on A. fumigatus, in this in vitro system, is sufficient to merit further investigation of this antibiotic-independent treatment technique.


    Acknowledgments
 
We thank the staff of the University of Pennsylvania Clinical Microbiology Laboratory for their technical assistance; Dr C. C. Clark of the Department of Orthopedic Surgery for help with equipment; the University of Pennsylvania Department of Radiation Oncology, Carmen Rodriguez, Tim Zhu, Teodor Vulcan and Michael Solonenko for use of and assistance with the light dosimetry and laser system. This work was supported by Department of Surgical Research Funds from the University of Pennsylvania Medical School.


    Notes
 
* Correspondence address. Department of Surgery, Thomas Jefferson University, 1025 Walnut Street, 605 College Building, Philadelphia, PA 19107, USA. Tel: +1-215-955-2996; Fax: +1-215-923-1420; E-mail: joseph.friedberg{at}mail.TJU.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . Carré, V., Gaud, O., Sylvain, I., Bourdon, O., Spiro, M., Blais, J. et al. (1999). Fungicidal properties of meso-arylglycosylporphyrins: Influence of sugar substituents on photoinduced damage in the yeast Saccharomyces cerevisiae. Journal of Photochemistry and Photobiology B—Biology 48, 57–62.

2 . Paardekooper, M., de Bruijne, A. W., van Steveninck, J. & van den Broek, P. J. (1995). Intracellular damage in yeast cells caused by photodynamic therapy with toluidine blue. Photochemistry and Photobiology 61, 84–9.[ISI][Medline]

3 . Zoladek, T., Nguyen, B. N., Jagiello, I., Graczyk, A. & Rytka, J. (1997). Diamino acid derivatives of porphyrins penetrate into yeast cells, induce photodamage, but have no mutagenic effect. Photochemistry and Photobiology 66, 253–9.[ISI][Medline]

4 . Denning, D. W. (2000). Aspergillus species. In Mandell, Douglas and Bennett's Principles and Practice of Infectious Diseases, 5th edn, (Mandell, G. L., Bennett, J. E. & Dolin, R., Eds), pp. 2674–85. Churchill Livingstone, Philadelphia.

5 . Vinogradov, S. A. & Wilson, D. F. (1994). Metallotetrabenzoporphyrins. New phosphorescent probes for oxygen measurements. Journal of the Chemical Society Perkin Transactions II, 103–11.

6 . Sigler, L. & Kennedy, M. J. (1999). Aspergillus, Fusarium, and other opportunistic moniliaceous fungi. In Manual of Clinical Microbiology, 7th edn, (Murray, P. R., Baron, E. J., Pfaller, M. A., Tenover, F. C. & Yolken, R. H., Eds), pp. 1212–41. ASM Press, Washington, DC.

7 . Espinel-Ingroff, A., Dawson, K., Pfaller, M., Anaissie, E., Breslin, B., Dixon, D. et al. (1995). Comparative and collaborative evaluation of standardization of antifungal susceptibility testing for filamentous fungi. Antimicrobial Agents and Chemotherapy 39, 314–9.[Abstract]

8 . Jackson, M., Flower, C. D. & Shneerson, J. M. (1993). Treatment of symptomatic pulmonary aspergillomas with intracavitary instillation of amphotericin B through an indwelling catheter. Thorax 48, 928–30.[Abstract]

9 . Vinogradov, S. A., Lo, L. W., Jenkins, W. T., Evans, S. M., Koch, C. K. & Wilson, D. F. (1996). Noninvasive imaging of the distribution of oxygen in tissue in vivo using near-infrared phosphors. Biophysical Journal 70, 1609–17.[Abstract]

10 . Kato, H., Okunaka, T. & Shimatani, H. (1996). Photodynamic therapy for early stage bronchogenic carcinoma. Journal of Clinical Laser Medicine and Surgery 14, 235–8.[Medline]

Received 16 August 2000; returned 2 January 2001; revised 9 February 2001; accepted 19 March 2001