A new light source for PDT of skin lesions

Rolf-Markus Szeimies, Sigrid Karrer, tAIfred Heine, Wolfgang Baumler, and Michael Landthaler

Department of Dermatology, University of Regensburg, Franz-Josef-Strauss-Allee 11, D-93053 Regensburg, 
Germany
tWaldmann Medizintechnik, Peter-Henlein-Strasse 5, D-78056 VS-Schwenningen

ABSTRACT

Since coherence of laser light is not necessary for PDT of skin tumors, attempts have been made to construct 
incoherent lamps. A recent development is PDT 1200 (Waldmann Medizintechnik/FRG), a light source based on a 
1200 watt metal halogen lamp. Emission of 580 to 740 nm radiation is achieved by using cut off filters. Power 
density can be varied from 30 mW/cm2 to 200 mW/cm2 in an area from 100 to 300 cm2. Biologic effectiveness 
was proved by comparison with the radiation of an argon-pumped dye laser emitting light at 630 nm. Three human 
cell lines were incubated with photofrin (5 1lg/ml and 10 1lg/ml) for 24 hours. After irradiation a MTT-test was 
performed to assess cell viability. Results clearly proved biological effectiveness of the light source PDT 1200. No 
significant difference in cell viability was detected using either concentration of sensitizer. Therefore, we believe 
that PDT 1200 is a Dromisins new liQht source fnr nhntadvnamic therapy of skin lesions.

Keywords: photofrin, photodynamic therapy, incoherent light source, tunable dye laser

1. INTRODUCTION

In recent years, photodynamic therapy has shown effectiveness in the curative and palliative treatment of cancer.i 2 
Based on tumor location like skin surface or hollow organs, different light application systems for PDT are 
necessary.34 A variety of systems are currently in use, using either laser sources for irradiation like the 
argon-pumped dye lasers or incoherent wavelength-filtered lamps. Since coherence of light is not mandatory for 
skin surface illumination, and irradiation with lamps is more reliable, simpler and cheaper than with lasers, the only 
problem that remains to be solved is to increase the intensity level achievable to that of lasers. Currently incoherent 
lamps are able to irradiate at an intensity level about 50 mW/cm2 compared to laser systems which can reach 
150-200 mW/cm2.4 On the other hand, it is difficult to illuminate a large surface area with a laser system because 
intensity decreases rapidly when the irradiation diameter is widened.

The aim of our study was to prove the biological effectiveness of a new incoherent lamp for PDT capable for 
irradiation at higher intensity levels and larger areas.

2. MATERIALS AND METHODS

Three human cell lines, HaCaT immortalized human keratinocytes,5 HF human dermal fibroblasts and J82 bladder 
cancer cells6 were maintained at 37  C in a humidified atmosphere. Cells were grown in 96 cell cultures with 7 x 
104 cells per dish. The cells were allowed to attach overnight, then the medium was removed. 100 ml of serum-free 
medium, containing photofrin at a concentration of either 5 1lg/ml or 10 1lg/ml was added to the cultures, and the 
cells were allowed to take up the dye for 24 h. Afterwards, extracellular dye was removed and light treatment was 
performed immediately. In change given to the information in our abstract, we changed the experimental protocol 
and used 5 versus 10 pg/ml photofrin, because it reflects better the treshold dose of dark toxicity of photofrin.7

Irradiation was performed with either an argon-pumped dye laser or the PDT 1200 lamp. The PDT 1200 lamp is a 
1200 watt metal halogen lamp (MSR 1200, Philips B.V., Eindhoven, The Netherlands). Emission of 600 to 800 nm 
radiation is achieved by using dichroic cut-off filters (DT Rot and Calflex-3000, Balzers Optik, Nurnberg, 
Germany). The wavelength band was limited because most of the photosensitizers of clinical interest have strong 
absorption bands at 630 nm. Shorter wavelenghts were not used, because they do not reach deeper layers in the 
skin. Infrared radiation was excluded, because of heating effects during irradiation.

Depending on distance between light source and skin surface, intensity could be varied from 30 mW/cm2 to 200 
mW/cm2 in an irradiated area from 100 to 300 cm2. Intensity measurements showed a homogen gaussian 
distribution with intensity loss of less than 10 % in the central light spot (95 cm2, distance 100 cm, Fig. l). An 
argon-pumped dye laser exciting Kiton red dye and producing up to 3.5 W of red light at x = 630 nm, was used for 
comparison. Fluence rate to which cells were

exposed was adjusted in both light sources to 30 mW/cm2. Depending on exposure time and according to 
preliminary dosedependent studies, a light dose of 30 J/cm2 was selected in this study.

__ x vv W111.

After photoirradiation, an MTT assay8 was immediately performed to check cell viability. After 24 h optical 
densities of the wells were read using a ELISA reader at 540 nm as a parameter of cell viability. Nine groups were 
forrned for each cell line. The first group served as a control and recieved no sensitizer or irradiation. The second 
group received irradiation with dye 1aser only, group three with PDT 1200 only. Group four was treated with 5 
mg/ml Photofrin, group five with 10 mg/ml Photofrin respectively. Group six was treated with S mg/ml Photofrin 
and dye laser, group seven with S mg/ml Photofrin and PDT 1200. Group eight was treated with 10 mg/ml 
Photofrin and dye laser, group nine with 10 mg/ml Photofrin and PDT 1200.

3. RESULTS

As expected, no significant change in cell viability was detected in cells irradiated only with laser or PDT 1200. A 
significant decrease in cell viability was seen in J82 cells incubated with Photofrin at both concentrations without 
irradiation (dark toxicity). However, fibroblasts and HaCaT cells at S 1lg/ml Photofrin incubation showed an 
increased cell viability above 100 percent. Light irradiation of Photofrin-incubated cells led to a highly significant 
decrease in cell viability at different concentrations compared to non-illuminated cells and controls. No significant 
difference in cell viability was detected between Laser and PDT 1200 irradiation of Photofrin-incubated cells at 
either concentration (Figs. 2, 3).

4. DISCUSSION

The incoherent lamp revealed a comparable biological effectiveness in the three cell lines, since cell viability after 
laser or PDT 1200 irradiation did not differ significantly. The significant decrease in cell viability in J82 cells 
incubated with Photofrin is possibly due to the long incubation period and high concentrations of sensitizer 
resulting in inhibition of microtubule assembly.9

Current research shows that the mechanism of phototoxicity by Photofrin is a result of production of highly reactive 
interrnediate singlet molecular oxygen leading mainly to membrane damage of sensitized and irradiated cells.~ l  
After longer incubation periods, Photofrin is mainly localized to the mitochondria where, after light activation, it 
harms membranes and reduces the activity of membrane-associated enzymes like succinic dehydrogenase.~ Lack of 
activity of this enzyme after PDT in vitro could easily be determined by an MTT assay.5

The incoherent lamp revealed a comparable biological effectiveness in the three cell lines, since cell viability after 
laser or PDT 1200 irradiation did not differ significantly. We believe therefore, that PDT 1200 is a promising new 
light source for the treatment of superficial skin lesions with PDT.

5. ACKNOWLEDGMENTS

This study was supported by Deutsche Forschungsgemeinschaft (Grant: La 620/1-2) and Bundesministerium fur 
Forschung und Technologie (Grant: 0706903A5). HaCaT cells were kindly provided by Prof. N. Fusenig, DKFZ 
Heidelberg/Germany .

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