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In Vitro Tests of the Validity of Singlet Oxygen Luminescence Measurements as a Dose Metric in Photodynamic Therapy

¹ Department of Medical Biophysics, Ontario Cancer Institute/University Health Network and University of Toronto, Toronto,
² Hamilton Regional Cancer Center and McMaster University, Hamilton, Ontario, Canada

Singlet oxygen (¹O₂) is widely believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). We showed recently that measurement of the weak near infrared luminescence of ¹O₂ is possible in cells in vitro and tissues in vivo. Here, we investigated the relationship between the integrated luminescence signal and the in vitro PDT response of AML5 leukemia cells sensitized with aminolevulinic acid-induced protoporphyrin IX (PpIX). Sensitized cell suspensions were irradiated with pulsed 523 nm laser light at average fluence rates of 10, 25, or 50 mWcm^-2 and, ¹O₂ luminescence measurements were made throughout the treatment. Cell survival was measured with either propidium iodide-labeled flow cytometry or colony-forming assay. The PpIX concentration in the cells, the photobleaching, and the pO₂ in the cell suspensions were also monitored. There were large variations in cell survival and ¹O₂ generation in different experiments due to different controlled treatment parameters (fluence and fluence rate) and other uncontrolled factors (PpIX synthesis and oxygenation). However, in all of the cases, cell kill correlated strongly with the cumulative ¹O₂ luminescence and allowed direct estimation of the ¹O₂ per cell required to achieve a specific level of cell kill. This study supports the validity and potential utility of ¹O₂ luminescence measurement as a dosimetric tool for PDT, as well as confirming the likely role of ¹O₂ in porphyrin-based PDT.

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