Simultaneous Two-photon Excitation of Photofrin in Relation to Photodynamic Therapy

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ABSTRACT

Photodynamic therapy (PDT), the use of light-activated drugs (photosensitizers), is an emerging treatment modality for tumors as well as various nononcologic conditions. Single-photon (1-γ) PDT is limited by low specificity of the photo-sensitizer, leading to damage to healthy tissue adjacent to the diseased target tissue. One solution is to use simultaneous two-photon (2-γ) excitation with ultrafast pulses of near-IR light. Due to the nonlinear interaction mechanism, 2-γ excitation with a focused beam is localized in three dimensions, allowing treatment volumes on the order of femtoliters. We propose that this will be valuable in PDT of age-related macular degeneration (AMD), which causes blindness due to abnormal choroidal neovasculature and which is currently treated by 1-γ PDT. Here, Photofrin has been used as the photosensitizer to demonstrate proof-of-principle of 2-γ killing of vascular endothelial cells in vitro. The 2-γ absorption properties of Photofrin were investigated in the 750–900 nm excitation wavelength range. It was shown that 2-γ excitation dominates over 1-γ excitation above 800 nm. The 2-γ absorption spectrum of Photofrin in the 800–900 nm excitation wavelength range was measured. The 2-γ cross section decreased from about 10 GM (1 GM = 10-5 cm4 s/photon) at 800 nm to 5 GM at 900 nm. Adherent YPEN-1 endothelial cells were then incubated with Photofrin for 24 h and then treated by PDT at 850 nm where the 1-γ contribution was negligible. Cell death was monitored with the use of 2-γ scanning laser microscopy. The light doses required for killing were high (6300 J cm-2 for ∼50% killing), but 2-γ cytotoxicity was unequivocally demonstrated. Although Photofrin is, per se, not a good choice for 2-γ PDT due to its low 2-γ cross section, this work provides baseline data to guide the development of novel photosensitizers with much higher 2-γ cross sections (>100 GM), which will be required for 2-γ PDT of AMD (and other conditions) to be clinically practical.

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