Development and application of biological techniques to two-photon photodynamic therapy

Abstract

Two-photon (2-γ) photodynamic therapy (PDT) as opposed to standard one-photon (1-γ) PDT with Visudyne has recently been suggested as a targeted treatment alternative for wet-form age-related macular degeneration (AMD) and other neovascular diseases. AMD is a major cause of severe vision loss in the older population. It occurs due to growth of new leaky blood vessels (neovasculature) from the choriocapillaris, which results in destruction of photoreceptors in the fovea and loss of central vision. Damage outside the diseased region is always a concern, due to photosensitizer accumulation and its 1-γ excitation. Highly targeted 2-γ excitation, due to its non-linear intensity dependence, intrinsically avoids out-of-focus damage to healthy tissues and so could be valuable for wet-AMD. We have previously developed a quantitative approach for comparing the 2-γ efficacy of photosensitizers in vitro. In this study, we report further the development of ex vivo and in vivo techniques. A mouse mesenteric vessel has been investigated as the ex vivo model of neovasculature. For the in vivo studies, we have explored a mouse dorsal skin-fold window chamber model. Two-photon PDT is delivered using tightly focused ~300 fs laser pulses from a Ti:sapphire laser operating at 850 nm with 90 MHz pulse repetition rate. Confocal microscopy coupled to the laser was used to visualize the vessel's/cell's response before, during and after the treatment. We are able to demonstrate quantitative biological techniques to evaluate efficacy of 2-γ PDT photosensitizers in vivo.