title: Quantitative in vitro demonstration of two-photon photodynamic therapy using photofrin and visudyne.
abstract: Photodynamic therapy (PDT), the combined action of a photosensitizer and light to produce a cytotoxic effect, is an approved therapy for a number of diseases. At present, clinical PDT treatments involve one-photon excitation of the photosensitizer. A major limitation is that damage may be caused to healthy tissues that have absorbed the drug and lie in the beam path. Two-photon excitation may minimize this collateral damage, as the probability of absorption increases with the square of the light intensity, enabling spatial confinement of the photosensitizer activation. A potential application is the treatment of the wet-form of age-related macular degeneration, the foremost cause of central vision loss in the elderly. Herein, the commercial photosensitizers Visudyne and Photofrin are used to demonstrate quantitative in vitro two-photon PDT. A uniform layer of endothelial cells (YPEN-1) was irradiated with a Ti:sapphire laser (300 fs, 865 nm, 90 MHz) using a confocal scanning microscope. Quantification of the two-photon PDT effect was achieved using the permeability stain Hoechst 33258 and a SYTOX Orange viability stain. Visudyne was found to be around seven times more effective as a two-photon photosensitizer than Photofrin under the conditions used, consistent with its higher two-photon absorption cross-section. We also demonstrate for the first time the quadratic intensity dependence of cellular two-photon PDT. This simple in vitro method for quantifying the efficacy of photosensitizers for two-photon excited PDT will be valuable to test specifically designed two-photon photosensitizers before proceeding to in vivo studies in preclinical animal models.
authors.affiliation: Department of Medical Biophysics, Ontario Cancer Institute, University of Toronto, Toronto, ON, Canada.
authors.forename: Mamta
authors.initials: M
authors.lastname: Khurana
authors.forename: Hazel A
authors.initials: HA
authors.lastname: Collins
authors.forename: Aliaksandr
authors.initials: A
authors.lastname: Karotki
authors.forename: Harry L
authors.initials: HL
authors.lastname: Anderson
authors.forename: David T
authors.initials: DT
authors.lastname: Cramb
authors.forename: Brian C
authors.initials: BC
authors.lastname: Wilson
mesh_terms: D000818:Animals; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000528:radiation effects; D017323:Dihematoporphyrin Ether / Q000737:chemistry* / Q000494:pharmacology*; D015394:Molecular Structure; D010777:Photochemistry; D010778:Photochemotherapy; D017785:Photons; D017319:Photosensitizing Agents / Q000494:pharmacology; D011166:Porphyrins / Q000737:chemistry* / Q000494:pharmacology*; D051381:Rats; D000077362:Verteporfin
chemical_list: D017319:Photosensitizing Agents; D011166:Porphyrins; D000077362:Verteporfin; D017323:Dihematoporphyrin Ether