Impact of photodynamic inactivation (PDI) using the photosensitizer chlorin e6 on viability, apoptosis, and proliferation of human corneal endothelial cells

Photodynamic inactivation (PDI) may be a potential alternative in case of therapy-resistant infectious keratitis. PDI using the photosensitizer chlorin e6 (Ce6) with high photosensitizing efficacy offers a valuable option, also for keratitis. The purpose of our study was to determine the impact of PDI with the photosensitizer Ce6 on viability, apoptosis, and proliferation of human corneal endothelial cells (HCECs), in vitro. Human corneal endothelial cell line was cultured in DMEM/Ham's F12 medium supplemented with 5 % fetal calf serum. HCECs cultures underwent illumination using red (670 nm) light for 13 min following exposure to 50-500 nM concentrations of Ce6 in the culture medium. Twenty-four hours after PDI, cell viability was evaluated by the Alamar blue assay, total DNA content of the cells and apoptosis using the APO-DIRECT (TM) Kit, and cell proliferation by the BrdU Cell Proliferation Assay Kit. Using Ce6 or illumination only, we did not detect significant changes of cell viability, apoptosis, and proliferation. Following PDI, viability and total DNA content of HCECs decreased significantly above 150 nM Ce6 concentration (P < 0.01; P < 0.05). The percentage of apoptotic HCECs increased significantly from 250 nM Ce6 concentration (P < 0.01), and proliferation of endothelial cells decreased significantly (P < 0.05) above 100 nM concentration of Ce6 after PDI. Photodynamic inactivation using Ce6 decreases viability and proliferation, and also triggers apoptosis of HCECs in vitro. PDI using the photosensitizer Ce6 may be a potential treatment alternative in infectious keratitis. However, to avoid endothelial cell damage, the photosensitizer must not penetrate the endothelium.