Lipofuscin of retinal pigment epithelium (RPE) cells is a complex heterogeneous system of chromophores which accumulates as granules during the cell's lifespan. Lipofuscin serves as a source of various cytotoxic effects linked with oxidative stress. Several age-related eye diseases such as macular degeneration of the retina, as well as some severe inherited eye pathologies, are accompanied by a significant increase in lipofuscin granule concentration. The accumulation of carotenoids in the RPE could provide an effective antioxidant protection against lipofuscin cytotoxic manifestations. Given the highly lipophilic nature of carotenoids, their targeted delivery to the vulnerable tissues can potentially be assisted by special proteins. In this study, we demonstrate how protein-mediated delivery of zeaxanthin using water-soluble Bombyx mori carotenoid-binding protein (BmCBP-ZEA) suppresses the photoinducible oxidative stress in RPE cells caused by irradiation of lipofuscin with intense white light. We implemented fluorescence lifetime imaging of the RPE cell culture ARPE-19 fed with lipofuscin granules and then irradiated by white light with and without the addition of BmCBP-ZEA. We demonstrate that after irradiation the mean fluorescence lifetime of lipofuscin significantly increases, while the presence of BmCBP-ZEA at 200 nM concentration suppresses the increase in the average lifetime of lipofuscin fluorescence, indicating an approx. 35% inhibition of the oxidative stress. This phenomenon serves as indirect yet important evidence of the efficiency of the protein-mediated carotenoid delivery into pigment epithelium cells.
Keywords: FLIM; bisretinoid fluorophores; bisretinoid oxidation and degradation products; carotenoid-binding protein; lipofuscin; retinal pigment epithelium; zeaxanthin delivery.