Sequestration of indocyanine green dye into lysosomes after uptake into retinal pigment epithelial cells

Abstract

Purpose : Retinal pigment epithelial (RPE) cells are readily labeled by indocyanine green (ICG) in the living human eye and can be fluorescently imaged using adaptive optics (AO) retinal imaging (PMID 30895942). Recent images of ICG labeled enlarged RPE cells have revealed that after entering the cell, the ICG dye resides within the cytoplasmic space (PMID 36100689). The goal of this study is to explore the mechanisms of ICG uptake and clearance in RPE cells.

Methods : Custom-modified microscopes outfitted with near infrared fluorescence detection capabilities were used to assess the subcellular localization of ICG in live cells through comparison with other fluorescent markers. Colocalization experiments were performed using several types of live cells. First, live cultured immortalized cells (COS-7) were briefly incubated with ICG and then image alongside different fluorescent markers to screen for candidate organelles which might interact with cytoplasmic ICG. Colocalization of ICG with cytoplasmic organelles was then confirmed using live primary human RPE cells which were briefly incubated with ICG in culture media immediately prior to imaging. Finally, the in vivo delivery of ICG to organelles in RPE cells was evaluated using primary murine RPE cells isolated from BALB/c mice at a time point of approximately 16 hours after systemic ICG administration (PMID 30895942).

Results : The increased resolution provided by the custom-modified microscopes relative to in vivo human AO-ICG imaging revealed that ICG consistently sequestered into organelles upon entering RPE cells. Screening experiments in COS-7 cells revealed a possible association with lysosomes but not other organelles. This colocalization result was further confirmed using fluorescently-labeled dextran alongside ICG incubation in primary human RPE cells, which showed colocalized dynamic movement of ICG and fluorescently-labeled dextran. Evaluation in murine RPE cells co-labeled with a lysosomal marker confirmed that systemically injected ICG ultimately localized to lysosomes within the RPE cells.

Conclusions : The subcellular localization of ICG with lysosomes demonstrated in primary RPE cells elucidates a possible mechanism of ICG uptake and clearance in RPE cells via lysosomal degradation. This data suggests that it may be possible to use ICG to visualize lysosomal activity in RPE cells in the living eye.

This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.