Solubilized Ubiquinol Improves Reactive Oxygen Species Scavenging in Donor Corneal Endothelial Cells

Abstract

Purpose : Oxidative stress in corneal endothelial cells (CECs) preserved under hypothermic conditions is thought to be a major driver of cell death prior to surgery as well as decreased graft survival after keratoplasty. To mitigate oxidative damage and reactive oxygen species (ROS) in CECs, we aimed to develop a highly dispersible and stable formulation comprising a complex of ubiquinol and gamma-cyclodextrin (γ-CD) for use in aqueous-phase ophthalmic products including corneal storage media, intraocular irrigation solution, and topical eye drops.

Methods : Molecular docking study in AutoDock Vina indicated that γ-CD has the strongest binding affinity with ubiquinol. Ubiquinol/γ-CD complex (UBCD) was prepared using the kneading method and its physiochemical properties were characterized. Stability of UBCD in Optisol-GS®, balanced salt solution, and Refresh® artificial tear eye drops for up to 1 week was determined by HPLC. High ROS-generating cells (A549) and B4G12 human corneal endothelial cells (HCEC-B4G12) were exposed to antimycin A, and UBCD ROS scavenging activity was measured using DHE fluorescence. Cytotoxic effect was determined up to 72 hours using MTS reagent. A NaviCyte vertical diffusion chamber was used to assess transcorneal penetrance of topically applied UBCD in human donor corneas. Lastly, UBCD penetrance of stored CECs was assayed using human corneas preserved for 7 days in Optisol-GS®.

Results : UBCD showed significantly higher stability compared to free ubiquinol by HPLC analysis. UBCD was more effective at lowering ROS at far lower concentrations compared to free ubiquinol. No evidence of cellular toxicity was detected in B4G12 cells. Topically applied UBCD penetrated the entire thickness of human donor corneas with significantly greater ubiquinol retention in the endothelium compared to free ubiquinol (p=0.059). UBCD demonstrated higher amounts of ubiquinol retained in the corneal endothelium of preserved donor corneas compared to free ubiquinol (p<0.05).

Conclusions : Findings indicate that UBCD can better preserve human CEC function during cold storage, and may be useful in preventing post-keratoplasty cell loss, increasing donor cornea preservation time, and reducing graft failure by scavenging ROS. UBCD’s higher bioavailability and stability in the aqueous phase will allow its incorporation into a variety of products for ophthalmic use to protect CECs against oxidative damage.

This is a 2021 ARVO Annual Meeting abstract.