Ruthenium-induced corneal collagen crosslinking under visible light

Abstract

Corneal collagen crosslinking (CXL) is a commonly used minimally invasive surgical technique to prevent the progression of corneal ectasias, such as keratoconus. Unfortunately, riboflavin/UV-A light-based CXL procedures have not been successfully applied to all patients, and result in frequent complications, such as corneal haze and endothelial damage. We propose a new method for corneal crosslinking by using a Ruthenium (Ru) based water-soluble photoinitiator and visible light (430 nm). Tris(bipyridine)ruthenium(II) ([Ru(bpy)₃]²⁺) and sodium persulfate (SPS) mixture covalently crosslinks free tyrosine, histidine, and lysine groups under visible light (400-450 nm), which prevents UV-A light-induced cytotoxicity in an efficient and time saving collagen crosslinking procedure. In this study, we investigated the effects of the Ru/visible blue light procedure on the viability and toxicity of human corneal epithelium, limbal, and stromal cells. Then bovine corneas crosslinked with ruthenium mixture and visible light were characterized, and their biomechanical properties were compared with the customized riboflavin/UV-A crosslinking approach in the clinics. Crosslinked corneas with a ruthenium-based CXL approach showed significantly higher young's modulus compared to riboflavin/UV-A light-based method applied to corneas. In addition, crosslinked corneas with both methods were characterized to evaluate the hydrodynamic behavior, optical transparency, and enzymatic resistance. In all biomechanical, biochemical, and optical tests used here, corneas that were crosslinked with ruthenium-based approach demonstrated better results than that of corneas crosslinked with riboflavin/ UV-A. This study is promising to be translated into a non-surgical therapy for all ectatic corneal pathologies as a result of mild conditions introduced here with visible light exposure and a nontoxic ruthenium-based photoinitiator to the cornea. STATEMENT OF SIGNIFICANCE: Keratoconus, one of the most frequent corneal diseases, could be treated with riboflavin and ultraviolet light-based photo-crosslinking application to the cornea of the patients. Unfortunately, this method has irreversible side effects and cannot be applied to all keratoconus patients. In this study, we exploited the photoactivation behavior of an organoruthenium compound to achieve corneal crosslinking. Ruthenium-based organic complex under visible light demonstrated significantly better biocompatibility and superior biomechanical results than riboflavin and ultraviolet light application. This study promises to translate into a new fast, efficient non-surgical therapy option for all ectatic corneal pathologies.

Keywords: Collagen; Cornea; Crosslinking; Keratoconus; Phototherapy; Riboflavin; Ruthenium.