No abstract

Protein crosslinking photosensitized by rose Bengal (RB2−) has multiple medical applications and understanding the photosensitization mechanism can improve treatment effectiveness. To this end, we investigated the photochemical efficiencies of monomeric RB2− (RBM2−) and dimeric RB2− (RBD2−) and the optimal pH for anaerobic RB2− photosensitization in cornea. Absorption spectra and dynamic light scattering (DLS) measurements were used to estimate the fractions of RBM2− and RBD2−. RB2− self‐photosensitized bleaching was used to evaluate the photoactivity of RBM2− and RBD2−. The pH dependence of anaerobic RB2− photosensitization was evaluated in ex vivo rabbit corneas. The 549 nm/515 nm absorption ratio indicated that concentrations > 0.10 mm RB contained RBD2−. Results from DLS gave estimated mean diameters for RBM2− and RBD2− of 0.70 ± 0.02 nm and 1.75 ± 0.13 nm, respectively, and indicated that 1 mm RB2− contained equal fractions of RBM2− and RBD2−. Quantum yields for RB2− bleaching were not influenced by RBD2− in RB2− solutions although accounting for RB2− concentration effects on the reaction kinetics demonstrated that RBD2− is not a photosensitizer. Optimal anaerobic photosensitization occurred at pH 8.5 for solutions containing 200 mm Arg. These results suggest potential approaches to optimizing RBM2−‐photosensitized protein crosslinking in tissues.

Oxygen-independent cornea crosslinking (CXL) using rose bengal (RB) and green light may have unique clinical applications. These studies were designed to gain insight into the arginine (arg)-enhanced anaerobic crosslinking process, to maximize crosslinking efficiency, and to test a clinically feasible method for oxygen-free CXL. Methods: Rabbit corneas were treated ex vivo using 1 mM RB and 532 nm light. RB photodecomposition, monitored by absorption spectrophotometry, was used to optimize arg concentration and to develop an irradiation and re-dying protocol. The minimal effective green light fluence was identified by linear tensile strength measurements. RB penetration into the stroma was determined by fluorescence microscopy. To favor the anaerobic pathway, a contact lens was used to minimize stromal oxygen level during irradiation. Stromal cell toxicity was evaluated by a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay. Results: RB photodecomposition reached 75% of its maximal effect at 200 mM arg and the optimal fluence increment was 32.7 J/cm 2. The minimal effective fluence for cornea stiffening was 65.4 J/cm 2. Placement of a contact lens promoted oxygen-independent cornea stiffening, similar to that obtained on isolated, oxygen-deprived cornea. RB penetration into the stroma with arg present was limited to ∼120 μm, about 25% deeper than without arg. Stromal cell toxicity was limited to the depth of RB and arg penetration. Conclusions: An oxygen-independent pathway in cornea for RB-CXL was characterized and optimized, including a possible clinical protocol for its use. Translational Relevance: Oxygen-independent RB-CXL is an efficient and effective process that can be developed further for unique clinical applications.