# Quantitative Measurements of Stromal and Epithelial Riboflavin With a Novel Transepithelial High-Concentration Riboflavin Soak-and-Rinse Protocol

## TL;DR

A new method for applying riboflavin to the cornea improves stromal absorption while reducing epithelial buildup, potentially making corneal crosslinking more effective.

## Contribution

A novel high-concentration riboflavin soak-and-rinse protocol is introduced to enhance transepithelial corneal crosslinking.

## Key findings

- A 20-minute soak with 0.8% riboflavin and BAK increased stromal riboflavin by 76% compared to lower concentrations.
- A 10-minute rinse reduced epithelial riboflavin by 5.9-fold compared to a 20-second rinse.
- The protocol achieved stromal riboflavin levels comparable to the standard epi-off method.

## Abstract

To enrich stromal riboflavin concentration and reduce epithelial riboflavin in transepithelial corneal collagen crosslinking (CXL).

Ex vivo experiments on rabbit corneas were performed. The control group followed the standard (epi-off) Dresden CXL: a 30-minute epi-off application of 0.1% riboflavin and 20% dextran. The transepithelial riboflavin solutions consisted of various riboflavin concentrations in 1% hydroxypropyl methylcellulose in 0.45% saline, with or without 0.01% benzalkonium chloride (BAK). The novel soak-and-rinse protocol consists of 0.8% riboflavin with 0.01% BAK and 1% hydroxypropyl methylcellulose (hypotonic) applied for 20 minutes, followed by a 10-minute saline rinse. Stromal and epithelial thicknesses were measured by optical coherence tomography; riboflavin concentrations were quantified by spectrophotometry on 3-mm stromal buttons and epithelial eluates. Statistical analysis employed one-way analysis of variance, linear regression, and one-tailed unpaired t-tests.

The 20-minute soak increased stromal riboflavin compared to the 10-minute soak. A 76% higher stromal concentration was achieved by adding BAK to the transepithelial 0.8% riboflavin solution (P < 0.05). The 10-minute rinse achieved a Dresden-equivalent stromal riboflavin level and reduced epithelial riboflavin by 5.9-fold compared to a 20-second rinse (P < 0.0001).

Stromal riboflavin concentrations equivalent to those achieved with the epi-off protocol can be achieved by transepithelial application of the novel high-concentration riboflavin formulation. The additional 10-minute rinse effectively reduced epithelial riboflavin levels, facilitating the delivery of ultraviolet light and oxygen into the stroma during CXL.

The novel transepithelial riboflavin soak-and-rinse protocol may potentially enhance the efficacy of transepithelial CXL by reducing epithelial consumption of ultraviolet light and oxygen and increasing the stromal CXL reaction.