Effect of the Synthetic NC-1059 Peptide on Diffusion of Riboflavin across an Intact Corneal Epithelium

Abstract

PURPOSE.To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma.METHODS. NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration.RESULTS. Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-lm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 lm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells.CONCLUSIONS. NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma. (Invest Ophthalmol Vis Sci.

New clinical pathways for keratoconus

“…A non-toxic reversible synthetic non-selective ion channel-forming peptide, NC-1059, has also been shown in a chick corneal model to enhance riboflavin permeability across an intact epithelium. 32 Two prospective, paired-eye studies in humans have documented small, but significant improvements in CDVA over fellow untreated eyes at 12 and 18 months, respectively, with a trend towards deterioration in topographic and acuity measures in non-treated control eyes. 33,34 Improvements in corneal curvature (B2D mean reduction in Kmax) were similar to those seen after epithelium-off CXL.…”

Nonlinear optical collagen cross-linking and mechanical stiffening: a possible photodynamic therapeutic approach to treating corneal ectasia

“…15 This currently requires the removal of the overlying corneal epithelium to allow penetration of riboflavin into the stroma, albeit alternative approaches not requiring epithelial removal are being intensively investigated. 16,17 Corneas are then exposed to 5.4 J∕cm 2 of 370 nm UVA light for two to 30 min depending on the illumination intensity (3.0 to 45 mW∕cm 2 ). 18,19 Photoactivation of riboflavin induces the formation of free radicals that interact with corneal proteins leading to covalent bonding within collagen fibrils.…”

Transepithelial Riboflavin Absorption in an Ex Vivo Rabbit Corneal Model

“…[7][8][9] Previous studies to quantify riboflavin penetration through an intact epithelium have used high-performance liquid chromatography (HPLC) [10][11][12] and fluorescence microscopy. [13][14][15][16][17] High-performance liquid chromatography, in which the sample is dissolved in a solvent for analysis, can accurately quantify the concentration of riboflavin in a whole block of tissue, but is unable to provide information about the concentration at different depths within the corneal stroma (unless lamella sections are prepared and separately dissolved). 18 Fluorescence microscopy, both single-photon excitation (i.e., confocal) and multiphoton excitation, [13][14][15][16][17] added advantage of quantifying concentration at depth within the cornea; both excitation techniques, however, suffer from signal attenuation with increasing scan depth, principally attributable to scattering and aberration.…”

“…[13][14][15][16][17] High-performance liquid chromatography, in which the sample is dissolved in a solvent for analysis, can accurately quantify the concentration of riboflavin in a whole block of tissue, but is unable to provide information about the concentration at different depths within the corneal stroma (unless lamella sections are prepared and separately dissolved). 18 Fluorescence microscopy, both single-photon excitation (i.e., confocal) and multiphoton excitation, [13][14][15][16][17] added advantage of quantifying concentration at depth within the cornea; both excitation techniques, however, suffer from signal attenuation with increasing scan depth, principally attributable to scattering and aberration. [14][15][16] We have previously reported 14 a novel time-lapse measurement approach to correct for two-photon fluorescence (TPF) signal attenuation; although applicable to epithelium-off absorption, this method has not proven suitable when imaging through an intact epithelium.…”