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Daniel M Gore, Paul French, David O'Brart, Chris Dunsby, Bruce D S Allan; NC-1059 peptide-assisted transepithelial riboflavin penetration in an ex-vivo rabbit corneal model. Invest. Ophthalmol. Vis. Sci. 2016;57(12):2911.
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© 2017 Association for Research in Vision and Ophthalmology.
NC-1059 is a synthetic non-selective ion channel-forming peptide. This study measured NC-1059-assisted riboflavin penetration through an intact epithelium in an animal model, compared to standard epithelium-off riboflavin delivery.
Rabbit heads transported in phosphate buffered saline were received within 5 hours post-mortem. Intact globes were enucleated and warmed to room temperature in balanced salt solution. A 200 µM NC-1059 peptide solution (sequence KKKK-AARVGLGITTVLVTTIGLGVRAA) was prepared by dissolving it in either 0.25 % or 0.1 % w/v riboflavin 5’-monophosphate solution.A 9 mm vacuum well secured on the cornea was filled with approximately 0.5 ml of 200 µM NC-1059 peptide in 0.1 % w/v riboflavin solution (n=3) or 0.25 % w/v riboflavin solution for 30 minutes (n=3). Riboflavin then was rinsed from the cornea for 1 minute. Epithelial-debrided globes soaked with peptide-free 0.1% w/v riboflavin solution served as controls (n=3).At the end of the soak, the globes were immediately frozen in liquid nitrogen. 35 µm corneal cross-sections were cut on a cryostat, mounted on a slide and imaged by two-photon fluorescence (TPF) microscopy. Riboflavin was excited by two-photon light of 890 nm wavelength, with fluorescence signal detected between 525-650 nm. TPF signals were converted to riboflavin concentration by use of a calibration curve which we produced by measuring the TPF signal within known concentrations of riboflavin on a well slide.
Mean (± SD) peak riboflavin concentration of 0.09 % (± 0.01) was observed within the anterior stroma in epithelium-off controls. The maximum stromal riboflavin concentration in the presence of 200 µM NC-1059 peptide solution and 0.1 % w/v riboflavin was 0.012 (± 0.005) %. The maximum stromal riboflavin concentration in the presence of 200 µM NC-1059 peptide solution and 0.25 % w/v riboflavin was 0.041 (± 0.01) %.
NC-1059 peptide enhances riboflavin penetration through an intact corneal epithelium, but does not match stromal concentrations achieved ‘epithelium-off’.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
Colour photographs of corneas soaked with NC-1059 peptide and riboflavin showing riboflavin penetration through the cornea into the anterior chamber. Upper row: Sagittal sections of globes mounted on a cryostat during section preparation. Lower row: 35 μm corneal sections laid on paper
Two-photon fluorescence images of tissue sections (grey scale). e, epithelium
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