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Ghazala Begum, Ann Logan, Richard J Blanch; An ex vivo corneal model to analyse the penetration of topical eye treatments. Invest. Ophthalmol. Vis. Sci. 2018;59(9):4453. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Topical delivery of drugs to the back of the eye remains a pharmaceutical challenge. The primary route of entry is through the cornea; however, this structure consists of densely packed cells with tight junctions preventing the entry of foreign molecules. Current methods of assessing corneal penetration are expensive and require large amounts of tissue and drugs. We have designed an ex vivo corneal model allowing for high throughput analysis of corneal penetration of siRNA as an emerging treatment for ocular diseases and the use of potential peptides to aid their entry.
5mm biopsy punches of porcine cornea were secured in between holders to create a seal and placed in a 96 well plate containing BSS. Fluorescein, Rhodamine B, Dextran blue and pH 12 sodium hydroxide were added to the epithelial side of the cornea as controls for corneal integrity over 4 hours. The corneal structure was examined by H&E. Further analysis of the epithelial layer was measured using the trans epithelial resistance (TER) monitor. 10µg of siRNA was added with and without penetrating peptides and analysed for penetration and corneal absorption.
Cornea was impermeable to Dextran blue but permeable to Rhodamine B and Fluorescein over the 4 hours (0.11±0.03 p<0.025). pH 12 sodium hydroxide treatment consistently led to significant changes (p<0.001) in the pH of the endothelial media with histological staining showing disintegrated epithelial layer with NaOH treatment. All other corneas showed strong epithelial integrity with tightly packed cells and clear structures after 4 hours. TER measurements indicated a reduction in the unit area of resistance after 120 mins approaching significance at 240mins 34.27±3.2Ωcm2 p<0.024. siRNA treatment was found to penetrate at approximately 0.03% of total added. Corneal absorption of siRNA was also assessed at 12.41±2.04% of total siRNA applied. Cell penetrating peptides affected the rates of penetration and absorption of siRNA.
We have successfully developed an ex vivo model to measure the rate of penetration of ocular treatments. Corneal integrity was meticulously assessed and was comparable with other ex vivo models. siRNA penetration was affected by peptides which could be due to altered charge influencing the electrostatic routes of penetration. The model described and the peptides used could have many positive consequences for pharmacological topical drug delivery.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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