May 2007
Volume 48, Issue 13
ARVO Annual Meeting Abstract  |   May 2007
Trans-Corneal HEPES Buffered Iontophoresis for Delivery of Oligonucleotides Into the Stroma
Author Affiliations & Notes
  • D. J. Gibson
    University of Florida, Gainesville, Florida
    Biochemistry and Molecular Biology,
  • G. S. Schultz
    University of Florida, Gainesville, Florida
  • Footnotes
    Commercial Relationships D.J. Gibson, None; G.S. Schultz, None.
  • Footnotes
    Support NIH/NEI Grant EY05587
Investigative Ophthalmology & Visual Science May 2007, Vol.48, 5879. doi:
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      D. J. Gibson, G. S. Schultz; Trans-Corneal HEPES Buffered Iontophoresis for Delivery of Oligonucleotides Into the Stroma. Invest. Ophthalmol. Vis. Sci. 2007;48(13):5879.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose:: To determine whether the electrochemically induced damage could be countered by the use of a biologically compatible buffer and to determine an accurate model that explains electromotive delivery.

Methods:: Fresh ex vivo rabbit globes were immobilized in a 50 ml beaker with 2.0% agarose. A silver loop electrode was placed encircling the eye and in contact with the sclera and a Delrin cup was placed on the eye, centered on the cornea. One milliliter of neutral 25mM HEPES buffer was added to the eye cup then a loading solution comprised of 250 µl of 20% sucrose in ddH2O and 50 µl of ssDNA (20nt oligonucleotide 1.6 mg/ml) was added, and finally a platinum loop electrode was then placed in the cup. Two series of treatments were used. First, a constant current of 1.5 mA with increasing treatment durations (3, 6, and 9 min) and the other series treated with different current levels (2.0, 3.0, 4.0, and 5.0 mA) for 3 and/or 6 min. The corneas were excised, fixed, paraffin embedded, and sectioned. Immunohistochemistry (IHC) was performed to determine oligonucleotide localization.

Results:: Even at the highest doses of charge (5.0 mA for 6.0 min = 1.08 Coulombs), the cornea never underwent liquefactive necrosis. IHC revealed significant penetration into stroma beginning with the 3.0 mA current for 3.0 min trial. Additionally, charge equivalent doses didn’t produce equivalent penetration.

Conclusions:: The use of a 25mM HEPES buffer in the eye cup prevents liquefactive necrosis caused by electrolytically induced alkalization. Additionally, in order for oligonucleotides to be delivered beyond the superficial epithelium, a certain electrical threshold must be overcome indicating that a classical electromechanical approach is appropriate. Iontophoresis posses the ability for trans-epithelial delivery of oligonucleotides into the stroma without histologically significant damage.

Keywords: gene transfer/gene therapy 

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