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D. J. Gibson, G. Schultz; Optimization of Electrode Material and Placement for in vivo Iontophoretic Delivery of Oligonucleotides Into the Stroma. Invest. Ophthalmol. Vis. Sci. 2008;49(13):3195. doi: https://doi.org/.
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To determine the material and placement of the receiving electrode that is both safe and optimal for in-vivo iontophoretic delivery of oligonucleotides into the cornea.
Live white New Zealand rabbits were anesthetized with isoflurane and their corneas were treated with proparacain eye drops. A Delrin cup was placed on the eye and centered on the cornea. One milliliter of neutral 25mM HEPES was added to the eye cup followed by 250 µl of 20% sucrose with a 50:50 mix of fluorsecinated:protected 20nt reporter ssDNA (25 µl each from 1.6 mg/ml stocks). A platinum loop electrode was then placed in the cup. Either a nickel plated copper loop (1 eye) or a platinum loop electrode (3 eyes) was placed encircling the Delrin cup and a gauze soaked with 25 mM HEPES was wrapped around the cup and placed in contact with the outer eyelid. A constant current of 5.0 mA was applied for 6.0 min. The eyes were observed with and without cyan LED (495 nm) excitation prior to euthanization at 0 or 24 h and the corneas were excised, fixed, paraffin embedded, and sectioned. Oligonucleotide localization was performed using fluorescent microscopy with and without immunohistochemistry (IHC).
In all trials, the corneas were protected by the HEPES as before. Irritation and oxidization staining were only present in the eye treated with the copper electrode. In all eyes, illumination with a cyan LED at 0h post treatment, but not after 24h, revealed that the entire area of treatment fluoresced intensely. Under the fluorescent microscope, the oligonucleotides were present throughout the cornea in the 0 hr eyes, both under fluorescein and rhodamine (IHC) excitation/emission conditions. After 24 h, only low levels of punctate IHC staining was present and appeared to be localized to epithelial cells and stromal fibroblasts.
Platinum electrodes connected to the subject by a 25 mM HEPES buffer allows ssDNA to be delivered to all 3 corneal layers without damage. The ssDNA is mostly cleared from the cornea within 24 h, but is localized to some epithelial and stromal cells. Now that the delivery and damage control are effective, this system is now ready for use in delivering biologically active macromolecule compounds.
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