High Field Computer Controlled Pulse Iontophoresis of Oligonucleotides Into the Cornea

Abstract

Introduction: : Iontophoresis of ribozymes or siRNAs requires higher electrical field strength or longer dosage time (dosage is proportional to field x time) to overcome the increased resistance to corneal penetration caused by the oligo’s size. The common method of applying a constant direct current causes significant damage to the cornea at the higher field required for timely delivery.

Purpose: : To determine if high field computer controlled pulse iontophoresis can be applied to the cornea without the damage that accompanies the application of a constant high field.

Methods: : Frozen ex vivo rabbit corneas were thawed, placed in an acetyl iontophoresis eye cup, and filled (endothelial side) with 1% agarose. The eye cup was then placed in a custom made iontophoresis chamber (Pt. electrodes) and both were filled with 0.25 x TAE. 550µl of a loading solution (67µM oligo, 20µg/ml Nile Blue, 40% sucrose) was added into the eye cup in direct contact with the corneal epithelium. The field was controlled by custom software, applied by a DATAQ DI–148U USB DAQ, and amplified by a common operational amplifier circuit. A field strength of ∼10V/cm (25.2V/2.5cm; the current ranged 4.0–11.6mA depending on duration) was applied constantly for 2 corneas (1 and 5 min.), and as a 1 second on / 2 seconds off pulse signal for two more (5 and 13 min.). An additional cornea served as a control against freeze/thaw, handling, and sectioning damage. The corneas were paraffin mounted, sectioned, and stained with H & E then histologically analyzed for damage.

Results: : Extensive damage including complete absence of the corneal epithelium was observed in the constant 5 min. and the pulsed 13 min. corneas. Additionally, the Nile Blue underwent a chromogenic shift, and the solution in contact with the cornea was highly viscous in both of these highly damaged cases. Less damage was observed in the pulsed 5 min. cornea than in the constant 1 min. cornea. Neither a chromogenic shift, nor viscous fluid were observed in either of these cases.

Conclusions: : High field computer controlled pulse iontophoresis can mediate the damage normally caused by an applied constant field while maintaining an equivalent or greater dose per degree of damage. Damage to the corneal epithelium may also be caused by basic conditions brought on by the generation of OH^– from the electrolysis of H₂0.

Keywords: gene transfer/gene therapy • herpes simplex virus • wound healing