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J.M. Nickerson, J.B. Davies, J.H. Boatright, V.T. Ciavatta; Trans-Scleral Delivery of DNA and Dyes by Electrical Fields . Invest. Ophthalmol. Vis. Sci. 2003;44(13):2328.
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Purpose: Iontophoresis has been used for drug delivery across the cornea for many years. We sought to test whether we could transfer small charged dyes and DNA across human sclera mediated by an electric field. Methods: Fragments of full-thickness human sclera were embedded in agarose and mounted in an agarose gel electrophoresis chamber. The scleral fragments completely spanned individual wells in the agarose gel. The wells were located about 1 cm from the vertically oriented scleral fragments and were loaded with DNA or dyes. Electrophoresis was carried out at about 2 V/cm for about 90 min. Digital time lapse photos of the gel were taken every 3 min during electrophoresis. The gels were stained afterwards with SYBR green to detect DNA. In some experiments with plasmid electrophoresis, a different apparatus was used with sclera mounted between two chambers. Results: Time lapse photography of agarose gel electrophoresis revealed that xylene cyanol passed through the sclera almost unimpeded, but with some spreading of the dye. Bromphenol blue was slowed down markedly by the sclera, but eventually it too passed through the tissue. Small DNAs, including a single stranded 45-mer and a double hairpin 68-mer oligonucleotide, passed through the sclera, though their rates of passage through the tissue were slowed and accompanied by band spreading. A 230 base pair double stranded DNA passed through the sclera more slowly. pBR322 did not pass through the sclera within the detection limits of SYBR green staining. However, in 2 experiments in 17 trials with the two-chamber apparatus, a small amount of plasmid could be detected that appeared to have crossed human sclera. Conclusions: Charged molecules such as xylene cyanol, bromphenol blue, and small DNAs up to 230 bp can be rapidly driven across human sclera by an electric field. Passage of larger molecules such as pBR322 was at best inefficient. This technique offers promise as a noninvasive DNA delivery tool, in small fragment gene therapy.
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