Delivery of several forms of DNA, DNA-RNA hybrids, and dyes across human sclera by electrical fields.
OBJECTIVE
Iontophoresis has been used for drug delivery across the cornea for many years. We sought to test whether small charged dyes and DNA can be transferred across human sclera by an electric field.


METHODS
Full-thickness human scleral fragments were embedded vertically in an agarose gel and positioned to completely span individual gel lanes. The scleral fragments were located approximately 1 cm downstream from the gel wells. DNA or dyes were loaded into the wells and electrophoresis was carried out at about 3.3 V/cm for approximately 2 h per run. Movement of DNA and dyes through the agarose and sclera was measured with either digital time-lapse photography or through DNA extraction and purification from the gel. SYBR green stain was used as a sensitive method to detect DNA.


RESULTS
Digital time-lapse photography of agarose gel electrophoresis revealed that two dyes, xylene cyanol and bromphenol blue, passed through the sclera in the presence of an electric field. Xylene cyanol was driven through the sclera virtually unimpeded except for some spreading of the dye. Bromphenol blue was slowed markedly by the sclera, but it too eventually passed through the tissue. Small DNAs, including a single stranded 51-mer and a double hairpin 68-mer oligonucleotide, passed through the sclera as detected by SYBR green staining. Linear double stranded DNAs ranging from 50 bp to 12,000 bp passed through the sclera. The larger the DNA, the slower the rate of passage through the sclera, and the greater the band spreading. pEGFP-1 (a 3 kb plasmid) passed through the sclera but was accompanied by a great amount of band spreading. Following completion of the initial electrophoresis run, the plasmid DNA was extracted from the smeared bands in the agarose distal to the sclera and re-run on a second gel without sclera. The initially smeared plasmid bands resolved into 2 distinct bands after extraction and purification and matched well with control plasmid bands.


CONCLUSIONS
Charged molecules such as xylene cyanol, bromphenol blue, and DNAs ranging from 51 bp oligonucleotides to a 3 kb plasmid can be driven across human sclera by an electric field and directly detected. Passage of plasmids was efficient, but the plasmid bands were diffuse after transit. This technique offers promise as a noninvasive DNA delivery tool, where gene therapy can be accomplished by small RNA or DNA synthetic oligonucleotides, larger double stranded fragments, or even plasmids.
Davies
John B
JB
Department of Ophthalmology, Emory University School of Medicine, Atlanta, GA 30322, USA.
Ciavatta
Vincent T
VT
Boatright
Jeffrey H
JH
Nickerson
John M
JM
D000293:Adolescent; D000328:Adult; D000368:Aged; D001557:Benzenesulfonates / Q000378:metabolism*; D052160:Benzothiazoles; D001692:Biological Transport; D001978:Bromphenol Blue / Q000378:metabolism*; D004247:DNA / Q000378:metabolism*; D003959:Diamines; D004574:Electromagnetic Fields; D004587:Electrophoresis, Agar Gel; D005456:Fluorescent Dyes / Q000378:metabolism; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D008875:Middle Aged; D009930:Organic Chemicals / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011804:Quinolines; D012313:RNA / Q000378:metabolism*; D012590:Sclera / Q000378:metabolism*; D012685:Sepharose
D001557:Benzenesulfonates; D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D001978:Bromphenol Blue; C048951:xylene cyanol; C098022:SYBR Green I; D012313:RNA; D004247:DNA; D012685:Sepharose