Gene Delivery Using Physical Methods - Challenges in Delivery of Therapeutic Genomics and Proteomics - page 105

Among the three mechanisms proposed for iontophoresis, electromigration is
expected to be the most dominant driving force for charged macromolecules such as
RNA, siRNA, and plasmid DNA. SiRNA and oligonucleotide (ODN), which are highly
negatively charged and have high charge-to-molecular weight ratios, are good candidates
for iontophoretic delivery [28] . �ow intraocular ODN penetration and transfection effi-
ciency after topical administration of ODN to the eye, even after combining with polox-
amer or liposomes, further motivates the iontophoretic study of these molecules [247] .
Iontophoresis has been a widely useful technique in corneal gene therapy, espe-
cially transcorneal iontophoresis [248] . Instillation of 6-FAM-labeled ODN on the
rabbit eye followed by transcorneal iontophoresis led to the detection of ODN within
the aqueous humor, the vitreous, and the retina 5, 10, and 20 min after treatment,
without any ocular toxicity [249] . The gene expression was confirmed with topical
application of a GFP-encoding plasmid followed by iontophoresis to produce protein
expression in cornea, the anterior chamber angle, and the ciliary subepithelial tis-
sues [250] . Cy3-labeled GAPDH siRNA was also successfully delivered into the cor-
neal epithelium of mouse eyes by 1-min application of 0.5 mA iontophoresis [250] .
In another study, single-stranded cyanine 5 (Cy5)-labeled antisense ODN targeting
VEGF-R2 receptor was delivered to all corneal layers of rat eyes using 0.3 mA ion-
tophoresis for 5 min (10A). Plasmid DNA, probably because of its large molecular
size, could not be iontophoretically delivered into the epithelial layer thus indicat-
ing size-dependent corneal iontophoretic effectiveness of the macromolecules to be
delivered [250] . Intraocular concentrations of ODNs in the iris/ciliary body were
achieved after trancorneoscleral iontophoresis within 1 h of treatment. ODNs were
observed in the retina and choroid at 6 h [251] . The study demonstrated that ionto-
phoresis of an antisense ODN directed against NOS2 protein downregulated its
expression in the iris/ciliary body of rat eyes with endotoxin-induced uveitis (EIU).
Nitrite production in the aqueous humor of the treated eyes was also significantly
reduced [11] . The mechanism of ODN entry in the eye was further studied with
fluorescently labeled phosphorotioate anti-VEGFR2 ODN [250] . The fluorescent
ODNs were rapidly detected in the corneal epithelium, corneal endothelium, and iris.
However, penetration to the more central corneal layers was found to be delayed. The
transpalpebral iontophoresis has also been used with low-current saline iontophoresis
(100 A/mm 2 for 10 min) for enhancing intraretinal delivery of fluorescent ODNs in
mouse pups [252] . The ODNs were injected into the vitreous either before or after
iontophoresis. The treatment showed no structural damage to the ocular tissues, with
enhanced penetration of ODNs in photoreceptor nuclei. The combination treatment
of iontophoresis and EP has also been found to be more effective in delivering and
retaining siRNA in the cornea compared to iontophoresis or EP [253] alone with pre-
dominantly corneal epithelial localization of siRNA.
3.9 Magnetofection
Various physical methods of gene delivery have been developed, and each one has
its own merits and demerits. EP is particularly important for introducing DNA to