Abstract
Abstract: :
Purpose: Oligonucleotides (ODN), designed for antisense or genoplasty strategies have the advantages to exert specific activities in both anterior and posterior segment eye diseases. One major limiting step of their clinical applications is the lack of appropriate and efficient system of delivery to the involved eye tissues. Particularly, anti VEGF R2 ODN could be of interest for the inhibition of corneal neo vascularization. Our purpose was to evaluate the potential of iontophoresis to deliver an anti VEGF R2 ODN in the anterior segment eye tissues and study its distribution, kinetics and fine localization after either topical application or trans-corneoscleral iontophoresis in normal rat eyes and in rats with neo vascularized corneas. Methods: CY5-labeled ODNs in PBS [60µM] were administered in the anterior segment of the rat eye by corneoscleral iontophoresis (300µA, 5min). Control animals received ODN applications without any current. Acutely, 90min and 24h after a single application, the eyes were either snap frozen and processed for cryo-section and histochemistry or dissected for flat mounting and examination of the corneas by confocal microscopy. Integrity of the ODN was evaluated on agarose gels. Results: Topically applied ODNs did not penetrate into the eye tissues. They remained confined in the superficial epithelial layers of the cornea acutely and at 90min only. When administered by transcorneoscleral iontophoresis, ODNs penetrated the anterior segment through the limbus, to all parts of the cornea, and to the iris. In the corneal epithelium, fluorescence was present in the intercellular space, in the cytoplasm and in nuclei. 24h after administration, the ODNs were mainly localized in the Descemet membrane of the cornea, and to a lesser extent in the corneal endothelium and epithelium. No lesion resulted from the current application. When iontophoretically administered to neovascularized eyes, ODNs were localized at 2h in the iris, the corneal stroma and endothelium. Double-staining with Von-Willebrand antibodies showed that ODNs concentrated in the cornea, in infiltrating cells and in endothelial cells. Extraction showed intact ODNs in the cornea and in the iris/ciliary body, 24h after administration. Conclusions: Iontophoresis allowed the intracellular delivery of intact ODNs in the epithelial and endothelial cells of the cornea and in the iris. ODNs could be delivered to the vascular endothelium in neo vascularized cornea, demonstrating the potential of this technique for the inhibition of corneal neo vessels.
Keywords: neovascularization • gene transfer/gene therapy • anterior segment