Investigation of the TAT transporter as a carrier for ocular peptide delivery and molecular therapy

Abstract

Abstract: : Purpose: To investigate the D/L–TAT peptide sequence originating from HIV as a molecular transporter to various ocular tissues Methods: FITC–labelled L–TAT and its retro–inverso form D–TAT peptides were synthesized and used at concentrations ranging from 0.1 to 1 mM. The following routes were tested for delivery efficiency: whole enucleated eyes, intra–vitreous and sub–retinal injections and corneal deposit in NMRI and VPP mice. Animals were sacrificed at 2 hrs, 16 hrs, 2 days or 1 week after treatment and the presence of the labelled–TAT was observed by fluorescence microscopy after eye fixation Results: In the cornea of enucleated eyes, nuclei in the epithelium, keratocytes and endothelium show strong fluorescence after 16 hrs incubation. Fluorescence is also seen in the stroma. In the retina, strong fluorescence is observed in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL). The outer segments also showed fluorescence. Intra–vitreous injection of 1 µl is also effective with fluorescence observed in the ganglion cell and inner nuclear layers. Fluorescence in the outer nuclear cell layer was weak, indicative of a gradient of TAT action. At the concentration used, no fluorescence was observed in the cornea. In subretinal injections (1 µl), labelled TAT was localized around the injection site with fluorescence observed in ONL, INL and GCL. No spreading of fluorescence was observed. This pattern was still present after 7 days. Repeated deposits of 1 mM D–TAT on the cornea of anesthetised animals twice a day during 3 days with analysis 24 hrs later showed no fluorescence in the corneal layers Conclusions: The L/D–TAT peptide represents a very effective molecular delivery system. D/L–TAT effectiveness is the highest in the enucleated eye and in intra–vitreous experiments which suggests good intracellular transport when time of exposure is adequate. Subretinal route is also effective but has limited expansion capacity. In addition, the D–TAT enantiomere showed long lasting activity. This molecular delivery system represents a potential tool for intraocular transportation

Keywords: retina • vitreous • neuroprotection