A newly discovered "Protein Transduction Domain": Is it possible to function as a biomolecule delivery system into the intraocular tissues ?

Abstract

Abstract: : Purpose: A new Protein Transduction Domain (PTD) of 11 amino acids(aa) was discovered from a transcription repressor of mouse. Intracellular delivery to many types of cells or tissues was observed in vitro and in vivo. The aim of this study was to evaluate the possible role of PTD as a biomolecule delivery motif across the blood retinal barrier and into the retinal tissue. Methods: PTD–Fluorescein isothiocyanate (FITC) was constructed with peptide synthesizer. Male Sprague–Dawley rats weighing 150g to 200g were used. PTD–FITC were dissolved in 1% PBS (pH 7.4), at 5uM for topical instillation and 1mM for systemic administration. To examine the penetrating patterns with topical application, the rat was sacrificed and enucleated respectively at 10, 30, 60, 120 minutes, and 24 hours after 10ul instillation. After fixation with 4% paraformaldehyde, the eyecups were embedded in OCT Compound, and frozen sections were made at 4um. After mounted and covered, sections were examined by confocal microscope. For the evaluation of those with systemic application, the same procedures were done in the same time–dependent manner after 1ml intraperitoneal injection. A randomly designed peptide (of 11 aa)–FITC and FITC alone were used for the control studies. Results: Except the sample at 10 minutes, Fluorescences were detected in the all samples with topical application. In the sample at 30 minutes, fluorescence was mainly localized to the sclerochoroidal tissue, but, weakly detected on the RPE and outer nuclear layer. At 120 minutes, fluorescence was more evenly distributed over the chorioretinal tissue, evenly to the ganglion cell layer. In the samples with the systemic injection, the fluorescences were more evenly distributed at 30, 60, 120 minutes. Fluorescences were not detected in the samples tested with two control compounds. Conclusions: These results suggest that the newly discovered peptide is capable of an excellent transduction into the ocular tissues and PTD–mediated biomolecule delivery may be a good therapeutic modality in the ophthalmological fields.

Keywords: protein structure/function • drug toxicity/drug effects • microscopy: confocal/tunneling