Cellular Uptake of Modified HIV–1 Tat Permeation Peptide Constructs Conjugated to Alexa Fluor–594in the Rodent Posterior Segment in an Ex–Vivo Model

Abstract

Abstract: : Purpose:Polypeptide sequences of charged amino acids known as permeation peptide domains have been shown to enhance uptake of impermeant molecules into cells. As such, they may be useful for translocating substrates into cells. The eye is an attractive target for this strategy given its accessibility. We examined the uptake of novel permeation peptide constructs based on the HIV–1 Tat basic domain in the rodent posterior segment in an ex–vivo model. Methods:Three different permeation peptide constructs were used. All three were conjugated at the C–terminus to Alexa Fluor–594 to enable visualization of cellular uptake. D–isomer amino acid peptides were used, as this has been shown to increase cellular uptake in in vitro models. D–Tat was based on the wildtype HIV–1 Tat sequence (Tat _47–60). D–Tat–Orn has substitution of ornithine for glutamine, a change also found to increase cellular uptake. A third construct was based on a peptide sequence associated with poor cellular uptake and served as a negative control. Eyes were enucleated from euthanized Brown Norway rats and the posterior segments incubated in micromolar concentrations of the above permeation peptide constructs for durations up to 1 hour at room temperature. After washes, the tissue was fixed, embedded in paraffin, cut into sections and stained with DAPI. Results:Sections were found to contain fluorescent staining of cells in both the retinal ganglion cell layer and inner nuclear layer of the retina. Labeling in the inner nuclear layer was specific, being restricted to a subset of the innermost cells. No labeling was noted in the outer nuclear or photoreceptor layers. Uptake occurred rapidly and was essentially complete within the first 20 minutes of incubation. Fluorescence was also noted in the ciliary epithelium. Specific cellular localization was confirmed using immunolabeling of retinal cellular markers. No uptake was seen with the negative control. Conclusions:Modified HIV–1 Tat permeation peptide constructs conjugated to Alexa Fluor–594 were taken up specifically by neurons in the inner retina, as well as by the ciliary epithelium. Future studies using animal models as well as human retina should reveal whether this also occurs in vivo or is species–specific. Since various moieties can be conjugated to these permeation peptide constructs, they may be useful for the delivery of therapeutic agents to the retina or ciliary epithelium or for in vivo imaging techniques.

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • ciliary body • microscopy: light/fluorescence/immunohistochemistry