Identification of Retinal Pigment Epithelial Cell-Targeting Peptide Ligands for Ocular Drug Delivery using One-Bead One-Compound Combinatorial Libraries

Abstract

Purpose : Intraocular drug delivery is currently limited to intravitreal injections because drugs given systemically lack cell-targeting capability. The goal of our study is to identify and characterize high-affinity peptide ligands that bind retinal pigment epithelial (RPE) cells to allow targeted ocular drug delivery from the systemic circulation.

Methods : One-bead-one-compound (OBOC) combinatorial libraries consists of 80-100 um TentaGel resin beads, each displaying ~100 pmol of a unique peptide sequence, which can be incubated with live cells in suspension, and positive beads with adherent cells recovered and decoded by microsequencing with Edman chemistry. We employed an OBOC library consisting of cyclic 7-amino-acid peptides with an arginine-glycine-aspartate (RGD) motif to target integrins which are overexpressed in RPE associated with choroidal neovascularization to identify candidate ligands that bind to the human RPE cell line ARPE-19, but not human umbilical vascular endothelial cells (HUVECs). We measured the binding affinity of biotinylated forms of candidate peptides to ARPE-19 cells using flow cytometry with a fluorophore-streptavidin conjugate. In vivo binding to healthy intact RPE cells was assessed by tail-vein injection of biotinylated candidate peptides with fluorophore-streptavidin conjugate in wild-type BALB/c mice, followed by live imaging using a custom scanning laser ophthalmoscope (SLO) mouse retinal imaging system.

Results : Five candidate peptides that specifically bind ARPE-19 cells and not HUVECs were identified by screening the OBOC library with the sequence cX₂X⁺GX^-X₆X₇c. Flow cytometry demonstrated two of these peptides to have up to 11-fold binding affinity to ARPE-19 cells as compared with a nonspecific control peptide with a scrambled sequence. In vivo SLO imaging of mouse eyes after systemic delivery of these two biotinylated peptides with Alexa Fluor 488-streptavidin conjugate showed potential increased persistence of fluorescence, compared with a nonspecific control peptide with Alexa Fluor 568-streptavidin conjugate that was injected concurrently.

Conclusions : OBOC combinatorial libraries may identify high affinity peptide ligands that target RPE cells, and potentially improve rational drug design for targeted delivery of pharmacologic agents or carriers to retinal tissues through the systemic circulation.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.