Novel peptide ligand directs liposomes toward EGF‐R high‐expressing cancer cells in vitro and in vivo

Abstract

Epidermal growth factor receptor (EGF-R) is an important target in anticancer therapy. Here we report how a novel EGF-R peptide ligand (D4: Leu-Ala-Arg-Leu-Leu-Thr) is identified using a computer-aided design approach from a virtual peptide library of putative EGF-R binding peptides by screening against the EGF-R X-ray crystal structure in silico and in vitro. The selected peptide is conjugated with a polyethylene glycol (PEG) lipid, and the lipid moiety of the peptide-PEG-lipid conjugate is inserted into liposome membranes by a postmodification process. D4 peptide-conjugated liposomes are found to bind to and enter cells by endocytosis specifically and efficiently in vitro in a process apparently mediated by EGF-R high-expressing cancer cells (H1299). In vivo, the D4 peptide-conjugated liposomes are found to accumulate in EGF-R-expressing xenograft tumor tissues up to 80 h after intravenous delivery, in marked contrast to controls. These results demonstrate how structure-based peptide design can be an efficient approach to identify highly novel binding ligands against important receptors. These data could have important consequences for the development of peptide-directed drug delivery systems with engineered specificities and prolonged times of action.

A novel small peptide as an epidermal growth factor receptor targeting ligand for nanodelivery in vitro

“…Screening of phage display libraries, 8,23 CAD, 10 hydropathic complementarity, 24 hydropathic analysis, and comparative sequence/structure analysis 7 have been used to design small peptide ligands. In this study, we demonstrated different methods of screening for small molecule peptide ligands.…”

“…LARLLT, which is a ligand that specifically binds to EGFR receptors, has been identified using a CAD approach. 10 The LARLLT peptide-conjugated liposomes could bind to the highly expressing EGFR cells (NCI-H1299) and efficiently enter the cells via endocytosis. 10 Our flow cytometric analysis showed that the binding rate of the FITC-AEYLR peptide with the NCI-H1299 cells was higher than that of the FITC-LARLLT peptide.…”

“…Currently, small peptides are pursued as targeting moieties because of their small size, low immunogenicity, and their ability to be incorporated into certain delivery vectors. [7][8][9][10][11][12] The epidermal growth factor receptor (EGFR) is a transmembrane protein on the cell surface that is overexpressed in a wide variety of human cancers. EGFR is an effective tumor-specific target because of its significant functions in tumor cell growth, differentiation, and migration.…”

“…[13][14][15] EGFR-targeted small molecule peptides such as YHWYGYTPQNVI have been successfully identified using phage display library screening; 8 by contrast, the peptide LARLLT has been generated using computerassisted design (CAD). 10 These peptides can be conjugated to the surfaces of liposomes that are then delivered selectively to tumors by the specific and efficient binding of these peptides to cancer cells that express high levels of EGFR.…”

“…17 We designed 5-to 6-mer synthetic peptides that were derived from the amino acid sequences of these sites (EY 1068 INQ, PDY 1148 QQD, and AEY 1173 LR, respectively). We then studied the ability of these peptides and NYQQN 18 to target EGFR in vitro, using LARLLT 10 as a positive control and RALEL (AEY 1173 LR, substituted and scrambled) as a negative control. …”