NGR Peptide 1

Peptides containing the asparagine-glycine-arginine (NGR) motif are recognized by CD13/aminopeptidase N (APN) receptor isoforms that are selectively overexpressed in tumor neovasculature. Spontaneous decomposition of NGR peptides can result in isoAsp derivatives, which are recognized by RGD-binding integrins that are essential for tumor metastasis. Peptides binding to CD13 and RGD-binding integrins provide tumor-homing, which can be exploited for dual targeted delivery of anticancer drugs. We synthesized small cyclic NGR peptide-daunomycin conjugates using NGR peptides of varying stability (c[KNGRE]-NH2, Ac-c[CNGRC]-NH2 and the thioether bond containing c[CH2-CO-NGRC]-NH2, c[CH2-CO-KNGRC]-NH2). The cytotoxic effect of the novel cyclic NGR peptide-Dau conjugates were examined in vitro on CD13 positive HT-1080 (human fibrosarcoma) and CD13 negative HT-29 (human colon adenocarcinoma) cell lines. Our results confirm the influence of structure on the antitumor activity and dual acting properties of the conjugates. Attachment of the drug through an enzyme-labile spacer to the C-terminus of cyclic NGR peptide resulted in higher antitumor activity on both CD13 positive and negative cells as compared to the branching versions.

Cyclic peptides are an attractive modality for the development of therapeutics and the identification of functional cyclic peptides that contribute to novel drug development. The peptide array is one of the optimization methods for peptide sequences and also useful to understand sequence-function relationship of peptides. Cell adherent cyclic NGR peptide which selectively binds to the aminopeptidase N (APN or CD13) is known as an attractive tumor marker. In this study, we designed and screened a library of different length and an amino acid substitution library to identify stronger cell adhesion peptides and to reveal that the factor of higher binding between CD13 and optimized cyclic peptides. Additionally, we designed and evaluated 192 peptide libraries using eight representative amino acids to reduce the size of the library. Through these optimization steps of cyclic peptides, we identified 23 peptides that showed significantly higher cell adhesion activity than cKCNGRC, which was previously reported as a cell adhesion cyclic peptide. Among them, cCRHNGRARC showed the highest activity, that is, 1.65 times higher activity than cKCNGRC. An analysis of sequence and functional data showed that the rules which show higher cell adhesion activity for the three basic cyclic peptides (cCX1 HNGRHX2 C, cCX1 HNGRAX2 C, and cCX1 ANGRHX2 C) are related with the position of His residues and cationic amino acids.

The conjugation of tunable peptides or materials with nanocarriers represents a promising approach for drug delivery to tumor cells. In this study, we report the development of a novel liposomal carrier system that exploits the cell surface binding synergism between photo-sensitive peptides (PSPs) and targeting ligands. The positive charges of the lysine residues on the cell-penetrating peptides (CPPs) were temporarily caged by the photolabile-protective groups (PG), thereby forming a PSP. Furthermore, this PSP enhances specific uptake into cancer cells after rapidly uncaging the PG via near-infrared (NIR) light illumination. In the circulatory system, the cell penetrability of PSP was hindered. In contrast, the asparagine-glycine-arginine (NGR) peptide moieties, selectively bind to CD13-positive tumors, were attached to the nanocarrier to facilitate the active accumulation of this liposomal carrier in tumor tissue. The dual-modified liposomes (PSP/NGR-L) were prepared by emulsification method, and the concentrations of DSPE-PEG2000-psCPP and DSPE-PEG5000-NGR in the liposomes were chosen to be 4% and 1% (molar ratio), respectively. The mean particle size of the PSP/NGR-L was about 95 nm, and the drug entrapment efficiency was more than 90%. Cellular uptake results demonstrated that the proposed PSP/NGR-L had an enhancement of cancer cell recognition and specific uptake. Furthermore, the PSP/NGR-L demonstrated a stronger antitumor efficacy in the HT-1080 tumor model in nude mice with the aid of NIR illumination.