Synthesis, characterization, and biological activity of poly(arginine)-derived cancer-targeting peptides in HepG2 liver cancer cells

Abstract

The solid-phase synthesis, structural characterization, and biological evaluation of a small library of cancer-targeting peptides have been determined in HepG2 hepatoblastoma cells. These peptides are based on the highly specific Pep42 motif, which has been shown to target the glucose-regulated protein 78 receptors overexpressed and exclusively localized on the cell surface of tumors. In this study, Pep42 was designed to contain varying lengths (3-12) of poly(arginine) sequences to assess their influence on peptide structure and biology. Peptides were effectively synthesized by 9-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis, in which the use of a poly(ethylene glycol) resin provided good yields (14-46%) and crude purities >95% as analyzed by liquid chromatography-mass spectrometry. Peptide structure and biophysical properties were investigated using circular dichroism spectroscopy. Interestingly, peptides displayed secondary structures that were contingent on solvent and length of the poly(arginine) sequences. Peptides exhibited helical and turn conformations, while retaining significant thermal stability. Structure-activity relationship studies conducted by flow cytometry and confocal microscopy revealed that the poly(arginine) derived Pep42 sequences maintained glucose-regulated protein 78 binding on HepG2 cells while exhibiting cell translocation activity that was contingent on the length of the poly(arginine) strand. In single dose (0.15 mM) and dose-response (0-1.5 mM) cell viability assays, peptides were found to be nontoxic in human HepG2 liver cancer cells, illustrating their potential as safe cancer-targeting delivery agents.

Cartilage penetrating cationic peptide carriers for applications in drug delivery to avascular negatively charged tissues

“…Varying lengths of arginine-based Cell Penetrating Peptides (CPPs) have been used for intra-cellular delivery of drugs and genetic materials [57, 58]. It has been observed that peptides of six or more arginine (or guanidinium groups) were necessary for uptake into the cells.…”

Heterogeneous Catalysis for Azide-Alkyne Bioconjugation in Solution Via Spin Column: Attachment of Dyes and Saccharides to Peptides and DNA

“…However, optimal coupling conditions may not be easily found, which may be exacerbated by the bulky 2,2,4,6,7-pentamethyldihydrobenzofurane (Pbf) protecting group commonly used for arginine during solid-phase synthesis (26). Further, solution-phase coupling schemes would likely require subsequent purification by HPLC (27, 28), while the reaction itself can be complicated by the high charge density and secondary structures displayed by poly-Arg (29, 30). …”

Solid-Phase Peptide Synthesis and Structural Analyses by Circular Dichroism Spectroscopy of the Cytotoxic D-(KLAKLAK)2 Sequence

“…These types of amphiphilic peptides have been shown to aggregate during the course of peptide synthesis on the non-polar polystyrene-based resins causing diminished purities and yields [5]. Therefore, the polar poly (ethylene) glycol (PEG) resin which was shown to afford excellent crude purities and yields of synthetically challenging amphiphilic peptide sequences was adopted for our solid-phase synthesis strategy [6] (Scheme 1).…”

“…Chlorobenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluorophosphate, HCTU, as activator and coupling reagent, N-methylmorpholine, NMM, as base in N,N-dimethylformamide, DMF, as solvent for the coupling reactions [5]. The deprotection steps of the fluorenylmethoxycarbonyl, Fmoc, group were completed with piperidine in DMF.…”