In recent years, biomedical chemists have been interested in macrocyclic peptides because of their properties and pharmaceutical potential. A macrocyclic backbone provides unique stability and these peptides can be used as scaffolds for drug design applications or for protein engineering.

A family of macrocyclic peptides named PawS-Derived Peptides (PDPs) is produced from precursors of seed storage albumins in species of the sunflower family. Here we examined the cell-penetrating properties of a novel and unusually large PDP recently found in the seeds of Zinnia elegans. PDP-23 is the first macrocyclic peptide known to possess two disulfide bonds. Being twice the size of most PDPs it adopts a unique conformation in which two β-hairpins, each stabilized by one of the disulfide bonds, fold on top of each other enclosing a hydrophobic core. The structure is highly thermostable and resistant to proteolytic degradation in serum and simulated gastric and intestinal fluids. However, the tertiary structure is not covalently cross linked and the peptide can change conformation in membrane mimicking environment, suggesting it may be able to pass membranes. In this work, PDP-23 was chemically synthesized and labelled with ALEXA-488 allowing monitoring of entry and peptide localization in various cell lines. Furthermore, the PDP-23 penetration study under the confocal microscope compared with SFTI and MCOI-II as cyclic cell penetrating peptides. The peptide cellular uptake was also quantified using flow cytometry. Moreover, the cytotoxic investigation revealed no toxic activity on the tested cells.