Influence of PEG Length on Conformational and Binding Properties of CCK Peptides Exposed by Supramolecular Aggregates

Five novel peptide amphiphiles, PA-[700] ÷ PA-[3000], with common formula (C18)2-PEGx-CCK8 are described as potential target selective nanocarriers towards tumor cells overexpressing cholecistokynin receptors. DLS measurements indicate that PA-[700] ÷ PA-[2000] self-assemble in in supramolecular aggregates with a hydrodynamic radius ranged between 63 and 83 nm, while PA-[3000] shows a RH of 104 nm with an increase of ≈ 30% with respect to the other peptide derivatives. Fluorescence studies suggested that, irrespective from the PEG length on the PA, the tryptophan residue located at the center of the CCK8 sequence, is completely surrounded from water molecules at high mobility. This result indicates a potential capability of all formulated nanovectors to recognize the over-expressed CCK-2 receptors. Also CD data suggest that CCK8 peptide in all PAs in their aggregate form, with the exception of PA-[700], adopt a conformation allowing the interaction with the receptor. Anyway biological data obtained by flow cytometry analysis indicate that the five PAs with different PEG spacers have a different behavior in the binding ability of PA towards to the CCK2-R, with higher binding properties shown by PA-[2000]. Finally, proteolitic cleavage experiments indicate that in PA-[2000] supramolecular aggregates the CCK8 peptide present the Tyr-Met and Trp-Met cleavable amide bonds available for the enzymatic process, while in PA-[3000] the longer PEG moiety masks the active site in CCK8, thus preventing both enzymatic cleavage of the Tyr-Met bond and receptor interaction. Based on these results, PEG2000 can be considered as the best spacer for CCK8 peptide amphiphiles.