Application of cysteinyl prolyl ester for the synthesis of cyclic peptides containing an RGD sequence and their biological activity measurement.

Cysteinyl RGD-peptidyl cysteinyl prolyl esters, which have different configurations at the cysteine and proline residues, were synthesized by the solid-phase method and cyclized by the native chemical ligation reaction. Cyclization efficiently proceeded to give cyclic peptides, regardless of the difference in the configuration. The peptides were further derivatized to the corresponding desulfurized or methylated cyclic peptides at the Cys residues. The inhibition activity to αvß6 integrin binding was then analyzed by ELISA. The results showed that the activity varied depending on the difference in the configuration and modification of the cysteinyl prolyl ester (CPC) moiety, demonstrating the usefulness of this method in the search for a good inhibitor of the protein-protein interaction.

Self-Assembled Polypeptide Nanogels with Enzymatically Transformable Surface as a Small Interfering RNA Delivery Platform.

Nanometer-size gel particles, or nanogels, have potential for delivering therapeutic macromolecules. A cationic surface promotes cellular internalization of nanogels, but undesired electrostatic interactions, such as with blood components, cause instability and toxicities. Poly(ethylene glycol) coating has been used to shield charges, but this decreases delivery efficiency. Technical difficulties in synthesis and controlling molecular weights make it unfeasible to, instead, coat with biodegradable polymers. Our proposed solution is cationized nanogels enzymatically functionalized with branched polysaccharide chains, forming a shell to shield charges and increase stability. Biodegradation of the polysaccharides by an endogenous enzyme would then expose the cationic charges, allowing cellular internalization and cargo delivery. We tested this concept, preparing maltopentaose functionalized cholesteryl poly(l-lysine) nanogel and using tandem enzymatic polymerization with glycogen phosphorylase and glycogen branching enzyme, to add branched amylose moieties, forming a CbAmyPL nanogel. We characterized CbAmyPL nanogels and investigated their suitability as small interfering RNA (siRNA) carriers in murine renal carcinoma (Renca) cells. The nanogels had neutral ζ potential values that became positive after degradation by α-amylase. Foster resonance energy transfer demonstrated that the nanogels formed stable complexes with siRNA, even in the presence of bovine serum albumin and after α-amylase exposure. The nanogels, with or without α-amylase, were not cytotoxic. Complexes of CbAmyPL with siRNA against vascular endothelial growth factor (VEGF), when incubated alone with Renca cells decreased VEGF mRNA levels by only 20%. With α-amylase added, however, VEGF mRNA knockdown by the siRNA/nanogels complexes was 50%. Our findings strongly supported the hypothesis that enzyme-responsive nanogels are promising as a therapeutic siRNA delivery platform.