We investigated the use of amphiphilic, protease-cleavable peptides as encapsulation moieties for hydrophobic metallodrugs, in order to enhance their bioavailability and consequent activity. Two hydrophobic, gold-containing anticancer agents varying in aromatic ligand distribution (Au(I)-N-heterocyclic carbene compounds 1 and 2) were investigated. These were encapsulated into amphiphilic decapeptides that form soluble filamentous structures with hydrophobic cores, varying supramolecular packing arrangements and surface charge. Peptide sequence strongly dictates the supramolecular packing within the aromatic core, which in turn dictates drug loading. Anionic peptide filaments can effectively load 1, and to a lesser extent 2, while their cationic counterparts could not, collectively demonstrating that loading efficiency is dictated by both aromatic and electrostatic (mis)matching between drug and peptide. Peptide nanofilaments were nontoxic to cancerous and noncancerous cells. By contrast, those loaded with 1 and 2 displayed enhanced cytotoxicity in comparison to 1 and 2 alone, when exposed to Caki-1 and MDA-MB-231 cancerous cell lines, while no cytotoxicity was observed in noncancerous lung fibroblasts, IMR-90. We propose that the enhanced in vitro activity results from the enhanced proteolytic activity in the vicinity of the cancer cells, thereby breaking the filaments into drug-bound peptide fragments that are taken up by these cells, resulting in enhanced cytotoxicity toward cancer cells.

Peptide materials are promising for various biomedical applications; however, a significant concern is their lack of stability and rapid degradation in vivo due to non-specific proteolysis. For materials specifically designed to respond to disease-specific proteases, it would be desirable to retain high susceptibility to target proteases while minimizing the impact of non-specific proteolysis. We describe N-terminal acetylation as a simple synthetic modification of amphiphilic self-assembling peptides that contain an MMP-9-cleavable segment and form soluble, nanoscale filaments. We found that the N-terminus capping of these peptides did not significantly impact their self-assembly behavior, critical aggregation concentration, or ability to encapsulate hydrophobic payloads. By contrast, their proteolytic stability in human plasma (especially for anionic peptide sequences) was considerably increased while susceptibility to hydrolysis by MMP-9 was retained when compared to non-acetylated peptides, especially during the first 12 h. We note, however, that due to the longer time scale required for in vitro studies (72 h), non-specific proteolysis of both anionic acetylated peptides leads to similar activity in vitro despite differing MMP-9 kinetics during the early stages. Overall, the enhanced stability against non-specific proteases, combined with the ability of these nanofilaments to enhance the effectiveness of gold-based drugs toward cancerous cells compared to healthy cells, brings these acetylated peptide filaments a step closer toward clinical translation.