Development of HAMLET-like Cytochrome c-Oleic Acid Nanoparticles for Cancer Therapy

Abstract

In the literature, HAMLET (Human Alpha-lactalbumin Made LEthal to Tumor cells) is described as a tumoricidal complex composed of oleic acid (OA) non-covalently bound to α-lactalbumin. We recently demonstrated that OA is the real drug in this complex and that the protein solely serves as the drug carrier. We hypothesized that by replacing α-lactalbumin with a bioactive protein it should be possible to synergistically increase the efficiency of the complex. Consequently, we developed a HAMLET-like complex composed of OA coupled to the apoptosis-inducing protein cytochrome c (Cyt c). As control we coupled OA to the non-toxic protein bovine serum albumin (BSA). The syntheses of HAMLET-like Cyt c-OA and BSA-OA complexes micelles were performed at pH 8 and 45°C and we loaded 10 and 53 molecules of OA per molecule of Cyt c and BSA, respectively. We found that OA binding promotes protein structural changes characteristic of the protein-OA interactions in HAMLET. Cyt c-OA and BSA-OA complexes had a circular shape and a diameter of 123 and 169 nm, respectively. Cell viability tests showed less than 10% of viability after cancer cell (HeLa and A-549) incubation for 6 h with Cyt c-OA, while normal cells (Cho-K1 and NIH/3T3) showed more than 20% of viability. BSA-OA killed both cell types with less efficiency and no selectivity. The Cyt c-OA complex showed 50% of caspase-3 and caspase-9 activation in a cell-free assay while BSA-OA lacked any caspase activation. Confocal micrographs showed morphological changes indicative of late- and early-apoptosis by the action of Cyt-OA and BSAOA, respectively. This study demonstrates that using Cyt c increases the potency of OA in HAMLET-like complexes.