Human α-Lactalbumin made lethal to tumor cells (HAMLET), and its bovine analogue BAMLET, have in the recent years shown promising results in cancer therapy. HAMLET contains several oleic acids which in turn stabilize a partially unfolded conformational state of the protein, inducing a higher surface activity compared to the native protein, and likely play a role in its cancer-detrimental function as well. Herein, we report the formation of gold-bovine α-lactalbumin nanoconstructs (Au–LA NCs) displaying conformational changes in the native protein and its concomitant ability to reduce HeLa cell viability approaching that of BAMLET. Modification of LA with gold was achieved via two synthesis protocols; (i) utilizing the intrinsic reduction potential of LA (Au–LAint) or (ii) the addition of an extrinsic reducing agent (Au–LAext). The gold–protein nanostructures formed were investigated using a palette of analytical probes including AFM, TEM, ζ-potential, UV–vis, circular dichroism, and fluorescence (steady-state and time-resolved). Toxicity toward HeLa cells was studied using a trypan blue assay and benchmarked against BAMLET. Whereas constructs from both synthetic protocols employed result in conformational changes of the protein and altered surface activity compared to the native protein, Au–LAint was found to display lipid-specific interaction and severe disruption of lipid monolayers. Au–LAint also revealed toxicity toward HeLa-cells comparable to that of BAMLET. The results imply that formation of Au–LA nanoconstructs could be a new route to making HAMLET-like materials, while also imparting a built-in optical probe from fluorescent or plasmonic gold species.

HAMLET/BAMLET (Human/Bovine α-Lactalbumin Made Lethal to Tumors) is a tumoricidal substance composed of partially unfolded human/bovine α-lactalbumin (HLA/BLA) and several oleic acid (OA) molecules. The HAMLET mechanism of interaction involves an insufficiently understood effect on the membrane or its embedded components. We examined the effect of BLAOA (bovine α-lactalbumin complexed with oleic acid, a HAMLET-like substance) and its individual components on cells and artificial lipid membranes using viability staining and metabolic dyes, fluorescence spectroscopy, leakage integrity assays and microscopy. Our results show a dose-dependency of OA used to prepare BLAOA on its ability to induce tumor cell death, and a correlation between leakage and cell death. BLAOA incorporates into the membrane, tightens the lipid packing and lowers their solvent accessibility. Fluorescence imaging reveals that giant unilamellar vesicles (GUVs) develop blebs and eventually collapse upon exposure to BLAOA, indicating that the lipid packing reorganization can translate into observable morphological effects. These effects are observed to be local in GUVs, and a tightly packed and solvent-shielded lipid environment is associated with leakage and GUV disruption. Furthermore, the effects of BLAOA on membrane are pH dependent, with an optimum of activity on artificial membranes near neutral pHs. While BLA alone is effective at membrane disruption at acidic pHs, OA is ineffective in a pH range of 4.5 to 9.1. Taken together, this supports a model where the lipid, fatty acid and protein components enhance each other's ability to affect the overall integrity of the membrane.

Helix A and -C of α-lactalbumin, a loosely folded amphitropic protein, perturb lipid monolayers by the formation of amyloid pore-like structures. To investigate whether these helices are able to disrupt fully formed bilayers, we designed peptides comprised of Helix A and -C, and investigated their membrane-perturbing properties. The peptides, designated A-Cage-C and A-Lnk-C, were prepared with tryptophan sites in the helical and the spacer segments in order to monitor which part were involved in membrane association under given conditions. The peptides associate with and disrupt negatively charged bilayers in a pH-dependent manner and α-helical tendencies increased upon membrane association. Both helices and the spacer segment were involved in membrane binding in the case of A-Lnk-C, and there are indications that the two helixes act in synergy to affect the membrane. However, the helices and the spacer segment could not intercalate when present as A-Cage-C at neutral conditions. At acidic pH, both helices could intercalate, but not the central spacer segment. AFM performed on bilayers under aqueous conditions revealed oligomers formed by the peptides. The presence of bilayers and acidic pHs were both drivers for the formation of these, suggestive of models for peptide oligomerization where segments of the peptide are stacked in an electrostatically favorable manner by the surface. Of the two peptides, A-Lnk-C was the more prolific oligomerizer, and also formed amyloid-fibril like structures at acidic pH and elevated concentrations. Our results suggest the peptides perturb membranes not through pore-like structures, but possibly by a thinning mechanism.

Human α-lactalbumin made lethal to tumor cells (HAMLET) is a tumoricidal complex consisting of human α-lactalbumin and multiple oleic acids (OAs). OA has been shown to play a key role in the activity of HAMLET and its related complexes, generally known as protein-fatty acid (PFA) complexes. In contrast to what is known about the fate of the protein component of such complexes, information about what happens to OA during their action is still lacking. We monitored the membrane, OA and protein components of bovine α-lactalbumin complexed with OA (BLAOA; a HAMLET-like substance) and how they associate with each other. Using ultracentrifugation, we found that the OA and lipid components follow each other closely. We then firmly identify a transfer of OA from BLAOA to both artificial and erythrocyte membranes, indicating that natural cells respond similarly to BLAOA treatment as artificial membranes. Uncomplexed OA is unable to similarly affect membranes at the conditions tested, even at elevated concentrations. Thus, BLAOA can spontaneously transfer OA to a lipid membrane. After the interaction with the membrane, the protein is likely to have lost most or all of its OA. We suggest a mechanism for passive import of mainly uncomplexed protein into cells, using existing models for OA's effect on membranes. Our results are consistent with a membrane destabilization mediated predominantly by OA insertion being a significant contribution to PFA cytotoxicity.

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