The cytotoxicity of BAMLET complexes is due to oleic acid and independent of the α-lactalbumin component

•We synthesized three different BAMLET complexes consisting of oleic acid coupled to bovine α-lactalbumin.•Oleic acid micelles alone are tumoricidal at equimolar concentrations of oleic acid bound in the BAMLET complexes.•α-Lactalbumin is non-toxic to cells even when delivered to their cytoplasm.•Both, BAMLET and oleic acid micelles showed no selective cytotoxicity to cancer cells.

Lipid–protein complexes comprised of oleic acid (OA) non-covalently coupled to human/bovine α-lactalbumin, named HAMLET/BAMLET, display cytotoxic properties against cancer cells. However, there is still a substantial debate about the role of the protein in these complexes. To shed light into this, we obtained three different BAMLET complexes using varying synthesis conditions. Our data suggest that to form active BAMLET particles, OA has to reach critical micelle concentration with an approximate diameter of 250 nm. Proteolysis experiments on BAMLET show that OA protects the protein and is probably located on the surface, consistent with a micelle-like structure. Native or unfolded α-lactalbumin without OA lacked any tumoricidal activity. In contrast, OA alone killed cancer cells with the same efficiency at equimolar concentrations as its formulation as BAMLET. Our data show unequivocally that the cytotoxicity of the BAMLET complex is exclusively due to OA and that OA alone, when formulated as a micelle, is as toxic as the BAMLET complex. The contradictory literature results on the cytotoxicity of BAMLET might be explained by our finding that it was imperative to sonicate the samples to obtain toxic OA.

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