Molecular insights into the interaction of 5-fluorouracil and Fe3O4 nanoparticles with beta-casein: An experimental and theoretical study

We investigated the potential carrier of milk beta-casein (β-CN) and its interactions with 5-fluorouracil (5-FU) and iron oxide nanoparticles (Fe3O4 NPs). We used different spectroscopic methods of fluorescence, UV-Visble, circular dichroism (CD), synchronous fluorescence, zeta potential assay, and computational studies to clarify the protein interaction with 5-FU and Fe3O4 NPs. The fluorescence data indicated both Fe3O4 NPs and 5-FU could quench the intrinsic fluorescence of β-CN. Fluorescence measurements showed that the single interaction of β-CN with 5-FU or Fe3O4 NPs was static, while reacted β-CN with both 5-FU and Fe3O4 NPs simultaneously showed a dynamic quenching. Synchronous fluorescence data in both tests revealed that the tryptophan (Trp) residue of β-CN had a dominant role in quenching and the polarity of its microenvironment more than tyrosine (Tyr) increased in interaction with 5-FU. All the binding sites and thermodynamic parameters were obtained at 25, 37, and 42 °C. The analysis of thermodynamic parameters and Job's plot techniques pointed to that both of these complexes with the 1:1 M ratio were exothermic (ΔH°<0) driven with the van der Waals and H-bonding interactions (in agreement with the docking results). The CD spectra in the region of far-UV and thermal denaturation study indicated minor changes in the secondary structure of β-CN in the presence of various concentrations of Fe3O4 NPs and 5-FU. Also, from the molecular dynamics (MD) analysis, as a result, the protein structure was stable during 100 ns. The outcomes highlighted that β-CN protein could form a great bind with 5-FU and Fe3O4 NPs ligands (supporting the zeta potential assay results) by independent binding sites. These results would be helpful insight to construct a potential magnetic nanocarrier β-CN base for 5-FU drug delivery.