With the development of nanotechnology, there is growing interest in using nanoparticles (NPs) for biomedical applications, such as diagnostics, drug delivery, imaging, and nanomedicine. The protein's structural stability plays a pivotal role in its functionality, and any alteration in this structure can have significant implications, including disease progression. Herein, we performed a combined experimental and computational study of the effect of gold NPs with a diameter of 5 nm (5 nm Au-NPs) on the structural stability of bovine serum albumin (BSA) protein in the absence and presence of NaCl salt. Circular dichroism spectroscopy showed a loss in the secondary structure of BSA due to the synergistic effect of Au-NPs and NaCl, and Thioflavin T fluorescence assays showed suppressed β-sheet formation in the presence of Au-NPs in PBS, emphasizing the intricate interplay between NPs and physiological conditions. Additionally, molecular dynamics (MD) simulations revealed that 5 nm Au-NP induced changes in the secondary structure of the BSA monomer in the presence of NaCl, highlighting the initial binding mechanism between BSA and Au-NP. Furthermore, MD simulations explored the effect of smaller Au-NP (3 nm) and nanocluster (Au-NC with the size of 1 nm) on the binding sites of the BSA monomer. Although the formation of stable BSA-Au conjugates was revealed in the presence of NPs of different sizes, no specific protein binding sites were observed. Moreover, due to its small size, 1 nm Au-NC decreased helical content and hydrogen bonds in the BSA monomer, promoting protein unfolding more significantly. In summary, this combined experimental and computational study provides comprehensive insights into the interactions among Au nanosized substances, BSA, and physiological conditions that are essential for developing tailored nanomaterials with enhanced biocompatibility and efficacy.

The use of organic solvents in drug delivery systems (DDSs) either to produce albumin nanoparticles or to manipulate the binding of target molecules to albumin, a promising nanocarrier material, presents challenges due to the conformational changes induced in the protein. In this study, we investigated the alterations in the conformation of bovine serum albumin (BSA) caused by acetonitrile (ACN) in aqueous solution by using a combination of spectroscopic analysis and molecular dynamics (MD) simulations. Ultraviolet (UV) absorption, fluorescence, and infrared (IR) absorption spectroscopy were used to analyze the BSA conformation in the solutions containing 0-60 vol % ACN. Additionally, MD simulations were conducted to elucidate the interactions between BSA and solvent components, focusing on the structural changes in the hydrophobic pocket with Trp residues of the albumin. Increasing the ACN concentration leads to significant changes in the BSA conformation, as evidenced by shifts in UV fluorescence wavelength, decreased intensity, and alterations in IR absorption bands. Furthermore, the formation of protein aggregates was observed at high ACN concentration (30 vol % ACN), shown by increased hydrodynamic diameter distribution. MD simulations further demonstrate that the presence of ACN molecules near the hydrophobic pocket with the Trp-213 residue increases the fluctuations in the positions of amino acids observed near the hydrophobic pocket with Trp-213. Moreover, the intrusion of water molecules into the hydrophobic pocket under 60% ACN conditions with highly decreased solvent polarity was correlated with the changes in the BSA secondary structure. These findings enhance our understanding of how solvent polarity affects the albumin conformation, which is crucial for optimizing albumin-based DDS applications.

Fluoride nanoparticles (NPs) are materials utilized in the biomedical field for applications including imaging of the brain. Their interactions with biological systems and molecules are being investigated, but the mechanism underlying these interactions remains unclear. We focused on possible changes in the secondary structure and aggregation state of proteins on the surface of NPs and investigated the principle underlying the changes using the amyloid β peptide (Aβ_16-20) based on infrared spectrometry. CeF₃ NPs (diameter 80 nm) were synthesized via thermal decomposition. Infrared spectrometry showed that the presence of CeF₃ NPs promotes the formation of the β-sheet structure of Aβ_16-20. This phenomenon was attributed to the hydrophobic interaction between NPs and Aβ peptides in aqueous environments, which causes the Aβ peptides to approach each other on the NP surface and form ordered hydrogen bonds. Because of the coexisting salts on the secondary structure and assembly of Aβ peptides, the formation of the β-sheet structure of Aβ peptides on the NP surface was suppressed in the presence of NH₄⁺ and NO₃^- ions, suggesting the possibility that Aβ peptides were adsorbed and bound to the NP surface. The formation of the β-sheet structure of Aβ peptides was promoted in the presence of NH₄⁺, whereas it was suppressed in the presence of NO₃^- because of the electrostatic interaction between the lysine residue of the Aβ peptide and the ions. Our findings will contribute to comparative studies on the effect of different NPs with different physicochemical properties on the molecular state of proteins.