Thermodynamic Parameters at Bio–Nano Interface and Nanomaterial Toxicity: A Case Study on BSA Interaction with ZnO, SiO₂, and TiO₂

Abstract

Understanding nanomaterial (NM)−protein interactions is a key issue in defining the bioreactivity of NMs with great impact for nanosafety. In the present work, the complex phenomena occurring at the bio/nano interface were evaluated in a simple case study focusing on NM−protein binding thermodynamics and protein stability for three representative metal oxide NMs, namely, zinc oxide (ZnO; NM-110), titanium dioxide (TiO 2 ; NM-101), and silica (SiO 2 ; NM-203). The thermodynamic signature associated with the NM interaction with an abundant protein occurring in most cell culture media, bovine serum albumin (BSA), has been investigated by isothermal titration and differential scanning calorimetry. Circular dichroism spectroscopy offers additional information concerning adsorption-induced protein conformational changes. The BSA adsorption onto NMs is enthalpy-controlled, with the enthalpic character (favorable interaction) decreasing as follows: ZnO (NM-110) > SiO 2 (NM-203) > TiO 2 (NM-101). The binding of BSA is spontaneous, as revealed by the negative free energy, ΔG, for all systems. The structural stability of the protein decreased as follows: TiO 2 (NM-101) > SiO 2 (NM-203) > ZnO (NM-110). As protein binding may alter NM reactivity and thus the toxicity, we furthermore assessed its putative influence on DNA damage, as well as on the expression of target genes for cell death (RIPK1, FAS) and oxidative stress (SOD1, SOD2, CAT, GSTK1) in the A549 human alveolar basal epithelial cell line. The enthalpic component of the BSA−NM interaction, corroborated with BSA structural stability, matched the ranking for the biological alterations, i.e., DNA strand breaks, oxidized DNA lesions, cell-death, and antioxidant gene expression in A549 cells. The relative and total content of BSA in the protein corona was determined using mass-spectrometry-based proteomics. For the present case study, the thermodynamic parameters at bio/nano interface emerge as key descriptors for the dominant contributions determining the adsorption processes and NMs toxicological effect.

Testing Strategies of the In Vitro Micronucleus Assay for the Genotoxicity Assessment of Nanomaterials in BEAS-2B Cells

“…Supporting our results, the time-dependent stability of TiO 2 was also observed in previously published work on TiO 2 in THP-1 cells [ 31 ]. The effect of culture medium on NMs stability and their in vitro effects was previously discussed e.g., by Precupas et al [ 57 ] and Prasad et al [ 58 ]. Further, our unpublished data showed that LHC-9, an alternative culture medium recommended for BEAS-2B cells that is characteristic of high protein agglomeration over time, negatively impacted stability of NMs at a concentration below 25 µg/mL.…”

Genotoxicity of Nanomaterials: Advanced In Vitro Models and High Throughput Methods for Human Hazard Assessment—A Review

“…In a second step this suspension is applied to the cultured cells in vitro. When NMs are suspended in CCM or other biological fluids, they change their particle surface, the associated particle-particle and particle-cell interactions [ 99 , 100 ]. This is dependent on the characteristics of the primary NM (composition, size, shape, surface chemistry) and the properties of the surrounding fluid (pH, ionic strength, protein content, temperature) [ 16 , 17 ].…”

Engineered Nanoparticle-Protein Interactions Influence Protein Structural Integrity and Biological Significance

“…These parameters are interrelated as ∆G = ∆H − T∆S, where ∆G represents a change in Gibbs free energy, ∆H represents a change in enthalpy, T represents absolute temperature and ∆S represents a change in entropy. These reveal the spontaneity of adsorption and binding of proteins with ENPs based on the free energy (ΔG) of the system [ 95 ].…”

“…Thermodynamic parameters like free energy and entropy of the ENPs-protein system are analyzed for their crucial role in deciding the spontaneity and possibility of ENPs-protein interactions. Techniques such as differential scanning calorimetry (DSC) and isothermal titration calorimetry (ITC) have been used for thermodynamic analysis of ENPs-protein interactions, comparing binding affinity of different ENPs for proteins and spontaneity of the binding process [ 95 ]. DSC is applied to calculate the change in free energy of the thermal denaturation of proteins.…”