Analytical strategy based on asymmetric flow field flow fractionation hyphenated to ICP-MS and complementary techniques to study gold nanoparticles transformations in cell culture medium

Highlights: • Analytical method development for the determination of AuNPs in DMEM by AF4-ICP-MS. • An oxidation process of the AuNPs is taking place in presence of cell culture medium. • Formation of protein corona and aggregates/agglomerates in DMEM was demonstrated. • AuNPs transformations in cell culture medium were confirmed by complementary techniques. • Detection of AuNPs transformations is of major interest for toxicity tests. -- Abstract: An analytical methodology based on asymmetric flow field flow fractionation (AF4) hyphenated to inductively coupled plasma mass spectrometry (ICP-MS) has been developed to study gold nanoparticles (AuNPs) in cell culture medium (Dulbecco's Modified Eagle Medium, DMEM, containing 10% fetal bovine serum, FBS, and antibiotics) used for in vitro toxicological studies. AF4-ICP-MS separation of AuNPs was performed using a regenerated cellulose membrane (molecular weight cut-off, MWCO, of 10 kDa). The carrier composition and the AF4 separation program were optimized. Under the optimum conditions, AuNPs of different types, i.e. phosphate buffered saline (PBS) and citrate stabilized, and sizes (10, 30 and 40 nm), without and with cell culture medium could be separated. The developed method allowed to detect transformations in AuNPs and dissolved gold species (Au³⁺) induced by this medium, such as an increase in the hydrodynamic volume and oxidation. Centrifugal ultrafiltration (CU), transmission electron microscopy (TEM) and Ultraviolet–visible (UV–vis) absorption spectrophotometry have been used as complementary techniques to study these processes. This information is of major interest to have a correct interpretation of the in vitro toxicological studies of NPs, which are more and more demanded due to the increasing concerns about the safe use of these materials and their impacts. This work demonstrates the potential of hyphenated techniques based on AF4 to achieve this relevant information.