Effects of Cell Culture Media on the Dynamic Formation of Protein−Nanoparticle Complexes and Influence on the Cellular Response

TLDR

In vitro nanoparticle toxicity testing is hampered by rapid changes in particle properties in biological fluids, and the dynamic formation of a protein corona around nanoparticles is a key event that can alter biological responses. The study aimed to determine how two common cell culture media, DMEM and RPMI, affect protein–nanoparticle interactions using citrate‑capped gold nanoparticles of varying sizes. The authors characterized corona formation and biological impact with dynamic light scattering, UV‑visible and plasmon resonance spectroscopy, SDS‑PAGE, mass spectrometry, and viability assays on HeLa and U937 cells. They found that DMEM produced a large, time‑dependent protein corona whereas RPMI yielded a thinner corona; complexes formed in RPMI were internalized more readily and caused greater cytotoxicity, highlighting the critical role of media choice in nanotoxicology studies.

Abstract

The development of appropriate in vitro protocols to assess the potential toxicity of the ever expanding range of nanoparticles represents a challenging issue, because of the rapid changes of their intrinsic physicochemical properties (size, shape, reactivity, surface area, etc.) upon dispersion in biological fluids. Dynamic formation of protein coating around nanoparticles is a key molecular event, which may strongly impact the biological response in nanotoxicological tests. In this work, by using citrate-capped gold nanoparticles (AuNPs) of different sizes as a model, we show, by several spectroscopic techniques (dynamic light scattering, UV−visible, plasmon resonance light scattering), that proteins−NP interactions are differently mediated by two widely used cellular media (i.e., Dulbecco Modified Eagle's medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), supplemented with fetal bovine serum). We found that, while DMEM elicits the formation of a large time-dependent protein corona, RPMI shows different dynamics with reduced protein coating. Characterization of these nanobioentities was also performed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and mass spectroscopy, revealing that the average composition of protein corona does not reflect the relative abundance of serum proteins. To evaluate the biological impact of such hybrid bionanostructures, several comparative viability assays onto two cell lines (HeLa and U937) were carried out in the two media, in the presence of 15 nm AuNPs. We observed that proteins/NP complexes formed in RPMI are more abundantly internalized in cells as compared to DMEM, overall exerting higher cytotoxic effects. These results show that, beyond an in-depth NPs characterization before cellular experiments, a detailed understanding of the effects elicited by cell culture media on NPs is crucial for standardized nanotoxicology tests.