Dispersion and Dosimetric Challenges of Hydrophobic Carbon-Based Nanoparticles in In Vitro Cellular Studies

Methodologies across the dispersion preparation, characterization, and cellular dosimetry of hydrophilic nanoparticles (NPs) have been developed and used extensively in the field of nanotoxicology. However, hydrophobic NPs pose a challenge for dispersion in aqueous culture media using conventional methods that include sonication followed by mixing in the culture medium of interest and cellular dosimetry. In this study, a robust methodology for the preparation of stable dispersions of hydrophobic NPs for cellular studies is developed by introducing continuous energy over time via stirring in the culture medium followed by dispersion characterization and cellular dosimetry. The stirring energy and the presence of proteins in the culture medium result in the formation of a protein corona around the NPs, stabilizing their dispersion, which can be used for in vitro cellular studies. The identification of the optimal stirring time is crucial for achieving dispersion and stability. This is assessed through a comprehensive stability testing protocol employing dynamic light scattering to evaluate the particle size distribution stability and polydispersity. Additionally, the effective density of the NPs is obtained for the stable NP dispersions using the volumetric centrifugation method, while cellular dosimetry calculations are done using available cellular computational modeling, mirroring approaches used for hydrophilic NPs. The robustness of the proposed dispersion approach is showcased using a highly hydrophobic NP model (black carbon NPs) and two culture media, RPMI medium and SABM, that are widely used in cellular studies. The proposed approach for the dispersion of hydrophobic NPs results in stable dispersions in both culture media used here. The NP effective density of 1.03-1.07 g/cm³ measured here for black carbon NPs is close to the culture media density, resulting in slow deposition on the cells over time. So, the present methodology for dispersion and dosimetry of hydrophobic NPs is essential for the design of dose-response studies and overcoming the challenges imposed by slow particle deposition.