Multi-endpoint assessments for in vitro nano-bio interactions and uptake of biogenic phosphorus nanomaterials using HEK293 cells

Abstract

Phosphorus (P)-based nanomaterials (NMs) are being explored as nanofertilisers due to higher uptake in plants, enhanced nutrient use efficiency, and minimal leaching. However, their colloidal behaviour in biologically relevant media and corresponding in vitro nano-bio interactions have been understudied topics for the evaluation of their potential cytotoxicity. Using HEK293, in this study, we investigated the interactions and any associated cytotoxicity of four different nanohydroxyapatites (nHAPs: (i) biogenic platelet-shaped, chemically synthesized (ii) nanorods, (iii) spherical, (iv) nanoneedles) and nanophosphorus (spherical biogenic nP derived from rock phosphate). The NM's interaction with cell culture media was studied by dynamic light scattering, transmission electron microscopy, and elemental and protein corona characterisation. The investigation on acute effects such as mitochondrial and lysosomal activities, membrane disintegrity, ROS generation, and cell viability was performed by following NIH guidelines. In addition to the dose-dependent approach, the cytotoxic effects of NM on changing the initial cell seeding density were also monitored. Our results suggest that physicochemically different P-based NMs interact differently with cell culture media resulting in NM transformation, minimal P release, and protein corona formation. Lipoprotein and albumins were the major components of the hard corona. No LC₅₀ values were attained for any of the cytotoxic endpoints up to 1000 µg mL⁻¹. Fluorescence and electron microscopy confirmed the uptake of NMs in the cells. This is the first report on the nano-bio interactions of differently shaped and sized P-based NMs using colloidal chemistry and two independent approaches-variable NM dose and variable cell densities. The findings suggest low cytotoxicity and hence the potential suitability of using the biogenic P-based NMs in agriculture as nanofertilisers.