Shape-engineered titanium dioxide nanoparticles (TiO2-NPs): cytotoxicity and genotoxicity in bronchial epithelial cells.

The aim of this study was to evaluate cytotoxicity (WST-1 assay), LDH release (LDH assay) and genotoxicity (Comet assay) of three engineered TiO2-NPs with different shapes (bipyramids, rods, platelets) in comparison with two commercial TiO2-NPs (P25, food grade). After NPs characterization (SEM/T-SEM and DLS), biological effects of NPs were assessed on BEAS-2B cells in presence/absence of light. The cellular uptake of NPs was analyzed using Raman spectroscopy. The cytotoxic effects were mostly slight. After light exposure, the largest cytotoxicity (WST-1 assay) was observed for rods; P25, bipyramids and platelets showed a similar effect; no effect was induced by food grade. No LDH release was detected, confirming the low effect on plasma membrane. Food grade and platelets induced direct genotoxicity while P25, food grade and platelets caused oxidative DNA damage. No genotoxic or oxidative damage was induced by bipyramids and rods. Biological effects were overall lower in darkness than after light exposure. Considering that only food grade, P25 and platelets (more agglomerated) were internalized by cells, the uptake resulted correlated with genotoxicity. In conclusion, cytotoxicity of NPs was low and affected by shape and light exposure, while genotoxicity was influenced by cellular-uptake and aggregation tendency.

Cloning and Expression Analysis of Human Amelogenin in Nicotiana benthamiana Plants by Means of a Transient Expression System.

Enamel is the covering tissue of teeth, made of regularly arranged hydroxyapatite crystals deposited on an organic matrix composed of 90% amelogenin that is completely degraded at the end of the enamel formation process. Amelogenin has a biomineralizing activity, forming nanoparticles or nanoribbons that guide hydroxyapatite deposit, and regenerative functions in bone and vascular tissue and in wound healing. Biotechnological products containing amelogenin seem to facilitate these processes. Here, we describe the production of human amelogenin in plants by transient transformation of Nicotiana benthamiana with constructs carrying synthetic genes with optimized human or plant codons. Both genes yielded approximately 500 µg of total amelogenin per gram of fresh leaf tissue. Two purification procedures based on affinity chromatography or on intrinsic solubility properties of the protein were followed, yielding from 12 to 150 µg of amelogenin per gram of fresh leaf tissue, respectively, at different purity. The identity of the plant-made human amelogenin was confirmed by MALDI-TOF-MS analysis of peptides generated following chymotrypsin digestion. Using dynamic light scattering, we showed that plant extracts made in acetic acid containing human amelogenin have a bimodal distribution of agglomerates, with hydrodynamic diameters of 22.8 ± 3.8 and 389.5 ± 86.6 nm. To the best of our knowledge, this is the first report of expression of human amelogenin in plants, offering the possibility to use this plant-made protein for nanotechnological applications.