Abstract
BackgroundInteractions between nanoparticles and cells are now the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles including those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. Autophagy plays a key role in human health and disease, particularly in cancer and neurodegenerative diseases. We herein investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels.ResultsTiO2-NPs were characterized by transmission electron microscopy (TEM) and dynamic light scattering techniques. Cellular uptake, as evaluated by TEM and NanoSIMS revealed that NPs internalization led to the formation of autophagosomes. TiO2-NPs treatment did not reduce cell viability of HaCaT cells nor increased oxidative stress. Cellular autophagy was additionally evaluated by confocal microscopy using eGFP-LC3 keratinocytes, western blotting of autophagy marker LC3I/II, immunodetection of p62 and NBR1 proteins, and gene expression of LC3II, p62, NBR1, beclin1 and ATG5 by RT-qPCR. We also confirmed the formation and accumulation of autophagosomes in NPs treated cells with LC3-II upregulation. Based on the lack of degradation of p62 and NBR1 proteins, autophagosomes accumulation at a high dose (25.0 μg/ml) is due to blockage while a low dose (0.16 μg/ml) promoted autophagy. Cellular viability was not affected in either case.ConclusionsThe uptake of TiO2-NPs led to a dose-dependent increase in autophagic effect under non-cytotoxic conditions. Our results suggest dose-dependent autophagic effect over time as a cellular response to TiO2-NPs. Most importantly, these findings suggest that simple toxicity data are not enough to understand the full impact of TiO2-NPs and their effects on cellular pathways or function.
Highlights
Interactions between nanoparticles and cells are the focus of a fast-growing area of research
NPs characterization Characterization of TiO2-NPs was done by transmission electron microscopy (TEM), zeta potential (Z-potential) measurement and dynamic light scattering (DLS) in water and cell culture medium (Fig. 1 and Table 1)
TiO2‐NPs are not cytotoxic and induce autophagosomes formation To define a non-cytotoxic level of NPs on skin cells, the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) and neutral red (NR) assays were used after treating HaCaT cells with TiO2-NPs for 1 and 24 h at 0.16-25 μg/ml (Fig. 2a, b)
Summary
Interactions between nanoparticles and cells are the focus of a fast-growing area of research. Though many nanoparticles interact with cells without any acute toxic responses, metal oxide nanoparticles includ‐ ing those composed of titanium dioxide (TiO2-NPs) may disrupt the intracellular process of macroautophagy. We investigated the in vitro biological effects of TiO2-NPs (18 nm) on autophagy in human keratinocytes (HaCaT) cells at non-cytotoxic levels. Owing to its unique features, nanoparticles (NPs) can enter cells and interact with cellular machinery. Current knowledge about the mechanisms underlying cell-NPs interactions is still limited. Though many NPs interact with cells without acute toxic responses, metal oxide NPs including. Tight regulation of autophagy is required; autophagy dysfunctions have been linked with numerous human pathologies including cancer, neurodegeneration, abnormal immune responses and premature ageing, so that autophagy regulation has become a new therapeutic strategy [12]. Autophagy dysfunction has been reported for diverse NPs such as liposomes, polymeric NPs, gold, iron oxide, zinc oxide, or titanium dioxide based NPs, carbon nanotubes, and quantum dots [9, 11]
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