Potential health risks and biomedical applications of engineered inorganic nanoparticles

Abstract

Synthetic amorphous silica (SAS) is widely used in different fields including the food industry as the additive E551. Since the beginning of its commercialization in the 1950s, SAS is made by two manufacturing methods resulting in the pyrogenic form (NM-203) produced by the thermal route or the hydrated products (precipitated silica, NM-200) produced by the wet route. These manufacturing processes lead to the production of SAS nanoparticles (NPs) that interact to form larger agglomerates and aggregates. In the first part of this work, we evaluated the toxicity of NM-200 and NM-203 in different human cell lines. Especially in THP-1 cells, a human monocytic cell line differentiated into macrophages, NM-203 exhibited greater cytotoxicity than NM-200. An in vitro NP-binding assay was performed using protein extracts obtained from THP-1 cells to identify cellular proteins interacting with high-affinity to SAS NPs and forming the so-called ‘Hard Protein Corona’ (hPC), the composition of which is crucial for the NP bioactivity. We have observed that NM-203 adsorbed a higher amount of proteins on their surface than NM-200 (two-fold increment), although the hPC composition determined by liquid chromatography analysis coupled to mass spectrometry (LC-MS/MS) was the same for both SAS NPs. Hydrophilic and hydrophobic interactions seem to promote the tight adsorption on the surface of SAS NPs of several proteins involved in crucial metabolic pathways. Many of these proteins exhibited a high frequency of intrinsically disordered regions that could drive the protein interaction to SAS NPs by hydrophobic interactions, especially for NM-203, which has a more hydrophobic surface due to the presence of siloxane bridges created during the thermal synthesis method. The interactomic analysis revealed that most of the proteins identified in hPC are constituents of specialized actin-rich structures called ‘podosomes’ that play important roles in phagocytosis, migration, and adhesion to the extracellular matrix. Confocal microscope and western blot analysis were conducted to evaluate the effects of SAS NP treatment on the abundance of representative podosome proteins. We have observed a de-localization and destructuration of actin filaments, with the formation of punctuate structures, and a reduced abundance of hnRNP K, a RNA-binding protein playing a role in the nuclear metabolism of RNAs but also highly represented in the podosomes. To evaluate the effects of SAS NPs on macrophage functionality we performed a phagocytic assay based on the internalization of fluorescent latex beads by the THP-1 cells. The confocal microscopy analysis showed a nearly complete block of the phagocytic process also at low dosage of both silica NPs. In conclusion, we have observed an increased cytotoxicity of NM-203 compared to NM-200 that could be attributed to a higher surface reactivity of NM-203. In the second part of my PhD project, we focused on the effects of engineered inorganic NPs in a yeast model of Parkinson's disease (PD) that overexpresses human alpha-synuclein (α-syn), an intrinsically disordered protein (IDP) representing the major component of Lewy bodies (LB), a histological hallmark of PD. In pathological conditions, α-syn undergo a series of lipid-dependent conformational changes leading to the formation of β-sheet-rich aggregates (α-syn oligomers), considered the most neurotoxic forms of α-syn. The overexpression of human α-syn in our yeast model of PD under the control of a galactose-inducible promoter caused a dose-dependent toxicity and a global cellular dysfunction, associated to the formation of α-syn intracellular foci, reminiscent of human LBs. Recent studies showed that SAS NPs promote the process of α-syn aggregation, whereas docking studies revealed that CeO2 NPs best fit in the active site of α-syn. A redox switch between Ce(3+) and Ce(4+) state on the NP surface confers an autoregenerative antioxidant activity to CeO2 NPs making these NPs promising for many biomedical applications. In our yeast PD model, the treatment with SAS NPs did not ameliorate cell viability, but at high doses we observed further toxic effects. Conversely, CeO2 NPs strongly counteracted α-syn-induced toxicity. Microscopic analysis shows that the treatment with CeO2 NPs re-localize α-syn at the plasma membrane level and strongly counteract α-syn foci formation. In line with these results, dot blot analysis revealed that CeO2 NP treatment did not affect the total protein expression levels, but drastically reduced the amount of toxic oligomeric forms of α-syn. We have also observed that the treatment with CeO2 NP restored the mitochondrial morphology, decreased ROS accumulation and strongly counteract the formation of aberrant aggresomes, proteinaceous cytoplasmic inclusions formed when the protein degradation system of the cell is overwhelmed. Analysis of the composition of the hPC formed on the surface of CeO2 NPs showed a direct interaction between these NPs and α-syn, in addition to other yeast proteins involved in metabolic pathways altered by α-syn oligomerization, as the Unfolded Protein Response (UPR) pathway. UPR is a stress response pathway induced by accumulation of misfolded proteins in the endoplasmic reticulum (ER). A chronic activation of this pathway is highly triggered in many neurodegenerative diseases and can lead to programmed cell death. Immunological analysis revealed an increase in the abundance of Kar2, the negative regulator of UPR, after few hours of the treatment with CeO2 NPs, indicating that CeO2 NPs can counteract the chronical activation of UPR pathway. These results are in line with the observations that the upregulation of BiP (the human homolog of Kar2) significantly prevented loss of dopaminergic neurons. In conclusion, CeO2 NPs represent a promising candidate with multi-target mode-of-action which could be used in drug discovery pipelines for the treatment of PD and related disorders.