Effects of Ultrasonic Dispersion Energy on the Preparation of Amorphous SiO2 Nanomaterials for In Vitro Toxicity Testing

Abstract

Synthetic amorphous silica (SAS) constitute a large group of industrial nanomaterials (NM). Based on their different production processes, SAS can be distinguished as precipitated, fumed, gel and colloidal. The biological activity of SAS, e.g., cytotoxicity or inflammatory potential in the lungs is low but has been shown to depend on the particle size, at least for colloidal silica. Therefore, the preparation of suspensions from highly aggregated or agglomerated SAS powder materials is critical. Here we analyzed the influence of ultrasonic dispersion energy on the biologic activity of SAS using NR8383 alveolar macrophage (AM) assay. Fully characterized SAS (7 precipitated, 3 fumed, 3 gel, and 1 colloidal) were dispersed in H2O by stirring and filtering through a 5 µm filter. Aqueous suspensions were sonicated with low or high ultrasonic dispersion (USD) energy of 18 or 270 kJ/mL, respectively. A dose range of 11.25–90 µg/mL was administered to the AM under protein-free conditions to detect particle-cell interactions without the attenuating effect of proteins that typically occur in vivo. The release of lactate dehydrogenase (LDH), glucuronidase (GLU), and tumor necrosis factor α (TNF) were measured after 16 h. Hydrogen peroxide (H2O2) production was assayed after 90 min. The overall pattern of the in vitro response to SAS (12/14) was clearly dose-dependent, except for two SAS which showed very low bioactivity. High USD energy gradually decreased the particle size of precipitated, fumed, and gel SAS whereas the low adverse effect concentrations (LOECs) remained unchanged. Nevertheless, the comparison of dose-response curves revealed slight, but uniform shifts in EC50 values (LDH, and partially GLU) for precipitated SAS (6/7), gel SAS (2/3), and fumed SAS (3/3). Release of TNF changed inconsistently with higher ultrasonic dispersion (USD) energy whereas the induction of H2O2 was diminished in all cases. Electron microscopy and energy dispersive X-ray analysis showed an uptake of SAS into endosomes, lysosomes, endoplasmic reticulum, and different types of phagosomes. The possible effects of different uptake routes are discussed. The study shows that the effect of increased USD energy on the in vitro bioactivity of SAS is surprisingly small. As the in vitro response of AM to different SAS is highly uniform, the production process per se is of minor relevance for toxicity.

Is aggregated synthetic amorphous silica toxicologically relevant?

“…This indeed resulted in de-aggregation of SAS but did not yield sizes with substantial differences whatever the energy delivered (≥ 882 J, see Additional file 2: Figure S4). A recent study also showed that different sonication energies used to disperse SAS did not strongly affect the size distribution and hence the biological effects [18]. Therefore, we used a mechanically vortexed suspension (AGGR) to obtain a size distribution different from DE-AGGR.…”

Serum Lowers Bioactivity and Uptake of Synthetic Amorphous Silica by Alveolar Macrophages in a Particle Specific Manner

“…To describe and predict the bioactivity of the multitude of SAS, in vitro assays using different cell types and incubation conditions have been published [ 13 , 14 , 15 , 16 , 17 ]. Recently, the effects of SAS from all major production processes were tested using rat alveolar macrophages (NR8383) in vitro [ 18 ]. A major finding was that SAS, irrespective of the production method, elicit highly uniform responses, e.g., with respect to the release of lactate dehydrogenase (LDH), glucuronidase (GLU), tumour necrosis factor alpha (TNFα) or induction of H 2 O 2 release [ 18 ].…”

“…Recently, the effects of SAS from all major production processes were tested using rat alveolar macrophages (NR8383) in vitro [ 18 ]. A major finding was that SAS, irrespective of the production method, elicit highly uniform responses, e.g., with respect to the release of lactate dehydrogenase (LDH), glucuronidase (GLU), tumour necrosis factor alpha (TNFα) or induction of H 2 O 2 release [ 18 ]. In these experiments, cells were exposed to SAS under protein-free standard conditions, i.e., in the absence of serum proteins.…”

Synthetic Ligand-Coated Starch Magnetic Microbeads for Selective Extraction of Food Additive Silicon Dioxide from Commercial Processed Food

“…Food grade silicon dioxide (SiO 2 ) has been known to have very low toxicity with a NOAEL (no observed adverse effect level) of over 2000 mg/kg when ingested orally [ 3 , 4 ]. However, various environmental factors such as heating, pH change, and potential interaction with food ingredients imposed during food processing may cause undesirable alterations in the physicochemical properties of food additive SiO 2 present in processed food, which may lead to changes in its toxicity [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. In fact, studies have shown that the particle size is an important parameter that determines the penetrating nature of SiO 2 in biological tissues, which may also increase its toxicity [ 10 , 12 ].…”

“…However, various environmental factors such as heating, pH change, and potential interaction with food ingredients imposed during food processing may cause undesirable alterations in the physicochemical properties of food additive SiO 2 present in processed food, which may lead to changes in its toxicity [ 5 , 6 , 7 , 8 , 9 , 10 , 11 ]. In fact, studies have shown that the particle size is an important parameter that determines the penetrating nature of SiO 2 in biological tissues, which may also increase its toxicity [ 10 , 12 ]. The growing concerns regarding the potential toxicity of nanoscale food additive SiO 2 have necessitated the effective means of extracting intrinsic forms of SiO 2 from a range of processed food for the analysis of its physicochemical properties and safety.…”