Wiemann Martin, Vennemann Antje, Stintz Michael, Retamal Marín Rodrigo R, Babick Frank, Lindner Gottlieb-Georg, Schuster Tobias B, Brinkmann Ulrich, Krueger Nils
IBE R&D Institute for Lung Health gGmbH, Mendelstr. 11, D-48149 Münster, Germany.
Research Group Mechanical Process Engineering, Institute of Process Engineering and Environmental Technology, Technische Universität Dresden, Münchner Platz 3, D-01062 Dresden, Germany.
Nanomaterials (Basel). 2018 Dec 22;9(1):11. doi: 10.3390/nano9010011.
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 EC 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.
合成无定形二氧化硅（SAS）构成了一大类工业纳米材料（NM）。根据其不同的生产工艺，SAS可分为沉淀法、气相法、凝胶法和胶体法。SAS的生物活性，如肺部的细胞毒性或炎症潜力较低，但已表明其取决于颗粒大小，至少对于胶体二氧化硅是如此。因此，从高度聚集或团聚的SAS粉末材料制备悬浮液至关重要。在此，我们使用NR8383肺泡巨噬细胞（AM）试验分析了超声分散能量对SAS生物活性的影响。将充分表征的SAS（7种沉淀法、3种气相法、3种凝胶法和1种胶体法）通过搅拌分散在水中，并通过5 µm过滤器过滤。水悬浮液分别用18或270 kJ/mL的低或高超声分散（USD）能量进行超声处理。在无蛋白条件下，将11.25⁻90 µg/mL的剂量范围施用于AM，以检测颗粒与细胞的相互作用，而不存在体内通常出现的蛋白质的衰减作用。在16小时后测量乳酸脱氢酶（LDH）、葡萄糖醛酸酶（GLU）和肿瘤坏死因子α（TNF）的释放。在90分钟后测定过氧化氢（H₂O₂）的产生。除了两种显示出非常低生物活性的SAS外，对SAS（12/14）的体外反应总体模式明显呈剂量依赖性。高USD能量逐渐降低了沉淀法、气相法和凝胶法SAS的颗粒大小，而低不良反应浓度（LOECs）保持不变。然而，剂量反应曲线的比较显示，沉淀法SAS（6/7）、凝胶法SAS（2/3）和气相法SAS（3/3）的EC值（LDH和部分GLU）有轻微但一致的变化。TNF的释放随较高的超声分散（USD）能量变化不一致，而在所有情况下H₂O₂的诱导均减少。电子显微镜和能量色散X射线分析显示SAS被摄取到内体、溶酶体、内质网和不同类型的吞噬体中。讨论了不同摄取途径的可能影响。该研究表明，增加的USD能量对SAS体外生物活性的影响出奇地小。由于AM对不同SAS的体外反应高度一致，生产工艺本身对毒性的相关性较小。
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