Freese Christian, Schreiner Daniel, Anspach Laura, Bantz Christoph, Maskos Michael, Unger Ronald E, Kirkpatrick C James
REPAIR-lab, Institute of Pathology, University Medical Center of the Johannes Gutenberg University Mainz, European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Mainz, Germany.
Fraunhofer ICT-IMM, Mainz, Germany.
Part Fibre Toxicol. 2014 Dec 24;11:68. doi: 10.1186/s12989-014-0068-y.
In general the prediction of the toxicity and therapeutic efficacy of engineered nanoparticles in humans is initially determined using in vitro static cell culture assays. However, such test systems may not be sufficient for testing nanoparticles intended for intravenous application. Once injected, these nanoparticles are caught up in the blood stream in vivo and are therefore in continuous movement. Physical forces such as shear stress and cyclic stretch caused by the pulsatile blood flow are known to change the phenotype of endothelial cells which line the luminal side of the vasculature and thus may be able to affect cell-nanoparticle interactions.
In this study we investigated the uptake of amorphous silica nanoparticles in primary endothelial cells (HUVEC) cultured under physiological cyclic stretch conditions (1 Hz, 5% stretch) and compared this to cells in a standard static cell culture system. The toxicity of varying concentrations was assessed using cell viability and cytotoxicity studies. Nanoparticles were also characterized for the induction of an inflammatory response. Changes to cell morphology was evaluated in cells by examining actin and PECAM staining patterns and the amounts of nanoparticles taken up under the different culture conditions by evaluation of intracellular fluorescence. The expression profile of 26 stress-related was determined by microarray analysis.
The results show that cytotoxicity to endothelial cells caused by silica nanoparticles is not significantly altered under stretch compared to static culture conditions. Nevertheless, cells cultured under stretch internalize fewer nanoparticles. The data indicate that the decrease of nanoparticle content in stretched cells was not due to the induction of cell stress, inflammation processes or an enhanced exocytosis but rather a result of decreased endocytosis.
In conclusion, this study shows that while the toxic impact of silica nanoparticles is not altered by stretch this dynamic model demonstrates altered cellular uptake of nanoparticles under physiologically relevant in vitro cell culture models. In particular for the development of nanoparticles for biomedical applications such improved in vitro cell culture models may play a pivotal role in the reduction of animal experiments and development costs.
一般来说，工程纳米颗粒对人体的毒性和治疗效果的预测最初是通过体外静态细胞培养试验来确定的。然而，这样的测试系统可能不足以测试用于静脉注射的纳米颗粒。一旦注射，这些纳米颗粒会在体内进入血流，因此处于持续运动状态。已知由脉动血流引起的剪切应力和循环拉伸等物理力会改变血管腔侧内皮细胞的表型，从而可能影响细胞与纳米颗粒的相互作用。
在本研究中，我们研究了在生理循环拉伸条件（1赫兹，5%拉伸）下培养的原代内皮细胞（人脐静脉内皮细胞）对无定形二氧化硅纳米颗粒的摄取，并将其与标准静态细胞培养系统中的细胞进行比较。使用细胞活力和细胞毒性研究评估不同浓度的毒性。还对纳米颗粒诱导炎症反应的特性进行了表征。通过检查肌动蛋白和血小板内皮细胞粘附分子染色模式以及通过评估细胞内荧光来评估不同培养条件下摄取的纳米颗粒数量，从而评估细胞形态的变化。通过微阵列分析确定26种应激相关基因的表达谱。
结果表明，与静态培养条件相比，拉伸条件下二氧化硅纳米颗粒对内皮细胞的细胞毒性没有显著改变。然而，在拉伸条件下培养的细胞摄取的纳米颗粒较少。数据表明，拉伸细胞中纳米颗粒含量的降低不是由于细胞应激、炎症过程的诱导或胞吐作用增强，而是内吞作用降低的结果。
总之，本研究表明，虽然拉伸不会改变二氧化硅纳米颗粒的毒性影响，但这种动态模型表明在生理相关的体外细胞培养模型下纳米颗粒的细胞摄取发生了改变。特别是对于生物医学应用纳米颗粒的开发，这种改进的体外细胞培养模型可能在减少动物实验和开发成本方面发挥关键作用。
