Institute of Pathology, University Medical Centre, Mainz, Germany.
Eur J Pharm Biopharm. 2013 Jun;84(2):275-87. doi: 10.1016/j.ejpb.2012.10.011. Epub 2012 Nov 24.
Drug and gene delivery via nanoparticles across biological barriers such as the alveolar-capillary barrier of the lung constitutes an interesting and increasingly relevant field in nanomedicine. Nevertheless, potential hazardous effects of nanoparticles (NPs) as well as their cellular and systemic fate should be thoroughly examined. Hence, this study was designed to evaluate the effects of amorphous silica NPs (Sicastar) and (poly)organosiloxane NPs (AmOrSil) on the viability and the inflammatory response as well as on the cellular uptake mechanisms and fate in cells of the alveolar barrier. For this purpose, the alveolar epithelial cell line (NCI H441) and microvascular endothelial cell line (ISO-HAS-1) were used in an experimental set up resembling the alveolar-capillary barrier of the lung. In terms of IL-8 and sICAM Sicastar resulted in harmful effects at higher concentrations (60 μg/ml) in conventional monocultures but not in the coculture, whereas AmOrSil showed no significant effects. Immunofluorescence counterstaining of endosomal structures in NP-incubated cells showed no evidence for a clathrin- or caveolae-mediated uptake mechanism. However, NPs were enclosed in flotillin-1 and -2 marked vesicles in both cell types. Flotillins appear to play a role in cellular uptake or trafficking mechanisms of NPs and are discussed as indicators for clathrin- or caveolae-independent uptake mechanisms. In addition, we examined the transport of NPs across this in vitro model of the alveolar-capillary barrier forming a tight barrier with a transepithelial electrical resistance of 560±8 Ω cm(2). H441 in coculture with endothelial cells took up much less NPs compared to monocultures. Moreover, coculturing prevented the transport of NP from the epithelial compartment to the endothelial layer on the bottom of the filter insert. This supports the relevance of coculture models, which favour a differentiated and polarised epithelial layer as in vitro test systems for nanoparticle uptake.
药物和基因通过纳米颗粒递送至生物屏障，如肺的肺泡毛细血管屏障，这在纳米医学中是一个有趣且日益相关的领域。然而，纳米颗粒（NPs）的潜在危险效应及其细胞和系统命运应该得到彻底检查。因此，本研究旨在评估无定形二氧化硅 NPs（Sicastar）和（聚）有机硅氧烷 NPs（AmOrSil）对肺泡屏障细胞的活力、炎症反应以及细胞摄取机制和命运的影响。为此，使用肺泡上皮细胞系（NCI H441）和微血管内皮细胞系（ISO-HAS-1），建立了一个类似于肺的肺泡毛细血管屏障的实验装置。就 IL-8 和 sICAM 而言，Sicastar 在较高浓度（60μg/ml）下在常规的单核培养中产生有害影响，但在共培养中没有，而 AmOrSil 则没有显示出显著的影响。在 NP 孵育细胞的内体结构的免疫荧光复染中，没有证据表明存在网格蛋白或小窝介导的摄取机制。然而，在这两种细胞类型中，NP 都被包含在 flotillin-1 和 flotillin-2 标记的囊泡中。Flotillins 似乎在 NP 的细胞摄取或转运机制中发挥作用，并被认为是网格蛋白或小窝非依赖性摄取机制的指标。此外，我们研究了 NP 通过形成紧密屏障的肺泡毛细血管屏障体外模型的转运，该屏障的跨上皮电阻为 560±8Ωcm2。与单核培养相比，共培养的 H441 内皮细胞摄取的 NP 要少得多。此外，共培养阻止了 NP 从上皮隔室向滤器插入物底部的内皮层的转运。这支持了共培养模型的相关性，共培养模型有利于形成分化和极化的上皮层，作为纳米颗粒摄取的体外测试系统。
