Louvain centre for Toxicology and Applied Pharmacology (LTAP), Institut de Recherche Expérimentale et Clinique (IREC), Université catholique de Louvain, Brussels, Belgium.
Interdisciplinary Research Institute in Bio- Nano- Sciences, Babes-Bolyai University (BBU), Cluj-Napoca, Romania.
Part Fibre Toxicol. 2021 Feb 18;18(1):9. doi: 10.1186/s12989-021-00402-5.
In vitro models are widely used in nanotoxicology. In these assays, a careful documentation of the fraction of nanomaterials that reaches the cells, i.e. the in vitro delivered dose, is a critical element for the interpretation of the data. The in vitro delivered dose can be measured by quantifying the amount of material in contact with the cells, or can be estimated by applying particokinetic models. For carbon nanotubes (CNTs), the determination of the in vitro delivered dose is not evident because their quantification in biological matrices is difficult, and particokinetic models are not adapted to high aspect ratio materials. Here, we applied a rapid and direct approach, based on femtosecond pulsed laser microscopy (FPLM), to assess the in vitro delivered dose of multi-walled CNTs (MWCNTs).
We incubated mouse lung fibroblasts (MLg) and differentiated human monocytic cells (THP-1) in 96-well plates for 24 h with a set of different MWCNTs. The cytotoxic response to the MWCNTs was evaluated using the WST-1 assay in both cell lines, and the pro-inflammatory response was determined by measuring the release of IL-1β by THP-1 cells. Contrasting cell responses were observed across the MWCNTs. The sedimentation rate of the different MWCNTs was assessed by monitoring turbidity decay with time in cell culture medium. These turbidity measurements revealed some differences among the MWCNT samples which, however, did not parallel the contrasting cell responses. FPLM measurements in cell culture wells revealed that the in vitro delivered MWCNT dose did not parallel sedimentation data, and suggested that cultured cells contributed to set up the delivered dose. The FPLM data allowed, for each MWCNT sample, an adjustment of the measured cytotoxicity and IL-1β responses to the delivered doses. This adjusted in vitro activity led to another toxicity ranking of the MWCNT samples as compared to the unadjusted activities. In macrophages, this adjusted ranking was consistent with existing knowledge on the impact of surface MWCNT functionalization on cytotoxicity, and might better reflect the intrinsic activity of the MWCNT samples.
The present study further highlights the need to estimate the in vitro delivered dose in cell culture experiments with nanomaterials. The FPLM measurement of the in vitro delivered dose of MWCNTs can enrich experimental results, and may refine our understanding of their interactions with cells.
体外模型在纳米毒理学中被广泛应用。在这些实验中，详细记录纳米材料进入细胞的部分，即体外传递剂量，是解释数据的关键因素。体外传递剂量可以通过量化与细胞接触的材料量来测量，也可以通过应用颗粒动力学模型来估计。对于碳纳米管（CNT），由于其在生物基质中的定量分析较为困难，且颗粒动力学模型不适用于高纵横比的材料，因此确定体外传递剂量并不明显。在这里，我们应用基于飞秒脉冲激光显微镜（FPLM）的快速直接方法来评估多壁 CNT（MWCNT）的体外传递剂量。
我们将小鼠肺成纤维细胞（MLg）和分化的人单核细胞（THP-1）在 96 孔板中孵育 24 小时，并用一组不同的 MWCNT 处理。通过 WST-1 测定法在两种细胞系中评估 MWCNT 的细胞毒性反应，通过测量 THP-1 细胞释放的 IL-1β来确定促炎反应。在不同的 MWCNT 中观察到细胞反应的差异。通过监测细胞培养液中浊度随时间的衰减来评估不同 MWCNT 的沉降速率。这些浊度测量结果显示，MWCNT 样品之间存在一些差异，但与细胞反应的差异并不平行。细胞培养孔中的 FPLM 测量结果表明，体外传递的 MWCNT 剂量与沉降数据不平行，并表明培养细胞有助于建立传递剂量。对于每个 MWCNT 样品，FPLM 数据允许将测量的细胞毒性和 IL-1β 反应调整为传递剂量。与未调整的活性相比，这种调整后的体外活性对 MWCNT 样品进行了另一种毒性排序。在巨噬细胞中，这种调整后的排序与表面 MWCNT 功能化对细胞毒性的影响的现有知识一致，并且可能更好地反映了 MWCNT 样品的内在活性。
本研究进一步强调了在纳米材料的细胞培养实验中估计体外传递剂量的必要性。MWCNT 体外传递剂量的 FPLM 测量可以丰富实验结果，并可能深化我们对其与细胞相互作用的理解。
