Department of Biological and Biochemical Sciences, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
Department of General and Inorganic Chemistry, Faculty of Chemical Technology, University of Pardubice, Pardubice, Czech Republic.
Int J Nanomedicine. 2022 Sep 13;17:4211-4225. doi: 10.2147/IJN.S374955. eCollection 2022.
Titanium dioxide nanoparticles, 25 nm in size of crystallites (TiO P25), are among the most produced nanomaterials worldwide. The broad use of TiO P25 in material science has implied a request to evaluate their biological effects, especially in the lungs. Hence, the pulmonary A549 cell line has been used to estimate the effects of TiO P25. However, the reports have provided dissimilar results on caused toxicity. Surprisingly, the physicochemical factors influencing TiO P25 action in biological models have not been evaluated in most reports. Thus, the objective of the present study is to characterize the preparation of TiO P25 for biological testing in A549 cells and to evaluate their biological effects.
We determined the size and crystallinity of TiO P25. We used four techniques for TiO P25 dispersion. We estimated the colloid stability of TiO P25 in distilled water, isotonic NaCl solution, and cell culture medium. We applied the optimal dispersion conditions for testing the biological effects of TiO P25 (0-100 µg.mL) in A549 cells using biochemical assays (dehydrogenase activity, glutathione levels) and microscopy.
We found that the use of fetal bovine serum in culture medium is essential to maintain sufficient colloid stability of dispersed TiO P25. Under these conditions, TiO P25 were unable to induce a significant impairment of A549 cells according to the results of biochemical and microscopy evaluations. When the defined parameters for the use of TiO P25 in A549 cells were met, similar results on the biological effects of TiO P25 were obtained in two independent cell laboratories.
We optimized the experimental conditions of TiO P25 preparation for toxicity testing in A549 cells. The results presented here on TiO P25-induced cellular effects are reproducible. Therefore, our results can be helpful for other researchers using TiO P25 as a reference material.
大小为 25nm 的锐钛矿型二氧化钛纳米颗粒（TiO2 P25）是世界上产量最大的纳米材料之一。TiO2 P25 在材料科学中的广泛应用意味着需要评估其生物效应，特别是在肺部。因此，已使用肺 A549 细胞系来估计 TiO2 P25 的影响。然而，报告在引起的毒性方面提供了不同的结果。令人惊讶的是，在大多数报告中，尚未评估影响生物模型中 TiO2 P25 作用的物理化学因素。因此，本研究的目的是对 A549 细胞中 TiO2 P25 生物学测试的制备进行表征，并评估其生物学效应。
我们确定了 TiO2 P25 的大小和结晶度。我们使用了四种技术来分散 TiO2 P25。我们评估了 TiO2 P25 在蒸馏水中、等渗 NaCl 溶液和细胞培养基中的胶体稳定性。我们应用了最佳分散条件，以使用生物化学测定法（脱氢酶活性，谷胱甘肽水平）和显微镜来测试 TiO2 P25（0-100μg·mL）在 A549 细胞中的生物学效应。
我们发现，在培养基中使用胎牛血清对于维持分散的 TiO2 P25 的足够胶体稳定性至关重要。在这些条件下，根据生化和显微镜评估的结果，TiO2 P25 无法引起 A549 细胞的明显损伤。当满足 TiO2 P25 在 A549 细胞中使用的定义参数时，在两个独立的细胞实验室中获得了 TiO2 P25 生物效应的相似结果。
我们优化了 TiO2 P25 在 A549 细胞中进行毒性测试的实验条件。这里呈现的 TiO2 P25 诱导细胞效应的结果是可重复的。因此，我们的结果可以为其他使用 TiO2 P25 作为参考材料的研究人员提供帮助。
