Abstract

Toxicity tests in rodents are still considered a controversial topic concerning their ethical justifiability. The chick embryo chorioallantoic membrane (CAM) assay may offer a simple and inexpensive alternative. The CAM assay is easy to perform and has low bureaucratic hurdles. At the same time, the CAM assay allows the application of a broad variety of analytical methods in the field of nanotoxicological research. We evaluated the CAM assay as a methodology for the determination of nanotoxicity. Therefore we calculated the median lethal dose (LD₅₀), performed in vivo microscopy and immunohistochemistry to identify organ-specific accumulation profiles, potential organ damage, and the kinetics of the in vivo circulation of the nanoparticles. Zinc oxide nanoparticles were intravascularly injected on day 10 of the egg development and showed an LD₅₀ of 17.5 µM (1.4 µg/mL_eggcontent). In comparison, the LD₅₀ of equivalent amounts of Zn²⁺ was 4.6 µM (0.6 µg/mL_eggcontent). Silica encapsulated ZnO@SiO₂ nanoparticles conjugated with fluorescein circulated in the bloodstream for at least 24 h. Particles accumulated mostly in the liver and kidney. In immunohistochemical staining, organ damage was detected only in liver tissue after intravascular injection of zinc oxide nanoparticles in very high concentrations. Zinc oxide nanoparticles showed a different pharmacokinetic profile compared to Zn²⁺ ions. In conclusion, the CAM assay has proven to be a promising methodology for evaluating nanotoxicity and for the assessment of the in vivo accumulation profiles of nanoparticles. These findings may qualify the methodology for risk assessment of innovative nanotherapeutics in the future.

摘要

啮齿动物的毒性测试仍然被认为是关于其道德合理性的有争议的话题。鸡胚绒毛膜尿囊膜 (CAM) 检测可能提供一种简单且廉价的替代方法。CAM 检测易于执行且官僚主义障碍较低。同时，CAM 分析允许在纳米毒理学研究领域应用广泛的分析方法。我们评估了 CAM 测定作为确定纳米毒性的方法。因此，我们计算了中位致死剂量 (LD ₅₀ )，进行了体内显微镜检查和免疫组织化学，以确定器官特异性积累谱、潜在器官损伤和体内动力学纳米粒子的循环。在卵发育的第 10 天血管内注射氧化锌纳米颗粒，其 LD ₅₀为 17.5 µM（1.4 µg/mL_卵含量）。相比之下，等量的 Zn ²⁺的 LD ₅₀为 4.6 µM（0.6 µg/mL_蛋含量）。二氧化硅包封的ZnO@SiO ₂纳米粒子与荧光素结合，在血液中循环至少24小时。颗粒主要积聚在肝脏和肾脏中。在免疫组织化学染色中，血管内注射非常高浓度的氧化锌纳米颗粒后，仅在肝组织中检测到器官损伤。与Zn相比，氧化锌纳米颗粒显示出不同的药代动力学特征²⁺离子。总之，CAM 测定已被证明是一种用于评估纳米毒性和评估纳米颗粒体内积累曲线的有前途的方法。这些发现可能会成为未来创新纳米疗法风险评估的方法。