CIGMH, DCV, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa , Caparica , Portugal.
Nanotoxicology. 2014 Aug;8(5):521-32. doi: 10.3109/17435390.2013.802821. Epub 2013 May 28.
Antisense therapy is a powerful tool for post-transcriptional gene silencing suitable for down-regulating target genes associated to disease. Gold nanoparticles have been described as effective intracellular delivery vehicles for antisense oligonucleotides providing increased protection against nucleases and targeting capability via simple surface modification. We constructed an antisense gold-nanobeacon consisting of a stem-looped oligonucleotide double-labelled with 3'-Cy3 and 5'-Thiol-C6 and tested for the effective blocking of gene expression in colorectal cancer cells. Due to the beacon conformation, gene silencing was directly detected as fluorescence increases with hybridisation to target, which can be used to assess the level of silencing. Moreover, this system was extensively evaluated for the genotoxic, cytotoxic and proteomic effects of gold-nanobeacon exposure to cancer cells. The exposure was evaluated by two-dimensional protein electrophoresis followed by mass spectrometry to perform a proteomic profile and 3-(4,5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, glutathione-S-transferase assay, micronucleus test and comet assay to assess the genotoxicity. This integrated toxicology evaluation showed that the proposed nanotheranostics strategy does not exhibit significant toxicity, which is extremely relevant when translating into in vivo systems.
反义疗法是一种适用于下调与疾病相关的靶基因的转录后基因沉默的强大工具。金纳米粒子已被描述为反义寡核苷酸的有效细胞内递药载体，通过简单的表面修饰提供了增加的对核酸酶的保护和靶向能力。我们构建了一种反义金纳米信标，由带有 3'-Cy3 和 5'-巯基-C6 的茎环寡核苷酸双标记，并测试了其在结直肠癌细胞中有效阻断基因表达的能力。由于信标构象，基因沉默可以直接通过与靶标杂交来检测荧光增加，这可用于评估沉默水平。此外，该系统还广泛评估了金纳米信标暴露于癌细胞时的遗传毒性、细胞毒性和蛋白质组学效应。通过二维蛋白质电泳和质谱分析进行蛋白质组学分析，以及 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴盐（MTT）测定、谷胱甘肽-S-转移酶测定、微核试验和彗星试验来评估遗传毒性。这种综合毒理学评价表明，所提出的纳米治疗策略没有表现出显著的毒性，当转化为体内系统时，这是极其相关的。
