Co-transfection of plasmid DNA and laser-generated gold nanoparticles does not disturb the bioactivity of GFP-HMGB1 fusion protein
Co-transfection of plasmid DNA and laser-generated gold nanoparticles does not disturb the bioactivity of GFP-HMGB1 fusion protein
复制标题
DOI：
10.1186/1477-3155-7-6
复制
发表时间：
2009-10-24
影响因子：
10.2
通讯作者：
Escobar, Hugo Murua
中科院分区：
文献类型：
--
作者：
Petersen, Svea;Soller, Jan T.;Escobar, Hugo Murua
超短脉冲激光烧蚀液体是制备纯金纳米粒子(AuNPs)的有力工具，避免了化学前驱体的产生，从而使其在生物医学应用中特别有趣。然而，由于其接受电子的特性，激光产生的AuNPs可能会影响生物分子的生化性质，这些生物分子经常吸附在纳米颗粒上。我们研究了这种激光产生的AuNPs对DNA分子生物功能的可能影响。我们测试了四个不同大小和带正电荷的AuNPs，方法是将它们与重组EGFP-C1-HMGB1 DNA表达载体孵育，这些重组DNA表达载体编码EGFP融合蛋白，并包含犬建筑转录因子HMGB1。我们能够证明，成功转基因的哺乳动物细胞仍然能够合成和处理融合蛋白。我们的观察表明，AuNP与编码重组犬HMGB1的质粒DNA孵育，既不阻止在有转染剂存在的情况下通过质膜介导对载体的摄取，也不影响合成的融合蛋白向细胞核的运输。重组GFP-HMGB1融合蛋白的生物学活性似乎也没有受到影响，因为可以观察到强烈的特征蛋白在细胞核中积累。我们还发现，转染率依赖于AuNP的大小。总之，我们的数据表明，在生物医学应用中，激光产生的AuNPs是一种很好的替代化学合成纳米颗粒的方法。
Ultrashort pulsed laser ablation in liquids represents a powerful tool for the generation of pure gold nanoparticles (AuNPs) avoiding chemical precursors and thereby making them especially interesting for biomedical applications. However, because of their electron accepting properties, laser-generated AuNPs might affect biochemical properties of biomolecules, which often adsorb onto the nanoparticles. We investigated possible effects of such laser-generated AuNPs on biological functionality of DNA molecules. We tested four differently sized and positively charged AuNPs by incubating them with recombinant eGFP-C1-HMGB1 DNA expression plasmids that code for eGFP fusion proteins and contain the canine architectural transcription factor HMGB1. We were able to show that successfully transfected mammalian cells are still able to synthesize and process the fusion proteins. Our observations revealed that incubation of AuNP with the plasmid DNA encoding the recombinant canine HMGB1 neither prevented the mediated uptake of the vector through the plasma membrane in presence of a transfection reagent nor had any effect on the transport of the synthesized fusion proteins to the nuclei. Biological activity of the recombinant GFP-HMGB1 fusion protein appears to have not been affected either, as a strong characteristic protein accumulation in the nucleus could be observed. We also discovered that transfection efficiencies depend on the size of AuNP. In conclusion, our data indicate that laser-generated AuNPs present a good alternative to chemically synthesized nanoparticles for use in biomedical applications.