Mykhaylyk Olga, Sanchez-Antequera Yolanda, Vlaskou Dialechti, Cerda Maria Belen, Bokharaei Mehrdad, Hammerschmid Edelburga, Anton Martina, Plank Christian
Institute of Experimental Oncology, Klinikum rechts der Isar der Technischen Universität München, Ismaninger Strasse 22, Munich, 81675, Germany,
Methods Mol Biol. 2015;1218:53-106. doi: 10.1007/978-1-4939-1538-5_5.
This chapter describes how to design and conduct experiments to deliver siRNA to adherent cell cultures in vitro by magnetic force-assisted transfection using self-assembled complexes of small interfering RNA (siRNA) and cationic lipids or polymers that are associated with magnetic nanoparticles (MNPs). These magnetic complexes are targeted to the cell surface by the application of a gradient magnetic field. A further development of the magnetic drug-targeting concept is combining it with an ultrasound-triggered delivery using magnetic microbubbles as a carrier for gene or drug delivery. For this purpose, selected MNPs, phospholipids, and siRNAs are assembled in the presence of perfluorocarbon gas into flexible formulations of magnetic lipospheres (microbubbles). Methods are described how to accomplish the synthesis of magnetic nanoparticles for magnetofection and how to test the association of siRNA with the magnetic components of the transfection vector. A simple method is described to evaluate magnetic responsiveness of the magnetic siRNA transfection complexes and estimate the complex loading with magnetic nanoparticles. Procedures are provided for the preparation of magnetic lipoplexes and polyplexes of siRNA as well as magnetic microbubbles for magnetofection and downregulation of the target gene expression analysis with account for the toxicity determined using an MTT-based respiration activity test. A modification of the magnetic transfection triplexes with INF-7, fusogenic peptide, is described resulting in reporter gene silencing improvement in HeLa, Caco-2, and ARPE-19 cells. The methods described can also be useful for screening vector compositions and novel magnetic nanoparticle preparations for optimized siRNA transfection by magnetofection in any cell type.
本章描述了如何设计和进行实验，通过磁力辅助转染，将小干扰RNA（siRNA）递送至体外贴壁细胞培养物中。该方法利用小干扰RNA（siRNA）与阳离子脂质或聚合物的自组装复合物，这些复合物与磁性纳米颗粒（MNP）相关联。通过施加梯度磁场，这些磁性复合物靶向细胞表面。磁性药物靶向概念的进一步发展是将其与超声触发递送相结合，使用磁性微泡作为基因或药物递送的载体。为此，将选定的磁性纳米颗粒、磷脂和小干扰RNA在全氟化碳气体存在下组装成磁性脂质球（微泡）的柔性制剂。文中描述了用于磁转染的磁性纳米颗粒的合成方法，以及如何测试小干扰RNA与转染载体磁性成分的结合。还描述了一种简单的方法来评估磁性小干扰RNA转染复合物的磁响应性，并估计磁性纳米颗粒的复合物负载量。提供了制备小干扰RNA的磁性脂质复合物和多聚复合物以及用于磁转染的磁性微泡的程序，以及用于分析靶基因表达下调的方法，并考虑了使用基于MTT的呼吸活性测试确定的毒性。还描述了用INF-7（融合肽）对磁性转染三联体进行修饰，从而改善HeLa、Caco-2和ARPE-19细胞中的报告基因沉默。所描述的方法对于筛选载体组合物和新型磁性纳米颗粒制剂以优化任何细胞类型中的磁转染小干扰RNA转染也可能有用。
