Oude Blenke Erik E, van den Dikkenberg Joep, van Kolck Bartjan, Kros Alexander, Mastrobattista Enrico
Department of Pharmaceutics, Utrecht Institute of Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, 3584 CG Utrecht, The Netherlands.
Nanoscale. 2016 Apr 28;8(16):8955-65. doi: 10.1039/c6nr00711b. Epub 2016 Apr 13.
Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.
卷曲螺旋相互作用是一种强大的蛋白质-蛋白质相互作用，参与许多生物过程，包括细胞内运输和膜融合。一种合成的异源二聚体卷曲螺旋形成肽对，即E3（EIAALEK）3和K3（KIAALKE）3，被用于使包裹剪接校正寡核苷酸或小干扰RNA（siRNA）的脂质体功能化。这些肽功能化的囊泡在溶液中高度稳定，但当用互补肽修饰的囊泡混合在一起时开始聚集，这表明肽迅速卷曲并使囊泡交联。当其中一种肽使用疏水性胆固醇锚定在细胞膜上时，用互补肽功能化的囊泡可以对接至这些细胞，而非功能化的细胞则未显示任何囊泡附着。尽管锚定的肽没有下游信号通路，但显微镜图像显示，四小时后，大多数对接的囊泡通过内吞作用被内化。最后，首次表明囊泡与细胞膜界面处的卷曲螺旋组装诱导主动摄取并导致核酸货物的胞质递送。siRNA和剪接校正寡核苷酸均实现了功能递送，分别导致相应细胞系中荧光素酶表达的沉默或恢复。这些结果表明，通过卷曲螺旋相互作用与细胞对接可诱导主动摄取，并以高度特异性的方式成功实现原本不能透过膜的核酸的细胞内递送。
