Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation
Functionalization with C-terminal cysteine enhances transfection efficiency of cell-penetrating peptides through dimer formation
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DOI：
10.1016/j.bbrc.2012.01.041
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发表时间：
2012-02-17
影响因子：
3.1
通讯作者：
Fant, Kristina
中科院分区：
文献类型：
--
作者：
Amand, Helene L.;Norden, Bengt;Fant, Kristina
细胞穿透肽能够以无毒的方式刺激哺乳动物细胞对大分子货物的吸收，因此有望成为高效且耐受性良好的基因传递载体。非共价肽- dna复合物（“肽复合物”）通过内吞作用进入细胞，但肽复合物稳定性差和内体包裹被认为是肽介导递送的主要障碍。我们探索了一种简单而高效的策略，通过添加一个末端半胱氨酸残基来改善肽基载体的功能。这使得肽通过二硫键形成二聚体，通过“螯合效应”增加其对核酸的亲和力，并且当键在细胞内减少时，让复合物解离以传递核酸。通过在经典CPP穿透蛋白及其类似物PenArg和EB1中引入单个c端半胱氨酸，我们发现这种微小的修饰极大地增强了HEK293T细胞中质粒DNA的转染能力。我们得出的结论是，这种影响主要是由于肽群的热力学稳定性增强，因为转染仍然需要破坏内核体的氯喹，并且对于固有DNA冷凝能力较低的肽，这种影响更为明显。有趣的是，对于EB1，添加一个半胱氨酸使肽能够在没有氯喹的情况下介导转染，这表明二聚化也可以改善内体逃逸特性。此外，EB1肽丛的细胞毒性大大降低，可能是由于其与DNA的强结合导致游离肽二聚体浓度降低。(C) 2012爱思唯尔公司版权所有。
Cell-penetrating peptides have the ability to stimulate uptake of macromolecular cargo in mammalian cells in a non-toxic manner and therefore hold promise as efficient and well tolerated gene delivery vectors. Non-covalent peptide-DNA complexes ("peptiplexes") enter cells via endocytosis, but poor peptiplex stability and endosomal entrapment are considered as main barriers to peptide-mediated delivery. We explore a simple, yet highly efficient, strategy to improve the function of peptide-based vectors, by adding one terminal cysteine residue. This allows the peptide to dimerize by disulfide bond formation, increasing its affinity for nucleic acids by the "chelate effect" and, when the bond is reduced intracellularly, letting the complex dissociate to deliver the nucleic acid. By introducing a single C-terminal cysteine in the classical CPP penetratin and the penetratin analogs PenArg and EB1, we show that this minor modification greatly enhances the transfection capacity for plasmid DNA in HEK293T cells. We conclude that this effect is mainly due to enhanced thermodynamic stability of the peptiplexes as endosome-disruptive chloroquine is still required for transfection and the effect is more pronounced for peptides with lower inherent DNA condensation capacity. Interestingly, for EB1, addition of one cysteine makes the peptide able to mediate transfection in absence of chloroquine, indicating that dimerisation can also improve endosomal escape properties. Further, the cytotoxicity of EB1 peptiplexes is considerably reduced, possibly due to lower concentration of free peptide dimer resulting from its stronger binding to DNA. (C) 2012 Elsevier Inc. All rights reserved.