非病毒基因递送系统通常设计为具有矛盾特性的载体系统,即细胞摄取前的足够稳定性和不稳定,以确保核酸被摄取到细胞后在转录过程中释放。我们先前报道了末端带有多臂PEG(maPEG-PLL)的聚(L-赖氨酸)形成的纳米纤维复合物,可通过maPEG之间的空间排斥抑制过度的DNA缩合,并在PCR扩增实验和无细胞基因表达系统中表现出有效的转录能力。在这项研究中,通过使用具有可在还原条件下破坏的二硫键(SS)的交联试剂在复合物内的PLL侧链之间引入交联来研究纳米纤维-复合物的可逆稳定而不损害有效转录能力。在硫酸葡聚糖和/或二硫苏糖醇的存在下,使用琼脂糖凝胶电泳和实时PCR评估复合物的稳定性和pDNA的反应性。我们成功地使用二硫代双(琥珀酰亚胺丙酸酯)(DSP)作为交联剂来可逆地稳定纳米纤维复合物。在使用培养细胞的实验中证实了可逆稳定的效果,DSP交联的复合物表现出优于聚乙烯亚胺复合物的基因表达,这是典型的聚合复合物。
Non-viral gene delivery systems are typically designed vector systems with contradictory properties, namely sufficient stability before cellular uptake and instability to ensure the release of nucleic acid cargoes in the transcription process after being taken up into cells. We reported previously that poly-(L-lysine) terminally bearing a multi-arm PEG (maPEG-PLL) formed nanofiber-polyplexes that suppressed excessive DNA condensation via steric repulsion among maPEGs and exhibited effective transcriptional capability in PCR amplification experiments and a cell-free gene expression system. In this study, the reversible stabilization of a nanofiber-
polyplex without impairing the effective transcriptional capability was investigated by introducing cross-links between the PLL side chains within the
polyplex using a cross-linking reagent with disulfide (SS) bonds that can be disrupted under reducing conditions. In the presence of dextran sulfate and/or dithiothreitol, the stability of the
polyplex and the reactivity of the pDNA were evaluated using agarose gel electrophoresis and real-time PCR. We succeeded in reversibly stabilizing nanofiber-polyplexes using dithiobis (succinimidyl propionate) (DSP) as the cross-linking reagent. The effect of the reversible stabilization was confirmed in experiments using cultured cells, and the DSP-crosslinked polyplexes exhibited gene expression superior to that of polyethyleneimine polyplexes, which are typical polyplexes.