Data relating to Nucleophile responsive charge-reversing polycations for pDNA transfection

Polycationic carriers promise low cost and scalable gene therapy treatments, however inefficient intracellular unpacking of the genetic cargo has limited transfection efficiency. Charge-reversing polycations, which transition from cationic to neutral or negative charge, can offer targeted intracellular DNA release. We describe a new class of charge-reversing polycation which undergoes a cationic-to-neutral conversion by a reaction with cellular nucleophiles. The deionization reaction is relatively slow with primary amines, and much faster with thiols. In mammalian cells, the intracellular environment has elevated concentrations of amino acids (∼10×) and the thiol glutathione (∼1000×). We propose this allows for decationization of the polymeric carrier slowly in the extracellular space and then rapidly in the intracellular milleu for DNA release. We demonstrate that in a lipopolyplex formulation this leads to both improved transfection and reduced cytotoxicity when compared to a non-responsive polycationic control.

聚阳离子载体（Polycationic carriers）有望实现低成本且可规模化的基因治疗方案，但遗传载荷的细胞内脱包效率低下，限制了转染效率。电荷反转聚阳离子（Charge-reversing polycations）——即从阳离子转变为中性或负电性的聚阳离子——可实现靶向性的细胞内DNA释放。我们报道了一类新型电荷反转聚阳离子，其可通过与细胞亲核试剂的反应实现阳离子向中性的转变。伯胺参与的脱阳离子反应速率相对较慢，而硫醇（thiols）参与的反应速率则显著更快。在哺乳动物细胞中，细胞内环境的氨基酸浓度约为胞外的10倍，谷胱甘肽（glutathione，一种硫醇类物质）的浓度约为胞外的1000倍。我们认为，这一特性可使聚合物载体在细胞外空间缓慢脱阳离子化，随后在细胞内环境中快速脱阳离子化，从而实现DNA释放。我们证实，与无响应性聚阳离子对照载体相比，采用脂质多聚复合物（lipopolyplex）制剂的该体系可同时提升转染效率并降低细胞毒性。