Saporin 是一种 28 621 Da 的蛋白质和植物毒素，具有 rRNA N-糖苷酶活性。由于皂草素具有强大的核糖体失活能力，因此通常将其作为抗癌剂进行研究。然而，其质膜通透性较差，极大地阻碍了其酶活性。为了克服这一障碍，我们使用了基于 pH 响应性伪肽聚（L-赖氨酸间苯二酰胺）的仿生细胞内递送平台，该平台以化学计量摩尔百分比 50% (PP50) 接枝了 L-苯丙氨酸。 PP50 与皂草素 (PP50/皂草素) 在弱酸性 pH 环境中共同孵育，以帮助细胞内递送并增加皂草素的治疗潜力。我们证明，PP50 极大地增强了皂草素在 2D 单层 A549 细胞和 3D A549 多细胞球体中的细胞毒性，同时单独施用时保持无毒。为了阐明细胞死亡的机制，我们评估了半胱天冬酶的激活、蛋白质合成的抑制、细胞凋亡的开始以及 PP50/皂草素进入的机制。发现蛋白质合成的抑制和半胱天冬酶 3/7、8 和 9 的激活发生在细胞凋亡和细胞死亡开始之前。 PP50/皂草素还被证明依赖于小胞饮作用和小窝介导的内吞作用来进入细胞。此外，当与 Cyanine5 标记的 PP50 (Cy5-PP50) 一起递送时，异硫氰酸荧光素标记的皂草素 (FITC-皂草素) 定位在细胞质和细胞核内。综上所述，这表明在用 PP50/皂草素处理的细胞中，多种途径被触发以引发细胞凋亡和细胞死亡。因此，这些结果使 PP50 成为蛋白质治疗药物内化的潜在细胞内递送平台。

Saporin is a 28 621 Da protein and plant toxin possessing rRNA N-glycosidase activity. Due to its potent ribosome-inactivating ability, saporin is commonly studied as an anticancer agent. However, its enzymatic activity is greatly hindered by its poor plasma membrane permeability. To overcome this barrier, we used a bioinspired intracellular delivery platform based on the pH-responsive pseudopeptide, poly(L-lysine isophthalamide) grafted with L-phenylalanine at a stoichiometric molar percentage of 50% (PP50). PP50 was co-incubated with saporin (PP50/saporin) in a mildly acidic pH environment to aid intracellular delivery and increase saporin's therapeutic potential. We demonstrated that PP50 greatly enhanced the cytotoxicity of saporin in the 2D monolayer of A549 cells and 3D A549 multicellular spheroids whilst remaining non-toxic when administered alone. To elucidate the mechanism of cell death, we assessed the activation of caspases, the inhibition of protein synthesis, the onset of apoptosis and the mechanism of PP50/saporin entry. Inhibition of protein synthesis and activation of caspases 3/7, 8 and 9 were found to occur before the onset of apoptosis and cell death. PP50/saporin was also shown to rely on micropinocytosis and caveolae-mediated endocytosis for cell entry. In addition, fluorescein isothiocyanate-labelled saporin (FITC-saporin) was localized within the cytoplasm and nuclei when delivered with Cyanine5-labelled PP50 (Cy5-PP50). Taken together, this suggests that multiple pathways are triggered to initiate apoptosis and cell death in cells treated with PP50/saporin. Therefore, these results make PP50 a potential intracellular delivery platform for the internalization of protein therapeutics.