Department of Chemical Engineering, Imperial College London, South Kensington Campus, London, SW7 2AZ, UK.
Biomater Sci. 2024 Sep 25;12(19):5010-5022. doi: 10.1039/d4bm00600c.
Saporin is a 28 621 Da protein and plant toxin possessing rRNA -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.
丝氨酸蛋白酶抑制剂是一种 28621 道尔顿的蛋白质和植物毒素，具有 rRNA-糖苷酶活性。由于其强大的核糖体失活能力，丝氨酸蛋白酶抑制剂通常被研究作为抗癌剂。然而，其酶活性受到其较差的质膜通透性的极大阻碍。为了克服这一障碍，我们使用了一种基于 pH 响应假肽的仿生细胞内递药平台，该假肽由等摩尔百分比为 50%的 L-苯丙氨酸接枝的聚（L-赖氨酸异苯丙酰胺）组成（PP50）。在温和的酸性 pH 环境中，将 PP50 与丝氨酸蛋白酶抑制剂（PP50/丝氨酸蛋白酶抑制剂）共同孵育，以辅助细胞内递药并增加丝氨酸蛋白酶抑制剂的治疗潜力。我们证明，PP50 大大增强了丝氨酸蛋白酶抑制剂在 2D 单层 A549 细胞和 3D A549 多细胞球体中的细胞毒性，而单独给药时则没有毒性。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/丝氨酸蛋白酶抑制剂进入细胞的机制。在细胞凋亡和细胞死亡发生之前，我们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。PP50/丝氨酸蛋白酶抑制剂的进入还依赖于微胞饮作用和网格蛋白介导的内吞作用。此外，当与 Cy5 标记的 PP50（Cy5-PP50）一起递送时，异硫氰酸荧光素标记的丝氨酸蛋白酶抑制剂（FITC-丝氨酸蛋白酶抑制剂）被定位在细胞质和细胞核内。综上所述，这表明在用 PP50/丝氨酸蛋白酶抑制剂处理的细胞中，多条途径被触发以启动细胞凋亡和细胞死亡。因此，这些结果表明，PP50 是一种用于蛋白质治疗剂内化的潜在细胞内递药平台。
