Abstract

Polyethylenimines (PEIs) have been previously introduced for siRNA delivery. In particular, in the case of higher molecular weight PEIs, this is associated with toxicity, while low molecular weight PEIs are often insufficient for siRNA complexation. The tyrosine-modification of PEIs has been shown to enhance PEI efficacy and biocompatibility. This paper evaluates a set of tyrosine-modified low molecular weight linear or branched polyethylenimines as efficient carriers of siRNA. Complexation efficacies and biophysical complex properties were analyzed by zeta potential, dynamic light scattering and circular dichroism measurements as well as gel electrophoresis. Biological knockdown was studied in 2 D cell culture and 3 D ex vivo tissue slice air-liquid interface culture. The results demonstrate that siRNAs were able to form stable complexes with all tested polymers. Complexation was able to protect siRNA from degradation by RNase and to mediate target gene knockdown, as determined on the mRNA level and in PC3-Luc3/EGFP and HCT116-Luc3/EGFP expressing reporter cells on the protein level, using flow cytometry and confocal microscopy. The direct comparison of the studied polymers revealed differences in biological efficacies. Moreover, the tyrosine-modified PEIs showed high biocompatibility, as determined by LDH release and mitochondria integrity (J-aggregate assay) as well as caspase 3/7 (apoptosis) and H₂O₂ levels (ROS). In 3 D tissue slices, complexes based on LP10Y proved to be most efficient, by combining tissue penetration with efficient gene expression knockdown.

摘要

先前已引入聚乙烯亚胺 (PEI) 用于 siRNA 递送。特别是，在较高分子量 PEI 的情况下，这与毒性有关，而低分子量 PEI 通常不足以进行 siRNA 络合。PEI 的酪氨酸修饰已显示可增强 PEI 功效和生物相容性。本文评估了一组酪氨酸修饰的低分子量线性或支化聚乙烯亚胺作为 siRNA 的有效载体。通过 zeta 电位、动态光散射和圆二色性测量以及凝胶电泳分析络合功效和生物物理复杂特性。在 2 D 细胞培养和 3 D离体研究中研究了生物敲除组织切片气液界面培养。结果表明，siRNA 能够与所有测试的聚合物形成稳定的复合物。络合能够保护 siRNA 不被 RNase 降解并介导靶基因敲低，如在 mRNA 水平和在蛋白质水平上表达报告细胞的 PC3-Luc3/EGFP 和 HCT116-Luc3/EGFP 中所确定的，使用流式细胞术和共聚焦显微镜. 所研究的聚合物的直接比较揭示了生物功效的差异。此外，酪氨酸修饰的 PEI 显示出高生物相容性，这由 LDH 释放和线粒体完整性（J-聚集测定）以及半胱天冬酶 3/7（细胞凋亡）和 H 2 _O 2_确定水平（活性氧）。在 3D 组织切片中，基于 LP10Y 的复合物被证明是最有效的，它结合了组织穿透和有效的基因表达抑制。