Chen Siyu, Pinto Carneiro Simone, Merkel Olivia M
Ludwig-Maximilians-University, Department of Pharmacy, Pharmaceutical Technology and Biopharmaceutics, Butenandtstraße 5-13, Munich, 81377, Germany.
Biomater Sci. 2025 Jan 28;13(3):659-676. doi: 10.1039/d4bm01290a.
Polymeric carriers have long been recognized as some of the most effective and promising systems for nucleic acid delivery. In this study, we utilized an anionic di-block co-polymer, PEG-PLE, to enhance the performance of lipid-modified PEI (C14-PEI) nanoplexes for delivering Cas9 mRNA and sgRNA targeting KRAS G12S mutations in lung cancer cells. Our results demonstrated that PEG-PLE, when combined with C14-PEI at a weight-to-weight ratio of 0.2, produced nanoplexes with a size of approximately 140 nm, a polydispersity index (PDI) of 0.08, and a zeta potential of around -1 mV. The PEG-PLE/C14-PEI nanoplexes at this ratio were observed to be both non-cytotoxic and effective in encapsulating Cas9 mRNA and sgRNA. Confocal microscopy imaging revealed efficient endosomal escape and intracellular distribution of the RNAs. Uptake pathway inhibition studies indicated that the internalization of PEG-PLE/C14-PEI primarily involves scavenger receptors and clathrin-mediated endocytosis. Compared to C14-PEI formulations, PEG-PLE/C14-PEI demonstrated a significant increase in luciferase mRNA expression and gene editing efficiency, as confirmed by T7EI and ddPCR, in A549 cells. Sanger sequencing identified insertions and/or deletions around the PAM sequence, with a total of 69% indels observed. Post-transfection, the KRAS-ERK pathway was downregulated, resulting in significant increases in cell apoptosis and inhibition of cell migration. Taken together, this study reveals a new and promising formulation for CRISPR delivery as potential lung cancer treatment.
长期以来，聚合物载体一直被认为是核酸递送中一些最有效且最具前景的系统。在本研究中，我们利用一种阴离子二嵌段共聚物PEG-PLE来提高脂质修饰的PEI（C14-PEI）纳米复合物在肺癌细胞中递送靶向KRAS G12S突变的Cas9 mRNA和sgRNA的性能。我们的结果表明，当PEG-PLE与C14-PEI以0.2的重量比组合时，产生的纳米复合物尺寸约为140 nm，多分散指数（PDI）为0.08，zeta电位约为 -1 mV。观察到该比例的PEG-PLE/C14-PEI纳米复合物既无细胞毒性，又能有效包封Cas9 mRNA和sgRNA。共聚焦显微镜成像显示RNA能有效从内体逃逸并在细胞内分布。摄取途径抑制研究表明，PEG-PLE/C14-PEI的内化主要涉及清道夫受体和网格蛋白介导的内吞作用。与C14-PEI制剂相比，通过T7EI和ddPCR证实，PEG-PLE/C14-PEI在A549细胞中的荧光素酶mRNA表达和基因编辑效率显著提高。Sanger测序鉴定了PAM序列周围的插入和/或缺失情况，共观察到69%的插入缺失。转染后，KRAS-ERK途径被下调，导致细胞凋亡显著增加并抑制细胞迁移。综上所述，本研究揭示了一种用于CRISPR递送的新型且有前景的制剂，可作为潜在的肺癌治疗方法。
