Interdisciplinary Nanoscience Center (iNANO), Aarhus University , DK-8000 Aarhus C, Denmark.
Department of Molecular Biology and Genetics, Aarhus University , DK-8000 Aarhus C, Denmark.
ACS Appl Mater Interfaces. 2017 Apr 12;9(14):12203-12216. doi: 10.1021/acsami.6b16556. Epub 2017 Apr 3.
PEGylation of cationic polyplexes is a promising approach to enhance the stability and reduce unspecific interaction with biological components. Herein, we systematically investigate the impact of PEGylation on physical and biological properties of chitosan/siRNA polyplexes. A series of chitosan-PEG copolymers (CS-PEG2k, CS-PEG5k and CS-PEG10k) were synthesized with similar PEG mass content but with different molecular weight. PEGylation with higher molecular weight and less grafting degree resulted in smaller and more compacted nanoparticles with relatively higher surface charge. PEGylated polyplexes showed distinct mechanism of endocytosis, which was macropinocytosis and caveolae-dependent and clathrin-independent. In vitro silencing efficiency in HeLa and H1299 cells was significantly improved by PEGylation and CS-PEG5k/siRNA achieved the highest knockdown efficiency. Efficient silence of ribonucleotide reductase subunit M2 (RRM2) in HeLa cells by CS-PEG5k/siRRM2 significantly induced cell cycle arrest and inhibited cell proliferation. In addition, PEGylation significantly inhibited macrophage phagocytosis and unspecific interaction with red blood cells (RBCs). Significant extension of in vivo circulation was achieved only with high molecular weight PEG modification (CS-PEG10k), whereas all CS/siRNA and CS-PEG/siRNA nanoparticles showed similar pattern of biodistribution with major accumulation in liver and kidney. These results imply that PEGylation with higher molecular weight PEG and less grafting rate is a promising strategy to improve chitosan/siRNA nanocomplexes performance both in vitro and in vivo.
聚乙二醇化阳离子聚合物多聚物是提高稳定性和降低与生物成分非特异性相互作用的一种很有前途的方法。在此，我们系统地研究了聚乙二醇化对壳聚糖/ siRNA 聚合物多聚物的物理和生物学性质的影响。用相似的聚乙二醇质量含量但不同分子量合成了一系列壳聚糖-聚乙二醇共聚物（CS-PEG2k、CS-PEG5k 和 CS-PEG10k）。分子量更高和接枝率更低的聚乙二醇化导致更小和更紧凑的纳米粒子，具有相对较高的表面电荷。聚乙二醇化聚合物多聚物表现出不同的内吞作用机制，这是巨胞饮和网格蛋白独立的、依赖于小窝蛋白的。在 HeLa 和 H1299 细胞中，聚乙二醇化显著提高了体外沉默效率，CS-PEG5k/siRNA 达到了最高的沉默效率。CS-PEG5k/siRRM2 对 HeLa 细胞中核糖核苷酸还原酶亚基 M2（RRM2）的有效沉默显著诱导了细胞周期停滞并抑制了细胞增殖。此外，聚乙二醇化显著抑制了巨噬细胞的吞噬作用和与红细胞（RBC）的非特异性相互作用。只有高分子量 PEG 修饰（CS-PEG10k）才能显著延长体内循环时间，而所有 CS/siRNA 和 CS-PEG/siRNA 纳米颗粒在体内分布模式相似，主要在肝和肾中积累。这些结果表明，高分子量 PEG 和较低接枝率的聚乙二醇化是提高壳聚糖/siRNA 纳米复合物在体外和体内性能的一种很有前途的策略。
