Mao Shirui, Neu Michael, Germershaus Oliver, Merkel Olivia, Sitterberg Johannes, Bakowsky Udo, Kissel Thomas
Department of Pharmaceutics and Biopharmacy, Philipps Universität Marburg, Ketzerbach 63, 35032 Marburg, Germany.
Bioconjug Chem. 2006 Sep-Oct;17(5):1209-18. doi: 10.1021/bc060129j.
Polyplexes between siRNA and poly(ethylene imine) (PEI) derivatives are promising nonviral carriers for siRNA. The polyplex stability is of critical importance for efficient siRNA delivery to the cytoplasm. Here, we investigate the effect of PEGylation at a constant ratio ( approximately 50%) on the biophysical properties of the polyplexes. Particle size, zeta potential, and stability against heparin as well as RNase digestion and reporter gene knockdown under in vitro conditions of different siRNA polyplexes were characterized. Stability and size of siRNA polyplexes were clearly influenced by PEI-PEG structure, and high degrees of substitution such as PEI(25k)-g-PEG(550)(30) resulted in large (300-400 nm), diffuse complexes (AFM) which showed condensation behavior only at high N/P ratios. All other polyplexes and the PEI control showed similar sizes (150 nm) and compact structures in AFM, with complete condensation reached at N/P ratio of 3. Stability of siRNA polyplexes against heparin displacement and RNase digestion could be modified by PEGylation. Protection against RNase digestion was highest for PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1), while siRNA/PEI provided insufficient protection. In knockdown experiments using NIH/3T3 fibroblasts stably expressing beta-galactosidase, it was shown that PEG chain length had a significant influence on biological activity of siRNA. Polyplexes with siRNA containing PEI(25k)-g-PEG(5k)(4) and PEI(25k)-g-PEG(20k)(1) yielded similar efficiencies of ca. 70% knockdown as lipofectamine controls. Confocal microscopy demonstrated enhanced cellular uptake of siRNA into cytosol by polyplexes formation with PEI copolymers. In conclusion, both the chain length and graft density of PEG were found to strongly influence siRNA condensation and stability and hence affect the knockdown efficiency of PEI-PEG/siRNA polyplexes.
小干扰RNA（siRNA）与聚乙烯亚胺（PEI）衍生物形成的多聚体是很有前景的非病毒siRNA载体。多聚体稳定性对于将siRNA有效递送至细胞质至关重要。在此，我们研究了以恒定比例（约50%）进行聚乙二醇化对多聚体生物物理性质的影响。对不同siRNA多聚体在体外条件下的粒径、zeta电位、抗肝素稳定性以及核糖核酸酶消化和报告基因敲低情况进行了表征。siRNA多聚体的稳定性和大小明显受PEI-PEG结构影响，高取代度如PEI(25k)-g-PEG(550)(30)会导致形成大的（300 - 400 nm）、分散的复合物（原子力显微镜观察），这种复合物仅在高N/P比时才表现出凝聚行为。所有其他多聚体和PEI对照在原子力显微镜下显示出相似的大小（150 nm）和紧密结构，在N/P比为3时达到完全凝聚。聚乙二醇化可改变siRNA多聚体抗肝素置换和核糖核酸酶消化的稳定性。PEI(25k)-g-PEG(5k)(4)和PEI(25k)-g-PEG(20k)(1)对核糖核酸酶消化的保护作用最强，而siRNA/PEI提供的保护不足。在使用稳定表达β-半乳糖苷酶的NIH/3T3成纤维细胞进行的敲低实验中，结果表明聚乙二醇链长度对siRNA的生物活性有显著影响。含有PEI(25k)-g-PEG(5k)(4)和PEI(25k)-g-PEG(20k)(1)的siRNA多聚体产生的敲低效率与脂质体对照相似，约为70%。共聚焦显微镜显示，通过与PEI共聚物形成多聚体，siRNA进入细胞质的细胞摄取增强。总之，发现聚乙二醇的链长度和接枝密度均强烈影响siRNA的凝聚和稳定性，进而影响PEI-PEG/siRNA多聚体的敲低效率。
