T7肽修饰氧化还原敏感型聚酰胺-胺作为siRNA递送载体的初步评价

Preliminary Evaluation of T7 Modified Redox-sensitive Polyamide Amine for siRNA Delivery

拟构建一种具有潜在肝肿瘤靶向作用的T7肽修饰氧化还原敏感聚酰胺-胺(T7-PEG-SS-PAMAM,T7-PSSP)复合物,并用于M2型丙酮酸激酶(PKM2)-siRNA的体外递送。聚合物T7-PSSP的结构特征通过~1H NMR进行确证。制备的T7-PSSP/siRNA复合物粒径为(119.8±0.76)nm,多分散系数(PDI)为0.19±0.12,ζ电位为(8.92±0.38)mV。琼脂糖凝胶电泳试验证明载体-基因复合物具有较好的稳定性,且对siRNA具有较好的保护作用。通过凝胶电泳法观察到,在谷胱甘肽还原剂的作用下,siRNA能快速从PSSP/siRNA复合物中释放出来,说明PSSP/siRNA复合物在还原环境中可有效释出携载的siRNA。体外摄取试验表明,T7肽的修饰可以显著提高siRNA在肝癌细胞HepG2中的摄取量。实时荧光定量RT-PCR结果显示,与阴性对照和其他复合物组相比,T7-PSSP/siRNA具有最高的PKM2基因转染和沉默效率。

In this study, a T7 modified redox-sensitive polyamide amine（T7-PEG-SS-PAMAM, T7-PSSP）conjugate with potential for liver cancer targeting was constructed for pyruvate kinase M2（PKM2）-siRNA delivery. 1H NMR was used to confirm the structural characters of the polymer. The particle size, polydispersity index and ζ potential of the T7-PSSP/siRNA complex were（119.8±0.76）nm, 0.19±0.12 and（8.92±0.38）mV, respectively. The results of agarose gel electrophoresis indicated that the carrier-gene complex had a good stability and could protect siRNA from RNase degradation. Flow cytometry quantitative analysis confirmed that the modification of T7 peptide could significantly increase the uptake of siRNA in HepG2 cells. According to the semi-quantitative analysis results, siRNA was rapidly released from the PSSP/siRNA complex under the action of glutathione reductant, indicating that the PSSP/siRNA complex could efficiently release the delivered siRNA in a reducing environment. Real-time fluorescence quantitative detection results showed that T7-PSSP/siRNA complex displayed the highest PKM2 gene transfection and silencing efficiency compared to the negative control and other complexes groups. The above results showed that this bifunctional siRNA delivery system, T7-PSSP/siRNA complex, had both tumor-targeted and redox-sensitive property, it could effectively release siRNA in the intracellular environment to enhance siRNA knockout effect, and thus could be used as a potential nanocarrier for efficient siRNA delivery.