Anti-EphA10 antibody-conjugated pH-sensitive liposomes for specific intracellular delivery of siRNA

Abstract

Xinlong Zang,1 Huaiwei Ding,2 Xiufeng Zhao,3 Xiaowei Li,1 Zhouqi Du,1 Haiyang Hu,1 Mingxi Qiao,1 Dawei Chen,1 Yuihui Deng,1 Xiuli Zhao1 1Department of Pharmaceutics, School of Pharmacy, 2Department of Pharmaceutical Chemistry, School of Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang, People’s Republic of China; 3Hongqi Hospital affiliated to Mudanjiang Medical University, Mudanjiang, People’s Republic of ChinaAbstract: Therapeutic delivery of small interfering RNA (siRNA) is a major challenge that limits its potential clinical application. Here, a pH-sensitive cholesterol–Schiff base–polyethylene glycol (Chol–SIB–PEG)-modified cationic liposome–siRNA complex, conjugated with the recombinant humanized anti-EphA10 antibody (Eph), was developed as an efficient nonviral siRNA delivery system. Chol–SIB–PEG was successfully synthesized and confirmed with FTIR and 1H-NMR. An Eph–PEG–SIB–Chol-modified liposome–siRNA complex (EPSLR) was prepared and characterized by size, zeta potential, gel retardation, and encapsulation efficiency. Electrophoresis results showed that EPSLR was resistant to heparin replacement and protected siRNA from fetal bovine serum digestion. EPSLR exhibited only minor cytotoxicity in MCF-7/ADR cells. The results of flow cytometry and confocal laser scanning microscopy suggested that EPSLR enhanced siRNA transfection in MCF-7/ADR cells. Intracellular distribution experiment revealed that EPSLR could escape from the endo-lysosomal organelle and release siRNA into cytoplasm at 4 hours posttransfection. Western blot experiment demonstrated that EPSLR was able to significantly reduce the levels of MDR1 protein in MCF-7/ADR cells. The in vivo study of DIR-labeled complexes in mice bearing MCF-7/ADR tumor indicated that EPSLR could reach the tumor site rather than other organs more effectively. All these results demonstrate that EPSLR has much potential for effective siRNA delivery and may facilitate its therapeutic application. Keywords: siRNA, cationic liposome, pH sensitive, endosomal escape, anti-EphA10 antibody, gene silencing

Keywords

• siRNA
• cationic liposome
• pH sensitive
• endosomal escape
• EphA10 antibody
• gene silencing