title: The effect of serum in culture on RNAi efficacy through modulation of polyplexes size.
abstract: Serum in the culture medium is one crucial factor that compromises RNAi efficiency of non-viral vectors. However, mechanistic roles of serum in siRNA delivery remain unknown. In this work, we took one cationic polymer, pullulan chemically modified by spermine (termed as pullulan-spermine, Ps), as a siRNA carrier model to investigate the effects of serum on key steps in siRNA delivery including formation of Ps and siRNA polyplexes (Ps-siRNA), cellular uptake, lysosomal escape, and cytotoxicity. We demonstrate that low serum concentration (1.25% and 2.5%) in culture medium results in large particles of Ps-siRNA, while high serum concentration (10%-40%) leads to small particles of Ps-siRNA. The larger particles initiated the internalization of siRNA more effectively in comparison to the smaller ones. The engulfed Ps-siRNA particles mainly locate in lysosomes. The large particles exhibited stronger abilities of destabilizing lysosomes than that of the small particles as large Ps-siRNA particles contain more amines and subsequently elicit a stronger proton sponge effect which results in more effective lysosomal escape of siRNA. Despite the lower RNAi efficiency, the small particle of Ps-siRNA in the high serum medium generates much lower cytotoxicity. These findings explain why serum significantly affects RNAi and also propose a strategy for improving RNAi efficiency and safety by modulating serum concentration and enhancing lysosomal destabilization.
authors.affiliation: Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100005, PR China.
authors.forename: Weiqi
authors.initials: W
authors.lastname: Zhang
authors.forename: Jian
authors.initials: J
authors.lastname: Liu
authors.forename: Yasuhiko
authors.initials: Y
authors.lastname: Tabata
authors.forename: Jie
authors.initials: J
authors.lastname: Meng
authors.forename: Haiyan
authors.initials: H
authors.lastname: Xu
mesh_terms: D001769:Blood; D045744:Cell Line, Tumor; D003470:Culture Media; D005434:Flow Cytometry; D006801:Humans; D008247:Lysosomes / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008855:Microscopy, Electron, Scanning; D034622:RNA Interference
chemical_list: D003470:Culture Media
keywords: Endo-lysosomal escape; Non-viral vectors; Pullulan-spermine; RNA interference; Serum tolerance