title: Investigation of the performance of PEG-PEI/ROCK-II-siRNA complexes for Alzheimer's disease in vitro.
abstract: Recent studies have showed inhibiting ROCK promoted axonal regeneration and suppressing ROCK-II decreased Aβ formation, suggesting ROCK is a potential target for the treatment of Alzheimer's disease. Because ROCK-II mRNA is abundantly expressed in brain, we targeted ROCK-II mRNA using a siRNA approach. To suppress ROCK-II mRNA expression, we synthesized PEG-PEI/ROCK-II-siRNA complexes and transfected C17.2 neural stem cells in vitro. The characteristics of the complexes were tested using a gel retardation assay. Particle size and zeta potential were examined using dynamic light scattering and the morphology of the complexes were observed by transmission electron microscopy. The toxicity was detected by an MTT assay and transfection efficiency was determined by flow cytometry. Laser confocal microscopy was employed to investigate the cell uptake of the complexes. RT-PCR and western blotting were used to verify the effect of gene silencing. Our results indicated that the characteristics of the complexes depended on the N/P ratios. At a high N/P ratio, PEG-PEI could completely condense the siRNA into small-sized uniform particles. However, high N/P ratios are accompanied with high cytotoxicity. Because of high transfection efficiency and low cytotoxicity, N/P=50 was chosen to transfect C17.2 cells in vitro. Laser confocal microscopy showed that ROCK-II-siRNA with green fluorescence was mainly distributed in the cytoplasm and synapses. Moreover, ROCK-II-siRNA was successfully released from the lysosome. RT-PCR and western blotting demonstrated effective gene silencing. These results indicated that PEG-PEI/ROCK-II-siRNA complexes effectively suppressed ROCK-II mRNA expression, providing the basis for future research in vivo.
authors.affiliation: Department of Neurology, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China.
authors.forename: Yunyun
authors.initials: Y
authors.lastname: Liu
authors.forename: Zhonglin
authors.initials: Z
authors.lastname: Liu
authors.forename: Yong
authors.initials: Y
authors.lastname: Wang
authors.forename: Yan-Ran
authors.initials: YR
authors.lastname: Liang
authors.forename: Xiaojun
authors.initials: X
authors.lastname: Wen
authors.forename: Jingyang
authors.initials: J
authors.lastname: Hu
authors.forename: Xingyi
authors.initials: X
authors.lastname: Yang
authors.forename: Jun
authors.initials: J
authors.lastname: Liu
authors.forename: Songhua
authors.initials: S
authors.lastname: Xiao
authors.forename: Du
authors.initials: D
authors.lastname: Cheng
mesh_terms: D000544:Alzheimer Disease / Q000188:drug therapy*; D000818:Animals; D015153:Blotting, Western; D002470:Cell Survival / Q000187:drug effects; D002850:Chromatography, Gel; D004337:Drug Carriers; D004563:Electrochemistry; D020868:Gene Silencing / Q000187:drug effects*; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D053758:Nanoparticles; D058953:Neural Stem Cells; D010316:Particle Size; D011092:Polyethylene Glycols; D011094:Polyethyleneimine / Q000031:analogs & derivatives; D034741:RNA, Small Interfering / Q000627:therapeutic use*; D060888:Real-Time Polymerase Chain Reaction; D054460:rho-Associated Kinases / Q000235:genetics*
chemical_list: D004337:Drug Carriers; D034741:RNA, Small Interfering; C544016:poly(ethylene glycol)-co-poly(ethyleneimine); D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D054460:rho-Associated Kinases