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Abstract #22928 Published in IGR 11-1
Cationic nano-copolymers mediated IKKβ targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro
Duan Y; Guan X; Ge J; Quan D; Zhuo Y; Ye H; Shao TMolecular Vision 2008; 14: 2616-2628
PURPOSE: To synthesize a ternary cationic copolymer called CS-g-(PEI-b-mPEG) and characterize its features as a non-viral siRNA carrier; in turn, to investigate the influence of small interfering RNA (siRNA) targeting I(kappa)B kinase subunit β (IKKβ) on the proliferation of human Tenon's capsule fibroblasts (HTFs) in vitro. METHODS: First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and grafted onto a chitosan (CS) molecule was synthesized. CS-g-(PEI-b-mPEG) was then compacted with 21nt siRNA at various copolymer/siRNA charge (N/P) ratios, and the resulting complexes were characterized by dynamic light scattering, gel electrophoresis, and serum incubation. Cell Titer 96® AQ(ueous) One Solution cell proliferation assay was used to investigate the cytotoxicity of this cationic copolymer. Second, siRNAs targeting IKKβ (IKKB-siRNAs) were delivered into the HTFs using CS-g-(PEI-b-mPEG) as the vehicle. Real-time reverse transcription polymerase chain reaction (RT-PCR) subsequently assessed the mRNA level of IKKβ, and western blot assay was used to determine protein expression. After IKKB-siRNA transfection, Cell Titer 96® AQ(ueous) One Solution cell proliferation assay was used to evaluate the proliferation of HTFs. RESULTS: The diameter of the CS-g-(PEI-b-mPEG)/siRNA complexes tended to decrease whereas their zeta potential tended to increase as the N/P ratio increased. The CS-g-(PEI-b-mPEG) copolymer showed good siRNA binding ability and high siRNA protection capacity. Furthermore, the copolymer presented remarkable transfection efficiency and showed much less cytotoxicity than 25 kDa PEI. IKKB-siRNAs were successfully delivered into HTFs using CS-g-(PEI-b-mPEG) as a vector. As a result, the expression of IKKβ was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-(kappa)B (NF-(kappa)B) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-(kappa)B pathway. CONCLUSIONS: All the results demonstrate that CS-g-(PEI-b-mPEG) is a promising candidate for siRNA delivery, featuring excellent biocompatibility, biodegradability, and transfection efficiency. The RNA interference (RNAi) strategy using cationic copolymers as siRNA carriers will be a safe and efficient anti-scarring method following glaucoma filtration surgery.
Dr. J. Ge, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou 510060, China. gejian@mail.sysu.edu.cn
Classification:
3.5 Molecular biology incl. SiRNA (Part of: 3 Laboratory methods)
2.3 Sclera (Part of: 2 Anatomical structures in glaucoma)