title: Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.
abstract: OBJECTIVE
  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 IkappaB kinase subunit beta (IKKbeta) 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 IKKbeta (IKKBeta-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 IKKbeta, 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 IKKbeta was downregulated at both the mRNA and protein levels, and the activation of nuclear factor-kappaB (NF-kappaB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-kappaB 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.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China.
authors.forename: Yongheng
authors.initials: Y
authors.lastname: Duan
authors.forename: Xipeng
authors.initials: X
authors.lastname: Guan
authors.forename: Jian
authors.initials: J
authors.lastname: Ge
authors.forename: Daping
authors.initials: D
authors.lastname: Quan
authors.forename: Yehong
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Hehua
authors.initials: H
authors.lastname: Ye
authors.forename: Tingting
authors.initials: T
authors.lastname: Shao
mesh_terms: D000704:Analysis of Variance; D049109:Cell Proliferation; D002478:Cells, Cultured; D048271:Chitosan / Q000737:chemistry; D003239:Connective Tissue Cells / Q000166:cytology / Q000378:metabolism; D015536:Down-Regulation; D005123:Eye / Q000166:cytology*; D005347:Fibroblasts / Q000166:cytology* / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D051550:I-kappa B Kinase / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular; D010316:Particle Size; D011092:Polyethylene Glycols / Q000737:chemistry; D011094:Polyethyleneimine / Q000737:chemistry*; D011108:Polymers / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics* / Q000378:metabolism; D014162:Transfection
chemical_list: D016328:NF-kappa B; D011108:Polymers; D034741:RNA, Small Interfering; D011092:Polyethylene Glycols; D011094:Polyethyleneimine; D048271:Chitosan; D051550:I-kappa B Kinase