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Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.

ABSTRACT: 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 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.

SUBMITTER: Duan Y

PROVIDER: S-EPMC2613073 | biostudies-literature | 2008

REPOSITORIES: biostudies-literature

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Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.

Duan Yongheng Y Guan Xipeng X Ge Jian J Quan Daping D Zhuo Yehong Y Ye Hehua H Shao Tingting T

Molecular vision 20081231

<h4>Purpose</h4>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.<h4>Methods</h4>First, a novel cationic copolymer composed of low molecular weight, linear poly(ethyleneimine) [PEI] blocked with polyethylene glycol (PEG) and gra ...[more]

PMID: 19137061

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Project description:PurposeTo investigate the efficacy and safety of cationic nano-copolymers CS-g-(PEI-b-mPEG) mediated IκB kinase beta (IKKβ) targeting siRNA in modulating wound healing in a monkey model of glaucoma filtration surgery.MethodsThe IKKβ targeting siRNAs were chemically synthesized and screened in cultured monkey Tenon's fibroblasts in vitro. Fourteen monkeys underwent trabeculectomy and were randomly allocated to one of three treatment regimens: subconjunctival injection of either CS-g-(PEI-b-mPEG)/IKKβ-siRNA (six eyes, 50nM, at the time of surgery and 7 days post surgery) or phosphate buffered saline (four eyes), or treated with mitomycin C (MMC; four eyes, 0.2 mg/ml). Bleb survival and characteristics, and intraocular pressure, were evaluated over a 60-day period. Histology of the surgical eyes was performed to evaluate ocular scarring and fibrosis in each group.ResultsSubconjunctival injection of CS-g-(PEI-b-mPEG)/IKKβ-siRNA was well tolerated in this model. Both siRNA and MMC significantly prolonged bleb survival compared with the PBS group (the medians for survival days were 45.5, 60, and 29.5 in the siRNA, MMC, and PBS groups, respectively, p<0.01). Higher blebs were observed in the siRNA group than in the PBS group (p<0.01), while the MMC group showed the highest blebs among three groups (p<0.01). The surgical eyes in both the siRNA and MMC groups had significantly larger bleb area compared with the PBS group (p<0.01), but there was no significant difference between the siRNA and MMC groups (p=0.214). There were no significant differences in IOP readings among the three groups on the designated days after surgery (all p>0.05). The histologic examination demonstrated that the eyes treated with siRNA showed a marked reduction in subconjunctival scar tissue compared with the eyes in the PBS group. The conjunctival epithelium appeared healthy without the acellularity that was present in the MMC group.ConclusionsSubconjunctival injection of cationic nano-copolymers mediated IKKβ targeting siRNA is associated with improved surgical outcome in a monkey model of trabeculectomy. This novel approach may potentially be a more controlled alternative as an anti-scarring agent in glaucoma filtration surgery.

Project description:Glaucoma filtration surgery is one of the most effective methods for lowering intraocular pressure in glaucoma. The surgery efficiently reduces intra-ocular pressure but the most common cause of failure is scarring at the incision site. This occurs in the conjunctiva/Tenon's capsule layer overlying the scleral coat of the eye. Currently used antimetabolite treatments to prevent post-surgical scarring are non-selective and are associated with potentially blinding side effects. Developing new treatments to target scarring requires both a better understanding of wound healing and scarring in the conjunctiva, and new means of delivering anti-scarring drugs locally and sustainably. By combining plastic compression of collagen gels with a soft collagen-based layer, we have developed a physiologically relevant model of the sub-epithelial bulbar conjunctiva/Tenon's capsule interface, which allows a more holistic approach to the understanding of subconjunctival tissue behaviour and local drug delivery. The biomimetic tissue hosts both primary human conjunctival fibroblasts and an immune component in the form of macrophages, morphologically and structurally mimicking the mechanical proprieties and contraction kinetics of ex vivo porcine conjunctiva. We show that our model is suitable for the screening of drugs targeting scarring and/or inflammation, and amenable to the study of local drug delivery devices that can be inserted in between the two layers of the biomimetic. We propose that this multicellular-bilayer engineered tissue will be useful to study complex biological aspects of scarring and fibrosis, including the role of inflammation, with potentially significant implications for the management of scarring following glaucoma filtration surgery and other anterior ocular segment scarring conditions. Crucially, it uniquely allows the evaluation of new means of local drug delivery within a physiologically relevant tissue mimetic, mimicking intraoperative drug delivery in vivo.

Dataset Information

Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.

Publications

Cationic nano-copolymers mediated IKKbeta targeting siRNA inhibit the proliferation of human Tenon's capsule fibroblasts in vitro.

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