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      Cationic nano-copolymers mediated IKKβ targeting siRNA inhibit the proliferation of human Tenon’s capsule fibroblasts in vitro

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          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 IκB 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 (IKKΒ-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-κB (NF-κB) in the HTFs was subsequently inhibited. Most impressively, the proliferation of HTFs was also effectively suppressed through the blocking of the NF-κB 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.

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          Most cited references39

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          In vitro cytotoxicity testing of polycations: influence of polymer structure on cell viability and hemolysis.

          A comparative in vitro cytotoxicity study with different water-soluble, cationic macromolecules which have been described as gene delivery systems was performed. Cytotoxicity in L929 mouse fibroblasts was monitored using the MTT assay and the release of the cytosolic enzyme lactate dehydrogenase (LDH). Microscopic observations were carried out as indicators for cell viability. Furthermore, hemolysis of erythrocytes was quantified spectrophotometrically. To determine the nature of cell death induced by the polycations, the nuclear morphology after DAPI staining and the inhibition of the toxic effects by the caspase inhibitor zVAD.fmk were investigated. All assays yielded comparable results and allowed the following ranking of the polymers with regard to cytotoxicity: Poly(ethylenimine)=poly(L-lysine)>poly(diallyl-dimethyl-ammonium chloride)>diethylaminoethyl-dextran>poly(vinyl pyridinium bromide)>Starburst dendrimer>cationized albumin>native albumin. The magnitude of the cytotoxic effects of all polymers were found to be time- and concentration dependent. The molecular weight as well as the cationic charge density of the polycations were confirmed as key parameters for the interaction with the cell membranes and consequently, the cell damage. Evaluating the nature of cell death induced by poly(ethylenimine), we did not detect any indication for apoptosis suggesting that the polymer induced a necrotic cell reaction. Cell nuclei retained their size, chromatin was homogenously distributed and cell membranes lost their integrity very rapidly at an early stage. Furthermore, the broad spectrum caspase inhibitor zVAD.fmk did not inhibit poly(ethylenimine)-induced cell damage. Insights into the structure-toxicity relationship are necessary to optimize the cytotoxicity and biocompatibility of non-viral gene delivery systems.
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            The Advanced Glaucoma Intervention Study (AGIS): 7. The relationship between control of intraocular pressure and visual field deterioration.The AGIS Investigators.

            (2000)
            To investigate the association between control of intraocular pressure after surgical intervention for glaucoma and visual field deterioration. In the Advanced Glaucoma Intervention Study, eyes were randomly assigned to one of two sequences of glaucoma surgery, one beginning with argon laser trabeculoplasty and the other trabeculectomy. In the present article we examine the relationship between intraocular pressure and progression of visual field damage over 6 or more years of follow-up. In the first analysis, designated Predictive Analysis, we categorize 738 eyes into three groups based on intraocular pressure determinations over the first three 6-month follow-up visits. In the second analysis, designated Associative Analysis, we categorize 586 eyes into four groups based on the percent of 6-month visits over the first 6 follow-up years in which eyes presented with intraocular pressure less than 18 mm Hg. The outcome measure in both analyses is change from baseline in follow-up visual field defect score (range, 0 to 20 units). In the Predictive Analysis, eyes with early average intraocular pressure greater than 17.5 mm Hg had an estimated worsening during subsequent follow-up that was 1 unit of visual field defect score greater than eyes with average intraocular pressure less than 14 mm Hg (P =.002). This amount of worsening was greater at 7 years (1.89 units; P <.001) than at 2 years (0.64 units; P =.071). In the Associative Analysis, eyes with 100% of visits with intraocular pressure less than 18 mm Hg over 6 years had mean changes from baseline in visual field defect score close to zero during follow-up, whereas eyes with less than 50% of visits with intraocular pressure less than 18 mm Hg had an estimated worsening over follow-up of 0.63 units of visual field defect score (P =.083). This amount of worsening was greater at 7 years (1.93 units; P <.001) than at 2 years (0.25 units; P =.572). In both analyses low intraocular pressure is associated with reduced progression of visual field defect, supporting evidence from earlier studies of a protective role for low intraocular pressure in visual field deterioration.
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              A two-stage poly(ethylenimine)-mediated cytotoxicity: implications for gene transfer/therapy.

              Poly(ethylenimine) (PEI) is a cationic macromolecule commonly used in gene transfer/therapy protocols with high transfection efficiency both in vitro and in vivo. PEI is also cytotoxic, but the molecular basis of its cytotoxicity is poorly understood. Here, we have demonstrated that branched (25 kDa) and linear (750 kDa) PEI can both induce membrane damage and initiate apoptosis in three clinically relevant human cell lines (Jurkat T cells, umbilical vein endothelial cells, and THLE3 hepatocyte-like cells). We have defined Phase I toxicity as early necrotic-like changes (30 min) resulting from compromised membrane integrity, assessed by considerable lactate dehydrogenase release and phosphatidylserine translocation from the inner plasma membrane to the outer cell surface. Phase II cytotoxicity (24 h) was due to activation of a "mitochondrially mediated apoptotic program," resulting from PEI-induced channel formation in the outer mitochondrial membrane. This led to the release of proapoptotic cytochrome c, subsequent activation of caspase 3, and alteration in mitochondrial membrane potential as a result of caspase translocation into the mitochondria. The reported observations have important implications for the design and execution of gene therapy protocols as well for controlling intracellular distribution of drugs with cationic-based polymer-delivery systems.
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                Author and article information

                Journal
                Mol Vis
                MV
                Molecular Vision
                Molecular Vision
                1090-0535
                2008
                31 December 2008
                : 14
                : 2616-2628
                Affiliations
                [1 ]State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, China
                [2 ]Institute of Polymer Science, School of Chemistry and Chemical Engineering, Sun Yat-sen University, Guangzhou, China
                Author notes

                The first two authors contributed equally to this work.

                Correspondence to: Professor Jian Ge, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou, 510060, China; Phone: +86-20-87331374; FAX: +86-20-87333271; email: gejian@ 123456mail.sysu.edu.cn
                Article
                299 2008MOLVIS0349
                2613073
                19137061
                f97b44ec-5594-452a-ab87-9948d9af3629
                Copyright @ 2008

                This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

                History
                : 20 October 2008
                : 05 December 2008
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                Vision sciences

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