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Hyperbranched Cationic Glycogen Derivative-Mediated I?B? Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats.

ABSTRACT: The role of the I?B/NF-?B signaling pathway in the uveoscleral outflow pathway was investigated with I?B? gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The I?B?-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the controls. The mRNA and protein expression of I?B?, NF-?Bp65, and MMP-2 were analyzed by real-time PCR, western blotting, and in situ gelatin zymography. Nuclear translocation of NF-?Bp65 was analyzed by immunofluorescence. Rat intraocular pressure (IOP) was monitored pre- and postinjection. Gene transfection efficiency and toxicity of the DMAPA-Glyp derivative were also evaluated. After RNA interference (RNAi), I?B? mRNA and protein expression were significantly inhibited. NF-?Bp65 mRNA and protein expression showed no significant differences. Nevertheless, nuclear translocation of NF-?Bp65 occurred in the DMAPA-Glyp+siRNA group. Both mRNA expression and activity of MMP-2 increased, with the largest increase in the DMAPA-Glyp+siRNA group. IOP in the DMAPA-Glyp+siRNA group fell to the lowest level on day 3 after RNAi. The levels of Cy3-siRNA in the ciliary muscle of the DMAPA-Glyp+siRNA group did not significantly decrease over time. At 7 and 14?d after RNAi, no significant pathological damage was detectable in the eyes injected with the DMAPA-Glyp derivative or the DMAPA-Glyp/siRNA complex. Taken together, our results suggest that downregulation of I?B? expression in the ciliary muscle plays a crucial role in reducing the IOP values of rats. I?B? may become a new molecular target for lowering IOP in glaucoma. The DMAPA-Glyp derivative is safe and feasible as an effective siRNA vector in rat eyes.

SUBMITTER: Zeng R

PROVIDER: S-EPMC7762656 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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Hyperbranched Cationic Glycogen Derivative-Mediated IκBα Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats.

Zeng Rui R Li Jinmiao J Gong Haijun H Luo Jiahao J Li Zijing Z Ou Zhaoxing Z Zhang Si S Yang Liqun L Lan Yuqing Y

BioMed research international 20201217

The role of the IκB/NF-κB signaling pathway in the uveoscleral outflow pathway was investigated with IκBα gene silencing mediated by the 3-(dimethylamino)-1-propylamine-conjugated glycogen (DMAPA-Glyp) derivative. The IκBα-siRNA-loaded DMAPA-Glyp complex was transfected into the ciliary muscles of rats by intracameral injection (labeled as the DMAPA-Glyp+siRNA group). The Lipofectamine™ 2000 (Lipo)/siRNA complex and the naked siRNA were set as the contro ...[more]

PMID: 33381584

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Project description:BackgroundThe purpose of this study was to synthesize and evaluate hyperbranched cationic glycogen derivatives as an efficient nonviral gene-delivery vector.MethodsA series of hyperbranched cationic glycogen derivatives conjugated with 3-(dimethylamino)-1-propylamine (DMAPA-Glyp) and 1-(2-aminoethyl) piperazine (AEPZ-Glyp) residues were synthesized and characterized by Fourier-transform infrared and hydrogen-1 nuclear magnetic resonance spectroscopy. Their buffer capacity was assessed by acid-base titration in aqueous NaCl solution. Plasmid deoxyribonucleic acid (pDNA) condensation ability and protection against DNase I degradation of the glycogen derivatives were assessed using agarose gel electrophoresis. The zeta potentials and particle sizes of the glycogen derivative/pDNA complexes were measured, and the images of the complexes were observed using atomic force microscopy. Blood compatibility and cytotoxicity were evaluated by hemolysis assay and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, respectively. pDNA transfection efficiency mediated by the cationic glycogen derivatives was evaluated by flow cytometry and fluorescence microscopy in the 293T (human embryonic kidney) and the CNE2 (human nasopharyngeal carcinoma) cell lines. In vivo delivery of pDNA in model animals (Sprague Dawley rats) was evaluated to identify the safety and transfection efficiency.ResultsThe hyperbranched cationic glycogen derivatives conjugated with DMAPA and AEPZ residues were synthesized. They exhibited better blood compatibility and lower cytotoxicity when compared to branched polyethyleneimine (bPEI). They were able to bind and condense pDNA to form the complexes of 100-250 nm in size. The transfection efficiency of the DMAPA-Glyp/pDNA complexes was higher than those of the AEPZ-Glyp/pDNA complexes in both the 293T and CNE2 cells, and almost equal to those of bPEI. Furthermore, pDNA could be more safely delivered to the blood vessels in brain tissue of Sprague Dawley rats by the DMAPA-Glyp derivatives, and then expressed as green fluorescence protein, compared with the control group.ConclusionThe hyperbranched cationic glycogen derivatives, especially the DMAPA-Glyp derivatives, showed high gene-transfection efficiency, good blood compatibility, and low cyto toxicity when transfected in vitro and in vivo, which are novel potential nonviral gene vectors.

Dataset Information

Hyperbranched Cationic Glycogen Derivative-Mediated I?B? Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats.

Publications

Hyperbranched Cationic Glycogen Derivative-Mediated I<i>κ</i>B<i>α</i> Gene Silencing Regulates the Uveoscleral Outflow Pathway in Rats.

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