In vivo transduction of Atoh1 gene into cochlear cells mediated by HA-PEI nanoparticle vectors
WANG Fangyuan
CHEN Zhiting
DENG Xiongwei
WU Yan
ZHANG Yue
YANG Fengbo
YANG Shiming
WU Nan
Abstract：Objective To test in vivo transfection efficiency of Atoh1-EGFP plasmids using hyaluronic acid modified branched polyethylenimine nanoparticles as a novel non-viral gene vector. Methods Plasmids were extracted following kit instructions. The gene-vector nanoparticles were synthesized (COOH/N/P=4:10:1) and then characterized before being trans-duced into the inner ear through intact round window membrane. At 7 days after transduction operation, transfection effi-ciency was evaluated by examination of both basilar membrane surface preparation and frozen sections under a confocal mi-croscope. Transfection results were finally verified by Western blot and RT-PCR at protein and nucleic acid levels respec-tively. Results We successfully synthesized a non-viral gene vector which loaded Atoh1 gene well and had a negative-ly-charged surface to reduce possible cytotoxicity. At 7 days after transfection, confocal microscope observation of basilar membrane showed EGFP in OHCs and IHCs. OHC transfection efficiency was 81.7 ± 4.71% in the basal turn and 33.8 ± 9.02%in the middle turn. Frozen section results revealed expression of EGFP mainly in the basal turn and only part of the middle turn of the cochlea. No signs of EGFP transduction were seen in either the top of the cochlea or in cells other than hair cells. Furthermore, Basilar membrane cells showed no obvious damage after operation. Both Western Blot and RT-PCR results verified successful transfection of the Atoh1 gene on the basilar membrane. Conclusion The Atoh1 gene can be suc-cessfully transduced into the inner ear by the HA modified bPEI nanoparticles and there is no obvious cytotoxicity to cochle-ar cells. The new nanoparticles are easy to produce at a low cost, which makes it an ideal non-viral vector for inner ear gene transfection.
Keywords：Hyaluronic acidbranched PolyetherimideAtoh1 geneinner ear gene delivery
Publication Date：2016-01-01
Online Publishing Date：2025-08-15(First online date of this platform, not the publication date of the document)
Pages：5( 803-807 )