Wu Nan, Li Min, Chen Zhi-Ting, Zhang Xiao-Bing, Liu Hui-Zhan, Li Zhou, Guo Wei-Wei, Zhao Li-Dong, Ren Li-Li, Li Jia-Nan, Yi Hai-Jin, Han Dong, Yang Wei-Yan, Wu Yan, Yang Shi-Ming
Department of Otolaryngology Head and Neck Surgery, Institute of Otolaryngology, Chinese PLA General Hospital, Beijing 100853, China.
J Biomed Nanotechnol. 2013 Oct;9(10):1736-45. doi: 10.1166/jbn.2013.1684.
Gene therapy is a promising clinical solution to hearing loss. However suitable gene carriers for the auditory system are currently unavailable. Given the unique structure of the inner ear, the route of delivery and gene transfer efficiency are still not optimal at present. This study presented a non-viral delivery system of in vivo delivery of Atoh1 gene (a potentially therapeutic gene for hearing loss) to rat cochlea. We treated polyamidoamine (PAMAM) dendrimers by activating and modifying with Na-carboxymethyl-beta-cyclodextrins (CM-beta-CD) in sequence. A novel gene carrier (CM-beta-CD modified activated PAMAM dendrimers, CMAP) was then constructed. CMAP nanoparticles could bind pRK5-Atoh1-EGFP plasmids to form vector-DNA complexes (dendriplexes) with a mean particle size of 132 +/- 20 nm and zeta potential of 31 +/- 3 mV. These dendriplexes were locally applied on the round window membrane and delivered to the inner ear by passive gradient permeation. Results showed that the Atoh1 gene was successfully transferred into the cells as indicated by the green fluorescence detected in the inner ear. A relatively selective gene transfer with high efficiency was achieved in the auditory hair cells but not much in other cell types in the cochlea. Auditory brainstem response was determined seven days after inoculation, indicating good tolerance. This approach may provide a novel tool for inner ear gene therapy and initiate the applications of biomaterials to treat auditory disorders.
基因治疗是一种有前景的治疗听力损失的临床方法。然而，目前尚无适用于听觉系统的基因载体。鉴于内耳的独特结构，目前给药途径和基因转移效率仍不理想。本研究提出了一种将Atoh1基因（一种潜在的治疗听力损失的基因）体内递送至大鼠耳蜗的非病毒递送系统。我们依次用羧甲基-β-环糊精（CM-β-CD）对聚酰胺胺（PAMAM）树枝状大分子进行活化和修饰处理。然后构建了一种新型基因载体（CM-β-CD修饰的活化PAMAM树枝状大分子，CMAP）。CMAP纳米颗粒可与pRK5-Atoh1-EGFP质粒结合形成平均粒径为132±20 nm、ζ电位为31±3 mV的载体- DNA复合物（树枝状复合物）。这些树枝状复合物局部应用于圆窗膜，并通过被动梯度渗透递送至内耳。结果表明，内耳中检测到的绿色荧光表明Atoh1基因已成功转入细胞。在听觉毛细胞中实现了相对选择性的高效基因转移，而在耳蜗的其他细胞类型中则没有太多转移。接种七天后测定听觉脑干反应，表明耐受性良好。这种方法可能为内耳基因治疗提供一种新工具，并开启生物材料治疗听觉障碍的应用。
