Neuronal gene delivery by negatively charged pullulan-spermine/DNA anioplexes.
Neuronal gene delivery by negatively charged pullulan-spermine/DNA anioplexes.
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DOI：
10.1016/j.biomaterials.2008.12.032
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发表时间：
2009-03
期刊：
影响因子：
14
通讯作者：
D. Thakor;Y. Teng;Y. Tabata
中科院分区：
文献类型：
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作者：
D. Thakor;Y. Teng;Y. Tabata
由于缺乏有效且无毒的载体，向神经元的非病毒基因转移仍然不可靠。在这里，我们通过质粒 DNA 和支链淀粉-精胺（一种由天然多糖和多胺制备的缀合物）的无盐络合实现了有效的神经元基因传递。具体而言，在低精胺氮：DNA 磷酸盐 (N:P) 比率下，形成的复合物具有 ζ 电位和直径分别约为−40mV 和 350nm。较高的 N:P 比率将 ze 电位增加至约 +10mV。所有复合物均稳定至少 1 周并保护 DNA 免遭降解。大鼠感觉神经元的体外转染发生在所有 N:P 比例下，但独特的是，阴离子复合物 (anioplexes) 的效率最高。随后的分析揭示了去唾液酸胎球蛋白 (1mg/ml) 和甲基-β-环糊精 (5mm) 对报告基因表达的抑制，表明利用糖蛋白特异性相互作用和脂筏进行摄取和细胞内运输。与商业阳离子脂质试剂形成鲜明对比的是，anioplexes 在高达 20μg/ml DNA 时并未表现出可测量的细胞毒性。此外，在血清和抗生素存在的情况下，转染效率得以维持。基于这些有利的特性，我们成功建立了两种用于培养的成体感觉神经元和组织外植体的转染方法。总的来说，这些数据表明带负电荷的支链淀粉-精胺/DNA复合物可以代表一种有效的基因传递技术，特别是对于神经元。
Nonviral gene transfer to neurons remains unreliable due to a lack of effective and nontoxic vectors. Here, we achieved effective neuronal gene delivery through salt-free complexation of plasmid DNA and pullulan–spermine, a conjugate prepared from a naturally derived polysaccharide and polyamine. Specifically, at low spermine nitrogen:DNA phosphate (N:P) ratios, complexes formed with ζ-potential and diameter of approximately−40mV and 350nm, respectively. Higher N:P ratios increased the ζ-potential to approximately +10mV. All complexes were stable for at least 1 week and protected DNA from degradation. In vitro transfection of rat sensory neurons occurred at all N:P ratios, but uniquely, efficiency was highest for anionic complexes (anioplexes). Subsequent analyses revealed the inhibition of reporter gene expression by asialofetuin (1mg/ml) and methyl-beta-cyclodextrin (5mm), indicating utilization of glycoprotein-specific interactions and lipid rafts for uptake and intracellular trafficking. In marked contrast to a commercial cationic lipid reagent, anioplexes did not exhibit measurable cytotoxicity at up to 20μg/ml DNA. Additionally, transfection efficiency was maintained in the presence of serum and antibiotics. Based on these favorable properties, we successfully established two transfection methods for cultured adult sensory neurons and tissue explants. Collectively, these data suggest that negatively charged pullulan–spermine/DNA anioplexes could represent an effective gene delivery technology, particularly for neurons.