Department of Pharmaceutics, School of Pharmacy and Center for Nano Drug/Gene Delivery and Tissue Engineering, Jiangsu University, Zhenjiang, People's Republic of China.
Int J Nanomedicine. 2012;7:1297-311. doi: 10.2147/IJN.S28010. Epub 2012 Mar 14.
The objective of this study was to investigate the use of cationized Pleurotus eryngii polysaccharide (CPEPS) as a nonviral gene delivery vehicle to transfer plasmid DNA encoding transforming growth factor beta-1 (pTGF-β1) into mesenchymal stem cells (MSCs) in vitro. Crude P. eryngii polysaccharide was purified, and then cationized by grafting spermine onto the backbone of the polysaccharide. Agarose gel electrophoresis, transmission electron microscopy, and a Nano Sense Zetasizer (Malvern Instruments, Malvern, UK) were used to characterize the CPEPS-pTGF-β1 nanoparticles. The findings of cytotoxicity analysis showed that when the nanoparticles were formulated with a CPEPS/pTGF-β1 weight ratio ≥ 10:1, a greater gel retardation effect was observed during agarose gel electrophoresis. The CPEPS-pTGF-β1 nanoparticles with a weight ratio of 20:1, respectively, possessed an average particle size of 80.8 nm in diameter and a zeta potential of +17.4 ± 0.1 mV. Significantly, these CPEPS-pTGF-β1 nanoparticles showed lower cytotoxicity and higher transfection efficiency than both polyethylenimine (25 kDa) (P = 0.006, Student's t-test) and Lipofectamine(TM) 2000 (P = 0.002, Student's t-test). Additionally, the messenger RNA expression level of TGF-β1 in MSCs transfected with CPEPS-pTGF-β1 nanoparticles was significantly higher than that of free plasmid DNA-transfected MSCs and slightly elevated compared with that of Lipofectamine 2000-transfected MSCs. Flow cytometry analysis demonstrated that 92.38% of MSCs were arrested in the G1 phase after being transfected with CPEPS-pTGF-β1 nanoparticles, indicating a tendency toward differentiation. In summary, the findings of this study suggest that the CPEPS-pTGF-β1 nanoparticles prepared in this work exhibited excellent transfection efficiency and low toxicity. Therefore, they could be developed into a promising nonviral vector for gene delivery in vitro.
本研究旨在探讨阳离子化杏鲍菇多糖（CPEPS）作为非病毒基因传递载体，将编码转化生长因子β-1（pTGF-β1）的质粒 DNA 转染到间充质干细胞（MSCs）中的应用。从粗杏鲍菇多糖中提取、纯化，然后通过将精胺接枝到多糖骨架上进行阳离子化。琼脂糖凝胶电泳、透射电子显微镜和 Nano Sense Zetasizer（Malvern Instruments，Malvern，英国）用于表征 CPEPS-pTGF-β1 纳米颗粒。细胞毒性分析结果表明，当纳米颗粒中 CPEPS/pTGF-β1 重量比≥10:1 时，在琼脂糖凝胶电泳中观察到更大的凝胶阻滞效应。重量比为 20:1 的 CPEPS-pTGF-β1 纳米颗粒的平均粒径为 80.8nm，zeta 电位为+17.4±0.1mV。重要的是，与聚乙烯亚胺（25kDa）（P=0.006，Student's t 检验）和 Lipofectamine（TM）2000（P=0.002，Student's t 检验）相比，这些 CPEPS-pTGF-β1 纳米颗粒具有更低的细胞毒性和更高的转染效率。此外，转染 CPEPS-pTGF-β1 纳米颗粒的 MSCs 中 TGF-β1 的信使 RNA 表达水平明显高于游离质粒 DNA 转染的 MSCs，略高于 Lipofectamine 2000 转染的 MSCs。流式细胞术分析表明，92.38%的 MSCs 在转染 CPEPS-pTGF-β1 纳米颗粒后被阻滞在 G1 期，表明向分化方向发展。综上所述，本研究结果表明，本工作制备的 CPEPS-pTGF-β1 纳米颗粒具有优异的转染效率和低毒性。因此，它们可能成为一种有前途的非病毒基因传递载体，用于体外基因传递。
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