Tianjin Key Laboratory of Biomedical Materials, Institute of Biomedical Engineering, Chinese Academy of Medical Sciences and Peking Union Medical College, Tianjin, China.
J Mater Sci Mater Med. 2010 Jan;21(1):223-9. doi: 10.1007/s10856-009-3849-3. Epub 2009 Aug 14.
Cationic amphiphilic drugs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and endosomes. It is therefore proposed that amphiphilic and cationic polysaccharide macromolecule, when used as gene delivery vectors, may have potential ability to direct the disassembly process of cell membrane organization, and penetrate across the cell membrane into cell and nucleus. In the current study, N-methylene phosphonic chitosan (NMPCS), an amphiphilic macromolecule, was synthesized by incorporating the methylene phosphonic group into the amino groups of chitosan (CS) using formaldehyde as the coupling agent, and characterized with a FTIR spectrometer. NMPCS/DNA or CS/DNA complexes were prepared using a complex coacervation method, and characterized by agarose gel electrophoresis retardation assay and dynamic light scattering (DLS). MTT assay was employed to evaluate the cytotoxicity of the polymers and pGL3-control luciferase plasmid was utilized as a reporter gene to assess the transgenic efficacy of the polymers. It was demonstrated that NMPCS was able to fully entrap the DNA at N/P ratio of 2:1, whereas CS entrapped the DNA completely at N/P ratio of 1:1. DLS showed that the NMPCS/DNA or CS/DNA complexes were of mean diameters ranging from 110 to 180 nm. Neither NMPCS nor CS induced significant loss of cell viability at the concentrations ranging from 1 to 50 microg/ml, whereas PEI at 5 microg/ml started to result in significantly decreased cell viability. The expression of transgene mediated by NMPCS was much higher (more than 100-folds) than that mediated by CS, indicating that NMPCS was a more efficacious gene ferrying vector than CS.
阳离子两亲性药物最近被证明可以通过破坏质膜和内体上的网格蛋白的组装-解体来抑制受体再循环。因此，当用作基因传递载体时，两亲性和阳离子多糖大分子可能具有指导细胞膜组织解体过程的潜在能力，并穿透细胞膜进入细胞和细胞核。在本研究中，通过使用甲醛作为偶联剂将亚甲基膦酸基团引入壳聚糖（CS）的氨基中，合成了两亲性大分子 N-亚甲基膦酸壳聚糖（NMPCS），并使用傅里叶变换红外光谱仪（FTIR）进行了表征。通过复凝聚法制备了 NMPCS/DNA 或 CS/DNA 复合物，并通过琼脂糖凝胶电泳阻滞试验和动态光散射（DLS）进行了表征。MTT 试验用于评估聚合物的细胞毒性，pGL3-对照荧光素酶质粒用作报告基因评估聚合物的转基因功效。结果表明，NMPCS 能够在 N/P 比为 2:1 时完全包封 DNA，而 CS 在 N/P 比为 1:1 时完全包封 DNA。DLS 显示 NMPCS/DNA 或 CS/DNA 复合物的平均直径范围为 110-180nm。NMPCS 或 CS 在 1-50μg/ml 的浓度范围内不会引起明显的细胞活力丧失，而 PEI 在 5μg/ml 时开始导致细胞活力显著降低。由 NMPCS 介导的转基因表达比由 CS 介导的表达高得多（超过 100 倍），表明 NMPCS 是比 CS 更有效的基因转染载体。
