New polyphosphoramidate with a spermidine side chain as a gene carrier
New polyphosphoramidate with a spermidine side chain as a gene carrier
复制标题
DOI：
10.1016/s0168-3659(02)00180-3
复制
发表时间：
2002-09-18
影响因子：
10.8
通讯作者：
Leong, KW
中科院分区：
文献类型：
--
作者：
Wang, J;Zhang, PC;Leong, KW
制备了一种新型阳离子聚合物(PPA-SP)，即含亚精胺侧链的聚磷酰胺，作为基因传递的非病毒载体。以聚1，2-丙二酸氢膦为原料，通过Atherton-Todd反应合成了PPA-SP。GPC/LS/RI法测得PPA-SP的重均相对分子质量为3.44×10~(4)，数均聚合度为90。平均每个聚合链的净正电荷为102。琼脂糖凝胶电泳法表明，PPA-SP能有效地凝聚DNA，并在N/P比(聚合物中的游离氨基与DNA中的磷酸基团)大于等于2时形成复合体。这种新型基因载体在N/P比大于2时对DNA具有明显的保护作用，在细胞培养中显示出比PLL和PEI更低的细胞毒性。PPA-SP对COS-7细胞的半数致死浓度为85微克/毫升，而对PLL和PEI细胞的半数致死浓度分别为20和42微克/毫升。在N/P比为5和10的生理盐水中制备的复合体的平均尺寸为250 nm，Zeta电位为26 mV。以荧光素酶基因为标记基因，优化了PPA-SP基因在HEK293细胞中的基因转移方案。当氯喹浓度为100um时，PPA-SP介导的基因表达显著增强。在优化的条件下，PPA-SP/DNA复合体的荧光素酶表达水平接近PEI/DNA复合体或Transfast介导的转染法。在非侵入性中枢神经系统基因传递模型中，PPA-SP/DNA复合体在舌内注射后，在小鼠脑干中产生与PEI/DNA复合体相当的bcl2表达。(C)2002 Elsevier Science B.V.保留所有权利。
A new cationic polymer (PPA-SP), polyphosphoramidate bearing spermidine side chain, was prepared as a non-viral vector for gene delivery. PPA-SP was synthesized from poly( 1,2-propylene H-phosphonate) by the Atherton-Todd reaction. The weight average molecular weight of PPA-SP was 3.44x10(4) with a number average degree of polymerization of 90, as determined by GPC/LS/RI method. The average net positive charge per polymer chain was 102. PPA-SP was able to condense plasmid DNA efficiently and formed complexes at an N/P ratio (free amino groups in polymer to phosphate groups in DNA) of 2 and above, as determined by agarose gel electrophoresis. This new gene carrier offered significant protection to DNA against nuclease degradation at N/P ratios above 2, and showed lower cytotoxicity than PLL and PEI in cell culture. The LD50 of PPA-SP was 85 mug/ml in COS-7 cells, in contrast to 20 and 42 mug/ml for PLL and PEI, respectively. The complexes prepared in saline at N/P ratios of 5similar to10 had an average size of 250 nm and zeta-potential of 26 mV. PPA-SP mediated efficient gene transfection in a number of cell lines, and the transfection protocol was optimized in HEK293 cells using a luciferase plasmid as a marker gene. Gene expression mediated by PPA-SP was greatly enhanced when chloroquine was used in conjunction at a concentration of 100 muM. Under the optimized condition, PPA-SP/DNA complexes yield a luciferase expression level closed to PEI/DNA complexes or Transfast mediated transfection. In a non-invasive CNS gene delivery model, PPA-SP/DNA complexes yielded comparable bcl-2 expression as PEI/DNA complexes in mouse brain stem following injection of the complexes in the tongue. (C) 2002 Elsevier Science B.V. All rights reserved.