Temperature-responsive polymeric carriers incorporating hydrophobic monomers for effective transfection in small doses.
Temperature-responsive polymeric carriers incorporating hydrophobic monomers for effective transfection in small doses.
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DOI：
10.1016/j.jconrel.2003.12.001
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发表时间：
2004-03
期刊：
影响因子：
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通讯作者：
N. Takeda;Emiko Nakamura;M. Yokoyama;T. Okano
中科院分区：
文献类型：
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作者：
N. Takeda;Emiko Nakamura;M. Yokoyama;T. Okano
合成了一系列温敏性三元无规共聚物聚[N-异丙基丙烯酰胺（PIPAAm）-co-甲基丙烯酸（二甲氨基）乙酯（DMAEMA）-co-甲基丙烯酸丁酯（BMA）]，并评价了它们在体外细胞培养中的基因转染效率。含有20mol%DMAEMA单元的对照共聚物IP-20 D（进料中IPAAm/DMAEMA/BMA的摩尔比=80/20/0，无BMA单元）在转染中是惰性的。相比之下，共聚物IP-20 D-10 B（进料中IPAAm/DMAEMA/BMA=70/20/10）即使在96孔板中每孔0.1 μg质粒DNA的小剂量条件下也有效地将质粒DNA转染到COS-1细胞培养物中，这表明掺入适量的疏水单元对转染效率至关重要。与对照均聚物聚-DMAEMA相比，当用IP-20 D-10 B载体转染时，基因表达显著得多，并且几乎等于高活性脂质载体LipofectAMINE PLUS™的基因表达。此外，IP-20 D-10 B的转染效率以热响应方式改变。通过在转染后阶段暂时将细胞培养物孵育温度降低至20 °C，基因表达比37 °C孵育温度下的基因表达加倍。DNA EtBr嵌入试验表明，通过降低孵育温度降低IP-20 D-10 B的DNA亲和力，这意味着热调节的基因表达可以提供更有效的DNA从聚合物载体释放。
A series of thermoresponsive ternary random copolymers, poly[N-isopropylacrylamide (PIPAAm)-co-(dimethylamino)ethylmethacrylate (DMAEMA)-co-butylmethacrylate (BMA)], was synthesized and their in vitro gene transfection efficiency in cell culture was evaluated. A control copolymer containing 20 mol% DMAEMA units, IP-20D (mole ratio of IPAAm/DMAEMA/BMA=80/20/0 in feed, no BMA units) was inert in transfection. In contrast, copolymer IP-20D-10B (IPAAm/DMAEMA/BMA=70/20/10 in feed) effectively transfected plasmid DNA into COS-1 cell cultures even under small dosing conditions of 0.1 μg of plasmid DNA per well in a 96-well plate, suggesting that incorporation of the appropriate amount of hydrophobic unit is crucial to transfection efficiency. Gene expression was much more significant when transfected by the IP-20D-10B carrier in comparison with control homopolymer poly-DMAEMA, and almost equal to that of the highly competent lipid carrier, LipofectAMINE PLUS™. Furthermore, the transfection efficiency of IP-20D-10B is altered in a thermally responsive manner. By temporarily lowering the cell culture incubation temperature to 20 °C in the posttransfection period, gene expression doubled over that for incubation temperature at 37 °C. The DNA EtBr intercalation assay suggested that DNA affinity for IP-20D-10B is decreased by lowering incubation temperature, implying that the thermally regulated gene expression could provide more efficient DNA release from the polymeric carrier.