Division of Medical Engineering and Materials, National Cerebral and Cardiovascular Center Research Institute, Japan.
Bioconjug Chem. 2013 Feb 20;24(2):159-66. doi: 10.1021/bc300317e. Epub 2013 Feb 8.
We have developed a substrate-mediated transfection method called "deposition transfection technology" using a poly(N,N-dimethylaminoethylmethacrylate) (PDMAEMA) homopolymer with both thermoresponsive and cationic characteristics. In this study, we enhanced deposition transfection efficiency by using tris(hydroxymethyl)aminomethane (Tris buffer) as a pH adjuster for transfection solution composed of PDMAEMA and plasmid DNA (pDNA). PDMAEMA with a molecular weight of 9.7 × 10(4) g mol(-1) was synthesized by photoinduced radical polymerization. The pH of PDMAEMA solution was increased gradually in the range from 8 to 11 by the addition of Tris, and then the solubility of PDMAEMA was significantly decreased and the dissolution time was extended from 15 to 40 min at Tris/PDMAEMA ratio of 1 and higher. On the other hand, while the polyion complexes (polyplexes) were formed by mixing PDMAEMA with luciferase-encoding plasmid DNA even under an excess amount of Tris at Tris/PDMAEMA ratio of 8, the binding affinity between PDMAEMA and pDNA was decreased with increasing Tris at Tris/PDMAEMA ratio of 2 and higher. When HeLa cells, smooth muscle cells, and cardiac fibroblasts were transfected by the deposition method using polyplex solution containing various amounts of Tris, the transgene expression dramatically increased at a Tris/PDMAEMA ratio of 2 in all cell types, which were more than 150-fold in HeLa cells, 40-fold in smooth muscle cells, and 30-fold in cardiac fibroblasts compared to those in the Tris-free condition. In addition, the enhanced transgene expression by Tris was sustained for over 10 days post-transfection as well as that observed in Tris-free condition. Thus, deposition transfection efficiency can be dramatically enhanced by using Tris buffer as a pH adjuster for polyplex solution.
我们开发了一种基于基质的转染方法，称为“沉积转染技术”，使用具有热敏性和阳离子特性的聚（N，N-二甲基氨基乙基甲基丙烯酸酯）（PDMAEMA）均聚物。在这项研究中，我们通过使用三羟甲基氨基甲烷（Tris 缓冲液）作为转染溶液的 pH 调节剂，来增强沉积转染效率，该转染溶液由 PDMAEMA 和质粒 DNA（pDNA）组成。分子量为 9.7×10^4 g mol^-1 的 PDMAEMA 是通过光引发自由基聚合合成的。通过添加 Tris，PDMAEMA 溶液的 pH 值逐渐从 8 增加到 11，然后 PDMAEMA 的溶解度显著降低，在 Tris/PDMAEMA 比例为 1 及以上时，溶解时间从 15 分钟延长至 40 分钟。另一方面，虽然在 Tris/PDMAEMA 比例为 8 时，即使存在过量的 Tris，PDMAEMA 与编码荧光素酶的质粒 DNA 混合也会形成聚离子复合物（多聚物），但随着 Tris/PDMAEMA 比例的增加，PDMAEMA 与 pDNA 之间的结合亲和力降低。当使用含有不同量 Tris 的多聚物溶液通过沉积法转染 HeLa 细胞、平滑肌细胞和心肌成纤维细胞时，在所有细胞类型中，Tris/PDMAEMA 比例为 2 时，转基因表达显著增加，HeLa 细胞中增加了 150 多倍，平滑肌细胞中增加了 40 倍，心肌成纤维细胞中增加了 30 倍，与无 Tris 条件相比。此外，Tris 增强的转基因表达可持续超过 10 天，与无 Tris 条件下观察到的一样。因此，使用 Tris 缓冲液作为多聚物溶液的 pH 调节剂可以显著提高沉积转染效率。
