Godbey W T, Wu K K, Hirasaki G J, Mikos A G
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892, USA.
Gene Ther. 1999 Aug;6(8):1380-8. doi: 10.1038/sj.gt.3300976.
We have developed a modified poly(ethylenimine) (PEI) transfection procedure that significantly increases PEI's transfection efficiency. While the basic transfection procedure had a transfection efficiency of 37%, our modified procedure yielded a 53% transfection efficiency. The altered procedure gives improved results because of two simultaneous actions: free polycations are removed from the transfecting solutions, and the composition of the PEI complexes that are administered to cells has been modified. The reduction in the amount of free polycations in transfecting solutions reduced the toxicity sometimes associated with the administration of polycations to cellular environments. The structural modification of PEI/DNA transfecting complexes involves improved PEI packing around the delivered plasmid to yield a greater buffering capacity without a change in the complex's surface charge concentration. These structural properties were confirmed by titration and zeta potential analyses. Whether the modified PEI/DNA complexes are more effective because of increased cellular uptake or an enhanced ability to escape from endolysosomes has been addressed. The increase in transfection efficiency was obtained when the buffering capacity of the PEI/DNA was increased without a change in surface charge concentration, which implies that it is the property of enhanced lysosomal buffering that is responsible for successful PEI transfection.
我们开发了一种改良的聚（乙烯亚胺）（PEI）转染程序，该程序显著提高了PEI的转染效率。虽然基本转染程序的转染效率为37%，但我们的改良程序产生了53%的转染效率。这种改变后的程序能产生更好的结果，原因有两个同时发生的作用：从转染溶液中去除了游离的聚阳离子，并且对施用于细胞的PEI复合物的组成进行了修饰。转染溶液中游离聚阳离子数量的减少降低了有时与向细胞环境中施用聚阳离子相关的毒性。PEI/DNA转染复合物的结构修饰涉及改善PEI围绕递送质粒的堆积，以在复合物表面电荷浓度不变的情况下产生更大的缓冲能力。这些结构特性通过滴定和zeta电位分析得到了证实。已经探讨了改良的PEI/DNA复合物是否由于细胞摄取增加或从内溶酶体逃逸的能力增强而更有效。当PEI/DNA的缓冲能力增加而表面电荷浓度不变时，转染效率得到了提高，这意味着增强的溶酶体缓冲特性是PEI成功转染的原因。
