Macropinocytosis Is the Major Pathway Responsible for DNA Transfection in CHO Cells by a Charge-Reversal Amphiphile

The cellular uptake of a functional charge-reversal amphiphile:DNA lipoplex is described. First, pharmacological inhibitors were applied to block different endocytosis pathways. By examining the resulting transfection activities, it was found that endocytosis was the pathway leading to transfection in Chinese hamster ovary (CHO) cells. When the specific pathway of macropinocytosis was inhibited, β-galactosidase expression was significantly depleted (90%); meanwhile the inhibition of clathrin-mediated pathway only brought a 30% decrease in expression; and the inhibition of caveolae-mediated pathway did not affect expression. Furthermore, a transfection kinetics study revealed that the cellular uptake responsible for gene expression was a slower process compared to clathrin-mediated endocytosis, consistent with fluid-phase uptake compared to receptor-mediated uptake. Next, a fluorescence colocalization study was used to visualize the DNA lipoplex uptake pathways. The colocalization of the DNA lipoplex and Cascade Blue, a fluid-phase uptake marker, was observed. Meanwhile, the colocalization of the DNA lipoplex and transferrin, a clathrin-mediated endocytosis marker, was also seen. However, no colocalization was observed with the endosome/lysosome marker Lysotracker. Our results indicate that macropinocytosis, not the commonly seen clathrin-mediated endocytosis for cationic lipids, is the major pathway leading to gene transfection in CHO cells for this charge-reversal amphiphile.