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

Abstract

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 co-localization study was used to visualize the DNA lipoplex uptake pathways. The co-localization of the DNA lipoplex and Cascade Blue®, a fluid-phase uptake marker, was observed. Meanwhile, the co-localization of the DNA lipoplex and transferrin, a clathrin-mediated endocytosis marker, was also seen. However, no co-localization 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.

The Role of Cytoskeleton Networks on lipid-mediated Delivery of DNA

“…The combined analysis of C 1 and C 2 suggests that actin filaments most likely play a role in the uptake of MC lipoplexes rather than in their following intracellular trafficking towards the nucleus. This conclusion is in very good agreement with recent findings showing that lipoplexes are taken up in living cells mainly through fluid-phase macropinocytosis [37,38]. Macropinocytosis relies on the formation of large endocytic vesicles generated by actin-driven invaginations of the plasma membrane, called membrane ruffles [39].…”

“…Cholesterol-sensitive macropinocytosis is the major pathway responsible for lipoplex internalization and is regulated by formation of large endocytic vesicles of irregular size and shape, generated by actin-driven invaginations of the plasma membrane (Figure 5A) [37,38]. Accordingly, if the actin network is destroyed, the ability of lipoplexes to penetrate within cells is dramatically reduced and they accumulate at the plasma membrane.…”

Gene delivery to the rat retina by non-viral vectors based on chloroquine-containing cationic niosomes

“…Due to its ability to internalize larger structures, macropinocytosis pathway has been proposed to mediate the internalization of non-viral gene delivery vehicles [37]. Moreover, MPC is considered as the major pathway responsible for DNA transfection in certain cell types [38]. In contrary, DPP80 nioplexes were internalized mainly by CME and, to a lesser extent, by MPC, while CvME had a much less participation in the cellular uptake process.…”

Lipoprotein-biomimetic nanostructure enables efficient targeting delivery of siRNA to Ras-activated glioblastoma cells via macropinocytosis

“…In addition, distinct in many ways from the better characterized endocytosis pathways such as clathrin and caveolae-mediated endocytosis, the relative large size (0.2–5 μm), a low level of lysosomal degradation and a rapid release of molecules to the cytosols make macropinocytosis a more efficient route for intracellular delivery484950.…”