Role of endocytosis in the transfection of L929 fibroblasts by polyethylenimine/DNA complexes

Abstract

Polyethylenimine (PEI) is one of the most efficient nonviral vectors for gene therapy. The aim of this study was to investigate the role of endocytosis in the transfection of synchronized L929 fibroblasts by PEI/DNA complexes. This was performed by confocal microscopy and flow cytometry, using the endocytosis marker FM4-64 and PEI/DNA complexes labeled either with the DNA intercalator YOYO-1, or with fluorescein covalently linked to PEI. Endocytosis appeared as the major if not the sole mode of entry of the PEI/DNA complexes into the L929 cells. The complexes followed a typical fluid phase endocytosis pathway and were efficiently taken up in less than 10 min in endosomes that did not exceed 200 nm in diameter. Later, the localization of the complexes became perinuclear and fusion between late endosomes was shown to occur. Comparison with the intracellular trafficking of the same complexes in EA.hy 926 cells (W.T. Godbey, K. Wu, A.G. Mikos, Proc. Natl. Acad. Sci. USA 96 (1999)) revealed that endocytosis of PEI/DNA complexes is strongly cell-dependent. In L929 cells, escape of the complexes from the endosomes is a major barrier for transfection. This limited the number of transfected cells to a few percent, even though an internalization of PEI/DNA complexes was observed in most cells. In addition, the entry of the complexes into the nucleus apparently required a mitosis and did not involve the lipids of the endosome membrane. This entry seems to be a short-lived event that involves only a few complexes.

Control of aggregate size of polyethyleneimine-coated magnetic nanoparticles for magnetofection

“…One of the most widely used positively charged modification agents is polyethyleneimine (PEI) which is capable of forming complexes with DNA and condensing them into compact nanoparticles [12,13]. Moreover, PEI can destabilize the endosomal membrane by the so-called "proton sponge effect" to enhance DNA release from endosomes and protect DNA against degradation [14]. To date, PEI is one of the most effi cient nonviral transfection agents.…”

Quantitative comparison between poly(L-arginine) and poly(L-lysine) at each step of polyplex-based gene transfection using a microinjection technique

“…The buffering capacities of polymeric gene carriers provide an index of endosomal escape. Although microscopic observations of endosomal escape of fluorescence-labeled polyplexes in living cells have been well reported using pH-responsive fluorescent molecules or endosome marker probes [14,15], quantitative and direct evaluations are difficult.…”

Efficient active transport of gene nanocarriers to the cell nucleus

“…Currently, the transport of nonviral DNA carriers through the cytoplasm is poorly characterized, but is thought to be inefficient and potentially rate limiting because of their need to ''randomly migrate'' to the nucleus (9). Confocal microscopy has been used to study intracellular trafficking of nonviral systems (6,10,11), allowing the locations of complexes at discrete times to be determined, yielding an insightful but qualitative description of the transport process. Fluorescence recovery after photobleaching has recently been used to quantify overall ''effective diffusion'' rates of DNA molecules in the cytoplasm (12).…”