Effects of Polyethyleneimine on Endocytosis and Lysosome Stability

Klemm, Ann R; Young, D M; Lloyd, John

doi:10.1016/s0006-2952(98)00098-7

Cited by 154 publications

(92 citation statements)

References 15 publications

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“…10 DNA is condensed by free PEI into complexes that form globular or toroidal structures. 8,33 Such complexes can enter cells via endocytosis, followed by fusion and then disruption of lysosomes, 34,35 which permits nuclear localization of the complex. 36,37 PEI complexes have been shown to enter pulmonary epithelial cells, as well as other lung tissue cells after intravenous injection.…”

Section: Discussionmentioning

confidence: 99%

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

et al. 2002

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Section: Discussionmentioning

confidence: 99%

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

et al. 2002

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“…The mechanism by which plasmids given in PEI complex of the specific N/P ratio resulted in the highest gene transfer into the cells remains to be further elucidated. Considering that PEI-complexed plasmids could be taken up into the cells by adsorptive endocytosis, 10 we cannot exclude the possibility that the positive zeta potential 8 of PEI/DNA complexes at the N/P ratio of 10/1 enhanced the adsorption of plasmids on the cell surface, resulting in more efficient endocytosis. However, the lower cellular uptake of plasmid DNA delivered in the N/P ratio of 20/1 with the highest zeta potential 11 implies that there might be additional factors other than zeta potential, contributing to the higher uptake of PEI-complexed plasmids at the N/P ratio of 10/1.…”

Section: And the Nucleus Was Stained With Diamino-2-phehylindole (Dapmentioning

confidence: 99%

Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA

Kim

et al. 2002

Gene Ther

106

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Although polyethylenimine (PEI) Cytokine gene therapy has been studied for the treatment of cancer and infectious diseases, and for the development of effective molecular adjuvants.1 Recently, cytokine genes have been administered in nonviral gene delivery systems to increase the stability and the efficiency of cytokine gene expression. Lipid-complexed forms of human interleukin-2 gene are currently in phase I clinical trial for prostate cancer.2 Polyvinyl pyrrolidoneformulated cytokine genes resulted in higher antitumor immunity against head and neck cancer. 3Of nonviral vectors, polyethylenimine (PEI) has been studied as a versatile, inexpensive, and useful transfection system. 4 In vivo, PEI was shown to be an efficient transfection vector in several organs, such as the liver, 5 and lung. 6 Recently, in vivo fates and the prolonged organ retention of plasmid DNA administered in PEI complexes have been reported. 7 However, in the cellular level there is lack of understanding on the efficiency of PEImediated plasmid DNA delivery into the cell and the nucleus compartment. Moreover, despite that various N/P ratios have been used in PEI studies, the underlying mechanisms by which the N/P ratios could affect the expression of plasmid DNA have not been well analyzed.In this study, we aimed to test whether PEI could enhance the expression of murine interleukin-2 (mIL-2) plasmids in macrophage cell lines, and if so, whether the N/P ratios of PEI/mIL-2 complexes could affect the cellular uptake, retention, and nuclear trafficking of plasmid DNA. Here, we report that the cellular expression of cytokine plasmid DNA given in PEI complexes could be highly increased in the N/P ratio-dependent manner, and that the N/P ratios could influence the cellular uptake, nuclear translocation, and subcellular retention of plasmid DNA.The mRNA expression of mIL-2 in macrophage cell lines could be orders of magnitude augmented by PEImediated delivery of mIL-2 plasmids (Figure 1a). Furthermore, the extent of enhancement depended on the N/P ratio. Empty pVAXI plasmid, used as a control, showed as low as 0.006 ± 0.004 fmole of mRNA/cell. Of various N/P ratios, 1/1 showed the lowest transfection efficiency, whereas 10/1 revealed the highest efficiency with more than four orders of magnitude higher mRNA expression levels relative to naked plasmid.To determine whether the lower expression of PEIcomplexed DNA at the N/P ratio of 20/1 than the 10/1 N/P ratio is due to the cytotoxicity of PEI, we performed the MTT assay for cell viability. Figure 1b shows that the cell viability of PEI-treated groups did not significantly

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“…The mechanism by which it occurs remains to be elucidated. However, since PEI-complexed plasmids were reported to be taken up by adsorptive endocytosis, 16 the possibility exists that PEI enhanced the adsorption of plasmids on the cell surface, resulting in more efficient endocytosis. In addition, positively charged synthetic DNA complexes were shown to activate the alternative complement system.…”

Section: The Density Of Each Band Was Measured Using a Gel-doc Image mentioning

confidence: 99%

Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes

Kim

Yoon

et al. 2001

Gene Ther

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Polyethylenimine (PEI) Recently, polyethylenimine (PEI) has drawn attention as a versatile, inexpensive, and useful nonviral transfection vector. 1 A variety of cell types such as monocytes, 2 dendritic cells, 3 myoblast cells, 4 and hepatocytes 5 were studied as target cells for PEI-mediated gene transfection. In vivo, PEI was shown to be an efficient transfection vector in several organs such as the kidney, liver, and lung. [6][7][8] However, these studies are mostly focused on the expression of the PEI/DNA limited to target organs in vivo or specific cell types in vitro. Although the knowledge of biodistribution fates of plasmid DNA may reveal the feasibility of the cellular level targeting in vivo, there is little understanding of the overall and quantitative organ distribution of plasmid DNA after administration in PEI complexes. Moreover, the safety of PEI/DNA complexes after repeated dosing has not been well documented, despite the potential requirement for repeated administration due to the episomal property of plasmids.In this study, we tested the distribution fate and safety of plasmid DNA after administering DNA in PEI complexes. Quantitative and competitive polymerase chain reaction (PCR) was employed for the assay of plasmid DNA in the biological samples. Here, we report that in comparison to naked DNA, the organ distribution was higher and remarkably prolonged after administration of plasmid DNA in PEI complexes, and that once a week dosing of PEI/DNA complexes over 3 weeks did not alter the histology of major organs such as the liver, lung, and kidney.PEI/DNA complexes with PEI nitrogen to DNA phosphate ratio (N/P ratio) of 10:1 were prepared in 5% glucose using 25 kDa PEI (Aldrich, St Quentin, France). The N/P ratio of 10:1 was chosen since it was shown to efficiently transfect cells in vivo. 9 The levels of plasmid DNA in biological samples were determined using competitive and quantitative PCR. Quantitative PCR is based on co-amplification of the sample template together with various amounts of internal standard (IS) competitor sharing with the target the primer recognition sites, but differing in size. The competitor was constructed to share the same sense and antisense primer used for target amplification. 10 As an example, Figure 1 shows the gel bands and internal standard curves obtained after running competitive PCR of DNA extracts from the liver 6 h after injection of naked DNA or PEI/DNA complexes. The IS for quantitative PCR was the 188-bp deleted mutant of pCMV␤. The construction of the competitor and PCR conditions were described previously. 10 The PCR products were 449 bp and 261 bp for the target and the IS, respectively. The band densities of the target gene PCR product diminished as the amounts of IS increased (Figure 1a and b). The quantitation of gel band density by densitometry generated internal standard curves of high linearity (Figure 1c). The curves also show that we could detect as low as fg-level plasmids by quantitative PCR. The amounts of plasmid DNA in the samples we...

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Effects of Polyethyleneimine on Endocytosis and Lysosome Stability

Cited by 154 publications

References 15 publications

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

Gene delivery to the lung using protein/polyethylenimine/plasmid complexes

Polyethylenimine-mediated cellular uptake, nucleus trafficking and expression of cytokine plasmid DNA

Prolonged organ retention and safety of plasmid DNA administered in polyethylenimine complexes

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