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

Abstract

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

Stable polyplexes based on arginine-containing oligopeptides for in vivo gene delivery

“…The radioactivity distribution pattern concurs with that of PEI-derived polyplexes, which are reported as one of the most effective nonviral vectors in vivo. 37 Luciferase expression levels revealed that all complexes were able to enhance the transfection efficiency to the same level. Introduction of an NLS sequence did not lead to further improvement of the transfection efficiency, suggesting that translocation to the nucleus is not rate-limiting.…”

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation

“…The underlying cause is probably a welldocumented phenomena of the intracellular plasmid degradation, spontaneous loss, and dilution during cell divisions in transiently transfected cells. 55,56 We have demonstrated that the sensor and the amplifier constructs were present and activated in the same cell 48 hr after the transfection ( Figure S3C). However, as expected, the plasmid copy number per cell dropped within 5 days after transfection, but nevertheless, the amplifier construct, which was introduced at the lowest amounts, and the inducible OFF-switch representing a safety mechanism, were still present at that time ( Figure S7C).…”

Phase I clinical trial of idiotypic DNA vaccine administered as a complex with polyethylenimine to patients with B-cell lymphoma

“…Many preclinical studies have shown a significant increase in immunogenicity of DNA-PEI conjugates, as well as a dramatic reduction in toxicity as the molecular weight of the polymer is reduced. 22,23,25,26,38 The use of PEI in clinical vaccination has been approved by the regional regulatory authorities (State Committee on Science and Technology of the Republic of Belarus).…”

“…Polyethylenimine (PEI) is a cationic polymer commonly used as a transfection reagent in vitro 22 and synthetic vehicle for transgene and DNA vaccine delivery in vivo. [23][24][25] It has been shown that T-cell immune responses can be increased by 20-fold with DNA-PEI complexes compared with responses elicited by intramuscular injections with naked DNA.…”