Immunostimulatory Characteristics Induced by Linear Polyethyleneimine–Plasmid DNA Complexes in Cultured Macrophages

Abstract

Intravenously injected plasmid DNA (pDNA) complexed with cationic liposome (lipoplexes) caused NF-kappaB-mediated cytokine production from macrophages, induced by CpG sequence in the pDNA. We have reported that cytokine production caused by linear polyethyleneimine (PEI)-pDNA complexes (PEI polyplexes) was much lower than that caused by lipoplexes (Kawakami, S., Ito, Y., Charoensit, P., Yamashita, F., and Hashida, M. [2006]. J. Pharmacol. Exp. Ther. 317, 1382-1390). As Toll-like receptor-9 recognizing CpG sequence is expressed in the endosomal compartment, we hypothesized that the buffering capacity of PEI enhanced the escape of PEI polyplexes from endosomes, and that consequently cytokine production was decreased. In this study, the mechanism of lower cytokine production induced by PEI polyplexes, compared with lipoplexes, was investigated using the murine macrophage-like cell line RAW 264.7. Although transfection efficacy and cellular association were similar for PEI polyplexes and lipoplexes, tumor necrosis factor-alpha and interleukin-6 production and NF-kappaB activation caused by polyplexes were significantly lower than with lipoplexes. As for intracellular distribution, PEI polyplexes spread into cytosol whereas lipoplexes accumulated in vesicles, suggesting enhancement of escape from endosomes by PEI. Bafilomycin A1, an inhibitor of early endosomes, enhanced cytokine production and NF-kappaB activation by PEI polyplexes but not by lipoplexes; however, chloroquine, an inhibitor of late endosomes, inhibited PEI polyplex-induced cytokine production and NF-kappaB activation, suggesting that the buffering effect of PEI on early endosomes decreases NF-kappaB-mediated cytokine production. In conclusion, we demonstrate that cytokine production and NF-kappaB activation induced by PEI polyplexes are significantly lower than with lipoplexes in cultured macrophages. The significantly low cytokine response of PEI polyplexes may be due to effective transition of PEI polyplexes from endosomes to cytosol.

The use of PEGylated poly [2-(N,N-dimethylamino) ethyl methacrylate] as a mucosal DNA delivery vector and the activation of innate immunity and improvement of HIV-1-specific immune responses

“…In our model, PEI alone or PEI/DNA polyplexes could not effectively induce cytokines secretion (Fig. 6a and c) and this is consistent with previous reports and related to its lower induction of NF-kB activation [40]. The homopolymer PDMAEMA also induced high level of cytokines production (Fig.…”

Generating the optimal mRNA for therapy: HPLC purification eliminates immune activation and improves translation of nucleoside-modified, protein-encoding mRNA

“…A complexing agent that allows endocytosed RNA to exit the endosome and enter the cytoplasm before acidification and TLR signaling would result in reduced TNF-α secretion, as was observed with TransIT-complexed mRNA. In a comparison of cationic liposomes to linear polyethyleneimine (PEI) (a cationic polymer) delivery of CpG containing DNA, it was found that PEI-DNA induced less TNF-α, which was associated with a faster exit from endosomes, while cationic lipid delivery resulted in DNA remaining in vesicular structures for extended periods of time and high levels of TNF-α (30). We similarly observed that cationic liposome complexed RNA induced higher levels of TNF-α compared to the cationic polymer with lipid TransIT.…”

Improving siRNA Bio-Distribution and Minimizing Side Effects

“…Among these mesoporous silica nanoparticles, polymers, dendrimers and cyclodextrins represent interesting alternative under active investigation [41]. Among synthetic polymers, polyethylenimine (PEI) is widely used due to its strong proton sponge effect that increases the endosomal escape of siRNA, a phenomenon useful to prevent the toll-like receptor-mediated immunological response that starts in endosome of macrophages [68]. However, synthetic polymer-based delivery systems, such as PEI, can cause toxicity because they are poorly degradable.…”