Cationic lipid-coated PEI/DNA polyplexes with improved efficiency and reduced cytotoxicity for gene delivery into mesenchymal stem cells

Abstract

Background: Effective gene transfection without serum deprivation is a prerequisite for successful stem cell-based gene therapy. Polyethylenimine (PEI) is an efficient nonviral gene vector, but its application has been hindered by serum sensitivity and severe cytotoxicity. Methods: To solve this problem, a new family of lipopolyplexes was developed by coating PEI/ DNA polyplexes with three serum-resistant cationic lipids, namely, lysinylated, histidylated, and arginylated cholesterol. The physical properties, transfection efficiency, cellular uptake, subcellular distribution, and cytotoxicity of the lipopolyplexes was investigated. Results: The outer coat composed of lysinylated or histidylated cholesterol remarkably improved the transfection efficiency of the polyplex with a low PEI/DNA ratio of 2 in the presence of serum. The resulting lysinylated and histidylated cholesterol lipopolyplexes were even more efficient than the best performing polyplex with a high PEI/DNA ratio of 10. Results from cellular uptake and subcellular distribution studies suggest that their higher transfection efficiency may result from accelerated DNA nuclear localization. The superiority of the lipopolyplexes over the best performing polyplex was also confirmed by delivering the therapeutic gene, hVEGF 165 . Equally importantly, the lipid coating removed the necessity of introducing excess free PEI chains into the transfection solution for higher efficiency, generating lipopolyplexes with no signs of cytotoxicity. Conclusion: Noncovalent modification of polyplexes with lysinylated and histidylated cholesterol lipids can simultaneously improve efficiency and reduce the toxicity of gene delivery under serum conditions, showing great promise for genetic modification of bone marrow stem cells.

ECM turnover-stimulated gene delivery through collagen-mimetic peptide-plasmid integration in collagen

“…While the release kinetics were likely not identical in the media containing serum, our current study suggests that GPP modification and multivalency still improve polyplex retention relative to unmodified polyplex retention on collagen even during preincubation with a range of serum concentrations for up to 2-weeks. In the unmodified polyplex samples, upon release into the serum-supplemented media, the unmodified polyplex’s high density of positive surface charges likely led to many interactions with negatively charged serum proteins such as albumin, leading to rapid aggregation and loss of viability within an hour [13, 59]. Due to the faster release kinetics of the gel containing the unmodified polyplex, compounded by the fact it initially held less polyplex, the collagen’s concentration of viable polyplex decreased at a significantly faster rate leading to lower levels of gene expression, which would also be exacerbated by the higher concentrations of serum expediting the aggregation of released polyplex.…”

“…It may further reduce the surface charge of the polyplex, reducing interaction with serum proteins and aggregation. In other works, neutral, hydrophilic polymers, such as poly(ethylene glycol) (PEG), or anionic polymers, such as alginate or hyaluronic acid, are used to help shield the high density of positive charges of the PEI [13, 59, 66]. In our system, collagen fragments may play a similar role and afford the released polyplex with both increased serum stability and resistance to aggregation and dissociation.…”

The Role of the Helper Lipid on the DNA Transfection Efficiency of Lipopolyplex Formulations

“…Synthetic, non-viral vectors offer advantages over viral vectors for in vivo gene therapy in that they are less immunogenic, have fewer packaging constraints and are safer 1 2 . Cationic lipoplexes and polyplexes predominate in the non-viral vector field but increasingly lipopolyplex formulations, which are combinations of lipids with peptides or polymers, are being explored as appreciation develops of their wider range of functionalities and higher transfection efficiencies 3 4 5 6 7 8 9 10 11 . Further detailed functional and structural studies are required to understand the properties of lipopolyplexes, how to formulate components and to develop improved formulations.…”

A simple and efficient method for transfecting mouse embryonic stem cells using polyethylenimine

“…PEI alone has been shown to transfect other types of murine stem cells, including mesenchymal stem cells [17–32] and neural stem cells [26, 33], but we are unaware of any reports demonstrating the successful use of PEI alone in mouse ESCs. One group set out to compare PEI chemical transfection efficiencies to nucelofection in mammalian cell lineages.…”