Influence of Polyplex Formation on the Performance of Star-Shaped Polycationic Transfection Agents for Mammalian Cells

Abstract

Genetic modification ("transfection") of mammalian cells using non-viral, synthetic agents such as polycations, is still a challenge. Polyplex formation between the DNA and the polycation is a decisive step in such experiments. Star-shaped polycations have been proposed as superior transfection agents, yet have never before been compared side-by-side, e.g., in view of structural effects. Herein four star-shaped polycationic structures, all based on (2-dimethylamino) ethyl methacrylate (DMAEMA) building blocks, were investigated for their potential to deliver DNA to adherent (CHO, L929, HEK-293) and non-adherent (Jurkat, primary human T lymphocytes) mammalian cells. The investigated vectors included three structures where the PDMAEMA arms (different arm length and grafting densities) had been grown from a center silsesquioxane or silica-coated γ-Fe 2 O 3 -core and one micellar structure self-assembled from poly(1,2-butadiene)-block PDMAEMA polymers. All nano-stars combined high transfection potential with excellent biocompatibility. The micelles slightly outperformed the covalently linked agents. For method development and optimization, the absolute amount of polycation added to the cells was more important than the N/P-ratio (ratio between polycation nitrogen and DNA phosphate), provided a lower limit was passed and enough polycation was present to overcompensate the negative charge of the plasmid DNA. Finally, the matrix (NaCl vs. HEPES-buffered glucose solution), but also the concentrations adjusted during polyplex formation, affected the results.

Non-Viral Transfection of Human T Lymphocytes

“…The zeta potential was measured for the polyplexes as a function of the N/P ratio (ratio of polymer N to DNA P), Table 1. Hepes-buffered glucose solution (HBG) was used as matrix for polyplex formation, because in our hands, this buffer originally proposed by Van Gaal et al [30] had previously been more efficient in transfecting mammalian cells that the commonly used unbuffered 150 mM NaCl [31]. As expected, the zeta potential of the polyplexes increases with increasing N/P ratio.…”

“…It is generally considered difficult to uncouple the two effects. However, in a recent paper [31], we have shown that independent of the N/P ratio the cytotoxicity of the transfection cocktail depends strongly on the absolute amount of polymer added. This phenomenon has been obscured in most previous investigations by the fact that the N/P ratio is adjusted by keeping the pDNA amount constant while increasing that of the polycation.…”

“…The different N/P ratios (20 for l-PEI, 10 for the nanostar) used in these experiments were chosen because they had previously been identified to be the most suitable for Jurkat cells in experiments involving a variant of the nanostar with 20 arms and l-PEI as reference material [11]. In term of transfection efficiency, the results obtained here for the nanostars are better than the ones previously determined for nanostars of similar chemistry, [11] including some micellar constructs [31,32], while viabilities were lower. The l-PEI results are in the expected single digit range.…”

Cationic polymers for non-viral gene delivery to human T cells

“…The Freitag group polymerized 20 “arms” of pDMAEMA off a silsesquioxane core and used this star polymer to deliver pDNA or siRNA, obtaining approximately 50% knockdown from siRNA delivery, and 10-50% transfection efficiency with 40-100% viability using pDNA [21]. This group also recently reported 13% transfection efficiency of primary T cells using this material with cell viability of 80% [22]. Most recently, the Stephan group reported a nanoparticle formulation with a poly(β-amino ester) core for DNA condensation and an antibody-conjugated polyglutamic acid shell for receptor-mediated uptake that was successfully used for in vitro and in vivo gene delivery to primary murine T cells, achieving ~3% transfection efficiency in vitro and ~1.5% in vivo [23].…”

Comparison of cell‐based versus cell‐free mammalian systems for the production of a recombinant human bone morphogenic growth factor

“…Lactate and glucose concentrations were quantified with commercial kits (UV-method) from R-Biopharm AG (Darmstadt, Germany). EGFP expression was measured as previously described by flow cytometry [31] or spectrophotometrically [30]. In HEK293trans cells, EGFP was also quantified in cell lysates.…”