Non-Viral Gene Delivery Vehicles
An initial screen of several cationic polymers (branched PEI, linear PEI, jetPEI, & Turbofect) and lipids (Lipofectamine LTX) in Jurkat T cells revealed that the vehicles could be separated into three significantly different cohorts (Figure 1A). For example, branched PEI and some of the other polymeric vehicle formulations provided no detectable luciferase expression, while Turbofect and a specific nitrogen:phosphate ratio (N:P = 10:1) of linear PEI yielded modest luciferase expression. The highest luciferase expression levels were obtained with Lipofectamine LTX (Lipo-Lo = 1.25 µL and 1μg DNA; Lipo-Hi = 2.75 µL and 1 μg DNA) and jetPEI (N:P = 5:1).
Subsequent transfections of pEF-GFP (Figure 1B) using the vehicles that provided the highest luciferase expression levels (Lipofectamine, Turbofect, and jetPEI) showed that the higher dose of Lipofectamine LTX (Lipo-Hi) provided the highest transfection efficiency for Jurkat T cells (25% EGFP+), while the transfection efficiencies obtained with Turbofect and jetPEI were significantly lower (4.5-4.8% EGFP+ cells). As observed with the luciferase assays, the higher amount of Lipofectamine (Lipo-Hi) appeared to provide a higher number of EGFP+ cells than the lower amount (Lipo-Lo), but the difference was not statistically significant. The histograms in Figure 1D-F also illustrate a distinct increase in the number of fluorescent cells obtained with Lipo-Hi compared to Lipo-Lo and Turbofect.
The effects of Lipofectamine, Turbofect, and jetPEI (5:1) on cell metabolic activity were also measured with an MTT assay (Figure 1C). Each vehicle significantly decreased cell metabolic activity approximately 30-40% relative to control samples of untransfected cells, but no significant differences were observed between the vehicles themselves.
Overall, Lipofectamine LTX appeared to be more efficient than Turbofect and branched or linear PEI. This observation is in agreement with previous studies that have compared the transfection efficiencies of cationic lipids and polymers in other cell lines.31,32 These studies attribute the higher transfection efficiency of the lipid vehicles to their higher affinity for cell membranes, which allows them to be endocytosed more quickly than cationic polymers like PEI.31Consequently, Lipofectamine (Lipo-Hi) was selected as the vehicle for our subsequent studies.