A mechanistic dissection of polyethylenimine mediated transfection of CHO cells: to enhance the efficiency of recombinant DNA utilization.

In this study, we examine the molecular and cellular interactions that underpin efficient internalization and utilization of polyethylenimine (PEI):DNA complexes (polyplexes) by Chinese Hamster Ovary (CHO) cells. Cell surface polyplex binding and internalization was a biphasic process, consisting of an initial rapid Phase (I), lasting approximately 15 min, followed by a slower second Phase (II), saturating at approximately 240 min post transfection. The second Phase accounted for the majority (60-70%) of polyplex internalization. While cell surface heparan sulphate proteoglycans (HSPGs) were rapidly cointernalized with polyplexes during Phase I, cell surface polyplex binding was not dependent on HSPGs. However, Phase II polyplex internalization and HSPG regeneration onto the surface of trypsinized cells occurred at similar rates, suggesting that the rate of recycling of HSPG-containing membrane to the plasma membrane limits Phase II internalization rate. Under optimal transfection conditions, polyplexes had a near neutral surface charge (zeta potential) and cell surface binding was dependent on hydrophobic interactions, being significantly inhibited by both chemical sequestration of cholesterol from the plasma membrane and addition of nonionic surfactant. Induced alterations in polyplex zeta potential, using ferric (III) citrate to decrease surface charge and varying PEI:DNA ratio to increase surface charge, served to inhibit polyplex binding or reduce secreted alkaline phosphatase reporter expression and cell viability, respectively. To increase polyplex hydrophobicity and internalization an alkylated derivative of PEI, propyl-PEI, was chemically synthesized. Using Design of Experiments-Response Surface Modeling to optimize the transfection process, the function of propyl-PEI was compared to that of unmodified PEI in both parental CHO-S cells and a subclone (Clone 4), which exhibited superior transgene expression via an increased resistance to polyplex cytotoxicity. The combination of propyl-PEI and Clone 4 doubled the efficiency of recombinant DNA utilization and reporter protein production. These data show that for maximal efficacy, strategies to increase polyplex internalization into cells must be used in concert with strategies to offset the inherent cytotoxicity of this process.

Cell line specific control of polyethylenimine-mediated transient transfection optimized with "Design of experiments" methodology.

We describe a design of experiments (DoE) response surface modeling strategy to optimize the concentration of basal variables underpinning polyethylenimine (PEI) mediated transfection of different CHO-K1 derived parental cell populations in a chemically defined medium, specifically the relative concentration of linear 25 kD PEI, host CHO cells and plasmid DNA. Using recombinant secreted alkaline phosphatase (SEAP) reporter activity as the modeled response, a discrete simple maximum was predicted for each CHO host cell population. Differences between the modeled optima derived from host cell specific differences in PEI cytotoxicity, such that the PEI:cell interaction effectively limited PEI-DNA polyplex load at a relatively constant PEI:DNA ratio. However, across the three CHO host cell populations, SEAP reporter production was not proportional to plasmid DNA input at the host cell specific predicted basal variable optima. A 10-fold variation in SEAP reporter output per mass of plasmid DNA delivered was observed. To determine the cellular basis of this difference in transient productivity, host CHO cells were transfected with fluorescently labeled polyplexes followed by flow cytometric analysis. Each CHO host cell population exhibited a distinct functional phenotype, varying in the extent of PEI-DNA polyplex binding to the cell surface and degree of polyplex internalization. SEAP production was directly proportional to the level of polyplex internalization and heparan sulfate proteoglycan level. Taken together, these data show that choice of host CHO cell line is a critical parameter, which should rationally precede cell line specific transient production platform design using DoE methodology.