Biopharmaceutical production through transient gene expression (TGE) is used within industry for the rapid supply of product for early stage testing. A key requirement of the process is the large scale transfection of mammalian cells, for which the cationic polymer, polyethylenimine (PEI), is widely used.
In this thesis, the mechanism of PEI mediated transfection of CHO-S cells is explored at the cell surface, a fundamental barrier to successful transgene delivery. By approaching the question from first principles, exploring the kinetics of transfection at the cell surface, bio-physical and bio-molecular interactions governing polyplex binding to the cell surface, three key findings were made.
Firstly, polyplex uptake was biphasic. Initial, rapid endocytosis of polyplex and heparan sulphate proteoglycans (HSPG) was followed by a slower phase of polyplex uptake, on depletion of cell surface HSPGs. Enzymatic depletion of cell surface HSPGs was found to reduce TGE by 25%, whereas sequestration of cholesterol using methyl-β-cyclodextrin abrogated TGE. Taken together, the data indicate that HSPGs mediate maximal TGE (via an early, rapid phase of endocytosis) but that the predominant mechanism of polyplex uptake is through the clustering of lipid rafts, occurring at depleted cell surface HSPG levels.
Secondly, the role of both electrostatic and hydrophobic interactions in polyplex binding to the cell surface was investigated. These experiments revealed that at statistically optimized conditions for TGE (with respect to PEI:DNA ratio) the net charge of the polyplex in chemically defined medium was approximately neutral. Under these conditions polyplexes bound to the cell surface, predominantly, via a hydrophobic interaction, independent of cell surface HSPGs. Accordingly polyplex binding to the cell surface was disrupted by both non-ionic surfactant and depletion of plasma membrane cholesterol by methyl-β-cyclodextrin. An increase in polyplex zeta potential at elevated polyplex PEI:DNA ratio increased polyplex binding to the cell surface, but was accompanied by increased cytotoxicity with elevated PEI internalization. A decrease in polyplex zeta potential using ferric (III) citrate resulted in decreased polyplex binding to the cell surface. Both alterations in polyplex charge reduced TGE. Taken together, these data indicate that hydrophobic binding of polyplexes to cell surface lipid rafts (bearing passenger HSPGs) is the primary molecular interaction that promotes subsequent lipid raft clustering and polyplex micro/macropinocytosis to facilitate maximal TGE.
Lastly, in order to engineer increased binding and endocytosis of recombinant DNA, alkylated PEIs varying in alkyl chain length and degree of substitution were chemically synthesized in order to increase polyplex hydrophobicity. Compared to unmodified PEI in TGE processes, optimized by Design of Experiments Response Surface Modelling, propyl-PEI was found to mediate more efficient TGE at similar reporter gene titre via a reduction in plasmid DNA load. Propyl-PEI formed polyplexes were found to mediate enhanced polyplex uptake relative to polyplexes formed of unmodified PEI.
