(353d) Effect of Polyplex Charge on Cellular Internalization and Gene Expression

Introduction: The cellular internalization of nanoparticles is governed by multiple particle characteristics, primarily size and surface properties such as charge, chemistry, and hydrophobicity. These characteristics as well as pharmacokinetics, biodistribution, and cell membrane interactions have been studied in a host of nanoparticles but less extensively in polymeric gene delivery vectors. A particular interest is the interaction between a charged particle and the cell membrane and its effect on internalization. Polycations electrostatically bind to DNA to form polymer complexes (polyplexes) that can deliver genetic materials to the cellular nucleus. Furthermore, polyanions can be complexed with conventional polyplexes to produce ternary polyplexes of varying surface charge. This project uses pharmacological inhibitors of several endocytosis pathways (clathrin-dependent, caveolin-dependent, and macropinocytosis) to study the effects of polyplex charge on cellular internalization pathways and gene delivery efficiency. The dynamics of polyanion/polycation/DNA complexation is also studied to determine the particle compositions that produce the highest uptake and gene delivery.

Methods: Ternary polyplexes were formed using 25-kDa branched polyethylenimine (PEI), 15-kDa poly(glutamic acid) (PGA), and the pGL3 luciferase reporter plasmid at various PGA:PEI:DNA weight ratios. Sizes and zeta potentials were determined using a Malvern zetasizer. Positively and negatively charged polyplexes were used to transfect cancer cells (HeLa and U-87) in vitro to assess the effect of charge on gene delivery. Small molecule inhibitors (genistein, methyl-β-cyclodextrin, chlorpromazine hydrochloride, amantadine hydrochloride, and amiloride) were used to inhibit clathrin-dependent, caveolin-dependent, or macropinocytosis endocytic pathways to observe the impact on gene delivery by positive and negative zeta potential polyplexes. Flow cytometry and florescence co-localization studies evaluate polyplex uptake efficiency and confirmed the internalization routes.

Results: DLS was used to show that the addition of PGA to a binary polyplex consisting of PEI and pGL3 doubled the particle size to around 600 nm. Addition of PGA also decreased the zeta potential from +16 mV to a minimum of -35 mV. Transfections in the presence of serum showed that positive zeta potential polyplexes containing PGA produced higher transgene expression than negative zeta potential polyplexes. Binary polyplexes (no PGA) produced the lowest expression at all PEI:pGL3 weight ratios. The 1.5:3:1 PGA:PEI:pGL3 weight ratio produced the highest expression so this formulation was chosen to represent positively charged polyplexes (+11mV) and the weight ratio resulting in -11 mV was selected to represent negatively charged polyplexes. Transfections in the presence of endocytosis inhibitors showed that inhibition of caveolin-dependent endocytosis and macropinocytosis resulted in drastically decreased transfection efficiency with varying changes in polyplex uptake. Inhibition of clathrin-dependent endocytosis had little effect on or slightly increased transgene expression with mildly decreased uptake. Similar results were observed with negatively charged polyplexes with the exception that inhibition of clathrin-dependent endocytosis significantly decreased gene delivery efficiency.

Conclusion/Implications: The change from positive to negative zeta potential with increasing amounts of PGA indicates that the positive polyplexes are electrostatically binding PGA until surface saturation occurs and charge becomes minimized. Addition of PGA increased polyplex size but did not increase the size with increasing PGA content. Negative zeta potential particles exhibited decreased gene expression compared to positive particles presumably due to repulsion at the cell membrane. Positive particles were able to minimize serum agglomeration due to PGA charge shielding, and the optimum weight ratios for gene delivery provide sufficient PEI/DNA interaction to complex and protect the DNA, but also allow efficient polyplex dissociation within the cell. Inhibition of caveolin-dependent and macropinocytosis uptake suggests that these pathways are effective in uptake of negative and positive polyplexes. Inhibition of clathrin-mediated uptake suggests that polyplexes do enter through clathrin-dependent pathways but clathrin tends to affect positive polyplex delivery efficiency less than negative charged polyplexes.