The Interaction of Polycationic Polymers with Supported Lipid Bilayers and Cells: Nanoscale Hole Formation and Enhanced Membrane Permeability

Abstract

Interactions of polycationic polymers with supported 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) lipid bilayers and live cell membranes (KB and Rat2) have been investigated using atomic force microscopy (AFM), cytosolic enzyme assays, confocal laser scanning microscopy (CLSM), and a fluorescence-activated cell sorter (FACS). Polycationic polymers poly-l-lysine (PLL), polyethylenimine (PEI), and diethylaminoethyl-dextran (DEAE-DEX) and sphere-like poly(amidoamine) (PAMAM) dendrimers are employed because of their importance for gene and drug delivery. AFM studies indicate that all the polycationic polymers cause the formation and/or expansion of preexisting defects in supported DMPC bilayers in the concentration range of 1−3 μg/mL. By way of contrast, hydroxyl-containing neutral linear poly(ethylene glycol) (PEG) and poly(vinyl alcohol) (PVA) do not induce hole formation or expand the size of preexisting defects in the same concentration range. All polymers tested are not toxic to KB or Rat2 cells up to a 12 μg/mL concentration (XTT assay). In the concentration range of 6−12 μg/mL, however, significant amounts of the cytosolic enzymes lactate dehydrogenase (LDH) and luciferase (LUC) are released. PEI, which possesses the greatest density of charged groups on its chain, shows the most dramatic increase in membrane permeability. In addition, treatment with polycationic polymers allows the small dye molecules propidium idodide (PI) and fluorescein (FITC) to diffuse in and out of the cells. CLSM images also show internalization of PLL labeled with FITC dye. In contrast, controls of membrane permeability using the neutral linear polymers PEG and PVA show dramatically less LDH and LUC leakage and no enhanced dye diffusion. Taken together, these data are consistent with the hypothesis that polycationic polymers induce the formation of transient, nanoscale holes in living cells and that these holes allow a greatly enhanced exchange of materials across the cell membrane.

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