|
|
| TR |
TITLE |
AUTHORS |
KEYWORDS |
MATERIALS & METHODS
|
MICROSCOPIC
TECHNIQUES |
SPECIES |
MORPHOLOGY |
CELL
LINE |
| 598 |
Interaction
of Polycationic Polymers with Supported Lipid Bilayers and Cells:
Nanoscale Hole Formation and Enhanced Membrane Permeability |
Seungpyo
Hong, Pascale R. Leroueil, Elizabeth K. Janus, Jennifer L. Peters, Mary-Margaret Kober,
Mohammad T. Islam, Bradford G. Orr, James R. Baker, Jr., and Mark M. Banaszak Holl |
polycationic
polymers, lipid bilayers, lactate dehydrogenase, luciferase, membrane permeabilit |
A concentration of 2 104 cells/mL
of Rat2 cells was seeded on MatTek glass bottom Petri dishes (35 mm) and incubated at 37 °C
under 5% CO2 for 24 h.
|
Confocal
Laser Scanning Microscopy |
rat |
|
Rat2 |
| 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 íg/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 íg/mL concentration (XTT assay). In the concentration
range of 6-12 íg/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. |
|
|
|
|
