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Model Amphipathic Peptides 87
A cationic amphipathic peptide, very similar to the I-derived series, WEAK-
LAKALAKALAKHLAKALAKALKACEA (KALA), was designed by Wyman
et al. 49 The KALA repeat was chosen to bind DNA, while the glutamic acid residues
were inserted to improve water solubility at physiological pH. Although cellular
uptake of the peptide was not demonstrated, KALA was shown to promote the uptake
of ODNs after formation of noncovalent KALA-ODN complexes. KALA modifies
especially the intracellular distribution and increases the nuclear accumulation of
ODNs by redistribution from punctate cytoplasmic regions into the nucleus. 49
Niidome et al. 50 studied binding of α-helical, cationic peptides to plasmid DNA
and their gene transfer ability. Peptides such as Ac-LARLLARLLARLLARLLAR-
LLARL-NHCH 3 form aggregates with plasmid DNA, which are internalized by an
endocytotic pathway. Obviously, the endosomal or lysosomal membrane-pertubing
activity of the peptide moiety allows rapid transfer of the peptide–DNA complex
from the endosomal compartment into the cytosol. Glutamic acid residues-containing
peptides such as GLFEAIAEFIEGGWEGLIEGCA (E5CA) or LAELLAELLAEL
undergo a conformational change when the pH shifts from neutral to 5 or 6, inducing
a transient permeabilization of the plasma membrane or membranes of the endosomal
compartment. 51-53 On the basis of these findings, the acidification of the extracellular
medium in the presence of the E5CA peptide was suggested in order to
improve cellular uptake of antisense ODNs into the cytosol and nucleus. 51
4.6 EXPERIMENTAL
4.6.1 CELL CULTURE
Cells were seeded at an initial density 5 × 10 4 cells/cm 2 in 24-well culture plates
and cultured at 37°C in a humidified 5% CO 2 containing air environment for 4 days
without replacing the medium. For CLSM, 10 4 cells on 22 × 22 mm coverslips were
cultured analogously.
4.6.2 UPTAKE EXPERIMENTS
After removal of the medium, the cell layers were rinsed two times at 37°C with
Dulbecco’s phosphate buffered saline (DPBS; Biochrom KG, Berlin) supplemented
with 1 g/l D-glucose (DPBSG) and subsequently exposed, unless indicated otherwise,
at 37°C for 30 min to 500 µl of the peptide solutions in DPBSG. Thereafter,
the incubation solutions were removed, the cells were washed two times with icecold
PBS, incubated with 500 µl of ice-cold PBS and treated with diazotized 2-nitroaniline
as described previously 11 in order to modify any surface-bound peptide.
In brief, 50 µl 0.6 M NaNO 2 was added to 400 µl ethanol/water 1/1 v/v containing
2-nitroaniline (0.06 M) and HCl (0.125 M). After standing for 5 min at ambient
temperature, 10 µl of this reagent was added to the ice-cold PBS covering the cell
layer and allowed to react for 10 min at 0°C. After aspiration of the diazo reagent,
the cells were washed two times with ice-cold PBS and finally lysed with 0.2 ml
0.1% Triton X-100 containing 10 mmol/l trifluoroacetic acid for 2 h at 0°C. The