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76 Cell-Penetrating Peptides: Processes and Applications
pmol/mg protein
250
200
150
100
50
0
LKB Ez
7 aorta
bovine
37°C
0°C
ECV
vein
human
S-EZ
aorta
porcine
FIGURE 4.4 Intact fractions of cell-associated peptide after exposing various cell types from
different species and organs for 30 min at 37 and at 0°C to 1.8 µM I, the all-D enantiomer
of I, and the Antennapedia peptide XV 9 and subsequent treatment with diazotized 2-nitroaniline.
Before exposure to the peptide at 0°C the cells were incubated in DPBSG for 60 min
at 0°C. Each bar represents the mean of three samples ± SD.
same way, the effect of hyperosmolar sucrose might be interpreted as the effect of
membrane shrinking due to hyperosmolar stress. 28
Taken together and based on data currently available, an unequivocal decision
for either of the mechanisms discussed appears impossible. In context, however,
these data imply that multiple, probably nonendocytic mechanisms are involved in
the uptake into mammalian cells of MAPs.
4.2.4 UPTAKE OF MAPs INTO VARIOUS CELL TYPES
FROM DIFFERENT SPECIES AND ORGANS
SK-N-SH
nerve
human
Hep G2
liver
human
After exposure to I of endothelial cells from other species, as well as other cell
types, an extensive uptake of I at 37 and 0°C with significant variability was observed
in all cases (Figure 4.4). These findings indicated that nonendocytic mechanisms
responsible for the cellular uptake of MAPs were generally operative.
4.3 INVESTIGATION OF STRUCTURAL REQUIREMENTS
FOR CELLULAR UPTAKE OF MAPs
4.3.1 INFLUENCE OF HYDROPHOBICITY, HYDROPHOBIC MOMENT, AND SIZE
OF THE HYDROPHILIC FACE ON CELLULAR UPTAKE OF HELICAL PEPTIDES
The cellular uptake of a series derived from I by exchange of Leu-11 against the
internal fluorescent probe Trp and by stepwise alterations of hydrophobicity, hydrophobic
moment, and hydrophilic face (with retention of positive charge and helixforming
propensity) was assessed by the earlier described HPLC protocol (Table 4.1;