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80 Cell-Penetrating Peptides: Processes and Applications
pmol/mgprotein
400
350
300
250
200
150
100
50
0
30 min peptide exposure
30 min peptide exposure
with 30 min re-exchange
IV VI
FIGURE 4.5 Intact fractions of cell-associated peptide after exposing LKB Ez 7 cells for
30 min at 37°C to 50 µM IV and VI with and without exposure to fresh DPBSG for 30 min
at 37°C prior to washing and treatment with diazotized 2-nitroaniline. Each bar represents
the mean of three samples ± SD.
comparable order as found with I was observed for the amphipathic peptides XI and
XIII. For the nonamphipathic counterparts, XII and XIV, containing charged residues
uniformly distributed around the helix, no internalization was detectable. The notion
that the net positive charge is of lower importance for entry into the cell interior
was further supported by the similar degree of internalization found for the Antennapedia
peptide XV, 9 which possesses two positive side chains more than I
(Table 4.2).
Common quantitation protocols, as the HPLC approach applied here, presuppose
removal of the vast excess of analyte in the incubation solution by a multiple wash
process. The wash steps are normally performed in the cold in order to suppress
bias of the results by energy-dependent efflux processes. Since, however, MAPs are
able to cross the plasma membrane in the cold, the possibility could not be ruled
out that the uptake results obtained might be biased by a rapid wash-out. Initial
evidence that the inability to detect cellular uptake of the nonamphipathic peptides
by the described HPLC protocol indeed reflected that only poor internalization
combined with a rapid wash-out was provided by uptake experiments performed at
50 µM concentration using peptides IV and VI. Under such conditions clear internalization
of these unstructured peptides, which were nontoxic even at this high
concentration, 29 was detected by the HPLC protocol (Figure 4.5). Likewise,
Figure 4.5 illustrates an extensive escape of these peptides from preloaded cells,
whereas for the amphipathic parent, peptide I, no significant efflux was measurable
under the same conditions. 10 This suggested that the low intracellular levels (relative
to those of the external medium) achieved with peptides IV and VI are at least
partially due to wash-out.