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78 Cell-Penetrating Peptides: Processes and Applications
Reference 29). In order to facilitate monitoring of toxic effects of the respective
peptides by leakage of fluorescein from the cell interior after preloading with
diacetylfluorescein, a generally slightly toxic peptide concentration of 5 µM was
chosen for these experiments. Under these conditions the parent peptide I induced
30% fluorescein leakage. 10 That the results obtained at the slightly toxic 5 µM peptide
concentration were not biased significantly by partial damage to the plasma membrane
was ascertained by additional experiments performed at the nontoxic 2 µM
peptide concentration that led to analogous results. 29
Clear cellular uptake and pore-forming activity were found in almost all cases
(Table 4.1). No unambigous correlation, however, either between physicochemical
parameters and uptake behavior pore-forming activity or between uptake behavior
and pore-forming activity was apparent from the data. Generally these findings argue
against a decisive role of any of the altered helix parameters on cellular uptake.
4.3.2 INFLUENCE OF MOLECULAR SIZE, CHARGE, AND PRIMARY STRUCTURE
Table 4.2 summarizes results of cell experiments performed with a series derived
from I by alterations in chain length, charge- and helix-forming propensity, and an
N-terminal carboxy fluorescein tag. Comparison with the results obtained with the
tryptophan-labeled series is justified by the similar uptake and toxicity found for
related pairs of tryptophan- and fluorescein-labeled analogs. 29
Reduction of chain length by two amino acid residues had no significant influence
upon the amount of internalized peptide (III, Table 4.2). Shortening the molecule
by four amino acid residues, either N or C terminally, however, resulted in a
substantial loss of uptake and membrane toxicity (IV, V, Table 4.2), suggesting that
a minimum chain length corresponding to four complete helix turns was essential.
Replacement of two of the five lysines of the molecule by glutamine residues
reduced the uptake markedly, accompanied by a clear reduction in membrane toxicity
(VII, Table 4.2). Surprisingly, further elimination of basic lysine side chains resulted
in significantly enhanced internalization (VIII, IX, Table 4.2), even when the noticeable
contributions of surface-bound peptide to the quantity of IX measured in the
cell lysate are taken into consideration. In the case of IX (and also of X) the lack
of side chain amino groups prevented the differentiation of surface-bound from
internalized peptide fractions by means of diazotized 2-nitroaniline. That IX has
been actually internalized at least comparably to I is suggested by the threefold
increase in the level of fluorescent metabolites in the cell lysate of IX-treated cells
compared to that found after exposure to I. 29 CLSM (see below) revealed a similar
image for both I- and IX-treated cells, again indicating comparable uptake. Reduction
of the helix-forming propensity by Ala–Gly replacement strongly impaired the
degree of internalization (VI, Table 4.2). Taken together, these results imply that
helical amphipathicity is the most decisive structural requirement of MAPs for
achieving high intracellular concentrations.
Additional uptake experiments performed with two pairs of helical amphipathic
and nonamphipathic peptides, unrelated to I and with a lower net charge (XI to XIV,
Table 4.2), confirmed the crucial role of amphipathicity and likewise the conclusion
that net positive charge is not decisive in this context. 29 Clear cellular uptake of a