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254 Cell-Penetrating Peptides: Processes and Applications
basal level (Table 11.1). At lower penetratin concentrations, membrane leakage
decreased in a dose-dependent manner with no detectable leakage under 10 µM
concentration. 14
The effect of penetratin on cellular viability has been studied by MTT assay. 34
No effect on lymphoblastoid cell line IB4 viability was detected after 72 h of
treatment with up to 30 µM of penetratin (Table 11.2). Mazel and colleagues used
D-penetratin as a vector for doxorubicin to increase the uptake of this cytotoxic
drug. 35 The vector peptide did not cause any drop of human erythroleukemia cell
line K562 (multidrug sensitive) and K562/ADR (multidrug resistant) viability as
judged by the MTT method. In contrast, Garcia–Echeverria and collegues detected
rounding up of the U2OS osteosarcoma cells after treatment with 50 µM penetratin,
suggesting that high concentrations of the peptide can be cytotoxic, at least to some
cells. 36
11.2.2.4 Transportan
The membrane toxicity of transportan lies somewhere between that of penetratin
and MAP as estimated by the DGP leakage assay. 14 Transportan induced efflux of
radioactivity from BMC at 5 µM concentration diluted in serum-free culture medium
(Table 11.1). At lower concentrations, no effect on plasma membrane permeability
could be detected.
11.2.2.5 Arginine-Rich Peptides
The structure–activity relationship studies of Tat (48–60) peptides, corresponding
to the Arg-rich domain of several transcription factors, revealed that peptides consisting
mainly of Arg could efficiently penetrate through the cell plasma membrane.
Futaki and co-workers studied the effect of basic peptides on cellular viability by
using the MTT assay. 37 The basic stretch (49–57) in Tat peptide was replaced with
only Arg residues, resulting in even more basic analogues than the original peptide.
This Arg 9–Tat (48–60) peptide decreased viability of mouse macrophage RAW264.7
cells to 70% after treatment with 100 µM for 24 h, while the original Tat (48–60)
sequence reduced the viability to 40% under identical conditions (Table 11.2).
These results indicate that Arg-rich peptides, which are highly basic, can be of
rather low toxicity to cells in culture and might thereby be suitable as carrier vectors.
The peptides that comprise only Arg residues are efficiently internalized by the cells.
Unfortunately, data about in vitro toxicity of these vector peptides are not yet
available. However, cellular cytotoxicity increases with peptide length and Arg
polymers of 12 kDa are cytotoxic at concentrations as low as 800 nM. 38
11.2.2.6 Conclusions
The longer and more amphipathic peptides seem to affect cell plasma membranes
more and thereby increase membrane permeability. As expected, MAP showed
highest cell toxicity as judged by MTT, dye exclusion, and leakage assays, followed
by transportan, penetratin, and Tat (48–60). On the first look, more membrane