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Toxicity and Side Effects of Cell-Penetrating Peptides 253
detected at slightly lower MAP concentrations with an approximate EC 50 value of
4 µM. The following study from the same group investigated cellular uptake and
membrane toxicity of MAP analogues designed by varying the parameters amphipathicity,
hydrophobicity and net charge. 33
Membrane toxicity was examined for some of the analogues by using the
fluorescein leakage assay. Shortening MAP by four amino acids from the C terminus
or substituting all Ala for Gly in full length peptide reduced the fluorescein leakage
about three times as compared to MAP at 4.5 µM concentration. However, the cell
penetration efficacy of these MAP analogues was dramatically impaired (decreasing
about tenfold). Cellular uptake was abolished when the peptide was shorter than 16
amino acids or when a negative net charge was introduced. 33 A later study reported
that a 50 µM extracellular concentration of these analogues did not induce membrane
perforation of LKB Ez7 (subculture of AEC cell), referring again to a very low
cellular uptake of these peptides. 20
The strong effect of MAP on cell membrane permeability was confirmed by the
DGP leakage assay. A peptide concentration of 1 µM initiated leakage of DGP from
human Bowes melanoma cells (BMC; Table 11.1). 14 Increased MAP concentrations
totally disrupted cell plasma membranes, raising the efflux of DGP to the same level
as for the positive control, 1% Triton X-100.
Oehlke and colleagues assessed the effect of MAP on AEC viability by the MTT
assay. 8 Like trypan blue exclusion and fluorescein leakage assays, MAP initiated a
decrease in viability at 4 µM concentration, with an EC 50 value of approximately
10 µM (Table 11.2). To date, the effects of other MAP analogues on cell viability
have not been studied.
There is some difference in toxicity threshold concentration of MAP estimated
by DGP leakage and MTT assay. It could be explained by different sensitivity of
the used cell lines (AEC and BMC) to MAP. However, the DGP leakage is a more
suitable assay for detection of subtle and temporal changes in the cell plasma
membrane permeability.
11.2.2.2 Tat
The Tat (48–60) peptide, opposite to MAP, seems to cause no or very little harm to
cells. Even at 20 µM peptide concentration, no increase in BMC membrane permeability
could be detected when studying the DGP leakage (Table 11.1). 14 The MTT
assay confirms very low cytotoxicity of Tat and its more basic analogues. Tat (48–60)
was shown to affect cell viability only at high concentrations. 30 HeLa cell viability
decreased to 80% when treated with 100 µM Tat (48–60) for 24 h (Table 11.2).
However, extension of the peptide chain at the N terminus by 10 amino acids,
resulting in Tat (37–60), decreased the HeLa cells’ viability to 40% under identical
experimental conditions. 30
11.2.2.3 Penetratin
Penetratin peptide causes only minor disturbances to plasma membranes. Treatment
of BMC with 20 µM penetratin for 15 min increased the DGP leakage to 5% over