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Toxicity and Side Effects of Cell-Penetrating Peptides 253<br />

detected at slightly lower MAP concentrations with an approximate EC 50 value of<br />

4 µM. The following study from the same group investigated cellular uptake and<br />

membrane toxicity of MAP analogues designed by varying the parameters amphipathicity,<br />

hydrophobicity and net charge. 33<br />

Membrane toxicity was examined for some of the analogues by using the<br />

fluorescein leakage assay. Shortening MAP by four amino acids from the C terminus<br />

or substituting all Ala for Gly in full length peptide reduced the fluorescein leakage<br />

about three times as compared to MAP at 4.5 µM concentration. However, the cell<br />

penetration efficacy of these MAP analogues was dramatically impaired (decreasing<br />

about tenfold). Cellular uptake was abolished when the peptide was shorter than 16<br />

amino acids or when a negative net charge was introduced. 33 A later study reported<br />

that a 50 µM extracellular concentration of these analogues did not induce membrane<br />

perforation of LKB Ez7 (subculture of AEC cell), referring again to a very low<br />

cellular uptake of these peptides. 20<br />

The strong effect of MAP on cell membrane permeability was confirmed by the<br />

DGP leakage assay. A peptide concentration of 1 µM initiated leakage of DGP from<br />

human Bowes melanoma cells (BMC; Table 11.1). 14 Increased MAP concentrations<br />

totally disrupted cell plasma membranes, raising the efflux of DGP to the same level<br />

as for the positive control, 1% Triton X-100.<br />

Oehlke and colleagues assessed the effect of MAP on AEC viability by the MTT<br />

assay. 8 Like trypan blue exclusion and fluorescein leakage assays, MAP initiated a<br />

decrease in viability at 4 µM concentration, with an EC 50 value of approximately<br />

10 µM (Table 11.2). To date, the effects of other MAP analogues on cell viability<br />

have not been studied.<br />

There is some difference in toxicity threshold concentration of MAP estimated<br />

by DGP leakage and MTT assay. It could be explained by different sensitivity of<br />

the used cell lines (AEC and BMC) to MAP. However, the DGP leakage is a more<br />

suitable assay for detection of subtle and temporal changes in the cell plasma<br />

membrane permeability.<br />

11.2.2.2 Tat<br />

The Tat (48–60) peptide, opposite to MAP, seems to cause no or very little harm to<br />

cells. Even at 20 µM peptide concentration, no increase in BMC membrane permeability<br />

could be detected when studying the DGP leakage (Table 11.1). 14 The MTT<br />

assay confirms very low cytotoxicity of Tat and its more basic analogues. Tat (48–60)<br />

was shown to affect cell viability only at high concentrations. 30 HeLa cell viability<br />

decreased to 80% when treated with 100 µM Tat (48–60) for 24 h (Table 11.2).<br />

However, extension of the peptide chain at the N terminus by 10 amino acids,<br />

resulting in Tat (37–60), decreased the HeLa cells’ viability to 40% under identical<br />

experimental conditions. 30<br />

11.2.2.3 Penetratin<br />

Penetratin peptide causes only minor disturbances to plasma membranes. Treatment<br />

of BMC with 20 µM penetratin for 15 min increased the DGP leakage to 5% over

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