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Toxicity and Side Effects of Cell-Penetrating Peptides 255
disturbance is caused by CPPs with higher yield of accumulation into the cells (ratio
between the intra- and extracellular concentration of CPP at the equilibrium), for
instance, MAP and transportan. This suggests that the membrane-disturbing effects
of CPPs, exerted at plasma membrane, depend primarily on their intracellular or
membrane concentration and not so much on the applied concentration.
It is difficult to estimate the exact intracellular concentration of CPPs. The
intramembrane concentration is even more difficult to assess, making correlation to
membrane toxicity practically impossible. Usually, intracellular CPP concentration
is determined by using fluorescein-labeled peptides. However, fluorescein is sensitive
to its surrounding environment, which can cause not only errors when estimating
the concentration, but also assumed change in intracellular localization pattern. 20
Usually the CPP efficiency to translocate into cells as well as the toxicity is
estimated on cells growing in culture where cells are at different stages in the cell
cycle. The amphipathic synthetic peptide NAc-GALFLGWLGAAGSTMGAWSQP-
KKKRKV-cysteamide has been shown to exert cell cycle-dependent toxicity. 39 This
peptide is more toxic to proliferating cells than confluent H9C2 cells, but minimally
or quiescent to differentiated cells. This fact must be considered when in vitro results
are the only basis for explaining in vivo results.
11.2.3 SIDE EFFECTS OF CPPS IN VITRO
Most described side effects of CPPs are based on in vitro toxicity data. This is mainly
due to the high importance of a nontoxic delivery vector and the relative ease of
finding a relevant assay. However, all the plausible side effects of CPPs cannot be
comprehended by studying cell toxicity.
The remarkable high positive net charge of most CPPs causes their tight binding
to polyanions. Indeed, penetratin binds heparin 40 and interacts strongly with polysialic
acid of the cell-surface glycosides. 41 Probably most CPPs can interact strongly
with DNA or RNA and the interaction with negatively charged polysialic acid has
been suggested to mediate the efficient uptake of pAntp by neurons. 40
The ability of CPPs to translocate into cells and their in vitro toxicity is relatively
well characterized, but still other biological activities are only known for Tat peptide
and transportan. The absence of data for other CPPs does not necessarily mean that
they are devoid of side effects.
11.2.3.1 Transportan
The chimeric molecule of transportan comprises the fragment of neuropeptide galanin
and a peptide from wasp venom — mastoparan. Indeed, transportan has some
characteristic features of galanin: it interferes with galanin ( 125 I-galanin) binding to
type 1 galanin receptors in BMC membranes, at rather low concentration (K D
17.4 nM). 42 Transportan, like mastoparan, modulates the activity of membranous Gproteins.
Surprisingly, in contrast to mastoparan, which activates G-proteins, transportan
possesses an inhibitory effect (EC 50 of 21.1 µM) on them. It is not clear
whether these effects of transportan are caused by direct binding of the peptide to the
receptor and G-proteins or by changing membrane properties and thereby modulating