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240 Cell-Penetrating Peptides: Processes and Applications
into the bilayer. A necessary, but not a satisfactory, criterion for any CPP is its own
translocation. In order to be classified as a useful CPP, the peptide should also be
able to carry cargo across a membrane. The size and nature of the cargo seems still
to be an open question. However, a minimum requirement should be that the cargo
is not just a part of the CPP peptide sequence itself, like an NLS-tag.
10.10 SPECULATIONS ON THE MECHANISMS
OF CPP TRANSLOCATION
The only common obvious property among CPPs is their ability to permeate many
types of living cells without damaging them. From cell culture studies it is believed
that the spectacular transport of CPPs and a sizable cargo is not mediated by a chiral
receptor, nor is it an energy-dependent process. Is there a common mechanism
responsible for translocation of CPPs over plasma membranes? It is likely that CPPs
of different types (Table 10.1) do not necessarily employ the same mechanism for
their translocation.
We can distinguish two types of CPPs: one has many charged groups distributed
along the molecule and another has relatively few charged residues terminating a
long hydrophobic stretch. There is no consensus of primary sequence among CPPs;
their origin and design are diverse (Table 10.1). The CPPs are more or less
amphiphatic but they should be water-soluble with a low tendency to self-aggregate.
Although some residues appear to be crucial, there is not always an all-or-none
situation. A truncated analog can function, although with a reduced potency. The
optimization of the properties is still based on empirical experiments. A number of
basic amino acids (R, K) seem to be required. Arginine-rich peptides like pure Arg
oligomers (n < 9) are even more potent than the parent CPPs. 59
That the CPPs interact in some way with a membrane is a rather trivial finding
from the model studies. However, the highly charged penetratin molecule interacts
mainly by electrostatic attraction to an acidic phospholipid membrane, whereas other
types of CPPs, like transportan and the other chimera, have a certain interaction
even with a neutral membrane. In the case of negatively charged membranes, the
interfacial concentration of a CPP will become very high. However, when the CPP
is linked to a cargo, the total net charge of the assembly will be relevant. In the case
of a polynucleotide cargo, the net charge might even hinder permeability if the
surface potential is negative. Still, polynucleotides seem to come through.
In contrast to membrane-active antimicrobial peptides (melittin, magainin, etc.),
so far no evidence exists for any pore formation leading to membrane leakage with
CPPs. Moreover, a transient pore would be based on an oligomeric structure, which
could hardly incorporate the many different types of cargos, including nanometer
beads. Based on some membrane model experiments with penetratin, an inverted
micelle model has been put forward. 6 However, the 31 P NMR spectra did not provide
convincing evidence of a spectral (isotropic) component from lipidic particles,
induced by penetratin. Nevertheless, it was suggested that penetratin, with its cargo,
could be accommodated within an aqueous cavity of an inverted micelle, and then
transiently internalized. Such a mechanism may not cause any leakage, but is as