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246 Cell-Penetrating Peptides: Processes and Applications
in vitro, such as in studies of cellular events, as well as in vivo for drug delivery. 1,2
The mechanism exploited by cell-penetrating peptides to traverse the plasma membrane
surrounding a cell is still unknown; however uncertainties regarding the mechanism
have not hindered application of CPPs as carriers of many polar compounds
lacking cellular uptake.
An ideal vector for transmembrane delivery would need to guarantee efficient
transport of cargo into the cell interior. It should enter the cells without interfering
with cellular processes or impairing the barrier function of plasma membrane surrounding
cells (i.e., the ideal CPP should be nontoxic and side effect-free). First,
the CPP (alone or conjugated with a cargo) must cross the plasma membrane — a
lipid bilayer. Direct interaction of the peptide with lipids in the membrane is thought
to be obligatory for cell penetration. 3 This interaction is accompanied by rearrangements
of the membrane that, depending on properties and concentration of a particular
CPP, could range from temporary to permanent and from subtle to profound.
Second, the most often used delivery peptides are derived from naturally occurring
proteins that carry different functions and possess various activities in cells. Whether
the respective CPP is completely devoid of parent protein activities has not been
sufficiently studied yet, nor if they gain new unexpected functions.
Most CPPs are positively charged under physiological conditions 3 and the impact
of nonspecific interaction with cellular anions cannot be underestimated at concentrations
achieved in cell-penetration assays. Here we give a brief overview about
published data concerning nondesired biological activities of CPPs.
11.2 TOXICITY STUDIES OF CPPs
The primary structure and especially the net charge of CPPs are important factors
influencing interaction with cellular membranes and also toxicity. Most CPPs are
positively charged and some obtain an α-helical conformation in the membranemimicking
environment. Some similarity can therefore be found with well-characterized
antimicrobial peptides, which constitute a defense system against invading
microorganisms in many organisms (for reviews see Ganz and Lehrer, 4 Boman, 6 and
Simmaco et al. 5 ). Both these types of peptides interact with the plasma membrane
and enter the cells, though antimicrobial peptides assemble and multimerize at the
membrane-forming pores. Pore formation is the main toxic mechanism of antimicrobial
peptides. Upon interaction with the target membrane they fold from an
unordered state into an amphipathic α-helix conformation. The target cell is lysed
by a two-step mechanism: binding the peptide to the cell surface followed by
membrane permeabilization (for a review see Dathe and Wieprecht 7 ). Some CPPs,
like the model amphipathic peptide (MAP), have features rather similar to antimicrobial
peptides and the cellular uptake of the two peptide classes could proceed by
a common mechanism. 8
Most cell-penetrating peptides comprise an Arg- and Lys-rich region called the
basic domain. Positively charged amino acids have been suggested to be pivotal in
the electrostatic interaction with negatively charged head groups of phospholipids
constituting cell plasma membranes. 9 Cell lytic peptides also contain a basic domain