crc press - E-Lib FK UWKS

Toxicity and Side Effects of Cell-Penetrating Peptides 247
as a typical amino acid motif which is presumably of similar function as that for
CPPs. In addition, CPPs usually have a hydrophobic part that is suggested to induce
integration of peptides into the cell membrane. 10 Some CPPs are amphipathic; like
antimicrobial peptides, they adopt an α-helical structure under membrane-mimicking
conditions. 11
The toxicity and cell-lytic activity of antimicrobial peptides necessitate studies
of CPPs’ toxicity on plasma membranes and on general cellular level since similarities
exist between these two classes of peptides. CPPs may act like toxic agents or
peptides on proteins and lipids of cellular membranes, on cell proliferation, and also
in vivo, causing cell death and inflammatory response. Further, some CPPs are
derived from biologically active proteins, so the activities of these peptides must be
evaluated before using them as carriers. Next we describe the most commonly used
methods for assaying in vitro toxicity of different CPPs.
11.2.1 IN VITRO TOXICITY
The term cytotoxicity denotes a complex process spanning from the death of a single
cell (general cytotoxicity) to the altering and gradual malfunction of the target
cell/tissue (organ/cell-specific cytotoxicity). For evaluating short-term effects of
bioactive compounds, a diversity of in vitro cell viability and cytotoxicity assays
has been developed. These in vitro treatments are cheaper, easier to quantify, and
ethically more acceptable than in vivo experiments. 12
On the other hand, in vitro toxicity assays suffer from inherent limitations. In
cell cultures it is usually difficult to recapitulate the respective in vivo situation with
its complex pharmacokinetics. In general, significant differences often exist in drug
exposure and concentration, metabolism, and excretion between in vitro and in vivo
situations. Furthermore, only metabolism in the liver turns some substances toxic,
which can be difficult to model in cell cultures. Analogously, the detoxification
process that converts substances toxic to cells in culture into nontoxic ones for a
living animal can be difficult to recreate in vitro. However, for initial testing of
cytotoxicity, in vitro methods are very useful and informative, especially when
studying compounds with putative membrane activity. In vitro toxicity assays track
changes in cellular homeostasis triggered by the compounds of interest by estimating
their general cytotoxicity or effect on cell viability and assessing integrity of the
plasma membrane or metabolic activity, respectively.
11.2.1.1 Cell Membrane Permeability
The cell plasma membrane, a lipid bilayer, constitutes the first barrier against toxic
agents, preventing hydrophilic molecules from entering the cell. The cellular homeostasis
of ions and organic molecules is regulated at the plasma membrane with help
from specific integral and peripheral proteins embedded in the lipid bilayer, i.e.,
transporters, receptors, channels, and pumps. Small and lipophilic substances are
not hindered by the barrier and diffuse passively through the membrane.
There are several ways in which toxic agents may change the cellular membrane
permeability. They may interfere with the functioning of membrane proteins, for