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278 Cell-Penetrating Peptides: Processes and Applications
accepted by the cell can be predicted. Studies of uptake kinetics are essential in
order to achieve a better understanding of the structure–activity relationship of CPPs.
Furthermore, data on the efficiency and limitations of CPPs for the transfer of
different cargoes in different cells could be amassed. Finally, studies of kinetics
could provide a very important contribution for elucidation of the mechanisms by
which CPPs pass membranes and enter cells.
It might, therefore, look quite surprising that few detailed kinetic studies of CPP
internalization into cells, cell compartments, or model lipid vesicles have been
undertaken. In a way, this is understandable since this field is relatively young.
Additionally, kinetic studies of CPP internalization are demanding in terms of finding
a reliable methodology. Despite the problems listed, some kinetic data have been
collected and analyzed for transportans (TP), penetratin, peptides derived from
nuclear transcription activator protein (Tat), and model amphipathic peptide (MAP
or KLAL); the internalization kinetics of TP has been most thoroughly studied. This
chapter reviews the kinetic studies of CPP internalization.
In the first part of the chapter, we summarize methods used for collecting the
kinetic data together with procedures for their analysis. Special attention is given to
the significance and reliability of kinetic parameters obtained by different methods.
In the second part, we discuss the results and conclusions derived from studies of
the most frequently used CPPs: TP, penetratin, Tat, and MAP and their analogs.
Within scope of the limited data available, we try to compare the rate and the yield
of internalization of different CPPs. In addition, we analyze the structure of the
cargo transported by CPP and its effect on the translocation rate.
It should be stressed that, at relatively high concentration (1 µM and higher),
CPPs can induce membrane leakage by membrane permeabilization. 1 CPP-induced
membrane leakage could lead to cell damage and, eventually, to cell death. In this
sense, some CPPs resemble the antimicrobial or lytic toxic peptides, for instance
the well-known components of bee and wasp venom, melittin and mastoparan. 2
However, CPPs can efficiently penetrate cell membrane at much lower concentrations
without inducing any damage to the biological membranes. In this chapter we
focus on “pure” pore-free internalization of CPPs into cells under physiologically
and, potentially, pharmacologically relevant conditions. Therefore, we mainly consider
kinetic data obtained at CPP concentrations that are presumably lower than
those required for membrane permeabilization.
13.2 METHODOLOGICAL CONSIDERATIONS
Any kinetic study of CPP internalization, with a goal to determine the rate of CPP
internalization, comprises the following steps: incubation of cells with a CPP at
suitable concentration, detection of the amount of CPP internalized into the cells as
a function of time, and subsequent analysis of the collected data. A hypothetical
scheme summarizing the steps of CPP uptake by cells is presented on Figure 13.1.
This scheme is an attempt to explain the available kinetic data on CPP uptake;
however, as such, it has not been adequately proven yet. Thus, the evaluation of this
scheme still remains an issue for further studies.