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Kinetics of Uptake of Cell-Penetrating Peptides 283
part remains in the water phase. Besides, molecular modeling studies have shown
that TP and TP analogues that possess the properties of CPP can be stable and deeply
inserted into lipid bilayer. 9
As opposed to TP, penetratin interacts mainly with membrane surface to which
it is adsorbed by electrostatic interactions, as revealed from spectroscopic studies. 5
This clearly demonstrates that no single common mechanism governs interaction
between different CPPs and membranes, and calls for detailed studies of this process,
for instance, similar to those undertaken with the family of Trp-pentapeptides. 14
13.2.5 ADDITIONAL CONSIDERATION
The mechanisms of internalization have not yet been clarified for CPPs. In order to
characterize kinetics of CPP uptake, it is necessary to determine that the internalization
data are obtained under conditions in which the CPP is not internalized by
endocytosis, mediated transport, pore formation, or a combination of these.
Exclusion of pore formation can be verified by leakage experiments in which
cells are first loaded with a suitable labeled-molecule and then leakage of the labeled
molecule from the cells is followed after the addition of CPP. 1 All types of proteinmediated
transport can be ruled out if the internalization is not saturable, i.e., if the
rate of internalization is linearly dependent on the increasing initial concentration
CPP and does not show tendency to reach plateau. This is additionally checked by
introducing a competitor peptide in excess, which should not substantially influence
the rate of internalization of the studied CPP. When the uptake is measured with a
labeled CPP, a nonlabeled CPP can be used as a competitor. 8 Active transport, which
is energy dependent, is suppressed by substances that deplete cell energy, 8,12 experiments
carried out at 4°C could yield additional information. Finally, endocytosis
can be inhibited by hyperosmolar solution of glucose, which blocks the formation
of clathrin-coated pits, 15 or by phenylarsine oxide treatment, which cross-links the
thiol groups of membrane surface proteins. 16
13.3 TYPES OF KINETIC EXPERIMENTS AND DATA ANALYSIS
The CPP uptake kinetics often follows the rules of simple first-order process
out
CPP ⎯ ⎯→CPP
Scheme 13.1
where k I is the first-order rate constant. This is seemingly controversial in light of
a complex uptake scheme described in Figure 13.1. However, knowing that CPPs
often accumulate in the cells, the simplification of the kinetic scheme seems reasonable.
In several cases such simplification has yielded valuable information in
studies of CPP uptake.
13.3.1 SINGLE TIME POINT MEASUREMENTS
l
k in
The simplest way to measure kinetics of CPP internalization is a so-called single
point experiment in which the amount of internalized CPP is determined at only