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Kinetics of Uptake of Cell-Penetrating Peptides 285
satisfactory, the proposed first-order rate mechanism is probably not obeyed. In this
case other equations might be applied, usually arising from more complex internalization
mechanisms or internalization models. Even if the fitting is good, one must always
be aware that the result applies only to the chosen concentration of CPP and that, in
another concentration range, CPP could internalize in accordance with a different model.
13.3.3 PROGRESS CURVES AT DIFFERENT CPP CONCENTRATIONS
The best approach is to follow the progress curves of internalization at several initial
concentrations of CPP. This is demanding in terms of material and time consumption
since a great number of experimental points is required, but the method yields
important advantages. First, the kinetic parameters obtained are valid for the whole
interval of used CPP concentrations and are therefore much more reliable. Even
more important, the procedure allows checking the order of the reaction and verifying
the complex kinetic models. If the first-order kinetics of internalization is proposed,
the rate constant should not be dependent on the initial concentration of CPP, while
the second-order kinetics requires linear relationship of the first-order rate constant
on the initial concentration of the CPP.
This can be verified by treating each progress curve separately using a suitable
computer program as described above for a fixed concentration experiment and
checking the concentration dependency of the obtained rate constants. Some computer
programs (see Dynafit, or a program of Stojan 18 ) allow treatment of all obtained
progress curves simultaneously, using either the explicitly derived equation for the
predicted mechanism or a numerical treatment of the system. For more complex
kinetic mechanisms, explicit equations are difficult or even impossible to obtain and
a numerical treatment is the only solution.
13.4 RESULTS FROM CPP UPTAKE KINETICS MEASUREMENTS
In this section we shall present and discuss results of kinetic studies performed so
far with four main classes of CPP: TP, penetratin, Tat, and MAP peptides.
13.4.1 TRANSPORTAN
For internalization studies, [ 125 I]–transportan and biotinyl–transportan were applied 8
and the internalization of TP into a number of different cell lines was inspected. For
kinetic studies, mainly Bowes cells were used. As revealed from visualization of the
internalized biotinyl–transportan by indirect immunofluorescence using confocal
microscopy, the process of internalization into different cells was rapid. The initial
process was very fast: the cells were intensely stained after 1 min incubation at 37°C
with 10 µM biotinyl–transportan. After the first 5 min, the peptide was localized
mostly in the plasma membrane and cytosolic membranous structures (endosomes,
endoplasmatic reticulum, Golgi). Staining of the nuclear membrane and nuclei was
slight, but clearly visible. After 15 to 30 min, biotinyl–transportan was preferentially
concentrated in the nuclear membrane and the nuclei.