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Kinetics of Uptake of Cell-Penetrating Peptides 291
concentrations the membrane leakage increased. Tat peptide did not induce any
leakage at all, even at highest concentrations used (20 µM), while a negligible effect
was detected for penetratin.
The effect of a cargo on the translocation velocity is of great interest. Cargoes
transported by CPPs are very different in structure and in size, ranging from organic
molecules of low molecular mass (e.g., biotin, fluorescein), over peptides, DNA,
and PNA fragments, to proteins of around 150 kDa. It is expected that a cargo could
have an influence on the rate of internalization of CPP–cargo construct. However,
as it was shown, small cargoes usually do not influence the rate of translocation
substantially. 8
The data on kinetics of translocation of CPP coupled to medium- and large-size
cargos are not available. From immunofluorescence studies utilizing confocal
microscopy, it could be deduced that TP attached to protein cargos of up to 150 kDa
internalizes more slowly than TP itself, as estimated visually from the dynamics of
fluorescence appearance in the cells. 8,23 Exact characterization of how much slower
this process is has not been carried out.
Biotinyl–TP could be detected in cells after a few minutes and reached maximal
intracellular concentration after 20 min of incubation. 8 Transportan attached to green
fluorescence protein (GFP, molecular mass 28 kDa), avidin–TRITC (molecular mass
66 kDa), and polyclonal antibody (molecular mass 150 kDa) were detected in the
cytoplasm substantially later, on average after 30 min of incubation. However, in
some cases, e.g., with noncovalently attached monoclonal antibody against biotin
to biotinyl–TP, the construct was detected in the cell after 2 h incubation. 23
Proteins have also been translocated by the Tat (47–57) carrier. 24 Successful
translocation of partially denatured β-galactosidase into different mouse cells was
achieved, but the experiments do not allow comparison of rate of translocation
between the Tat (47–57)–β-galactosidase construct and Tat (47–57). Interestingly,
β-galactosidase was refolded inside the cells and regained its full physiological role.
13.5 CONCLUSIONS AND FUTURE PERSPECTIVE
Methods of kinetics have already proven their usefulness by providing valuable data
on the rate of CPP internalization and by establishing the link between rate and yield
of cellular uptake, as well as structure of CPP. Complete study of cellular penetration
kinetics with all CPPs and various cargo molecules would give additional valuable
information and allow constructing kinetic models for cell penetration of each
individual CPP, leading to a better understanding of principles that govern the
internalization of CPPs. Establishing methodological procedures for convenient and
reliable real-time monitoring of internalization is an important first step.
ACKNOWLEDGMENTS
This work was supported by grants from the EC Biotechnology Project BIO4-98-0227,
Swedish Research Councils TFR and NFR, and grants from the Ministry of Science,
Education, and Sport of the Republic of Slovenia.