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Kinetics of Uptake of Cell-Penetrating Peptides 281
In principle, the CPP or cargo molecules could be labeled with a variety of
different radionuclids, e.g., 35 S, 32 P, or 3 H. Some tritiated short peptides have been
used in internalization studies, 12 but these peptides do not classify as CPPs and are
not reviewed here. Radioactive chelated technetium ( 99m Tc) coupled to Tat peptide
was transported into the cells. 6 To our knowledge, labeling of CPPs with other
radionuclides has not been performed.
The use of a radioactively labeled CPP or CPP–cargo construct has an advantage
over other methods due to high sensitivity. The main disadvantage of this detection
method is the necessity for separation of the cell-internalized fraction of the labeled
compound from the bulk remaining in the incubation solution. This prevents the
design of kinetic experiments enabling continuous monitoring of the amount of cellinternalized
CPP or CPP–cargo construct. Additionally, by this method, the fraction
of CPP or CPP–cargo construct firmly inserted into the cell membranes cannot easily
be differentiated from the fraction in cytoplasm and inner organelles.
Different fluorophores have been used to label CPPs and to trace their translocation
into cells. Fluorescein and fluorescein derivatives, 4,6,11 NBD (7-nitrobenz-
2-oxo-1,3-diazol), 5 and Abz (2-amino benzoic acid) 1,7 have been most frequently
used. After the incubation of a cell with the labeled CPP, the solution is removed
and the concentration of the labeled CPP inside the cells determined either directly 7
or by fluorescent flow cytometry. 5 Protocols have been developed to determine the
amount of internalized CPP from cell lysate using HPLC. 4
With fluorescently labeled CPPs, it is possible to quench fluorescence in the
incubation solution after selected incubation time by addition of suitable quencher
while the fluorescence of the labeled CPP inside the cells remains unchanged due
to inability of quencher to penetrate into the cells. In this way, the fluorescence of
NBD–penetratin in the incubation solution was irreversibly quenched by addition
of sodium dithionite and the concentration of NBD–penetratin internalized into the
cells was determined without any additional separation procedure of cells and incubation
solution. 5
Lorenz et al. have devised a method of confocal laser scanning microscopy
(CLSM) to quantify the cytosolic portion of CPP uptake 13 (see Chapter 4). In short,
attached cells are incubated with fluorescently labeled peptide and the time course
of the fluorescence inside and outside the cells is monitored. The signal is corrected
for the superimposition of the background fluorescence resulting from nonideal
confocal properties of the microscope. With this method, it is possible to monitor
the uptake of a CPP in real time.
Not only CPPs, but also cargo molecules in a CPP–cargo construct can be labeled
with fluorophore. The amount of a labeled cargo transported into the cells by a CPP
could be determined by one of the methods described above.
Recently, a new convenient strategy has been developed for characterization of
uptake kinetics of CPPs. CPP–cargo constructs were synthesised where small pentapeptide
cargo molecules, carrying a fluorophore, Abz, were coupled to different
CPPs (TP, penetratin, Tat, and MAP) labeled with the fluorescence quencher 2-nitrotyrosine
via disulphide bond, a bond readily reduced in the intracellular milieu. 1 The
intensity of the resulting fluorescence inside the cells is proportional to the amount