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280 Cell-Penetrating Peptides: Processes and Applications
surface with electrostatic and hydrophobic interactions (Figure 13.1). Most CPPs
are positively charged at physiological pH and thus could interact with negatively
charged groups on the cell surface. In order to determine the rate of internalization,
the internalized fraction of CPP must be separated from noninternalized, and its
amount estimated as a function of the incubation time. The loosely bound CPP can
be removed from the cell surface, e.g., by a short-time incubation of the cells in icecold
acidic solution (e.g., 5 min in the mixture of 0.2 M acetic acid and 0.5 M NaCl,
pH 2.5; see Pooga et al. 8 ).
Surface-bound CPP can be removed also by short treatment of cells with trypsin, 7
or the amount of this fraction can be separately quantified by modification of the
adsorbed CPP 4 and subtracted from the amount of total cell-associated CPP in order
to calculate the amount of internalized CPP. If the surface-bound CPP comprises
only a very small part of the total amount of cell-associated CPP (5% or less), the
kinetics of the uptake of labeled CPP is not substantially affected by this small
fraction of the loosely membrane-attached CPP. In this case, any treatment of cells
to remove loosely membrane-bound CPP can be omitted. 9
Detached cells can be collected by centrifugation and washed; subsequently, the
amount of the internalized CPP inside the cells is determined. An alternative technique
is by centrifugation of cells for 15 sec at 6000 × g through a mixture of 40%
dinonylphthalate and 60% dibutylphthalate 8 or 75:25 mixture of silicon and mineral
oil. 6 The bottoms of the centrifugation tubes containing the precipitated cells are
then cut off and the amount of CPP in the cells and in the remaining incubation
solution can be determined separately. This technique is preferable if the outflow of
the CPP from the cells is fast. Sometimes a separation of the cells from the incubation
solution is not necessary. This largely depends on the method by which the CPP is
detected. Some approaches exploiting detection of CPP by fluorescence make it
possible to bypass the separation procedure. They are briefly described below.
13.2.3 DETECTION OF CPP
Different methods can be used to detect CPPs and to assess their concentrations (see
Chapter 12). In general, CPPs cannot be directly observed in their intact form with
methods suitable for kinetic measurements. Therefore, the peptide must be specially
modified to allow reliable detection of rather small amounts that are usually internalized.
In most internalization kinetic studies, the CPP itself or the cargo molecules
(in CPP–cargo constructs) have been labeled by radioactive isotopes or fluorophore
molecules.
Radioactive labeling of a CPP can be performed with 125 I-iodination, 8 e.g., using
the chloramine-T method, 10 which can be exploited only when Tyr is present in the
sequence. Labeling of cargo molecule has the advantage that the delivery of a cargo
molecule into the cells can directly be traced and that there is a possibility to choose
from a large variety of commercially available labeled compounds that could be
used as cargo. One example is the synthesis of a construct where a commercially
available radiolabeled antineoplastic agent, 14 C-doxorubicin, was used as cargo coupled
to penetratin. 11