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282 Cell-Penetrating Peptides: Processes and Applications
of the internalized and liberated cargo, while the fluorescence of CPP–cargo construct
in the incubation solution remains quenched. In this way, a continuous realtime
quantitative monitoring of the cargo internalization into cells via CPP was
achieved.
The method has a number of advantages. Convenient and reliable kinetic data
are obtained in each single experiment where all experimental points obtained for
the internalization process are measured in virtually identical conditions (same set
of cells, same incubation solution, etc.), thus decreasing the scattering of data. It is
assumed that the disulphide bond keeping the quencher in close proximity to fluorophore
is reduced by gluthatione in the cytoplasm. If so, the portion of CPP–cargo
construct that remains firmly associated with cell membranes has less chance to be
reduced and made visible. Consequently, mainly the concentration of free cargo in
the cytosol is determined. This approach is a method of choice for studying kinetics
of internalization of CPP–cargo constructs in micromolar and submicromolar concentration
ranges.
Lower sensitivity of methods based on measuring fluorescence is a main drawback
in comparison with methods based on radioactivity measurements. The detection
limit for fluorescence methods is close to the concentrations where some CPPs
(MAP and TP, see Hällbrink et al. 1 ) could disturb membranes. For these CPPs,
following the internalization at lower concentrations (below 0.1 µM) seems much
safer. This can be reliably achieved by use of radioactively labeled CPP.
13.2.4 QUANTIFICATION OF CPPS IN MEMBRANES
The quantification of the fraction of CPP or CPP–cargo construct inserted into the
membrane and the amount actually free in the cytosol (Figure 13.1) is an important
issue in kinetic studies. In constructing kinetic models to describe the dynamics of
internalization of a CPP into cells, the insertion of the CPP and CPP–cargo construct
into the membrane should be considered an important intermediate step of the uptake
that must be directly or indirectly taken into the account during model prediction.
Interaction of the CPP and CPP–cargo constructs with membranes is also an important
physiological issue. CPPs are potentially aimed to be used for the internalization
of drugs and it is important to know (or predict) the quantity of the drug transported
and available in the cytosol, and the amount temporarily deposited in membranes.
Different techniques exist for assessing the fraction of peptide adsorbed to membranes,
e.g., treating the membrane with acids, treatment of cells with trypsin, or
modification of the absorbed CPP, as mentioned above.
The determination of a membrane-associated fraction of a CPP is sometimes a
difficult issue. Attempts have been made to evaluate this fraction by determination
of the partition coefficients of CPPs between water and lipid environment. n-Octanol
is often used as a rough approximation modeling the lipid part, 8 with known drawbacks.
14 Transportan peptide provides a clear example for this. As determined by
Soomets et al., water–octanol partition coefficient for TP was determined to be 15, 9
but the confocal micrography revealed the presence of TP mainly in the cellular
membrane structures. 8 This can be explained by the amphiphilic character of TP,
enabling insertion of the hydrophobic side into the membrane, while the hydrophilic