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82 Cell-Penetrating Peptides: Processes and Applications
relative fluorescence intensity
300
250
200
150
100
50
0
I all-D-I II VI III IV V VII VIII IX X
(a)
cytosol
nucleus
FIGURE 4.7 Fluorescence intensity of internalized peptides in the cytosol and nucleus after
exposure of LKB Ez7 cells for 30 min at 37°C to 1 µM I and I-derived peptides (A) and
subsequent washing with cold PBS (B), normalized to the fluorescence intensity of the external
peptide solution. Each bar represents the mean from three cells ± S.E.M.
lower positive charge, chain length, or amphipathicity than the parent compound I,
substantially reduced fluorescence intensities were found, whereas high fluorescence
intensities were observed in cells treated with those peptides proven to be extensively
internalized by HPLC measurements (Figure 4.7b). These findings further confirm
the conclusion that wash processes can substantially bias results of peptide uptake
experiments.
Quantitative deductions from the fluorescence intensities measured by CLSM
were only possible in combination with parallel HPLC determinations, since the
high relative intensities of the cytosolic fluorescence in Figure 4.7b are biased by
alterations of the peptide’s environment and aggregation status. 32
HPLC analysis of the wash fluids revealed that the peak corresponding to the
intact peptide represented more than 90% of the detected fluorescence in all cases,
indicating that metabolites or fluorescent contaminants were unlikely to have contributed
significantly to the wash-out fraction. CLSM monitoring of cells exposed
to the I-unrelated helical amphipathic and nonamphipathic pairs XI to XIV
(Table 4.2) as well as to the natural vector peptides derived from the Antennapedia
homeodomain (XV; see Reference 9) and from the Kaposi-fibroblast growth factor
(Fluos-AAVALLPAVLLALLAP-NH 2 (XVI); Reference 33) led to similar results to
those obtained with the MAP series (Figure 4.8). The on-line CLSM protocol
revealed extensive internalization in all cases, even for nonamphipathic peptides XIII
and XV (undetectable within the cell by the HPLC protocol) and significant losses
of cytosolic and nuclear fluorescence after washing, particularly in the cases of the
nonamphipathic peptides (Figure 4.8).
Exposure to other cell types of I-derived peptides resulted in a consistently high
intracellular fluorescence in all cases, which was distinctive for each cell type
relative fluorescence intensity
350
300
250
200
150
100
50
0
cytosol
nucleus
I all-D-I II VI III IV V VII VIII IX X
(b)