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60 Cell-Penetrating Peptides: Processes and Applications
The total uptake displayed a roughly linear dependence of the amount of total
construct in the experiment, showing that the transport mechanism was not saturated
and that the upper limit of construct import (if such limits exist) was not reached
within the concentration range used. At saturation, the cargo concentration inside
the cells was about 400-fold higher than that which remained extracellularly, showing
that transportan is indeed very efficient in delivering small peptides to the cytoplasm.
In this assay, rate of transportan uptake was shown to be close to the rate obtained
with the peptide itself. Moreover, transportan uptake was shown not to be disturbed
by the presence of 10% fetal bovine serum in the incubation medium.
3.6.2 DELIVERY OF PNA
Synthetic molecules that can specifically inhibit translation or transcription hold
great promise as potential antisense and antigene drugs. The novel DNA mimics
polyamide/peptide nucleic acid (PNA); 25 locked (LNA) 26 and morpholino nucleic
acids 27 have probably the highest potential as antisense reagents, since they reveal
high stability in biological fluids as well as in vivo conditions in general. Moreover,
they show low toxicity and strong, specific pairing with complementary singlestranded
RNA/DNA.
Among these DNA analogues, PNA with a noncharged achiral polyamide backbone
to which the nucleobases are attached is one of the best studied. 28 Unfortunately,
the uptake of unmodified PNA by most cell types in culture as well as in vivo is
poor, especially at low or medium concentrations. 29 Consequently, in order to reach
intracellular PNA concentration necessary for biological action, high or extremely
high extracellular (20 to 100 µM) concentrations are usually applied. 30
To circumvent this problem, we used transportan to facilitate the cellular uptake
of PNA. A disulfide bond was formed between an extra cysteine on ε-NH Lys 13 of
transportan and the biotinyl-Cys of PNA oligomer. Usually 4 to 8 h incubation with
10 to 20 µM biotinyl-PNA is needed to yield intracellular concentration detectable
by indirect immunofluorescence. In contrast, transportan-PNA constructs (TP-PNA)
enter cells at remarkable speed; 5 to 10 min incubation of Bowes melanoma cells
with 2 to 3 µM TP-PNA is sufficient for internalization of detectable amounts of
biotinyl-PNA. Prolonging incubation to 2 to 4 h leads to accumulation of PNA in
the cells and concentrations as low as 0.1 µM extracellular TP-PNA lead to a cellular
concentration detectable by fluorescence. 29
The cellular internalization of unmodified PNA at high concentrations results
predominantly in granular staining, suggesting that the cells take up “naked” PNA
by constitutive (or induced) endocytosis. After 4 to 8 h incubation, the delivered
PNA is trapped in vesicular structures like endosomes or lysosomes and stays
inactive. Later the staining spreads in the cell and is localized more diffusely,
implying that part of the PNA has been liberated from lysosomes.
The cellular distribution of transportan-delivered PNA resembles that of transportan
only at very initial steps of internalization (up to 5 min). Later their localizations
do not overlap, confirming that, in the cell interior, the cargo is liberated
from the carrier peptide after disulfide bond reduction and can follow its own signals
for intracellular routing.