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106 Cell-Penetrating Peptides: Processes and Applications
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Cytochalasin
Early endosome
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Bafilomycin
Nocodazol
Late endosome
FIGURE 5.3 Mechanism of action of several cell-trafficking inhibitors. Penetration of a DNA
formulation is blocked by cytochalasin B, an inhibitor of microfilament depolarization. The
complexes then enter early endosomes, followed by formation of transport vesicles, which
can be blocked by bafilomycin A, an inhibitor of vacuolar ATPase. Maturation of the transport
vesicles into late endosomes can be inhibited by addition of chloroquin, a weak base. Finally,
fusion of the late endosomes with lysosomal compartments can be blocked by nocodazole,
which induces microtubule depolymerization.
pathway. 50,51 Cell-penetrating peptides can enter the nucleus through a mechanism
independent of the importin pathway or by active transport dependent on energy.
The question of energy is therefore an important parameter to be considered when
characterizing the nuclear import mechanism. The endosomal pathway is energydependent,
so incubation at 4°C constitutes one of the means of blocking that
pathway; however, this may be limited to specific cell lines and may affect cell death
or modify cellular composition.
5.5.2 MECHANISM OF NUCLEAR TRANSPORT
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Transport vesicle
Chloroquin
Nocodazol
Lysosome
DNA release
Several examples using NLS-containing peptides have been demonstrated to facilitate
transfection of DNA. 6-10 However, only a fraction of these show a real effect
of the NLS as a nuclear-facilitating agent. This effect should be investigated directly
on nuclear import and nuclear accumulation of plasmid should be monitored to
distinguish from secondary effects. In most cases, the NLS may contribute through
nonspecific electrostatic interactions that enhance DNA complex formation or aggregation.
It is therefore important to establish a correlation between transfection
efficiency and interaction with the import machinery.
In all cases, control experiments should be performed with cell-penetrating
peptides lacking an NLS or containing mutated or scrambled NLSs, to confirm the
specific role of the NLS. SV40-derived NLS peptides were used and in control a
reverse NLS showed no transfection 33 or nuclear import following microinjection
into zebrafish eggs using luciferase reporter gene. 84 A specific M9-NLS sequence
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