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Signal Sequence-Based Cell-Penetrating Peptides 105
nucleus, it rapidly condenses into a chromatin-like structure. Multiple NLSs might
therefore inhibit nuclear transport of two NLSs docked onto a nuclear pore. 87
5.5 DESIGN AND EVALUATION OF NLS-CONTAINING
CELL-PENETRATING PEPTIDES
The “ideal” NLS cargo complex is recognized by the nuclear import machinery
through specific NLS sequences; this complex is driven into the nucleus where it
enhances transgene expression. Efficient nuclear transport of cell-penetrating peptides
requires essential steps that are controlled by several regulatory pathways. As
such, these parameters need to be taken into account when designing a new peptide
vector. Of particular relevance are accessibility of the NLS and availability of the
import factors, regulation of affinity of the NLS for its import receptor, phosphorylation,
existence of cytosolic or nucleoplasmic retention signals, and regulation of
NPC permeability, as well as the possible regulation of the affinity of the cargo for the
hydrophobic central channel. In this section, technical aspects will be described in order
to evaluate and improve NLS-containing cell-penetrating peptides. 5,9,18,27
5.5.1 MECHANISM OF CELL DELIVERY
The transfection or transport of cargo into cells involves a number of selective steps
in addition to nuclear import. Understanding the behavior of cell-penetrating peptide
and DNA or cargo complexes in cells is essential to improve their efficiency. Intracellular
transport of drugs or cargo into the nucleus can occur through endocytosis
or through an independent pathway. A large number of methods has been proposed
to determine the mechanism of inhibition of specific stages of the endosomal pathway
(Figure 5.3). 5-9
Cytochalasin B, bafilomycin A, and chloroquine are drugs that interfere with
the endosomal pathway and whose effects are observed at concentrations ranging
from 5 to 25 µM. 90 Cytochalasin B depolymerizes microfilaments involved in phagocytosis
without affecting other endocytotic processes such as receptor-mediated
endocytosis. 91 The effects of cytochalasin B can be observed in a dose-dependent
manner with a maximal effect at 50 µg/ml. Bafilomycin A and chloroquine interfere
with the acidification of early and late endosomes, respectively. Bafilomycin A is a
specific inhibitor of the vacuolar proton pump ATPase, and therefore prevents acidification
of early endosomes and blocks formation of endosomal transport vesicles
between early and late endosomes. 90,92 Chloroquine acts as a weak organic base and
neutralizes the pH of late endosomes. 90,93 If the cell-penetrating internalization process
occurs through the endosomal pathway, at least one of these inhibitors should
dramatically affect expression of the transgene. Bafilomycin A inhibits and chloroquine
increases expression of transgene delivered by cationic lipid or MPS vectors.
(For review, see Brisson et al. 90 )
In stark contrast with this internalization process, transfection mediated by the
peptide carrier MPG (Figure 5.4) is absolutely not affected by either of these inhibitors,
suggesting that MPG-dependent internalization is independent of the endosomal