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94 Cell-Penetrating Peptides: Processes and Applications
5.1 INTRODUCTION
Over the past 10 years, substantial progress has been made in the development of
cell-penetrating peptide-based drug delivery systems. This was directly correlated
with the dramatic acceleration in the production of new therapeutic molecules
because the cell delivery systems described until then were restricted by very specific
issues. As such, the goal became to design short synthetic peptides which would
incorporate cellular addresses and intramolecular routing signals and to formulate
synthetic peptide cargo complexes which could overcome both extracellular and
intracellular limitations. 1-5 Cell-penetrating peptides present several advantages in
that they are modulable, lack immunogenicity, easy to produce, and can incorporate
a number of specific attributes required for efficient cargo delivery. They have been
shown efficiently to introduce drugs, antisense DNA, PNA, oligonucleotides, and
small proteins into cells in vivo and in vitro. 4-9
In the design of cell-penetrating peptides, the two main barriers to overcome
are the cell membrane and the nuclear membrane. Signal sequence-based cellpenetrating
peptides described in the literature can be subdivided into two types:
natural peptides and chimeric peptides. The main cell routing elements are termed
import signals and nuclear localization sequences (NLSs) and are presumed to
function in most eukaryotic cells. Efficient uptake into cells in the absence of specific
receptors or transporters and escape from the endosomal or lysosomal compartments
are additional obstacles to be surmounted and therefore key parameters to be considered
in the rational design and engineering of cell-penetrating peptides.
Although the nucleus represents a major barrier, it also constitutes one of the
major targets for drug and gene therapy. Indeed, cell-penetrating peptides containing
NLS have been extensively used for drug and cargo delivery, with the NLS either
directly incorporated in the formulation or cross-linked to the cell-penetrating peptide
or even to its cargo. 6-10 This chapter will highlight characteristics of NLS-based
peptides designed to address the nucleus and will discuss why understanding the
mechanism of nuclear import and of structural features of cell-penetrating peptides
is essential for improvement of in vivo drug therapy.
5.2 SIGNAL SEQUENCE: THE NUCLEAR
LOCALIZATION SEQUENCE
5.2.1 INTRODUCTION: THE NUCLEAR LOCALIZATION SEQUENCE
Although the nucleus constitutes one of the major cellular compartments to be
targeted, nuclear delivery of drugs and DNA is limited by the selectivity of the
nuclear envelope. Indeed, in the case of gene therapy, for example, expression of
transcripts can only take place following delivery into the nucleus. However, transport
of therapeutic DNA from the cytoplasm into the nucleus constitutes one of the
major limiting steps in nondividing cells. Fortunately, over the last few years, the
structure and molecular mechanisms of nuclear transport have been characterized
in detail, thereby enabling more appropriate design of cell-penetrating peptides