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164 Cell-Penetrating Peptides: Processes and Applications
8.1 INTRODUCTION
Cellular uptake of therapeutic agents requires assistance of agents facilitating the
crossing of the natural barrier, which is the plasma membrane. A large number of
such agents have been developed in the recent past, mainly devoted to the transfer
of nucleic acids and proteins. Among them, peptides appear to be very powerful
tools for the transfer of DNAs; two main strategies are concerned: (1) covalent
linkage between cargo and vector, thus forming a conjugate, and (2) formation of a
complex between the two partners.
Within the peptides used in the first strategy, the first efficient vector developed
is polylysine, which can act as vector for oligonucleotides. 1-5 Later, more sophisticated
molecules such as the L-oligomers were proposed as vectors of cytotoxic
agents with the aim to develop an anticancer strategy. 6,7 Simultaneously, fusion
peptides 8 and peptides based on the KDEL sequence 9,10 were proposed as efficient
vectors in the antisense strategy.
Although still based on peptide sequences, vectors issued from the homeodomain
of Antennapedia were shown to translocate toward the nuclei of nervous cells in
culture. 11-15 This α-helical peptide appears to be a very powerful agent, facilitating
membrane crossing for both peptides 16,17 and oligonucleotides. 18 These properties
led to a wider search for other translocating peptide sequences; among them, the
so-called Tat-derived peptides from HIV1 were found. 19-21 Also, other peptides
derived from natural sequences have been proposed. These are a peptide-based on
neuropeptide Y, 22 shown to be an efficient vector for daunorubicin and doxorubicin
although it requires the presence of a receptor, and a peptide issued from human
calcitonin that acts through excised bovine nasal mucosa. 23
As to the second strategy that involves the formation of complexes, besides the
well documented approaches based on viruses, 24-27 liposomes, 28 cationic lipids, 29 or
PEI, 30 peptidic vectors have also been investigated. All of them share the common
presence of cationic residues, but differ mainly from each other by distribution of
the basic residues along the peptide chain and thus by their ability to form an
amphipathic structure. 31-42 According to the conformation involved in the formation
of the amphipathic structure, these vectors are able to translocate charged molecules
such as DNA or hydrophobic drugs.
All these peptides are short (below 40 residues) and constitute a series of vectors
which validate the peptide approach. However, none of the peptidic vectors reported
so far can be considered perfect. In order to attempt and define a new generation of
nontoxic vector peptides with specific targeting, a prerequisite is to identify the
mechanism leading to the cellular internalization process of vectors in the free form
and when associated with the cargo. Two main processes with different mechanisms
leading to cellular internalization must be examined: through the endosome pathway
or by direct penetration into the cytoplasm.
In both cases, an obligatory step in such an approach is devoted to structural
investigations for identification of structural requirements and their optimization for
design of efficient vectors. The cellular internalization mechanism can be summarized
as a three-step process; (1) uptake by the cellular membrane, (2) translocation
through the membrane, and (3) release on the cytoplasmic side into the cell interior.