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366 Cell-Penetrating Peptides: Processes and Applications
act to manipulate protein function or levels has driven much of our early understanding
of metabolic and signaling pathways within the cell. However, the usefulness
of small molecules is often compromised by their narrow bioavailability
profiles — either too polar for transport across the lipid membrane or too nonpolar
for effective delivery. Furthermore, unlike “information-rich” proteins, small molecule
therapy often suffers from lack of target specificity and is complicated by side
effects and toxicity. 2
Likewise, in recent years development of molecular techniques for gene delivery
with the concomitant expression of specific gene products that can alter cellular
phenotype, or with therapeutic biological activity, has been responsible for major
advances in our understanding of cellular processes, although they have offered
surprisingly little benefit in the management of genetic disorders. 3,4 Delivery of
genetic material into eukaryotic cells through use of viral vectors such as adenoviruses
or by nonviral mechanisms such as microinjection, electroporation, or chemically
through use of cationic lipids or calcium phosphate, for instance, are problematic.
The low efficiency of different nonviral vector systems and their
immunogenicity, toxicity, and inability to target many cell types, coupled with
subsequent transient and varied gene expression, have limited their efficacy in vivo.
In fact, the often massive transgene overexpression induced through these systems
has been considered a contributing factor in spurious experimental results.
17.2 PROTEIN TRANSDUCTION TECHNOLOGY
The ability to deliver full-length functional proteins into cells is problematic due to
the bioavailability restriction imposed by the cell membrane. That is, the plasma
membrane of the cell forms an effective barrier that limits the diffusion and intercellular
uptake of molecules to those sufficiently nonpolar and smaller than approximately
500 Da in size. Efforts to increase the effectiveness of protein uptake by
utilizing receptor-mediated endocytosis or through packaging of macromolecules
into caged liposomal carriers suffers due to poor cellular uptake. Intracellular sequestration
of these molecules into the endocytic pathway, preventing their release into
the cellular environment, makes them incapable of interacting with their cellular
targets.
The recent observation that certain intracellular proteins added exogenously into
media are able to pass efficiently through the plasma membrane of cells has led to
identification of a novel class of proteins from which peptide sequences termed
“protein transduction domains” (PTDs) have been derived. 5,6 To date, several proteins
have been demonstrated to possess transducing capabilities; the three most widely
studied are the Drosophila antennapedia transcription protein (AntHD), 7-9 the herpes
simplex virus structural protein VP22, 10 and the HIV-1 transcriptional activator Tat
protein. 5,6 Furthermore, identification of short basic peptide sequences from these
proteins (Antp: RQIKIWFQNRRMKWKK, Tat-(47–57): YGRKKRRQRRR),
which confers cellular uptake has led to identification and synthesis of numerous
new PTDs. 11-13
Significantly, when covalently cross-linked to full-length proteins such as βgalactosidase
or horseradish peroxidase, these PTDs are able to stimulate their