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8 Cell-Penetrating Peptides: Processes and Applications
carrying a pyridine sulfenyl-activated thiol function at its 5′end with the free sulfhydryl
group of a cystein residue added to the C-terminal end of the Tat-transporting
peptide, as outlined in Figure 1.3. 24 This strategy allowed the coupling of activated
ODN to the carrier peptide with a good yield, provided high salt concentration and
acetonitrile were added during the coupling reaction to prevent precipitation.
It is generally assumed that the intracellular environment is reductive, thus
favoring the dissociation of the cargo ODN from the transporting peptide after
cellular internalization. 25 As shown in Figure 1.4, the chemical conjugation of a
19-mer rhodamine-labeled ODN to the Tat peptide allows cellular internalization
and nuclear delivery of the transported ODN. Whether dissociation of the ODN
cargo from the transporting Tat peptide actually took place after cell uptake was
impossible to assess in these experiments. Indeed, short ODN have a strong nuclear
tropism, once introduced in the cell cytoplasm, 26 and will therefore co-localize in
nuclei with Tat peptides.
Along the same lines, a 20-mer phosphorothioate ODN analogue complementary
to the ribosome-binding site of the P-glycoprotein mRNA was conjugated to the Tat
cell-penetrating peptide (CPP) through a disulfide bridge as well. 27 Interestingly, the
biological activity of this ODN was more efficient in the presence of serum, unlike
most nucleic acid delivery agents. 28 A specific inhibition of P-glycoprotein expression
was attained at submicromolar concentration, while much higher levels were
required in the absence of a delivery vehicle in the same experimental model. 29 This
is, to our knowledge, the only system in which the usefulness of Tat-conjugation
has been documented for the administration of antisense ODN. Coupling antisense
ODN and PNA to the Antennapedia CPP has also been documented on several
models, as will be reviewed in a separate chapter in this book. Impressively, PNAs
conjugated to the Antennapedia CPP penetrate the blood–brain barrier after in vivo
administration to rats. 30
Altogether, CPPs appear to be valuable tools for the administration of antisense
ODN and PNA even in an in vivo setting. This is particularly important since very
few synthetic nucleic acid delivery vehicles have yet proven to be useful in vivo.
Although no experimental evidence has been produced to our knowledge, conjugation
to CPP could be useful as well for the delivery of decoy oligonucleotides,
ribozymes, aptamers, or RNAi (responsible for double-stranded RNA-mediated
interference).
As mentioned earlier, we initially aimed at conjugating transported ODN and
the Tat peptide through a disulfide bridge, a strategy also used by the group of
FIGURE 1.3 (opposite) Peptide (or oligonucleotide [ODN]) Tat-CPP-conjugate synthesis
scheme. Peptide (left) or ODN (right) were synthesized with an additional sulfhydryl group
to allow an easy coupling step to the Tat CPP. Cystein can be incorporated at one (or the
other) end of the transported peptide while the SH group can be linked to the 5′ (or 3′)
terminus of an ODN during the synthesis. This thiol group can then be easily activated using
2,2′-dithiopyridine. 24 After isolation of the activated compounds, the coupling through disulfide
bridge formation with the Tat peptide can be performed. This strategy allows the quantitative
formation of the heterodimer (peptide-TatCPP or ODN-TatCPP) only. For fluorescent
microscopy or FACS analysis purposes, chimeras can be specifically labeled through an amino
group by using a succinimidyl dye.