ENGINEERING OF CELL-PENETRATING PEPTIDE-CONJUGATED INTRACELLULAR DELIVERY SYSTEMS - Biologically-Responsive Hybrid Biomaterials - page 202

membrane translocating sequences (MTSs) [8]. Model peptides are CPPs that
mimic the translocation properties of known CPPs; example is the model
amphipathic peptide (MAP) [9]. Designed CPPs encompass the chimeric
peptides that are produced by the fusion of hydrophilic and hydrophobic domains
from different sources; examples are transportan, fusion of galanin and
mastoparan [10]; and MPG, chimera of peptide from fusion sequence of HIV-1
gp41 protein and peptide from the nuclear localization sequence of SV40 T-
antigen [11]. In addition, synthetic peptides such as polyarginines also show
potential for translocation [12]. All the CPPs are highly positively charged,
contributed by basic residues lysine or arginine; lysine is the primary contributor
in transportan and MAP, while arginine contributes in TAT peptide, penetratin
and pVEC. In terms of the cellular uptake and cargo delivery kinetics, MAP has
the fastest uptake, followed by transportan, TATp (48-60), and penetratin.
Similarly, MAP has the highest cargo delivery efficiency, followed by
transportan, TATp (48-60), and penetratin.
TATp, the most frequently used CPP, is derived from the transcriptional
activator protein encoded by human immunodeficiency virus type 1 (HIV-1)
[13]. Green [14] and Frankel [15] demonstrated that the 86-mer trans-activating
transcriptional activator, Tat, protein encoded by HIV-1, was efficiently
internalized by cells in vitro when introduced in the surrounding media. Later it
was shown that residues 49-57 were responsible for membrane translocation,
with the positive charge contributing largely to transduction ability of TAT [16].
These CPPs have opened a new path in drug delivery, allowing the translocation
of various cargo molecules into cells.
The mechanism of cellular translocation of CPPs is still subject to
controversial discussion and in some cases, the mechanism is cell-type or cargo
specific. Earlier it was concluded that CPPs enter cells very rapidly within
minutes even at 4°C and thus involves energy-independent mechanism [5, 11,
17, 18]. This work relied on fluorescence imaging or flow cytometry analysis of
chemically fixed cells. Then it was pointed out that certain fixation procedures
may cause artifacts leading to an overestimation of cellular uptake [19, 20].
Richard et al. demonstrated that the distribution pattern of TAT (48-60) and R9
as well as their conjugates was different in living versus fixed cells [21]. It can
be concluded that CPPs can enter the cells by two distinct routes: energy-
dependent vesicular mechanisms, referred to as endocytosis, or via a direct
process involving translocation of the lipid bilayer. Clathrin-mediated
endocytosis was suggested in [22] where TATp showed co-localization with the
classical endocytic marker, transferring. This was substantiated further in [23],
where TAT PTD and Antp PTD showed uptake only at 37°C. Studies also