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290 Cell-Penetrating Peptides: Processes and Applications
cargo and labeled with Abz. 1 Cellular uptake of Tat (49–57) was analyzed according
to Michaelis kinetics in parallel with penetratin and two model poly-L-Arg peptides,
allowing only approximate comparison of rate of internalization of these peptides
and not giving proper kinetic parameters. As it was shown, Tat (49–57) was internalized
almost twice as slowly as penetratin. 22 This is not in accordance with the
results of Hällbrink et al. who found that a first-order rate constant for Tat was
1.7-fold higher than that for penetratin. 1
A number of Tat analogues were synthesized and tested in T-Jurkat cells. 22 One
family of analogues comprises a truncated Tat (49–57) (the shortest peptide was Tat
51–57); another family of peptides was obtained by L-Ala-scan technique. All
analogues exhibited diminished cellular uptake. These results suggest that the cationic
residues of the Tat peptide play a principal role in its uptake, although detailed
kinetic parameters for these analogues were not obtained.
13.4.5 MAP AND MAP ANALOGUES
As shown by Oehlke et al., the internalization of the so-called model amphipathic
peptide (MAP) proceeded roughly linearly throughout a period of 60 min. 4 With longer
incubation periods, the amount of internalized peptides starts to decrease due to enzymatic
cleavage of MAP within the incubation solution. The rate of internalization is
dependent on the concentration of MAP and is approximately in accordance with firstorder
kinetics. A similar result was obtained by Hällbrink et al., 1 who observed approximately
first-order internalization kinetics of small peptide cargo attached to MAP. 1
Scheller et al. have tested internalization of 15 MAP analogues with stepwise
alterations of hydrophobicity, hydrophobic moment, and hydrophilic face, but with
unchanged positive charge and helix forming propensity. 3 Here, the fastest internalization
into the aortic endothelial cells was achieved with an analogue denoted IX
that was taken up approximately 25-fold faster than the original MAP (for details
see Chapter 4).
13.4.6 COMPARISON OF CPPS AND THE EFFECT OF CARGO
ON THE RATE OF INTERNALIZATION
Hällbrink et al. have undertaken the only complete comparative study on the rate
and yield of penetration of small cargo with TP, penetratin, Tat, and MAP, respectively.
1 The approximately first-order kinetics of internalization of cargo with all
tested CPPs was observed. The fasted uptake was achieved with MAP (t 0.5 = 7 min),
followed by TP (t 0.5 = 12 min), Tat (t 0.5 = 34 min), and penetratin (t 0.5 = 56 min).
The efficiency of cargo delivery into the cells, expressed by the apparent cellular
transport equilibrium constant (K io), matches the rate of cellular uptake. The fastest
penetrator, MAP, was also the most effective delivery vector (K io = 417), followed
by TP (K io = 353), Tat (K io = 107), and penetratin (K io = 71). Unfortunately, the high
efficiency and rate of cargo delivery into the cells is compensated by the potency
of membrane leakage induction. In case of MAP peptide, intensive leakage of
radioactive 2-[ 3 H]-deoxyglucose-6-phosphate from the cells was followed already
at 1 µM concentration. TP was not effective at this concentration, but at higher