Membrane Crossover by Cell-Penetrating Peptides: Kinetics and Mechanisms – From Model to Cell Membrane Perturbation by Permeant Peptides - Intracellular Delivery: Fundamentals and Applications - page 184

reached after 15 min for transportan and after 1 h for penetratin. Similar results were
obtained by Drin (Drin et al. 2003 ), using a NBD-labeled fluorescent penetratin
analogue. The kinetics plateau was reached after 1 h incubation at 37°C of non-
adherent human K562 leukemia cells with 1 mM NBD-penetratin. We have reported
also kinetics of internalization at 37°C and 4°C of a biotin-labeled and photoactivat-
able penetratin analogue (Jiao et al. 2009 ). The kinetics was determined in CHO-K1
cells and CHO-pgA745 (GAG-deficient) cells. It was shown that at 37°C the plateau
was reached after 1 h incubation of 5 mM penetratin with CHO-K1 and after 30 min
with CHO-pgA745 (Jiao et al. 2009 ). Another study with a Tat-conjugated cargo
also reported similar kinetics using a fluorescence assay (Cheung et al. 2009 ). In
addition, Pep-1 (Ac-KETWWETWWTEWSQPKKKRKV-cysteamine), a rationally
designed cell-penetrating peptide that can establish hydrophobic interactions with
cargo molecules (thus that does not require a covalent link with these latter) is able
to convey b-galactosidase into cells with a similar time course (Henriques et al.
2005 ). However, faster (in the range of seconds) and slower (tens of minutes) inter-
nalization kinetics could be measured for different cell-penetrating peptides
(Eiríksdóttir et al. 2010a ) using a releasable luciferin assay (Jones et al. 2006 ). These
results led the authors to classify cell-penetrating peptides according to the internal-
ization kinetics, which reflect their uptake pathways, as translocation for the fast
kinetics and endocytosis for the slower one (Eiríksdóttir et al. 2010a ).
However all these studies were done with a population of cells, thus the kinetics
observed are not the kinetics of single cells but an average of the internalization
kinetics in a population.

5 Pathways of Internalization in Cells

Regarding the internalization pathways of cell-penetrating peptides, there is a huge
discrepancy between reported studies. The important point that is now spreading in
literature is that any single chemical modification of the peptide sequence severely
impacts the internalization pathways of the resulting compounds (Maiolo et al.
2005 ; El Andaloussi et al. 2007 ; Aussedat et al. 2008 ; Walter et al. 2009 ), as well
as the cell-type (Mueller et al. 2008 ). These observations are quite understandable
as any modification in the peptide or in membrane components must also affect
peptide/membrane interactions.
Although still controversial for some cell-penetrating peptides, it is nonetheless
quite clear that there are multiple internalization pathways for cell-penetrating pep-
tides (Nakase et al. 2008 ; Jiao et al. 2009 ; Alves et al. 2010 ). It came out that the
discrepancies between reported studies should have arisen from the experimental
conditions used, principally the concentration of peptide and the chemicals used to
inhibit internalization pathways, that could also have side-effects (Ivanov 2008 ).
All pinocytosis pathways have indeed been suggested for cell-penetrating peptide
internalization, especially macropinocytosis (Jones 2007 ) that is a reported mecha-
nism for penetratin (Amand et al. 2008 ), oligoarginine (Nakase et al. 2004, 2007 ),